WO2009100218A2 - Kruppel-like transcription factor klf4/gklf and uses thereof - Google Patents

Kruppel-like transcription factor klf4/gklf and uses thereof Download PDF

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WO2009100218A2
WO2009100218A2 PCT/US2009/033219 US2009033219W WO2009100218A2 WO 2009100218 A2 WO2009100218 A2 WO 2009100218A2 US 2009033219 W US2009033219 W US 2009033219W WO 2009100218 A2 WO2009100218 A2 WO 2009100218A2
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klf4
tumor
cells
ntcl
staining
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WO2009100218A3 (en
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John Michael Ruppert
Susan M. Ruppert
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Uab Research Foundation
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/57415—Specifically defined cancers of breast
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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Definitions

  • Cellular oncogenes have been isolated by characterization of transforming retroviruses from animal tumors, by examination of the breakpoints resulting from chromosomal translocation, and by expression cloning of tumor DNA molecules using mesenchymal cells such as NIH3T3.
  • Several human tumor types exhibit loss-of-function mutations in a tumor suppressor gene that lead to activation of a specific oncogene in a large proportion of tumors.
  • c-MYC expression is regulated by the APC colorectal tumor suppressor; expression of GLI is activated by loss-of-function of PTC in human basal cell carcinoma and in animal models; E2F is activated by loss-of-function of the retinoblastoma susceptibility protein pi 05Rb; and RAS GTPase activity is regulated by the familial neurofibromatosis gene NFl.
  • the comparative genomic hybridization assay and related methods have shown that numerous uncharacterized loci in tumors undergo gene amplification.
  • KLF Kruppel-Like Factor/Epithelial Zinc Finger
  • Figure 1 shows Notch (Ntc) maturation and signaling.
  • Figure IA shows structure of Ntc proteins.
  • Ntc EC extracellular domain (-180 kDa) generated from full length by cleavage at site Sl in Golgi.
  • NtcTM transmembrane form (—120 kDa) generated by cleavage at S2 by TACE.
  • Ntc IC intracellular form ( ⁇ 90 kDa), generated by cleavage at S3 by ⁇ -secretase.
  • LP leader peptide
  • TM transmembrane domain
  • ANK 6 ankyrin repeat domain;
  • Ntc IC displaces histone deacetylases (HDACl) and co-repressors (Hairless, CoR) from CSL. Then Mastermind- Like (MAML) proteins and histone acetylases (p300, CAF) can bind.
  • HDACl histone deacetylases
  • MAML mastermind- Like
  • p300, CAF histone acetylases
  • the complex activates transcription through CSL binding sites in cellular promoters such as Hes (Hairy/Enhancer of split) genes.
  • Alternate Pathways signaling mediated by proteins other than CSL.
  • KLF4 can activate Ntc expression, and can alter Ntc signaling by induction of the CSL- antagonist Hairless. This results in inhibition of Classical Pathway signaling as indicated by CSL activity and expression of Hesl (bottom left), and instead promotes Alternate Pathway signaling (bottom right).
  • Figure 3 shows Notchl is a direct transcriptional target of KLF4 in breast epithelial MCFlOA cells.
  • Figure 3 A shows KLF4-ER- or Vector (pB ⁇ uro)-transduced RK3E cells were treated with cycloheximide (CHX), or 4-hydroxytamoxifen (4OHT) as indicated above the lanes, and total RNA was isolated two hours later.
  • Northern blot analysis used a labeled PCR fragment of rat Ntcl (upper panel). The membrane was stripped and reprobed with a cloned human /3-tubulin (/3-Tub) cDNA fragment as a loading control (below).
  • FIG. 3B shows KLF4-ER- or Vectorexpressing RK3E cells were either not treated (left panels) or treated with 4OHT (right panels) for 10 hours. Indirect immunofluoresence staining used ⁇ -Ntcl C-20 antibody (Santa Cruz), Phalloidin and DAPI. Scale bar is 20 ⁇ m.
  • Figure 3C shows a schematic of the human Ntcl promoter (- 9903 to +240 bp relative to the initiation codon, ATG). Vertical lines indicate consensus KLF4 binding sites.
  • a broken arrow indicates the putative start site of transcription as determined by homology with the mouse gene.
  • K-P indicate the positions and sizes in bp of PCR fragments scanned by CbJP.
  • Figure 3D-F show ChIP. MCFlOA cells expressing HA-KLF4 were treated with formaldehyde. Sonicated, cross-linked chromatin was isolated and immunoprecipitated (IPd) with the indicated antibodies. The IPs were analyzed by PCR to amplify fragments K-P (panels F and D) from the Ntcl promoter or a fragment from the human GAPDH promoter (panel E). Lanes 2 and 6 show PCRs performed with 0.02% of the input material used for IP. In lane 1, template DNA was omitted (negative control).
  • Figure 3F the efficiencies of IP of fragments K-N relative to that of fragment O are indicated below the panels.
  • Figure 3G (left) is a schematic showing deletion mutants ⁇ 1- ⁇ 5 of the human Notchl promoter cloned in pGL3-Pro. These were transiently transfected with pTK-/3-gal (control) into HEK293 cells. The relative transcription efficiency of the mutants is shown in the histogram on the right. The experiment was done three times in triplicate and SE bars are shown.
  • Figure 3H shows KLF4 induces Notchl ligands by an indirect (slow-onset) mechanism.
  • Figure 4A shows rapid upregulation of Notchl following induction of KLF4 in the skin of 'tet-on ! K14-rtTA; TREEXF4 transgenic mice.
  • KLF4 was induced by addition of dox to the drinking water, and skin sections were obtained from two mice at each of the times shown. The sections were stained with rabbit oKLF4 or ⁇ Ntcl-C20. The top and bottom panels represent nearly adjacent sections of one tissue sample. Scale bars, 50 ⁇ m.
  • Figure 4B-D show expression of Notchl and KLF4 are strongly correlated in primary human breast tumors.
  • Figure 4B shows staining of sub-adjacent sections of a representative breast tumor with the indicated antibodies. Scale bar, lOO ⁇ m.
  • Figure 4C shows Pearson correlation analysis of KLF4 and Notchl in 89 cases of breast cancer. Scores were obtained using a 0.0-4.0 scale, with weighting based on the percentage of cells exhibiting each score. Cytoplasmic KLF4 was likewise correlated with Ntcl, with similar P and r values. Tumors with the same scores appear as one data point.
  • Figure 4D shows contingency analysis of Notchl and KLF4 (Fisher's exact test, two-tailed).
  • Figure 5 shows transformation by KLF4 requires Ntcl signaling.
  • Figure 5 A shows RK3E epithelial cells stably transduced with antisense (AS) Ntcl 10 or Vector were superinfected with KLF4, Ras, c-Myc or GUI retroviruses and allowed to form foci for four weeks. The Wright-stained dishes shown are representative of three or more independent experiments. Transformation by RAS is expected to require Ntcl (see Miele L., Nat. Med. 2001).
  • Figure 5B shows the results in A were quantitated by counting of foci. SE bars are shown.
  • FIG. 5C shows RK3E cells were transfected with increasing amounts of pcDNA3-Ntcl AS, and a constant amount of Ntcl , Hesluc reporter, and pTK-jS-Gal (a control for transfection efficiency). Luciferase activity was normalized to ⁇ - Gal. The experiment was performed in triplicate and SD bars are shown.
  • Figure 5D shows RK3E cells were transduced by retroviruses endcoding Ntcl IC or KLF4-ER and allowed to form foci for two weeks in the presence of vehicle (DMSO) or 7SI (L-685,458). Transformation by the S3 cleavage product Ntcl IC is expected to be ⁇ Si-independent (see Fig. IA).
  • Figure 6 shows neoplastic transformation of RK3E cells by full length Notch 1 (Ntc 1).
  • Figure 6A shows FL Focus formation by RK3E cells infected with Vector, Ntcl IC or Ntc 1 FL retroviral supernatants.
  • Figure 6B shows immunoblot of extracts from cloned cell lines derived from individual Ntcl foci showing increased expression of Ntcl FL (lanes 3- 9) as compared with Vector cells (lanel) or Ntcl IC cells (lane 2). The same samples were probed for jS-actin (control; lower panel).
  • Figure 6C shows Ntcl FL transformation correlates with increased Ntcl expression.
  • RK3E cells were transduced, selected in puro, and either cultured at subconfluence (Ntcl FL *) or incubated for 2 weeks at confluence until a transformed morphology was observed (Ntcl FL ).
  • Whole cell extracts were analyzed by immunoblot with ⁇ -Ntcl C20 which recognizes all three species (top panel). The filter was stripped and reprobed with V 1744 which is specific for Ntcl IC (middle panel). Additional species (uncharacterized) that interact with C-20 appear in Ntcl FL and Ntcl IC transformed cells (asterisk). Amidoblack staining of spotted cell extract was used as a loading control (LC).
  • Ntcl FL requires cleavage to Ntcl IC by ⁇ -secretase for a transformed growth phenotype.
  • Ntcl IC and Ntcl FL expressing cells were plated at low confluence in six well plates and allowed to form colonies in the presence of 7SI or vehicle (DMSO). This colony morphology assay was described previously (Foster, KW et al., Cell Growth Differ 1999). 7SI reduced the density of Ntcl FL but not Ntcl IC colonies. This strongly supports specificity since the product of the blocked enzymatic step, Ntcl IC , should be resistant to 7SI.
  • Figure 7 shows KLF4 blocks classical pathway activation by Notchl
  • Figure 7A shows KLF4 suppresses classical Ntcl signaling. Hesl-luc, 8x wt CSL and 8x mutant CSL reporters were used in transient transfection assay of RK3E cells. The experiment was done in triplicate (bars, SD). Similar results were obtained in HEK293 cells.
  • Figure 7B shows two mechanisms of transformation by Notchl .
  • KLF4 is low/absent (left panel)
  • Notchl induces transformation by signaling through the classical pathway.
  • Activated Ntcl IC relocates to the nucleus where it displaces Hairless (Hr) and Co- repressors (CoR) from CSL.
  • MAML and Co-activators (CoA) are recruited to the complex which then activates targets such as the Hes genes.
  • Notchl induces transformation through an alternative pathway.
  • Figure 8 shows dominant negative (DN) MAML and DN CSL block transformation of RK3E by NtclIC but not by KLF4 or RAS.
  • Figure 8 A shows to detect expression of DN proteins, lysates from RK3E cell lines stably expressing Vector or DN MAMLs were probed with ⁇ -MAMLl . The position of MAMLDN2 protein is indicated by an arrow (top panel). As MAMLDNl is not recognized by ⁇ -MAMLl, GFP antibody was used to detect MAMLDNI-GFP fusion protein (middle panel). /3-actin served as a control for protein loading (bottom panel).
  • Figure 8B shows semi-quantitative RTPCR was done with total RNA isolated from the indicated cell lines using mouse-CSL specific primers.
  • FIG. 8C shows classical Notchl signaling is silenced in RK3E cells that stably expressing DN MAML or DN CSL. Hesl-luc reporter activity was measured in MAML DNl, MAML DN2, CSL DN or Vector (pQCXIN) cells 48 hrs. after co-transfection of reporter with Ntcl or Vector. Experiments were done twice in triplicate (bars, SE).
  • Figure 8D shows focus formation assay. Cells stably carrying the retroviral vector indicated above the dishes were superinfected with Ntcl IC , KLF4, RAS or pBPuro retroviral supernatant as indicated on the left. Plates are representative of three independent experiments done in triplicate. Insets show KLF4 foci at higher magnification.
  • Figure 8E shows quantification of foci. The histogram shows number of foci in DN cell lines normalized to Vector (bars, SD).
  • Figure 9 shows maturation of MCFlOA is blocked by KLF4 or Ntcl ic .
  • MCFlOA cells were plated on a layer of Matrigel, and underwent morphogenesis over a period of 10-14 days to form smooth, round, hollow spheres of epithelium (Vector panels).
  • Cells stably transduced with erbB2 formed extended, multi-lobular structures with smooth, round borders, consistent with hyperplasia and epithelial differentiation.
  • cells transduced with KLF4 or Ntcl retained the disorganized structure observed at 4-5 days, with rough, irregular borders, indicating an arrest of epithelial differentiation. The developmental arrest persisted until 19 days, when the experiment was terminated.
  • Scale bars lOOjLtm.
  • Figure 10 shows phenotypes of KLF4 proteins.
  • Figure 1OA shows to detect expression of endogenous KLF4 transcripts, total RNA was isolated from the indicated cell lines and analyzed by Northern blot (left panel). A /3-tubulin probe served as a control for loading.
  • rabbit oKLF4 upper panel
  • normal rabbit Ig lower panel
  • Bound Ig was detected using a red- fluorescent secondary ab. Signal corresponding to KLF4 is shown superimposed on the cell nuclei (TRITC+DAPI). Scale bars, 50 ⁇ m.
  • Figure 1OB shows mammary epithelial cell lines (MCFlOA or MCFlOAT) were transduced using the indicated retrovirus supernatants (Vector, KLF4 ala5 , KLF4 ala6 ) and selected in puromycin. Surviving colonies were stained with modified Wright's stain after 10 days in selection. MCF10A-KLF4 DN colonies surviving puromycin were estimated as ⁇ 1% of the Vector control. MCF10A-KLF4 WT cells grew similarly, or formed even more dense colonies, as compared to the Vector control. Similar results were obtained in three independent experiments.
  • Figure 1OC shows RK3E cells were transduced using the retrovirus supernatants Vector, KLF4 ala5 , or KLF4 ala6 (Construct 1) and selected in puro. Surviving cells were transduced with c-MYC, ErbB2, GLIl, N-RAS, Notch 1 or KLF4 retroviruses (Construct 2) and incubated in puromycin at confluence for 3 weeks to allow the oncogene to induce transformation (there is no drug selection for the Construct 2 in focus assays). The background observed when Construct 2 is Vector is shown in the inset (CtI). Two independent experiments were performed in triplicate. Similar results were observed for KLF4-ala6.
  • Figure 1OD shows Results of one experiment, including standard deviation bars, are shown. The ⁇ 10-fold suppression for ErbB2 shown in C is consistently observed (2 expts, 6 dishes total).
  • FIG 11 shows RAR ⁇ RXR ⁇ and NUR77 are direct transcriptional targets of KLF4.
  • Figure 1 IA shows KLF4-ER or vector expressing cells were treated for two hours with 4OHT and CHX, RNA was isolated and assayed by microarray (Affimetrix chip RAE U230A). Two completely independent experiments were done. The histogram shows the induction of the indicated genes.
  • p21 is a known target of KLF4.
  • Figure HB shows RNAs prepared as in A were used as templates for RT-PCR using oligonucleotides specific to rat RARy and GAPDH (control).
  • Figure 11C shows RK3E cells stably expressing KLF4-ER or Vector were induced with 4OHT for the times indicated above the lanes.
  • Figure 12 shows RXR-selective agonist (9cUAB30) specifically blocks KLF4 induced transformation.
  • Figure 12A shows K14-rtTA;TRE-KLF4 transgenic mice were induced with dox for 2 weeks. Food was admixed with placebo or with 9cUAB30. Induction of dysplasia-SCC was scored by gross and microscopic exam. No abnormality was observed in 8/9 animals treated with 9 ⁇ JAB30.
  • FIG. 12B shows retroviral supernatants were generated using the expression vectors indicated on the left and applied to RK3E cells. Dishes were incubated for 3-4 weeks with addition of 9cUAB30 or DMSO to the culture media every two days. Transformed foci were identified by Wright stain. Similar results were obtained in 3 independent experiments, each performed in replicate.
  • Figure 13 shows efficacy of 9cUAB30 in therapy of established breast cancers.
  • Breast cancers were induced in female rats using MNU and therapy was initiated at day 0, when the tumor size was -200 mm 2 as described in the text. (Bars, SD).
  • Figure 14 shows KLF4-high tumor cells are sensitive to ⁇ SI or 9cUAB30.
  • Figure 14A shows cells were plated at 5,000 cells/well in 24 well plates and treated with DMSO (0.2%) control, 1.0 M 9cUAB30, or 10 M 7SI in media/10% FBS. Drugs were added 24 hours after plating and drugs/medium was changed every other day. ATP levels were determined after 5 days of treatment.
  • KLF4 expression levels H, high; I, intermediate; L, low
  • Ntcl IC expression was shown by western blot (NT, not reported) (Stylianou S. et al., 2006).
  • Figure 14B shows dose-response of MCF7 was determined at 5 days.
  • Figure 14C shows in an experiment independent from the work shown in panel A, drugs were applied singly or in combination and MCF7 growth was determined after 5 days. For all data the mean of 4 replicates is shown. Bars, SD.
  • Figure 15 shows the genotype determines the tumor type following oral DMBA treatment of genetically altered mice.
  • Figure 15A shows Kaplan-Meier analysis. Insets show the number of animals in each treatment group (N), the number of tumors of all types (T), the average multiplicity of tumors (T/N). The % of animals in each group that developed breast cancer or SCC is indicated (Tumor Incidence).
  • Figure 15B shows characterization of Klf4 ab (Hl 80). SCC was induced by expressing human KLF4 in the skin of transgenic mice (Tet-on SCC). Hl 80 showed prominent nuclear and cytoplasmic staining, as published for this model (Foster KW et al., Oncogene 2005).
  • Hl 80 also showed staining of endogenous Klf4 in normal mouse skin and adnexa (Normal skin). IgG was used as a negative control. Arrows indicate the dermoepidermal junction (DEJ).
  • Figure 15C Staining of breast cancers from the MMTV-ErbB2;DMBA model. Klf4 and Ntc 1 were co-positive in Case #8 (see Table 3) but negative in Case #10. Scale bars, 100 ⁇ m.
  • FIG 16 shows KLF4 is expressed in primary human pancreatic cancers. Paraffin sections were stained with KLF4 antibody (Hl 80, Santa Cruz). Results were confirmed using an anti-KLF4 rabbit antibody (Foster KW et al., Oncogene 2005). DAB staining is indicated. As published for other tumor types, some tumors show mainly cytoplasmic staining (Case 3), others show nuclear staining (Case 5), and others appear mixed (Cases 2, 4). Normal rabbit antibody (Dako) was used as a negative control (upper right). Scale bar, 50 ⁇ m.
  • FIG 17 shows KLF4 expression during pancreatic tumor progression in the PDX-Cre; LSLRasG12D; Smad41oxP/loxP mouse model.
  • KLF4 immunostaining was low or absent in normal pancreatic ducts (Normal pancreas, arrows) or acini (arrowhead).
  • a pancreatic islet is shown under the scale bar (50 ⁇ m).
  • KLF4 was prominent in the early neoplastic ducts (PanlN) and was consistently detected where neoplastic ductal cells appeared more invasive (Pancreatic tumor).
  • Figure 18A shows Western blot analysis of human pancreatic cancer lines. A single filter was probed in sequence with each of the antibodies shown. Molecular weight markers are shown on the right. As a control, RK3E cells transduced with a KLF4 vector or an empty vector were analyzed in parallel (lanes 11-12).
  • Figure 18B shows siRNA- mediated knockdown of KLF4. The KLF4-high cell line Pane 10.05 was tranfected with the indicated siRNA, and extracts were analyzed at 96h. A single filter was sequentially analyzed with the indicated antibodies. siRNA-Ctl is a commercially supplied control (Dharmacon) without similarity to human mRNAs.
  • Ntcl knockdown was likewise observed when siRNA#2 or siRNA#3 were transfected alone.
  • Figure 18C shows Growth effects of KLF4-Ntcl knockdown in pancreatic cancer cells. siRNAs were transfected in 6-well plates. Cell number at 96h posttransfection was estimated using the Bradford assay to quantitate total cellular protein. The results shown are the mean of two sequential, independent experiments performed by different operators (JMR and QL). Using a novel transfection protocol that we developed, transfection efficiencies for the two cell lines were estimated as >90% based upon a KIFl 1 control that gives mitotic arrest and cell death.
  • Figure 19 shows cytoxicity of KLF4 inhibitors in pancreatic cancer.
  • Cells were plated at 1000 cells per well and allowed to equilibrate for 24h. Cells were then treated as indicated for 5 days.
  • the SI was L685,458, which is a nontoxic, oncogene-specific antagonist of KLF4-induced transformation (RK3E cells).
  • RK3E cells KLF4-induced transformation
  • Some other ⁇ SIs tested were not compatible with long-term (3 weeks) cell culture assays. Cell number (normalized to DMSO control) was determined using the Pierce ATPLite assay (luciferase assay of ATP).
  • Figures 19A-C show experiments performed once in triplicate.
  • Figure 19D shows results of one triplicate experiment. Similar results were obtained in an independent experiment. Data for each cell line (4-5 conditions) were analyzed by 1 way ANOVA with post-Tukey test. Bars, SD.
  • Figure 20 shows subjacent sections of primary human breast tumor stained with the indicated antibodies. Within the section were areas representing either infiltrating ductal carcinoma (Tumor, upper row) or adjacent, uninvolved epithelium (lower row). Scale bar, 50 ⁇ m.
  • Figure 21 shows immunoblot analysis of Nl IC following siRNA-mediated suppression of KLF4 in BT474 and ZR75-1 human breast cancer cell lines.
  • the upper and lower portions of a filter representing a single SDS-PAGE gel were queried in parallel with Nl IC -Nter or KLF4 (Hl 80) antibodies, respectively.
  • the lower portion was reprobed with ⁇ -tubulin antibody (loading control).
  • Figure 22 shows KLF4-ER cells transfected with Notchl -specific siRNAs, and Nl IC expression was determined by immunoblot 72 hours later. A portion of the filter was queried in parallel with -tubulin antibody (loading control).
  • Figure 23 shows quantification of transformed foci from the experiment shown in Fig. 24.
  • Figure 24 shows RK3E cells transfected with Notchl -specific siRNAs and then transduced with KLF4-ER retrovirus 24 hours later. Transformed foci were scored at 20 days. ErbB2 served as a control oncogene and showed no inhibition by siRNAs. Three independent experiments were performed in duplicate.
  • Figure 25 shows inhibition of canonical Notchl signaling by KLF4.
  • Figure 25 A shows semi-quantitative RT-PCR used to analyze expression of Notchl and Notchl - regulated genes following activation of KLF4.
  • Figure 25B shows immunblot analysis of extracts prepared in parallel with the RNAs used in panel A. The filter was queried with Hesl antibody, then stripped and reprobed with /3-actin antibody (loading control).
  • Figure 25C shows RK3E cells transfected with the indicated luciferase reporter and an internal reference in combination with Nl IC , KLF4, and/or empty vector. Following normalization to adjust for differences in transfection efficiency or cell number, the basal activity of each reporter was assigned a value of 1.0 (lanes 1, 7, and 13). The data represent one experiment performed in duplicate. Similar results were obtained in independent experiments.
  • Figure 26 shows immunostaining of human breast cancers with antibodies to KLF4, and Notchl (N1-C20, and NlIC-Nter). Staining of tumor cells is indicated by a brown precipitate. Cases shown are representative of a total of 8 cases with positive staining and 4 cases with low/negative staining. No staining was observed when normal rabbit immunoglobulin was used as the primary antibody (not shown). Scale bar, 50 ⁇ m.
  • compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
  • compositions and methods relating to the identification, diagnosis, and/or treatment of tumors based on expression of Gut-Enriched Kriippel-Like Factor (GKLF) protein/ Kruppel-like factor 4 (KLF4) are disclosed herein.
  • GKLF Gut-Enriched Kriippel-Like Factor
  • KLF4 Kruppel-like factor 4
  • a significant number of transcription factors use a conserved zinc finger domain to bind their target DNAs.
  • the human genome encompasses over 700 genes that contain a particular C 2 H 2 -type of zinc finger, which employs two cysteine and two histidine amino acid residues to coordinate the single zinc atom in the finger-like structure.
  • a further subgroup of the C 2 H 2 -zinc finger proteins exhibits homology to the Drosophila melanogaster segmentation gene product, Kr ⁇ ppel.
  • KLFs Kriippel-like factors
  • KLF family members are characterized by their three Cys 2 His 2 zinc fingers located at the C-terminus, separated by a highly conserved H/C link. DNA binding studies demonstrated that the KLFs have similar affinities for different GC -rich sites, or sites with CACCC homology, and can compete with each other for the occupation of such sites. KLFs also share a high degree of homology between the specificity protein (Sp) family of zinc-finger transcription factors and bind similar, if not the same sites, in a large number of genes.
  • Sp specificity protein
  • KLF4 also known as Gut-enriched Kruppel-Like Factor (GKLF) acts as a transcriptional activator or a transcriptional repressor depending on the promoter context and/or cooperation with other transcription factors.
  • GKLF Gut-enriched Kruppel-Like Factor
  • KLF4 transactivates the iNOS promoter in cooperation with p65 (ReIA), and the p21Cipl/Wafl promoter in cooperation with p53, but it directly suppresses the p53 promoter and inhibits ornithine decarboxylase promoter activity by competing with specificity protein- 1 (Sp-I).
  • Sp-I specificity protein- 1
  • KLF4 also interacts with the p300/CBP transcription co-activators.
  • KLF5 also known as Intestinal enriched Kruppel-Lke Factor (DCLF) or basic transcription element binding protein 2 (Bteb2) has been assigned purely transcriptional activation activity but, similar to KLF4, binds p300 which acetylates the first zinc finger conferring a trans-activating function.
  • DCLF Intestinal enriched Kruppel-Lke Factor
  • Bteb2 basic transcription element binding protein 2
  • KLF4 & KLF5 the amino acids that are predicted by the Klevit model to interact with DNA are identical and the two compete for the same CACCC element found in a wide variety of promoters.
  • KLF4 & KLF5 can act antagonistically during cellular proliferation, differentiation, and promoter activation, either via direct competition or via alterations in their own gene expression.
  • KLF4 inhibits proliferation through activation of p21Cipl/Wafl, and direct suppression of cyclin Dl and cyclin Bl gene expression. Both KLF4 & KLF5 proteins act on the klf4 promoter where KLF4 increases expression and KLF5 decreases expression of klf4 mRNA.
  • KLF4 A full-length mouse cDNA clone encoding KLF4 was initially isolated from a NIH3T3 cDNA library by reduced-stringency screening with a DNA probe containing the zinc finger region of an immediate early gene product, Zif268 or Egrl.
  • Mouse KLF4 contains 483 amino acids, has a predicted molecular weight of 53 kD, and is 90% identical to human KLF4.
  • the carboxyl terminus of KLF4 contains three C 2 H 2 -zinc fingers.
  • KLF4 is a nuclear protein whose cellular address depends on two nuclear localization signals. A survey of the tissue distribution in adult mice revealed that KLF4 is highly expressed in terminally differentiated, post-mitotic epithelial cells of the intestinal tract, a finding consistent with the antiproliferative effect of KLF4.
  • KLF4 is a member of the SpI -like family of transcription factors (Kaczynski, J., et al. 2003; Bieker, J. J. 2001).
  • the 53kDa protein contains an N-terminal transactivation domain, a repression domain, a nuclear localization signal, and three C-terminal zinc fingers (ZFD) (Geiman, D. E., et al. 2000; Yet, S. F., et al. 1998; Ruppert, J. M., et al. 1988).
  • KLF4 By use of expression cloning in RK3E cells KLF4 was identified as an oncogene (Foster, K. W., et al. 1999). KLF4 is expressed in post-mitotic cells of the skin and gut, and may function as a tumor suppressor in certain contexts (Garrett-Smha, L. A., et al. 1996; Shields, J. M., et al. 1996; Dang, D. T., et al. 2003; Zhao, W., et al. 2004). Klf4 functions in the suprabasal layers of the skin to form the water permeability barrier. Klf4-/- mice develop normally, but die soon after birth from dehydration (Segre, J. A., et al. 1999).
  • KLF4 mRNA and protein is up-regulated early in progression of breast cancer, oral squamous cell carcinoma (SCC), and cutaneous SCC (Foster, K. W., et al. 1999; Foster, K. W., et al. 2000; Foster, K. W., et al. 2005; Huang, C. C, et al. 2005).
  • KLF4 is down-regulated in several other tumor types (Foster, K. W., et al. 2000; Huang, C. C, et al. 2005).
  • dysplasia results from increased KLF4 in proliferation-competent cells of the basal layer (Foster, K. W., et al. 1999).
  • Breast and skin share a common embryologic origin, and tumors of these tissues show similar genetics (infrequent mutation of RAS, more frequent alterations of p53, pl6INK4A, c-MYC, ErbB family members) (Ruppert, J. M., et al. 1997; Forastiere, A., et al. 2001; Mao, L., et al. 2004).
  • Klf4 and just three other genes were found sufficient to induce the pluripotent embryonic stem cell phenotype (Takahashi, K., et al. 2006). These pluripotent cells contributed to embryonic development when injected into blastocysts, and when injected into athymic mice they formed tumors with tissues from all three germ layers. Consistent with this observation, KLF4 functions as an oncogene in several contexts (Foster, K. W., et al. 1999; Rowland, B. D., et al. 2005; Suzuki, T., et al. 2002; Li, Y., et al. 2005).
  • KLF4-deficient gut epithelium revealed a role of KLF4 in cell fate (Sancho, E., et al. 2003). These mice show selective loss of goblet cells, which derive from stem cells following several binary cell fate decisions (Sancho, E., et al. 2003). Just as KLF4 promotes the goblet cell fate, loss of CSL or Hesl or even induction of Ntcl induces goblet cells, linking the KLF4 and Ntcl pathways in cell fate specification (Sancho, E., et al. 2003; Zecchini, V., et al. 2005).
  • KLF4 induces a profile (Ntcl high, Hesl low, c-MYC low, p21/Wafl high) that is the pattern expected of goblet cells, indicating that KLF4 functions as a regulator of cell fate in epithelia (Segre, J. A., et al. 1999; Sancho, E., et al. 2003; Katz, J. P., et al. 2005; Katz, J. P., et al. 2002).
  • Notchl Ntcl
  • the Notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms. Notch is present in all metazoans, and vertebrates possess four different notch receptors, referred to as Notchl to Notch4.
  • the Notch receptor is a single-pass (i.e. it crosses the membrane once, in contrast to many other transmembrane proteins which loop back and forth between the extracellular and intracellular spaces) transmembrane receptor protein. It is a hetero-oligomer composed of a large extracellular portion which associates in a calcium dependent, non-covalent interaction with a smaller piece of the Notch protein composed of a short extracellular region, a single transmembrane-pass, and a small intracellular region.
  • the Notch protein sits like a trigger spanning the cell membrane, with part of it inside and part outside. Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to alter gene expression.
  • Notch signaling also has a role in neuronal function and development, stabilizing arterial endothelial fate and angiogenesis, regulating crucial cell communication events between endocardium and myocardium during both the formation of the valve primordial and ventricular development and differentiation.
  • Notch signaling also has a role in cardiac valve homeostasis and has implications in other human disorders involving the cardiovascular system. Additionally, Notch signaling has a role in timely cell lineage specification of both endocrine and exocrine pancreas, influencing binary fate decisions of cells that must choose between the secretory and absorptive lineages in the gut, expanding the HSC compartment during bone development and participates in commitment to the osteoblastic lineage suggesting a potential therapeutic role for Notch in bone regeneration and osteoporosis.
  • Notch signaling has a role in regulating cell-fate decision in mammary glands at several distinct development stages, and some non-nuclear mechanisms, such as controlling the actin cytoskeleton through the tyrosine kinase AbI.
  • Notch signaling is often repressed in many cancers, and faulty Notch signaling is implicated in many diseases including T-ALL (T-cell acute lymphoblastic leukemia), CADASIL (Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy), MS (Multiple Sclerosis), Tetralogy of Fallot, Alagille syndrome, and myriad other disease states.
  • T-ALL T-cell acute lymphoblastic leukemia
  • CADASIL Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy
  • MS Multiple Sclerosis
  • Tetralogy of Fallot Alagille syndrome
  • Maturation of the Notch receptor involves cleavage at the prospective extracellular side during intracellular trafficking in the Golgi complex. This results in a bipartite protein, composed of a large extracellular domain linked to the smaller transmembrane and intracellular domain. Binding of ligand promotes two proteolytic processing events; as a result of proteolysis, the intracellular domain is liberated and can enter the nucleus to engage other DNA-binding proteins and regulate gene expression.
  • Notch and most of its ligands are transmembrane proteins, so the cells expressing the ligands typically need to be adjacent to the Notch expressing cell for signaling to occur.
  • the Notch ligands are also single-pass transmembrane proteins and are members of the DSL (Delta/Serrate/LAG-2) family of proteins.
  • TACE Tumor Necrosis Factor Alpha Converting Enzyme
  • KLF4 is a regulator of Ntcl transcription and signaling.
  • Ntcl first identified as a translocation partner in T cell acute lymphoblastic leukemia (T-ALL) (Ellisen, L. W., et al. 1991), is a transmembrane (TM) receptor that interacts with membrane-anchored ligands (Nickoloff, B. J., et al. 2003; Weinmaster, G., et al. 1991; Weinmaster, G., et al. 1992; Reaume, A. G., et al. 1992; del Amo, F. F., et al. 1993; Lardelli, M., et al.
  • Ntcl is synthesized as a -270 kDa precursor, NtclFL (Fig. IA) (Nickoloff, B. J., et al. 2003; Selkoe, D., et al. 2003). Maturation involves removal of the leader peptide (LP) and cleavage by a furin-like convertase at the Sl site in the Golgi (Logeat, F., et al. 1998). In turn, cleavage at S2 yields the -120 kDa NtcTM, a substrate for cleavage at S3 by ⁇ -secretase (Baron, M. 2003). Formation of the active intracellular form, Ntc IC , is promoted by Ntcl ligands and blocked by -ySIs (Li, T., et al. 2003; Zhang, Z., et al. 2000).
  • Ntcl does not bind DNA directly, but interacts with other proteins such as CSL, also called CBFl (Lecourtois, M., et al. 1995; Baron, M., et al. 2002).
  • CSL exists in a repressor complex with histone deacetylases (HDACs) and co-repressors including Hairless (Morel, V., et al. 2001; Kao, H. Y., et al. 1998; Lai, E. C. 2002; Zhou, S., et al. 2000; Zhou, S., et al. 2001) (Fig. IB).
  • HDACs histone deacetylases
  • Ntc IC histone acetyltransferases
  • CBP/P300 histone acetyltransferases
  • the Ntc IC -CSL complex activates transcription from CSL binding sites in the promoters of target genes such as Hesl (Iso, T., et al. 2003) (Fig. IB).
  • NtclIC proteins (Fig. IA-B) are produced in T-ALL by translocation or by point mutation (>50%) (Weng, A. P., et al. 2004).
  • Ntcl IC was shown to transform El A-immortalized rat kidney cells (RK3E cells) similar to KLF4 (Foster, K. W., et al. 1999; Capobianco, A. J., et al. 1997; Ascano, J. M., et al. 2003). Mapping studies have failed to identify a single minimal transforming region of NtclIC (Fryer, C. J., et al. 2002; Aster, J. C, et al. 2000; Jeffries, S., et al. 2000; Dumont, E., et al. 2000).
  • KLF4 and Ntcl are co-expressed in differentiating layers of the skin (Lin, M. H., et al. 2003). KLF4 and Ntcl are highly expressed in most breast cancers and head and neck SCC, but are low in other tumor types (Huang, C. C, et al. 2005; Nickoloff, B. J., et al. 2003; Brennan, K., et al. 2003; Lin, M. H., et al. 2003; Leethanakul, C, et al. 2000; Nicolas, M., et al. 2003).
  • KLF4 and Ntcl appear to function as oncogenes in breast cancer and cutaneous/oral SCC, but are candidate tumor suppressors in the cervix, gut, and hair follicle (Nicolas, M., et al. 2003; Maillard, L, et al. 2003; Rangarajan, A., et al. 2001; Talora, C, et al. 2002).
  • Evidence supports a role for Ntcl signaling in breast cancer (Brennan, K., et al. 2003; Politi, K., et al. 2004; Kiaris, H., et al. 2004).
  • Ntcl expression is prominent in human breast cancers (Brennan, K., et al. 2003; Weijzen, S., et al. 2002; Reedijk, M., et al. 2005).
  • Ntcl FL the form upregulated in human tumors
  • KLF4 directly induces Ntcl FL transcription and Ntcl FL and Ntcl 10 are both oncogenes.
  • Antibodies specific for Human KLF4 and KLF5 are commercially available from, for example, Abeam, Abnova Corporation, ABR- Affinity BioReagents, Aviva Systems Biology, CeMines, CHEMICON, GeneTex, IMGENEX, Lifespan Biosciences, Novus Biologicals, R&D Systems, Santa Cruz Biotechnology, Inc.
  • a monoclonal antibody directed against residues 479-1197 of Kruppel-like factor 4 SEQ ID NO: 6
  • Such antibody can be used to monitor a treatment, further evaluate effectiveness of the treatment in an individual.
  • the monoclonal antibody detects the localization and level of KLF4 protein, and wherein decreases of KLF4 protein level indicate effective response of the individual to the treatment.
  • a kit for monitoring a treatment thereby evaluating effectiveness of the treatment in an individual comprising the monoclonal antibody disclosed herein and a suitable carrier.
  • Antibodies specific for Human Notchl are commercially available from, for example, Abeam, AbD Serotec, ABR- Affinity BioReagents, AlphaGenix, Inc., AnaSpec, BD Biosciences Pharmingen, BioLegend, Biomeda Corporation, Calbiochem, CHEMICON, Developmental Studies Hybridoma Bank, Epitomics, Inc., GeneTex, Lab Vision, Lifespan Biosciences, Novus Biologicals, Proteintech Group, Inc., and Sigma- Aldrich. i. Antibodies Generally, Abeam, AbD Serotec, ABR- Affinity BioReagents, AlphaGenix, Inc., AnaSpec, BD Biosciences Pharmingen, BioLegend, Biomeda Corporation, Calbiochem, CHEMICON, Developmental Studies Hybridoma Bank, Epitomics, Inc., GeneTex, Lab Vision, Lifespan Biosciences, Novus Biologicals, Proteintech Group, Inc., and Sigma- Aldrich. i. Antibodies Generally
  • antibodies is used herein in a broad sense and includes both polyclonal and monoclonal antibodies.
  • immunoglobulin molecules also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with human KLF4 or KLF5.
  • the antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sd. USA, 81:6851-6855 (1984)).
  • the disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies.
  • disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro, e.g., using the HIV Env-CD4-co-receptor complexes described herein.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.).
  • DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.
  • In vitro methods are also suitable for preparing monovalent antibodies.
  • Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
  • Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
  • the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen.
  • Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • antibody can also refer to a human antibody and/or a humanized antibody.
  • Many non-human antibodies e.g., those derived from mice, rats, or rabbits
  • are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • Human antibodies e.g., those derived from mice, rats, or rabbits
  • human antibodies can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole et al. ⁇ Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol., 147(l):86-95, 1991). Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J MoI. Biol, 227:381, 1991; Marks et al., J. MoI. Biol, 222:581, 1991).
  • the disclosed human antibodies can also be obtained from transgenic animals.
  • transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Set USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol, 1:32> (1993)).
  • the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge.
  • Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein. iii. Humanized antibodies
  • Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
  • a humanized form of a non-human antibody is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.
  • a humanized antibody residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen).
  • CDRs complementarity determining regions
  • donor non-human antibody molecule that is known to have desired antigen binding characteristics
  • Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues.
  • Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).
  • Fc antibody constant region
  • humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No.
  • ⁇ -secretase inhibitors (7SI) for use on tumors having the disclosed oncogene expression patterns.
  • Gamma secretase is a multi-subunit protease complex, itself an integral membrane protein, that cleaves single-pass transmembrane proteins at residues within the transmembrane domain.
  • the most well-known substrate of gamma secretase is amyloid precursor protein, a large integral membrane protein that, when cleaved by both gamma and beta secretase, produces a short 39-42 amino acid peptide called amyloid beta whose abnormally folded fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients.
  • Gamma secretase is also critical in the related processing of the Notch protein.
  • the gamma secretase complex has not yet been fully characterized but minimally consists of four individual proteins: presenilin, nicastrin, APH-I (anterior pharynx- defective 1), and PEN-2 (presenilin enhancer 2).
  • Presenilin an aspartyl protease, is the catalytic subunit; mutations in the presenilin gene have been shown to be a major genetic risk factor for Alzheimer's disease.
  • Gamma secretase is an internal protease that cleaves within the membrane- spanning domain of its substrate proteins, including amyloid precursor protein (APP) and Notch. Substrate recognition occurs via nicastrin ectodomain binding to the N-terminus of the target, which is then passed via a poorly understood process between the two presenilin fragments to a water-containing active site at which the catalytic aspartate residue resides.
  • APP amyloid precursor protein
  • gamma secretase inhibitors 7SI are known in the art and contemplated for use in the disclosed compositions and methods.
  • An example of a ⁇ SIs undergoing clinical trials for the treatment of breast cancer is MK0752 (Merck, Whitehouse Station, NJ; ClinicalTrials.gov Identifier: NCT00106145).
  • Additional examples of ⁇ SIs include MRK003-ONC (Merck), benzodiazepine, dibenzazepine, and Compound Z.
  • MRK003-ONC Merck
  • benzodiazepine dibenzazepine
  • Compound Z Compound Z.
  • 7,208,488 (04/24/07), 7,208,602 (04/24/07), 7,232,820 (06/19/07), 7,253,158 (08/07/07), 7,253,195 (08/07/07), 7,253,306 (08/07/07), 7,256,186 (08/14/07), 7,262,208 (08/28/07), and 7,262,223 (08/28/07) are incorporated herein by reference in their entireties for the teaching of 7SIS.
  • ⁇ -secretase 7-secretase has been intensively studied, and numerous small molecule inhibitors have been identified (Citron, M. 2004).
  • Aspartyl protease transition state analogues such as pepstatin and L685458 inhibit ⁇ -secretase.
  • Other potent and specific ⁇ -secretase inhibitors include sulfonamides and benzodiazepines (Tian, G., et al. 2002).
  • the ⁇ -secretase complex is an unusual protease that acts within the membrane to release intracellular fragments from Type I integral membrane proteins, a process termed regulated intramembrane proteolysis (RIP).
  • 7-secretase is composed of the transmembrane proteins presenilin 1 (PSl) and presenilin 2 (PS2) as well as nicastrin and Aph-1 (Ye, Y., et al. 1999; Struhl, G., et al. 1999; De Strooper, B., et al. 1999; Song, W., et al. 1999; Shiraishi, H., et al. 2006; Fortini, M. E. 2002).
  • PSl presenilin 1
  • PS2 presenilin 2
  • Ntc Unlike for Ntc, for none of these other substrates has the knockout phenotype been recapitulated by PSl knockout or 7SI treatment. In addition, there are no known Ntc-independent effects of ⁇ SIs in animals or humans, and these drugs are consistently described as well-tolerated at efficacious doses (Citron, M. 2004; Wong, G. T., et al. 2004; Siemers, E. R., et al. 2006). The observed selectivity for Ntc can be due to partial inhibition of ⁇ -secretase or to functional redundancy within the other pathways regulated by ⁇ -secretase.
  • retinoid X receptor agonists (rexinoids) for use on tumors having the disclosed oncogene expression patterns.
  • the retinoid X receptor is a type of nuclear receptor which is activated by 9-cis retinoic acid.
  • RXR heterodimerizes with subfamily 1 nuclear receptors including CAR, FXR, LXR, PPAR, PXR, RAR, TR, and VDR.
  • the RXR heterodimer in the absence of ligand is bound to hormone response elements complexed with corepressor protein. Binding of agonist ligands to RXR results in dissociation of corepressor and recruitment of coactivator protein which in turn promotes transcription of the downstream target gene into mRNA and eventually protein.
  • RXR selective retinoids such as 9-cis-retinoic acid (9cRA)
  • 9cRA 9-cis-retinoic acid
  • the rexinoid can be 9-cis-UAB30 (9cUAB30), which is very effect in the chemoprevention of breast cancer and has very low toxicity.
  • 9cUAB30 9-cis-UAB30
  • Atigadda et al, J. Med. Chem. 2003 is hereby incorporated by reference herein for the teaching of 9cUAB30 and methods of making and using same.
  • KLF4 activates transcription of nuclear hormone receptors (RAR ⁇ , RXR ⁇ , Nur77), and retinoid agonist can block KLF4 oncogenic activity.
  • RAR ⁇ nuclear hormone receptors
  • retinoid agonist can block KLF4 oncogenic activity.
  • a retinoid agonist for RXRs 9-cis-UAB30 (9cUAB30) was developed. This rexinoid is 50-fold less toxic than the pan-agonist 9-cis-retinoic acid (9cRA), 15 -fold less toxic than the RXR-agonist Targretin, and does not elevate triglycerides.
  • 9cUAB30 is a highly efficacious, nontoxic chemopreventive agent (Grubbs, C.
  • 9cUAB30 is also a candidate therapeutic, as indicated by its induction of apoptosis and inhibition of BrdU incorporation in established tumors (Grubbs, C. J., et al. 2005).
  • RARs and RXRs are members of the nuclear hormone receptor (NR) superfamily (Bastien, J., et al. 2004; Nagpal, S. et al. 2000). For both RAR and RXR, three isotypes ( ⁇ , ⁇ , ⁇ ) are encoded by separate genes. All-trans retinoic acid (ATRA) and 13cRA are agonists for the RARs. 9cRA is an agonist for both RARs and RXRs. Retinoid receptors act through RA response elements (RAREs) within gene promoters to regulate transcription (Chambon, P. 1996; Giguere, V., et al. 1987; Petkovich, M., et al. 1987).
  • RAREs RA response elements
  • NRs can localize to the nucleus, bind to RAREs, and recruit co- repressors (Bastien, J., et al. 2004; Dilworth, F. J., et al. 2001; Aranda, A., et al. 2001; Glass, C. K., et al. 2000).
  • Liganded NRs can recruit a variety of co-activators, chromatin remodellers, and modifiers that recruit the RNA Polymerase II machinery (Dilworth, F. J., et al. 2001). In some cases growth suppression by retinoid agonists may be due to repression of AP-I (Karin, M., et al. 1997; Chen, C.
  • RARjS is lost in tumors such as breast cancer and SCC by gene deletion, mutation, or promoter methylation (Niles, R. M., et al. 2004; Hayashi, K., et al. 2001; Sirchia, S. M., et al. 2002; Nakayama, T., et al. 2001), and mice deficient in RXR ⁇ in the prostate develop high grade lesions (Huang, J., et al. 2002).
  • RXRs can heterodimerize with other partners including vitamin D3 receptors, PPARs, and orphan nuclear receptors including NUR77, Norl and Nurrl (Niles, R. M., et al. 2004; Winoto, A., et al. 2002; Sohn, S. J., et al. 2003).
  • the RXR receptor of the heterodimers can be transcriptionally silent (e.g., RXR: VDR) or be permissively activated upon binding of either the RXR ligand or a ligand for the binding partner (e.g., RXR:PPARs).
  • RAR/RXR heterodimers are thought to require binding of an RAR ligand before the RXR ligand can enhance transcription.
  • the two receptors Upon binding of a ligand to both RAR and RXR and release of co-repressors, the two receptors synergize to activate transcription (Bastien, J., et al. 2004; Nettles, K. W., et al. 2005).
  • RXR-Nurrl heterodimers are robustly activated by RXR ligands (Law, S. W., et al. 1992; Forman, B. M., et al. 1995; Perlmann, T., et al. 1995; Wallen-Mackenzie, A., et al.
  • NUR77 a target of KLF4
  • the binding of NUR77 to BCL2 converts the latter from a blocker of apoptosis to a promoter of cell death (Lin, B., et al. 2004).
  • Natural ligands for NUR77 are unknown, but synthetic l,l-Bis(3'-indolyl)-l-(psubstituted phenyl)methanes function as NUR77 agonists and induce apoptosis in breast cancer cells (Chintharlapalli, S., et al. 2005). While 9cRA and SRl 1237 promote RXR ⁇ homodimerization, resulting in a block of NUR77-mediated apoptosis (Cao, X., et al. 2004), the effects of 9cUAB30 and its derivatives have not been determined. If these promote heterodimerization by binding to RXR ⁇ and/or NUR77 they may activate apoptosis.
  • RA influences epithelial development and differentiation (Fisher, G. J., et al. 1996). In skin the most abundant RAR is RAR ⁇ (Fisher, G. J., et al. 1996; Darwiche, N., et al. 1995).
  • KLF4 and Ntcl these are most abundant in suprabasal keratinocytes (Huang, C. C, et al. 2005; Lin, M. H., et al. 2003).
  • RAR ⁇ retinoid receptors
  • the disclosed cancer therapeutics can be combined, conjugated or coupled with or to carriers and other compositions to aid administration, delivery or other aspects of the inhibitors and their use.
  • composition will be referred to herein as carriers.
  • Carriers can, for example, be a small molecule, pharmaceutical drug, fatty acid, detectable marker, conjugating tag, nanoparticle, or enzyme.
  • compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject, along with the composition, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically- acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly or subcutaneously.
  • Other compounds can be administered according to standard procedures used by those skilled in the art.
  • compositions can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions can potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These can be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • the carrier molecule can be covalently linked to the disclosed cancer therapeutics.
  • the carrier molecule can be linked to the amino terminal end of disclosed peptides.
  • the carrier molecule can be linked to the carboxy terminal end of disclosed peptides.
  • the carrier molecule can be linked to an amino acid within disclosed peptides.
  • the herein provided compositions can further comprise a linker connecting the carrier molecule and disclosed cancer therapeutics.
  • the disclosed inhibitors can also be conjugated to a coating molecule such as bovine serum albumin (BSA) (see Tkachenko et al., (2003) J Am Chem Soc, 125, 4700-4701) that can be used to coat microparticles, nanoparticles of nanoshells with the cancer therapeutics.
  • BSA bovine serum albumin
  • Protein crosslinkers that can be used to crosslink the carrier molecule to the cancer therapeutics, such as disclosed peptides, are known in the art and are defined based on utility and structure and include DSS (Disuccinimidylsuberate), DSP (Dithiobis(succinimidylpropionate)), DTSSP (3,3'-Dithiobis (sulfosuccinimidylpropionate)), SULFO BSOCOES (Bis[2- (sulfosuccinimdooxycarbonyloxy) ethyl] sulfone), BSOCOES (Bis[2- (succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST (Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene
  • nanoparticle refers to a nanoscale particle with a size that is measured in nanometers, for example, a nanoscopic particle that has at least one dimension of less than about 100 ran.
  • nanoparticles include paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohoms, nano-onions, nanorods, nanoropes and quantum dots.
  • a nanoparticle can produce a detectable signal, for example, through absorption and/or emission of photons (including radio frequency and visible photons) and plasmon resonance.
  • Microspheres can also be used with the methods disclosed herein.
  • Microspheres containing chromophores have been utilized in an extensive variety of applications, including photonic crystals, biological labeling, and flow visualization in microfluidic channels. See, for example, Y. Lin, et al., Appl. Phys Lett. 2002, 81, 3134; D. Wang, et al., Chem. Mater. 2003, 15, 2724; X. Gao, et al., J. Biomed. Opt. 2002, 7, 532; M. Han, et al., Nature Biotechnology. 2001, 19, 631; V. M. Pai, et al., Mag. & Magnetic Mater. 1999, 194, 262, each of which is incorporated by reference in its entirety. Both the photostability of the chromophores and the monodispersity of the microspheres can be important.
  • Nanoparticles such as, for example, silica nanoparticles, metal nanoparticles, metal oxide nanoparticles, or semiconductor nanocrystals can be incorporated into microspheres.
  • the optical, magnetic, and electronic properties of the nanoparticles can allow them to be observed while associated with the microspheres and can allow the microspheres to be identified and spatially monitored.
  • the high photostability, good fluorescence efficiency and wide emission tunability of colloidally synthesized semiconductor nanocrystals can make them an excellent choice of chromophore.
  • nanocrystals that emit different colors i.e. different wavelengths
  • Colloidally synthesized semiconductor nanocrystals (such as, for example, core-shell CdSe/ZnS and CdS/ZnS nanocrystals) can be incorporated into microspheres.
  • the microspheres can be monodisperse silica microspheres.
  • the nanoparticle can be a metal nanoparticle, a metal oxide nanoparticle, or a semiconductor nanocrystal.
  • the metal of the metal nanoparticle or the metal oxide nanoparticle can include titanium, zirconium, hafnium, vanadium, niobium, tantalum, chromium, molybdenum, tungsten, manganese, technetium, rhenium, iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel, palladium, platinum, copper, silver, gold, zinc, cadmium, scandium, yttrium, lanthanum, a lanthanide series or actinide series element (e.g., cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium,
  • the metal can be iron, ruthenium, cobalt, rhodium, nickel, palladium, platinum, silver, gold, cerium or samarium.
  • the metal oxide can be an oxide of any of these materials or combination of materials.
  • the metal can be gold, or the metal oxide can be an iron oxide, a cobalt oxide, a zinc oxide, a cerium oxide, or a titanium oxide. Preparation of metal and metal oxide nanoparticles is described, for example, in U.S. Pat. Nos. 5,897,945 and 6,759,199, each of which is incorporated by reference in its entirety.
  • cancer therapeutics can be immobilized on silica nanoparticles (SNPs).
  • SNPs have been widely used for biosensing and catalytic applications owing to their favorable surface area-to-volume ratio, straightforward manufacture and the possibility of attaching fluorescent labels, magnetic nanoparticles (Yang, H.H. et al. 2005) and semiconducting nanocrystals (Lin, Y. W., et al. 2006).
  • the nanoparticle can also be, for example, a heat generating nanoshell.
  • nanoshell is a nanoparticle having a discrete dielectric or semi-conducting core section surrounded by one or more conducting shell layers.
  • U.S. Patent No. 6,530,944 is hereby incorporated by reference herein in its entirety for its teaching of the methods of making and using metal nanoshells.
  • Targeting molecules can be attached to the disclosed compositions and/or carriers.
  • the targeting molecules can be antibodies or fragments thereof, ligands for specific receptors, or other proteins specifically binding to the surface of the cells to be targeted. b. Liposomes
  • Liposome refers to a structure comprising an outer lipid bi- or multi-layer membrane surrounding an internal aqueous space. Liposomes can be used to package any biologically active agent for delivery to cells.
  • liposomes Materials and procedures for forming liposomes are well-known to those skilled in the art. Upon dispersion in an appropriate medium, a wide variety of phospholipids swell, hydrate and form multilamellar concentric bilayer vesicles with layers of aqueous media separating the lipid bilayers. These systems are referred to as multilamellar liposomes or multilamellar lipid vesicles ("MLVs”) and have diameters within the range of 10 nm to 100 ⁇ m. These MLVs were first described by Bangham, et al., J MoI. Biol. 13:238-252 (1965). In general, lipids or lipophilic substances are dissolved in an organic solvent.
  • the lipid residue forms a film on the wall of the container.
  • An aqueous solution that typically contains electrolytes or hydrophilic biologically active materials is then added to the film.
  • Large MLVs are produced upon agitation.
  • the larger vesicles are subjected to sonication, sequential filtration through filters with decreasing pore size or reduced by other forms of mechanical shearing.
  • pressurized extrusion Barenholz, et al., FEBS Lett. 99:210-214 (1979)
  • Liposomes can also take the form of unilamnellar vesicles, which are prepared by more extensive sonication of MLVs, and consist of a single spherical lipid bilayer surrounding an aqueous solution.
  • Unilamellar vesicles can be small, having diameters within the range of 20 to 200 nm, while larger ULVs can have diameters within the range of 200 nm to 2 ⁇ m.
  • ULVs Unilamellar vesicles
  • Small ULVs can also be prepared by the ethanol injection technique described by Batzri, et al, Biochim et Biophys Acta 298:1015-1019 (1973) and the ether injection technique of Deamer, et al., Biochim et Biophys Acta 443:629-634 (1976). These methods involve the rapid injection of an organic solution of lipids into a buffer solution, which results in the rapid formation of unilamellar liposomes. Another technique for making ULVs is taught by Weder, et al. in "Liposome Technology", ed. G. Gregoriadis, CRC Press Inc., Boca Raton, FIa., Vol. I, Chapter 7, pg. 79-107 (1984). This detergent removal method involves solubilizing the lipids and additives with detergents by agitation or sonication to produce the desired vesicles.
  • Papahadjopoulos, et al., U.S. Pat. No. 4,235,871 describes the preparation of large ULVs by a reverse phase evaporation technique that involves the formation of a water-in- oil emulsion of lipids in an organic solvent and the drug to be encapsulated in an aqueous buffer solution. The organic solvent is removed under pressure to yield a mixture which, upon agitation or dispersion in an aqueous media, is converted to large ULVs.
  • Suzuki et al., U.S. Pat. No. 4,016,100 describes another method of encapsulating agents in unilamellar vesicles by freezing/thawing an aqueous phospholipid dispersion of the agent and lipids.
  • liposomes can also be multivesicular. Described in Kim, et al., Biochim et Biophys Acta 728:339-348 (1983), these multivesicular liposomes are spherical and contain internal granular structures. The outer membrane is a lipid bilayer and the internal region contains small compartments separated by bilayer septum. Still yet another type of liposomes are oligolamellar vesicles ("OLVs”), which have a large center compartment surrounded by several peripheral lipid layers. These vesicles, having a diameter of 2-15 ⁇ m, are described in Callo, et al., Cryobiology 22(3):251-267 (1985).
  • OLVs oligolamellar vesicles
  • Fatty acids i.e., lipids
  • the fatty acid is a polar lipid.
  • the fatty acid can be a phospholipid
  • the provided compositions can comprise either natural or synthetic phospholipid.
  • the phospholipids can be selected from phospholipids containing saturated or unsaturated mono or disubstituted fatty acids and combinations thereof.
  • These phospholipids can be dioleoylphosphatidylcholine, dioleoylphosphatidylserine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, dioleoylphosphatidic acid, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylserine, palmitoyloleoylphosphatidylethanolamine, palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic acid, palmitelaidoyloleoylphosphatidylcholine, palmitelaidoyloleoylphosphatidylserine, palmitelaidoyloleoylphosphatidylethanolamine, palmitelaidoyloleoylphosphatidylglycerol, palmi
  • These phospholipids may also be the monoacylated derivatives of phosphatidylcholine (lysophophatidylidylcholine), phosphatidylserine (lysophosphatidylserine), phosphatidylethanolamine (lysophosphatidylethanolamine), phophatidylglycerol (lysophosphatidylglycerol) and phosphatide acid (lysophosphatidic acid).
  • the monoacyl chain in these lysophosphatidyl derivatives may be palimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoyl or myristoleoyl.
  • the phospholipids can also be synthetic.
  • Synthetic phospholipids are readily available commercially from various sources, such as AVANTI Polar Lipids (Albaster, Ala.); Sigma Chemical Company (St. Louis, Mo.). These synthetic compounds may be varied and may have variations in their fatty acid side chains not found in naturally occurring phospholipids.
  • the fatty acid can have unsaturated fatty acid side chains with C14, C16, C18 or C20 chains length in either or both the PS or PC.
  • Synthetic phospholipids can have dioleoyl (18:1)-PS; palmitoyl (16:0)-oleoyl (18:1)-PS, dimyristoyl (14:0)-PS; dipalmitoleoyl (16:1)-PC, dipalmitoyl (16:0)-PC, dioleoyl (18:1)- PC, palmitoyl (16:0)-oleoyl (18:1)-PC, and myristoyl (14:0)-oleoyl (18:1)-PC as constituents.
  • the provided compositions can comprise palmitoyl 16:0.
  • compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject's cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).
  • cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art.
  • the compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes.
  • the transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.
  • a method of treating or preventing a tumor in a subject comprising administering to the subject a therapeutically effective amount of a 7-secretase inhibitor CySI). Also provided herein is a method of treating or preventing a tumor in a subject comprising administering to the subject a therapeutically effective amount of a retinoid X receptor (RXR) agonist (rexinoid). Also provided herein is a method of treating or preventing a tumor in a subject comprising administering to the subject a therapeutically effective amount of a ⁇ -secretase inhibitor CySI) and a retinoid X receptor (RXR) agonist (rexinoid).
  • RXR retinoid X receptor
  • the 7SI can be any 7SI known in the art, including MK0752, MRK003-ONC, L- 685,458, or dibenzazepine (DBZ).
  • the rexinoid can be any retinoid known in the art, including 9cUAB30.
  • the tumor of the disclosed method can be any tumor with a detectably higher level of Kruppel-like factor 4 (KLF4) as compared to normal reference cells.
  • the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
  • the tumor of the disclosed method can be any tumor with predominantly cyotosolic expression of Kriippel-like factor 4 (KLF4) as compared to normal reference cells.
  • the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher cytosolic expression of KLF4 in the tumor cells as compared to that of normal reference cells.
  • the tumor of the disclosed method can be any tumor with a detectably higher level of Notchl (Ntcl) as compared to normal reference cells.
  • the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of Ntclin the tumor cells as compared to that of normal reference cells.
  • the tumor of the disclosed method can be any tumor with predominantly nuclear expression of Ntcl as compared to normal reference cells.
  • the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher nuclear expression of Ntcl in the tumor cells as compared to that of normal reference cells.
  • the tumor of the disclosed method can be a breast cancer.
  • the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget's disease of the breast.
  • the breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2 -negative (triple-negative breast cancer).
  • the subject has been diagnosed with the breast tumor by mammogram or biopsy.
  • the subject has undergone a lumpectomy or mastectomy, hi some aspects, the subject has been identified has having a genetic risk of developing a breast tumor.
  • the tumor of the disclosed method can be pancreatic cancer.
  • the tumor can be an adenocarcinoma.
  • the tumor of the disclosed method can be squamous cell carcinoma.
  • the tumor can be in the skin, mouth, esophagus, prostate, lungs, or cervix.
  • Also provided herein is a method of treating or preventing a tumor in a subject comprising the steps of (a) detecting staining of Kriippel-like factor 4 (KLF4) in the tumor cells, (b) detecting a difference in the amount of KLF4 staining in the breast tumor cells as compared to that of normal reference cells, and (c) administering to the subject a therapeutically effective amount of a ⁇ -secretase inhibitor ( ⁇ SI).
  • the method comprises detecting a higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
  • the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
  • Also provided herein is a method of treating or preventing a tumor in a subject comprising the steps of (a) detecting staining of Kriippel-like factor 4 (KLF4) in said breast tumor cells, (b) detecting a difference in the localization of KLF4 staining in the breast tumor cells as compared to that of normal reference cells, and (c) administering to the subject a therapeutically effective amount of a ⁇ -secretase inhibitor (7SI).
  • the method comprises detecting a predominantly cytosolic staining of KLF4 in the tumor cells, hi some aspects, the method comprises detecting a predominantly nuclear staining of KLF4 in the tumor cells.
  • the disclosed methods can further comprise administering to the subject a therapeutically effective amount of a retinoid X receptor (RXR) agonist (rexinoid).
  • RXR retinoid X receptor
  • the disclosed method can further comprise detecting a difference in the amount of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells.
  • the method comprises detecting a higher level of Ntcl in the tumor cells as compared to that of normal reference cells.
  • the disclosed method can further comprise detecting a difference in the localization of Notch 1 (Ntcl) staining in the tumor cells as compared to that of normal reference cells.
  • the method comprises detecting a predominantly nuclear staining of Ntcl in the tumor cells.
  • the method comprises detecting a predominantly cytosolic staining of Ntcl in the tumor cells.
  • the tumor of the disclosed method can be a breast cancer.
  • the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget' s disease of the breast.
  • the breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative breast cancer).
  • the subject has been diagnosed with the breast tumor by mammogram or biopsy.
  • the subject has undergone a lumpectomy or mastectomy.
  • the subject has been identified has having a genetic risk of developing a breast tumor.
  • the tumor of the disclosed method can be pancreatic cancer.
  • the tumor can be an adenocarcinoma.
  • the tumor of the disclosed method can be squamous cell carcinoma.
  • the tumor can be in the skin, mouth, esophagus, prostate, lungs, or cervix.
  • the reference cells of the disclosed method can be from normal breast, such as from breast reduction or mammoplasty.
  • the reference cells of the disclosed method can be from human tumor cell lines.
  • the reference cells of the disclosed method can be from the subject.
  • the reference cells can be from normal breast tissue of the subject. 3. Treatment Selection
  • Also provided is a method of selecting a treatment for a subject diagnosed with a tumor comprising examining the expression of Kr ⁇ ppel-like factor 4 (KLF '4) in the tumor, wherein a difference in the amount of KLF4 in one or more tumor cells as compared to that of normal reference cells indicates that the selected treatment is ⁇ -secretase inhibitor
  • the method comprises detecting a higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
  • the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
  • the tumor of the disclosed method can be a breast cancer.
  • the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (EDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget's disease of the breast.
  • the breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative breast cancer).
  • a higher level of KLF4 in the tumor cells as compared to that of normal reference cells indicates that the selected treatment is 7SI.
  • the method can further comprise detecting a difference in the amount of Notch 1 (Ntc 1) staining in the tumor cells as compared to that of normal reference cells.
  • Ntc 1 Notch 1
  • a higher level of Ntc 1 in the tumor cells as compared to that of normal reference cells indicates that the selected treatment is -ySI.
  • the method can further comprise detecting a difference in the localization of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells.
  • Ntcl Notchl
  • predominantly nuclear staining of Ntcl in the tumor cells indicates that the selected treatment is 7SI.
  • a predominantly cytosolic staining of Ntcl in the tumor cells indicates that the selected treatment is 7SI.
  • Also provided is a method of selecting a treatment for a subject diagnosed with a tumor comprising examining the expression of Kriippel-like factor 4 (KLF4) in said breast tumor, wherein a difference in the localization of KLF4 in one or more breast tumor cells as compared to that of normal reference cells indicates that the selected treatment is 7-secretase inhibitor CySI).
  • KLF4 Kriippel-like factor 4
  • a predominantly cytosolic staining of KLF4 in the tumor cells indicates that the selected treatment is 7SI. In some aspects, a predominantly nuclear staining of KLF4 in the tumor cells indicates that the selected treatment is 7SI.
  • the tumor of the disclosed method can be a breast cancer.
  • the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget's disease of the breast.
  • the breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2 -negative (triple-negative breast cancer).
  • the method can further comprise detecting a difference in the amount of Notchl (Ntc 1) staining in the tumor cells as compared to that of normal reference cells.
  • Ntc 1 a higher level of Ntc 1 in the tumor cells as compared to that of normal reference cells indicates that the selected treatment is ⁇ SI.
  • the method can further comprise detecting a difference in the localization of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells.
  • Ntcl Notchl
  • a predominantly nuclear staining of Ntcl in the tumor cells indicates that the selected treatment is 'ySI.
  • a predominantly cytosolic staining of Ntcl in the tumor cells indicates that the selected treatment is 7SI.
  • the method comprises detecting a higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
  • the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of EXF4 in the tumor cells as compared to that of normal reference cells.
  • the tumor of the disclosed method can be a breast cancer.
  • the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (EBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget's disease of the breast.
  • the breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2 -negative (triple-negative breast cancer).
  • a higher level of KLF4 in the tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI.
  • the method can further comprise detecting a difference in the amount of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells.
  • Ntcl Notchl
  • a higher level of Ntcl in the tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI.
  • the method can further comprise detecting a difference in the localization of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells.
  • Ntcl Notchl
  • a predominantly nuclear staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 'ySI.
  • a predominantly cytosolic staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 7SL
  • KLF4 Kruppel-like factor 4
  • a predominantly cytosolic staining of KLF4 in the tumor cells identifies the subject as a candidate for treatment with a 'ySI.
  • a predominantly nuclear staining of KLF4 in the tumor cells identifies the subject as a candidate for treatment with a 7SI.
  • the tumor of the disclosed method can be a breast cancer.
  • the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget' s disease of the breast.
  • the breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative breast cancer).
  • the method can further comprise detecting a difference in the amount of Notchl (Ntcl) staining in said tumor cells as compared to that of normal reference cells.
  • Ntcl Notchl
  • a higher level of Ntcl in the tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI.
  • the method can further comprise detecting a difference in the localization of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells.
  • Ntcl Notchl
  • a predominantly nuclear staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 7SI.
  • a predominantly cytosolic staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 7SI.
  • a method of determining the prognosis of an individual diagnosed as having a tumor comprising examining the expression of Kriippel-like factor 4 (KLF4) and Notchl (Ntcl) in one or more tumor cells, wherein a difference in the amount, localization, or both of KLF4 and a difference in the amount, localization, or both of Ntcl staining in the tumor cells as compared to that of normal reference cells indicates a lower likelihood of survival.
  • KLF4 Kriippel-like factor 4
  • Ntcl Notchl
  • the tumor of the disclosed method can be a breast cancer.
  • the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget' s disease of the breast.
  • the breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative breast cancer).
  • a higher level of KLF4 in the tumor cells as compared to that of normal reference cells indicates a lower likelihood of survival.
  • the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
  • a predominantly cytosolic staining of KLF4 in the tumor cells indicates a lower likelihood of survival. In some aspects, a predominantly nuclear staining of KLF4 in the tumor cells indicates a lower likelihood of survival. In some aspects, a higher level of Ntcl in the tumor cells as compared to that of normal reference cells indicates a lower likelihood of survival. In some aspects, a predominantly cytosolic staining of Ntcl in the tumor cells indicates a lower likelihood of survival. In some aspects, a predominantly nuclear staining of Ntcl in the tumor cells indicates a lower likelihood of survival. In some aspects, a predominantly cytosolic staining of KLF4 and a predominantly nuclear staining of Ntcl indicates a lower likelihood of survival.
  • KLF4 and Notchl can be detected in cells using standard methods known in the art.
  • KLF4 and/or Notchl can be detected in cells using immunodetection methods.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding immunodetection methods.
  • Immunoassays in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Many types and formats of immunoassays are known and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmune precipitation assays (RIPA), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAP/ FLAP).
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • RIPA radioimmune precipitation assays
  • immunobead capture assays Western blotting, dot blotting, gel-shift assays
  • immunoassays involve contacting a sample suspected of containing a molecule of interest (such as the disclosed biomarkers) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest (such as antibodies to the disclosed biomarkers) with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • a molecule of interest such as the disclosed biomarkers
  • an antibody to a molecule of interest such as antibodies to the disclosed biomarkers
  • the sample- antibody composition such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as the disclosed biomarkers or their antibodies) in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process.
  • a molecule of interest such as the disclosed biomarkers or their antibodies
  • the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label. See, for example, U.S.
  • a label can include a fluorescent dye, a member of a binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence.
  • a fluorescent dye also known herein as fluorochromes and fluorophores
  • enzymes that react with colorometric substrates (e.g., horseradish peroxidase).
  • colorometric substrates e.g., horseradish peroxidase
  • each antigen can be labeled with a distinct fluorescent compound for simultaneous detection. Labeled spots on the array are detected using a fluorimeter, the presence of a signal indicating an antigen bound to a specific antibody.
  • Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5- Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-
  • Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4- methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4- 1 methylcoumarin; 9- Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350TM; Alexa Fluor 430TM; Alexa Fluor 488TM; Alexa Fluor 532TM; Alex
  • Fluoro-Gold Hydroxystilbamidine
  • Fluor-Ruby FluorX; FM 1-43TM; FM 4-46; Fura RedTM (high pH); Fura RedTM/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 1OGF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type' non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo- 1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotric
  • a modifier unit such as a radionuclide can be incorporated into or attached directly to any of the compounds described herein by halogenation.
  • radionuclides useful in this embodiment include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, fluorine-18.
  • the radionuclide can be attached to a linking group or bound by a chelating group, which is then attached to the compound directly or by means of a linker.
  • radionuclides useful in the apset include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling techniques such as these are routinely used in the radiopharmaceutical industry.
  • the radiolabeled compounds are useful as imaging agents to diagnose neurological disease (e.g., a neurodegenerative disease) or a mental condition or to follow the progression or treatment of such a disease or condition in a mammal (e.g., a human).
  • the radiolabeled compounds described herein can be conveniently used in conjunction with imaging techniques such as positron emission tomography (PET) or single photon emission computerized tomography (SPECT).
  • PET positron emission tomography
  • SPECT single photon emission computerized tomography
  • Labeling can be either direct or indirect.
  • the detecting antibody the antibody for the molecule of interest
  • detecting molecule the molecule that can be bound by an antibody to the molecule of interest
  • Detection of the label indicates the presence of the detecting antibody or detecting molecule, which in turn indicates the presence of the molecule of interest or of an antibody to the molecule of interest, respectively.
  • an additional molecule or moiety is brought into contact with, or generated at the site of, the immunocomplex.
  • a signal- generating molecule or moiety such as an enzyme can be attached to or associated with the detecting antibody or detecting molecule.
  • the signal-generating molecule can then generate a detectable signal at the site of the immunocomplex.
  • an enzyme when supplied with suitable substrate, can produce a visible or detectable product at the site of the immunocomplex.
  • an additional molecule (which can be referred to as a binding agent) that can bind to either the molecule of interest or to the antibody (primary antibody) to the molecule of interest, such as a second antibody to the primary antibody, can be contacted with the immunocomplex.
  • the additional molecule can have a label or signal-generating molecule or moiety.
  • the additional molecule can be an antibody, which can thus be termed a secondary antibody. Binding of a secondary antibody to the primary antibody can form a so-called sandwich with the first (or primary) antibody and the molecule of interest.
  • the immune complexes can be contacted with the labeled, secondary antibody under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes can then be generally washed to remove any non-specifically bound labeled secondary antibodies, and the remaining label in the secondary immune complexes can then be detected.
  • the additional molecule can also be or include one of a pair of molecules or moieties that can bind to each other, such as the biotin/avadin pair. In this mode, the detecting antibody or detecting molecule should include the other member of the pair.
  • a molecule which can be referred to as a first binding agent
  • a second binding agent that has binding affinity for the first binding agent, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (thus forming tertiary immune complexes).
  • the second binding agent can be linked to a detectable label or signal-genrating molecule or moiety, allowing detection of the tertiary immune complexes thus formed. This system can provide for signal amplification.
  • Immunoassays that involve the detection of as substance, such as a protein or an antibody to a specific protein, include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection.
  • Label-free assays are generally diagnostic means of determining the presence or absence of a specific protein, or an antibody to a specific protein, in a sample.
  • Protein separation methods are additionally useful for evaluating physical properties of the protein, such as size or net charge.
  • Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein, or antibody to a specific protein, in a sample.
  • in vivo detection is useful for evaluating the spatial expression patterns of the substance, i.e., where the substance can be found in a subject, tissue or cell.
  • the molecular complexes ([Ab- Ag] n) generated by antibody-antigen interaction are visible to the naked eye, but smaller amounts may also be detected and measured due to their ability to scatter a beam of light.
  • the formation of complexes indicates that both reactants are present, and in immunoprecipitation assays a constant concentration of a reagent antibody is used to measure specific antigen ([Ab-AgJw), and reagent antigens are used to detect specific antibody ([Ab-AgJn).
  • reagent species is previously coated onto cells (as in hemagglutination assay) or very small particles (as in latex agglutination assay), "clumping" of the coated particles is visible at much lower concentrations.
  • assays based on these elementary principles are in common use, including Ouchterlony immunodiffusion assay, rocket Immunoelectrophoresis, and immunoturbidometric and nephelometric assays.
  • the main limitations of such assays are restricted sensitivity (lower detection limits) in comparison to assays employing labels and, in some cases, the fact that very high concentrations of analyte can actually inhibit complex formation, necessitating safeguards that make the procedures more complex.
  • Group 1 assays date right back to the discovery of antibodies and none of them have an actual "label" (e.g. Ag- enz).
  • Other kinds of immunoassays that are label free depend on immunosensors, and a variety of instruments that can directly detect antibody— antigen interactions are now commercially available. Most depend on generating an evanescent wave on a sensor surface with immobilized ligand, which allows continuous monitoring of binding to the ligand.
  • Immunosensors allow the easy investigation of kinetic interactions and, with the advent of lower-cost specialized instruments, may in the future find wide application in immunoanalysis.
  • Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving.
  • electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique.
  • the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel.
  • the matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage.
  • the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels.
  • a porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely.
  • agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes.
  • Polyacrylamide which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.
  • Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode.
  • the net charge carried by a protein is in addition independent of its size - i.e., the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.
  • SDS Sodium dodecyl sulphate
  • DTT dithiothreitol
  • Determination of molecular weight is done by SDS-PAGE of proteins of known molecular weight along with the protein to be characterized.
  • the Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye- front.
  • a simple way of determining relative molecular weight by electrophoresis (Mx) is to plot a standard curve of distance migrated vs. loglOMW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel.
  • proteins are fractionated first on the basis of one physical property, and, in a second step, on the basis of another.
  • isoelectric focusing can be used for the first dimension, conveniently carried out in a tube gel
  • SDS electrophoresis in a slab gel can be used for the second dimension.
  • One example of a procedure is that of O'Farrell, P.H., High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), herein incorporated by reference in its entirety for its teaching regarding two-dimensional electrophoresis methods.
  • Laemmli U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680 (1970), which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods, discloses a discontinuous system for resolving proteins denatured with SDS.
  • the leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine.
  • the resolving gel and the stacking gel are made up in Tris-HCl buffers (of different concentration and pH), while the tank buffer is Tris-glycine. All buffers contain 0.1% SDS.
  • Western blot analysis allows the determination of the molecular mass of a protein and the measurement of relative amounts of the protein present in different samples. Detection methods include chemiluminescence and chromagenic detection. Standard methods for Western blot analysis can be found in, for example, D.M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent 4,452,901, each of which is herein incorporated by reference in their entirety for teachings regarding Western blot methods.
  • proteins are separated by gel electrophoresis, usually SDS-PAGE.
  • the proteins are transferred to a sheet of special blotting paper, e.g., nitrocellulose, though other types of paper, or membranes, can be used.
  • the proteins retain the same pattern of separation they had on the gel.
  • the blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose.
  • An antibody is then added to the solution which is able to bind to its specific protein.
  • probes for the detection of antibody binding can be conjugated antiimmunoglobulins, conjugated staphylococcal Protein A (binds IgG), or probes to biotinylated primary antibodies (e.g., conjugated avidin/ streptavidin).
  • the power of the technique lies in the simultaneous detection of a specific protein by means of its antigenicity, and its molecular mass. Proteins are first separated by mass in the SDS-PAGE, then specifically detected in the immunoassay step. Thus, protein standards (ladders) can be run simultaneously in order to approximate molecular mass of the protein of interest in a heterogeneous sample.
  • the herein disclosed oncogenes can be detected by cell separatation methods such as including fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • separation techniques include, but are not limited to, those based on differences in physical (density gradient centrifugation and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rhol23 and DNA-binding dye Hoechst 33342).
  • Cells may be selected based on light-scatter properties as well as their expression of various cell surface antigens.
  • the purified stem cells have low side scatter and low to medium forward scatter profiles by FACS analysis. Cytospin preparations show the enriched stem cells to have a size between mature lymphoid cells and mature granulocytes.
  • Monoclonal antibodies are particularly useful.
  • the antibodies can be attached to a solid support to allow for crude separation.
  • the separation techniques employed should maximize the retention of viability of the fraction to be collected.
  • the separation techniques employed should maximize the retention of viability of the fraction to be collected.
  • Various techniques of different efficacy may be employed to obtain "relatively crude” separations. Such separations are where up to 10%, usually not more than about 5%, preferably not more than about 1%, of the total cells present are undesired cells that remain with the cell population to be retained.
  • the particular technique employed will depend upon efficiency of separation, associated cytotoxicity, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.
  • Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, or other convenient technique.
  • Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • the antibodies may be conjugated with markers, such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type. Any technique may be employed which is not unduly detrimental to the viability of the remaining cells.
  • the cells may now be separated by a fluorescence activated cell sorter (FACS) or other methodology having high specificity.
  • FACS fluorescence activated cell sorter
  • Multi-color analyses may be employed, with the FACS which is particularly convenient.
  • the cells may be separated on the basis of the level of staining for the particular antigens.
  • cells are initially separated by a coarse separation, followed by a fine separation, with positive selection of one or more markers associated with the stem cells and negative selection for markers associated with lineage committed cells. 7. Nucleic Acid Detection
  • KLF4 and Notch 1 mRNA can be detected in cells using standard methods known in the art.
  • specific mRNAs can be detected using Northern blot analysis, nuclease protection assays (NPA), in situ hybridization, or reverse transcription-polymerase chain reaction (RT-PCR).
  • NPA nuclease protection assays
  • RT-PCR reverse transcription-polymerase chain reaction
  • each of these techniques can be used to detect specific RNAs and to precisely determine their expression level.
  • Northern analysis is the only method that provides information about transcript size, whereas NPAs are the easiest way to simultaneously examine multiple messages.
  • In situ hybridization is used to localize expression of a particular gene within a tissue or cell type, and RT-PCR is the most sensitive method for detecting and quantitating gene expression.
  • Northern analysis presents several advantages over the other techniques. The most compelling of these is that it is the easiest method for determining transcript size, and for identifying alternatively spliced transcripts and multigene family members. It can also be used to directly compare the relative abundance of a given message between all the samples on a blot.
  • the Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., intactness of the RNA sample and how efficiently it has transferred to the membrane).
  • RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe.
  • Nonisotopic or high specific activity radiolabeled probes can be used including random-primed, nick- translated, or PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides. Additionally, sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an exon) may be used as probes.
  • the Nuclease Protection Assay (including both ribonuclease protection assays and Sl nuclease assays) is an extremely sensitive method for the detection and quantitation of specific mRNAs.
  • the basis of the NPA is solution hybridization of an antisense probe (radiolabeled or nonisotopic) to an RNA sample. After hybridization, single-stranded, unhybridized probe and RNA are degraded by nucleases. The remaining protected fragments are separated on an acrylamide gel. Solution hybridization is typically more efficient than membrane-based hybridization, and it can accommodate up to 100 ⁇ g of sample RNA, compared with the 20-30 ⁇ g maximum of blot hybridizations. NPAs are also less sensitive to RNA sample degradation than Northern analysis since cleavage is only detected in the region of overlap with the probe (probes are usually about 100-400 bases in length).
  • NPAs are the method of choice for the simultaneous detection of several RNA species. During solution hybridization and subsequent analysis, individual probe/target interactions are completely independent of one another. Thus, several RNA targets and appropriate controls can be assayed simultaneously (up to twelve have been used in the same reaction), provided that the individual probes are of different lengths. NPAs are also commonly used to precisely map mRNA termini and intron/exon junctions.
  • ISH In situ hybridization
  • ISH is a powerful and versatile tool for the localization of specific mRNAs in cells or tissues. Unlike Northern analysis and nuclease protection assays, ISH does not require the isolation or electrophoretic separation of RNA. Hybridization of the probe takes place within the cell or tissue. Since cellular structure is maintained throughout the procedure, ISH provides information about the location of mRNA within the tissue sample.
  • the procedure begins by fixing samples in neutral-buffered formalin, and embedding the tissue in paraffin. The samples are then sliced into thin sections and mounted onto microscope slides. (Alternatively, tissue can be sectioned frozen and post- fixed in paraformaldehyde.) After a series of washes to dewax and rehydrate the sections, a Proteinase K digestion is performed to increase probe accessibility, and a labeled probe is then hybridized to the sample sections. Radiolabeled probes are visualized with liquid film dried onto the slides, while nonisotopically labeled probes are conveniently detected with colorimetric or fluorescent reagents.
  • RT-PCR has revolutionized the study of gene expression. It is now theoretically possible to detect the RNA transcript of any gene, regardless of the scarcity of the starting material or relative abundance of the specific rnRNA.
  • cDNA complementary DNA
  • RT-PCR is somewhat tolerant of degraded RNA. As long as the RNA is intact within the region spanned by the primers, the target will be amplified.
  • Relative quantitative RT-PCR involves amplifying an internal control simultaneously with the gene of interest.
  • the internal control is used to normalize the samples. Once normalized, direct comparisons of relative abundance of a specific mRNA can be made across the samples. It is crucial to choose an internal control with a constant level of expression across all experimental samples (i.e., not affected by experimental treatment).
  • Commonly used internal controls e.g., GAPDH, ⁇ -actin, cyclophilin
  • GAPDH, ⁇ -actin, cyclophilin often vary in expression and, therefore, may not be appropriate internal controls. Additionally, most common internal controls are expressed at much higher levels than the mRNA being studied. For relative RT-PCR results to be meaningful, all products of the PCR reaction must be analyzed in the linear range of amplification. This becomes difficult for transcripts of widely different levels of abundance.
  • RT-PCR is used for absolute quantitation. This technique involves designing, synthesizing, and accurately quantitating a competitor RNA that can be distinguished from the endogenous target by a small difference in size or sequence. Known amounts of the competitor RNA are added to experimental samples and RT-PCR is performed. Signals from the endogenous target are compared with signals from the competitor to determine the amount of target present in the sample. 8. Administration
  • compositions can be administered in any suitable manner.
  • the manner of administration can be chosen based on, for example, whether local or systemic treatment is desired, and on the area to be treated.
  • the compositions can be administered orally, parenterally (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection), , by inhalation, extracorporeally, topically (including transdermally, ophthalmically, vaginally, rectally, intranasally) or the like.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • compositions required can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. Thus, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are effected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage can vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counter indications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • a typical daily dosage of the ⁇ -secretase inhibitor (7SI) used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • the efficacy of the therapeutic 7-secretase inhibitor (7SI) can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a composition disclosed herein is efficacious in treating or inhibiting an tumor in a subject by observing that the composition reduces the tumor or prevents a further increase in tumor size.
  • treatment is meant the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder, hi addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • terapéuticaally effective means that the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • carrier means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • KLF4/GKLF RNA and protein were developed (Foster, K. W., et al. 2000; Pandya, A. Y., et al. 2004; Foster, K. W., et al. 2005; Huang, C. C, et al. 2005).
  • KLF4 mRNA and protein was shown to be upregulated in most cases of human breast cancer, unlike several other tumor types (Foster, K. W., et al. 2000).
  • KLF4 is likewise upregulated in DCIS, identifying this as an early change.
  • a polyclonal rabbit ⁇ -KLF4 was developed (Foster, K. W., et al. 2005; Huang, C. C, et al. 2005) against a human peptide that is much superior at differentiating nuclear and cytoplasmic KLF4 as compared to the original monoclonal ⁇ -KLF4 (Foster, K. W., et al. 2000; Pandya, A. Y., et al. 2004).
  • a polyclonal ⁇ -Ntcl was characterized (C20, Santa Cruz) which reacts well with cytoplasmic Ntcl (NtclTM) and poorly with nuclear Ntcl (activated form, Ntcl ).
  • Ntcl acts in the nucleus
  • western blot shows that the major species present in cells and tissues is the inactive precursor, NtclTM that must be cleaved by ⁇ -secretase to yield Ntcl IC (Fig. 1). It is unknown how NtclTM in tissues correlates with the active Ntcl ic .
  • KLF4 in the cytoplasm KLF4 in the nucleus
  • NtclTM Ntcl precursor
  • KLF4 was prognostic in a subgroup of patients with early stage disease (Pandya, A. Y., et al. 2004).
  • the number of deaths in this subgroup (10 compared to 23 in the earlier study) limited the analysis of early stage disease.
  • the 153 patients were next analyzed as to KLF4 and Ntcl.
  • KLF4 can promote Ntcl signaling as indicated by increased conversion of NtclTM precursor to Ntcl IC (thereby lowering cytoplasmic Ntcl), leading to an aggressive phenotype. This latter effect may be more prevalent than just the poor prognosis sub-group identified.
  • KLF4 induces Ntcl and its ligands in epithelial cells
  • a conditional allele was generated by fusing KLF4 to a fragment of the estrogen receptor (i.e., KLF4-ER) (Foster, K. W., et al.
  • Ephexin AF254801.1 +3.9 Mediates Ephrin/EphA signaling
  • fragments of the Ntcl promoter function as KLF4- regulated enhancers (Fig. 3G). Nearly all of the activity was contained within a 524 base, promoterproximal fragment ( ⁇ 5, Fig. 3G) that corresponds to ChIP fragment O (Fig. 3C).
  • KLF4 induced the Ntcl ligands Dill, D114, and Jagl (Fig. 3H). This induction was observed only late (i.e., 48hr), indicating that the ligands are indirectly induced.
  • Jagl is a marker of poor outcome in breast cancer (Reedijk, M., et al. 2005). d. Concordance of KLF4 and Ntcl in mouse skin and human breast tumors
  • Ntcl is known to correlate with poor outcome in breast cancer (Pandya, A. Y., et al. 2004; Reedijk, M., et al. 2005; Parr, C, et al. 2004).
  • KLF4 is the first transcription factor regulator of Ntcl, and probably the major determinant of Ntcl transcription in breast cancer.
  • Ntcl antagonists including 7SIs 5 block transformation by KLF4 and Ntcl FL but not Ntcl IC
  • Ntcl Ic antisense (AS) RNA was stably expressed by retroviral transduction (Fig. 5 A-B). These cells grew similarly to the Vector cells. Ntcl IC -AS efficiently blocked transformation by activated Ras, consistent with the report that Ntcl IC is required for transformation by RAS in other cells (Weijzen, S., et al. 2002). Ntcl IC -AS also efficiently blocked KLF4-induced, but not c-MYC- or GIi 1 -induced transformation.
  • the Hesl-luc reporter assay assesses Ntcl activity in cells, and confirmed that Ntcl IC -AS specifically inhibits Ntcl IC in RK3E cells (Fig. 5C). Activity of the pTK-/3-gal control was unaffected.
  • the KLF4-ER transformation assay (Foster, K. W., et al. 2005) was also used to examine the role of ⁇ -secretase, required for S3 cleavage of Ntcl (Fig. IA).
  • the ⁇ SI L-685,458 (Compound X; Sigma) permitted transformation of RK3E by Ntcl IC , but transformation by KLF4-ER was efficiently blocked (Fig. 4D).
  • Ntc 1 IC can transform a cell line similar to RK3E (Capobianco, A. J., et al. 1997).
  • Ntcl IC and Ntcl FL induced foci in RK3E, unlike a Vector control (Fig. 6A-C).
  • the Ntcl FL transgene in these cells is WT in the PEST region (a motif conferring more rapid turnover) and heterodimerization domains that are mutated in T-ALL (Weng, A. P., et al. 2004).
  • Ntcl FL or Ntcl IC were cultured in the presence of ⁇ SIs. Colonies formed by Ntcl FL cells appeared flat in the presence of 7SI, whereas colonies of Ntcl IC cells piled up and formed dense colonies of cells that were morphologically transformed, similar to the vehicle control (Fig. 6D). As these control oncogenes represent the substrate (Ntcl ) and the product (Ntcl ) of ⁇ -secretase, these results strongly support the idea that ⁇ SIs act specifically on 7-secretase to inhibit transformation. f. Ntcl IC induces transformation through a different downstream pathway in the presence of KLF4
  • Ntcl IC transforms cells through CSL or by an alternate route (Brennan, K., et al. 2003) (Fig. IB).
  • KLF4 potently inhibited both basal activity and Ntcl IC -induced activity of a Hesl-luc reporter in HEK293 cells (Fig. 7A, Hesl). This effect is likely due to inhibition of CSL activity, as Ntcl -dependent activity of an 8x wt CSL promoter-reporter was inhibited by KLF4 (Fig. 7A, 8x wt CSL).
  • KLF4-ER cells treated with 4OHT +/- CHX also showed direct induction of the CSL-antagonist Hairless (Table 2, Fig. 7B). Inhibition of CSL reporter activity was due to induction of Hairless as shown using Hairless shRNAs (Fig. 75C).
  • the effects of KLF4 on CSL are summarized in Fig. 7D.
  • Fig. 8A-D DN alleles of CSL and MAMLl block transformation by Ntc 1 IC , but do not block transformation by KLF4, indicating that Ntcl can transform cells through two different routes (Fig. IB) and that KLF4 shunts Ntcl away from the classical pathway in favor of the alternate pathway. Since Ntcl acts very differently depending upon the presence/absence of KLF4, efficacy of 'ySIs in patients may depend upon levels of KLF4 rather than on levels of Ntcl alone.
  • KLF4 suppression of the classical Ntcl pathway has been demonstrated so far in RK3E and HEK293 cells, several in vivo observations suggest the effect is quite general. Reducing CSL-dependent pathway activity by deletion of the CSL or Hesl genes favors goblet cell formation in the gut, a known activity of KLF4 (Sancho, E., et al. 2003; Katz, J. P., et al. 2002; van Es, J. H., et al. 2005). Also, the early lesions induced by KLF4 in a transgenic mouse model of cutaneous SCC resemble lesions resulting from conditional knockout of CSL (Foster, K. W., et al.
  • KLF4 blocks maturation of human MCFlOA breast epithelial cells and promotes tumor formation in MMTV-ErbB2 mice
  • Immortalized MCFlOA cells can be cultured in 2D monolayers on plastic, or else induced to recapitulate the polarized, acinar structures of breast epithelium by culture on a layer of basement membrane components (Matrigel) (Seton-Rogers, S. E., et al. 2004; Mills, K. R., et al. 2004; Seton-Rogers, S. E., et al. 2004; Debnath, J., et al. 2003a; Debnath, J., et al. 2003b; Debnath, J., et al. 2002; Muthuswamy, S. K., et al. 2001).
  • Matrigel basement membrane components
  • ErbB2 but not ErbBl, reinitiates proliferation of preformed acini and induces a hyperplastic, multiacmar phenotype, with luminar repopulation, but with retention of other differentiated features such as apico-basal polarity of these epithelial cells (Muthuswamy, S. K., et al. 2001).
  • ErbB2-induced hyperplastic lesions can be converted to fully transformed cells by TGF 1 S (Seton-Rogers, S. E., et al. 2004; Seton-Rogers, S. E., et al. 2004).
  • KLF4 induced a block to differentiation/maturation (Fig. 9).
  • Fig. 9 When cultured on Matrigel, instead of the well-organized spheres and smooth, regular basal cell layer characteristic of untransformed cells, KLF4 induced grape-like clusters of cells with irregular basal layers. Clusters retained their roughened, dysplastic morphology throughout the period of observation of 19 days, suggesting that the maturation block is permanent.
  • the similar maturation block by KLF4 and Ntcl demonstrates clearly that
  • KLF4 has a property of an oncogene in human breast cells because Ntcl was recently shown to induce soft agar growth and transformation of the same MCFlOA (Stylianou, S., et al. 2006).
  • MMTV-ErbB2 was used to test the activity of KLF4 in vivo the latent tumor model.
  • KLF4 mutants generated by site-directed mutation of conserved or charged residues to alanine, were screened for dominant negative (DN) activity.
  • Two mutants termed KLF4 Ala5 and KLF4 Ala6 , were found to block transformation of RK3E cells by KLF4, but not by other oncogenes (e.g., RAS, GLIl).
  • KLF4 DN s was analyzed in MCFlOA cells and RAS-transformed MCFlOAT cells (Dawson, P. J., et al. 1996).
  • MCFlOA cells express endogenous KLF4 (Foster, K. W., et al. 2000 ; Huang, C. C, et al. 2005) (Fig. 10A).
  • Retroviral transduction with HA-KLF4 WT vector yields cells that grow normally and stably express the exogenous protein.
  • KLF4 Ala5 or KLF4 ⁇ la6 vectors induced only rare puroR colonies (Fig. 10B).
  • KLF4 DN is not tolerated by MCFlOA, consistent with an essential role of endogenous KLF4 in the growth of these cells.
  • KLF4 DN s do not inhibit the Ras-transformed derivative, MCFlOAT (Fig. 10B). i. KLF4 is required for transformation by c-MYC and ErbB2
  • ErbB2 A648C induces transformation of RK3E (Li, X., et al. 2005).
  • ErbB2 requires ErbB3 to drive the proliferation of breast cancer cells (Holbro, T., et al. 2003) and cooperates with ErbB3 in NIH3T3 transformation (Alimandi, M., et al. 1995). Transformation of RK3E by ErbB2 was blocked by either KLF4 DN (experiments performed twice in triplicate for a total of 12 dishes - 2 expts x 2 alleles x 3 replicates) (Fig. 10C). KLF4 DN cells showed >10 foldreduced transformation by ErbB2 retrovirus compared to control cells.
  • Microarray data identified ErbB3, an essential ErbB2 signaling partner, as a direct transcriptional target of KLF4 (Table 2). Not only are these coexpressed in rumors, but also in normal skin ErbB2, ErbB3, and KLF4 are co-expressed in all cell layers, and increased in superficial, postmitotic cells (Huang, C. C, et al. 2005; Piepkorn, M., et al. 2003; Lebeau, S., et al. 2005). Like induction of Ntcl, KLF4-ErbB signaling links KLF4 to a pathway that is important in breast cancer. j.
  • KLF4 induces nuclear receptors (NRs) in RK3E epithelial cells KLF4-ER directly induced transcription of RAR ⁇ in each of the two microarray experiments (Fig. 1 IA; Table 2). Another NR, Nur77 (Lin, B., et al. 2004) was one of the most strongly induced transcripts on the array. Array results were confirmed by RT-PCR, using GAPDH as a control for input and Ntcl as a positive control (Fig. 1 IB-C). RXR ⁇ was induced by KLF4 in as little as 3 hrs, similarly to RAR ⁇ , Nur77, and Ntcl (Fig. HC). k. 9cUAB30 blocks skin tumor initiation by KLF4
  • NRs nuclear receptors
  • 9 ⁇ JAB30 is a highly effective antagonist of KLF4-induced transformation in vivo.
  • 9cUAB30 As an inhibitor of KLF4 in vitro transformation assays were performed in the presence/absence of 9cUAB30 (Fig. 12B).
  • 9cUAB30 inhibited transformation by KLF4, a result now demonstrated in three independent experiments, all performed in duplicate/triplicate. There was no effect on the size or number of foci induced by other oncogenes, including the breast cancer oncogenes Ntcl or ErbB2, and no effect on the BCC oncogene GUI. This striking result indicates that 9cUAB30 specifically targets the KLF4 pathway.
  • KLF4 is highest in ZR75-1 and MCF7, lowest in MDA-MB-231 and MDA-MB- 453, and intermediate in other lines in a panel of breast tumor cell lines (Foster, K. W., et al. 2000).
  • MCF7 cells which require KLF4 for rapid growth in culture (Rowland, B. D., et al. 2005), and Ntcl for growth in soft agar (Stylianou, S., et al. 2006), were more sensitive than any other line to 7SI (50% inhibition) and the second-most sensitive line to 9cUAB30 (22% inhibition). Effects were quite reproducible as shown in dose-response studies (Fig. 14B) and in the combined drug study (Fig. 14C). o. Development of a human-like model of breast cancer using oral DMBA in genetically-engineered mice
  • DMBA Three groups of FVB/N females were treated with DMBA: 1) MMTV- ErbB2 +/" ; p53 +/ ⁇ 2) MMTVErbB2+/-, and 3) p53 +/ ⁇ Dosing was 1 mg/gavage, 1 dose/wk from age 50 days, treated for 8 weeks. No genotyping was needed because all breeders were homozygous (e.g., MMTV-ErbB2 +/+ ) or else WT FVB. Kaplan-Meier analysis shows that the genotype of DMBA-treated animals determined the tumor type, with MMTV- ErbB2 +/" promoting breast cancer (84% incidence) and p53 +/" promoting SCC (75% incidence).
  • the MMTV-ErbB2 +/" ; DMBA group showed only a 20% incidence of SCC. Nearly all breast tumors were adenocarcinoma, with tumor and stromal cells similar in appearance to human breast cancer (Fig. 15C).
  • Hl 80 specifically detects the human KLF4 protein by western blot and stained the nuclei of SCC cells of tet- on KLF4 transgenic mice (Foster, K. W., et al. 2005) (Fig. 15B). H180 also detects endogenous mouse Klf4 (normal skin, Fig. 15B). Using this antibody approximately 50% of breast tumors in DMBA-treated mice were co-positive for Klf4 and Ntcl (Fig. 15C, Table 3). The association of Klf4 and Ntcl in these tumors is similar as in human breast cancer (Fig. 4).
  • a common concern over use of carcinogens is that Ras mutations will be frequent.
  • Table 3 Immunostaining analysis of the MMTV-ErbB2 +/ ⁇ ; DMBA model.
  • p. KLF4 is required for in vitro growth of human pancreatic cancer cell lines with high KLF4 levels
  • siRNA-KLF4 #2/#3 A mixture of two siRNAs (siRNA-KLF4 #2/#3 ) was sufficient to block nearly all endogenous KLF4 expression by western (Fig. 18B).
  • Cells transfected with the control siRNA siRNA-Ctl; not similar to any human mRNA; Dharmacon
  • siRNA-KLF 4#2/#3 was then transfected into Pane 08.13, Pane 10.05, or the KLF4- positive, 7S I- sensitive breast tumor cell line MCF7.
  • Whole cell protein extracts were prepared at 96h post-transfection. Compared to controls, siRNA-KLF4 #2/#3 appeared to suppress the final cell number. By microscopy there were many single cells instead of clusters of dividing cells.
  • MCF7 cells which do not require KLF4 for cell growth, served as a control.
  • KLF4 knockdown cells showed reduced levels of active Ntcl IC by western blot, strongly supporting the notion that KLF4 regulates Ntcl in pancreatic cancer (Fig. 18B). Further, transfection of siRNA#2 or #3 alone OR as a mixture was sufficient to give the same growth defect, and consistently suppressed Ntcl compared to controls (siRNA#l, siRNA-Ctl). q. KLF4+ human pancreatic cancer cells are ⁇ SI-sensitive
  • KLF4 protein levels correlate with drug sensitivity in pancreatic cancer
  • 1000 cells were plated per well in triplicate 96- wells, and the cell number determined after a 5 day treatment with vehicle (DMSO), 7SI (L-685,458), 9cUAB30, or both drugs.
  • Cell counts were determined using an ATP-dependent luciferase reaction (Pierce ATPlite Assay) which in gave a strictly linear response from 100-100,000 cells per well. The day 5 cell counts in were always less than 25,000 cells, well within the linear range.
  • the KLF4-high lines Pane 10.05 and Pane 08.13 were sensitive to 7SI, while the KLF4-low lines Panel and S2013 were not.
  • 9cUAB30 was less effective than "ySI, but showed statistically significant inhibition of Pane 10.05 or Pane 08.13 at 4.0-12.0 ⁇ M (p ⁇ 0.05; Fig. 19B-C).
  • 9cUAB30 inhibits transformation, but shows no cytoxicity in cell growth assays, indicating that it targets some other process besides cell proliferation.
  • the combination of ⁇ 1+9CUABSO was also compared to 'ySI alone. In 2 of 3 experiments Pane 10.05 cells showed less growth in than in 7SI alone (Fig. 19C-D).
  • KLF4/GKLF protein was upregulated in most cases of primary human pancreatic cancer (21 of 37 cases, 57%, Fig. 16), unlike in several other tumor types.
  • KLF4 was similarly analyzed in a genetically engineered mouse model of PanIN/PDAC. KLF4 was never detected in multiple sections of normal pancreatic ducts and acini (PDX-Cre control mice), but prominently upregulated in PanlN-like lesions (Fig. 17). This result identified KLF4 as an early change, similarly as shown in oral SCC, breast cancer, and cutaneous SCC. KLF4 was also expressed in mouse lesions that are histologically similar to invasive human PDAC, but was not detected in ductal epithelium entrapped within or else distant from invasive lesions. 2.
  • Example 2 i. Results a. KLF4 upregulates Notchl in RK3E cells
  • KLF4-ER KLF4-estrogen receptor
  • 4OHT 4-hydroxytamoxifen
  • RK3E cells stably expressing either KLF4-ER or Vector were treated for 2 hours with 4OHT, with 4OHT in combination with the protein synthesis inhibitor cycloheximide (CHX), or with vehicle alone.
  • Notchl mRNA was upregulated an average of 7.5 fold in KLF4-ER cells treated with 4OHT+CHX, but not in the control. The increased expression of Notchl mRNA was confirmed by Northern blot (Fig. 3A, top panel, lanes 4 and 5). In contrast, Notch 2, 3, 4 and Dll/Jag ligands were unchanged at 2 hours of induction.
  • Antibody Nl -C20 directed against the C-terminus of Notchl, showed strong immunofluorescent staining in KLF4-ER cells, but not Vector cells, treated with 4OHT for 10 hours (Fig. 3B).
  • antibody Nl IC -Nter specific for the amino- terminus of N1 IC generated by -secretase cleavage, showed accumulation of N1 IC with increasing time in 4OHT (Fig. 3A, middle panel). This accumulation was diminished by inclusion of a ⁇ -secretase inhibitor ( ⁇ SI) in the medium (Fig. 3A, bottom panel, lane 3).
  • ⁇ SI ⁇ -secretase inhibitor
  • N1 FL to N1 IC As cleavage of N1 FL to N1 IC is dependent upon the expression of Notch ligands and components of ⁇ -secretase, these were examined for regulation by KLF4.
  • Semi-quantitative RT-PCR showed that mRNAs for the Notch ligands Dill, D114 and Jagl increased by 48 hours of induction. As there was no detectable increase at 2 hours, these ligands can be indirect targets of KLF4. Immunoblotting showed upregulation of Dill and Jagl proteins but not D114, indicating that the latter can be regulated post-transcriptionally.
  • the ⁇ -secretase component, Presenillinl (PSl) showed little increase.
  • KLF4 activity indicates that KLF4 was induced throughout the skin, with heterogeneity in the levels of expression (Foster KW, et al. 2005). Indeed, SCC-like lesions were apparent at 14 days and Notchl and KLF4 were uniformly positive (Fig. 4 A, right). KLF4 expression became increasingly nuclear with progression, as observed in human SCC (Foster KW, et al. 2005; Huang CC, et al. 2005). The close temporal correlation of KLF4 and Notchl expression supports regulation of Notch 1 by KLF4. c.
  • KLF4 and Notchl are upregulated in the majority of breast cancers, and KLF4 immunostaining identifies tumors with an aggressive phenotype (Pandya AY, et al. 2004).
  • KLF4 and Notchl are upregulated in the majority of breast cancers, and KLF4 immunostaining identifies tumors with an aggressive phenotype (Pandya AY, et al. 2004).
  • paraffin sections of primary breast cancers were immunostained.
  • N1-C20 and Nl IC -Nter antibodies were first characterized by staining subjacent sections representing 12 cases of infiltrating ductal carcinoma and adjacent uninvolved epithelium.
  • N1-C20 Although staining by the two Notch antibodies within and between sections was similar, N1-C20 predominantly stained the cytoplasm while Nl IC -Nter mainly stained the nucleus. Consistent with these results, immunoblotting studies indicated that N1-C20 recognizes Nl , the transcriptionally active nuclear form, less efficiently than other forms of Notch 1, such as Nl TM.
  • KLF4 antibody gave uniform staining of the 8 Notchl-high tumors but showed little staining in 4 Notchl-low tumors (Fig. 26).
  • KLF4-stained slides and Notchl -stained slides were scored independently. As described previously, intensity scores were determined using a 0.0-4.0 scale, and weighted according to the percentage of cells exhibiting that intensity (Pandya AY, et al. 2004). Median values were: KLF4 (nuclear) 0.5, Notchl (cytoplasmic) 0.75.
  • Chromatin immunoprecipitation was used to determine if KLF4 could bind to the human Notchl promoter (Fig. 3C).
  • MCFlOA cells were transduced with pBpuro-HA-KLF4 retrovirus and a puromycin (puro)-resistant pool selected.
  • the Notchl promoter fragment P was specifically immunoprecipitated from these cells by HA antibodies but not by control IgG (Fig. 3F, lanes 1-4).
  • a GAPDH promoter fragment was brought down by TFIIB antibodies, but not by HA or control IgG (lanes 5-8).
  • Further scanning of the Notchl promoter by ChIP showed that fragments L, M, N and O, within a ⁇ 2. 1 kb region encompassing the start of the Notchl open reading frame, were bound by KLF4.
  • fragment K was not bound (Fig. 3F, lanes 9- 12).
  • promoter-reporter assays were performed in HEK293 cells in which endogenous KLF4 is low.
  • the 2.1 kb fragment ⁇ l (-1866/+240) and a series of truncations 2 to 5 were cloned into the luciferase reporter vector, pGL3-Pro (Fig. 3G).
  • Each construct was co-transfected with either KLF4 or Vector and a control for transfection efficiency. Relative to the Vector, ⁇ l was activated 4-fold by KLF4 (Fig. 3G).
  • Notch 1 is a specific downstream effector of transformation by KLF4, endogenous Notchl was knocked down. Attempts to generate stable knockdown lines using lentiviral shRNA vectors were unsuccessful. For oncogene-transduced cells to establish transformed foci, they must continue to divide as the surrounding cells become confluent during the first 2-3 days following retroviral transduction. Thus, the suppression of oncogene transforming activity during the immediate post-transduction period should suffice to reduce the overall size and/or number of transformed foci. For example, transient expression of a dominant-negative allele of Snail 1 can block transformation by GIi 1 (Li X, et al. 2006). Transient transfection of RK3E cells with Notch 1 siRNAs was therefore used to reduce the expression of Nl IC relative to control (Fig. 22, compare lanes 1-3 with lane 4).
  • Notch 1 mRNA was rapidly induced within 3 hours (Fig.25 A, lane 8).
  • the Notch target genes Nrarp and Cyclin Dl were upregulated by 12 hours (Fig. 25A), similar to the latency of N1 IC , as shown in Fig. 3A.
  • Fig. 25A several other targets of Notch signaling were not altered by 48 hours, including Hesl, Hes5, Herpl, Herp2, Herp3, Ascll and c-Myc (Fig. 25B). Therefore, in this context Notch 1 signaling appeared to be restricted to a subset of its target promoters.
  • a Hesl-luciferase reporter that is widely used to assay activity of the canonical Notchl pathway was used.
  • RK3E cells were co-transfected with KLF4, Nl IC , or a combination of the two expression constructs, along with an internal control. Luciferase activity was measured 48 hours later (Fig. 25C). While N1 IC activated the Hesl promoter as expected (Fig. 25C, lanes 3 and 5), KLF4 suppressed Nl IC -dependent activation (compare lanes 3 and 4, and lanes 5 and 6). Similar results were obtained in HEK293 cells.
  • MAML DN2 The expression of MAML DN2 was detected by immunoblotting with a MAML-specific antibody (Fig. 8A).
  • MAML DN1 lacks the epitope recognized by this antibody, and was expressed as a GFP-fusion protein detectable by GFP antibodies (Fig. 8A).
  • RT-PCR with species-specific primers was used to detect expression of CSL DN (Fig. 8B).
  • the activation of the Hesl-luciferase reporter was next examined in the presence of exogenous N1 IC by transient transfection into each of these pools of cells (Fig. 8C).
  • RK3E and HEK293 cells were grown in DMEM with 10% fetal bovine serum.
  • RK3E/pBpuro, RK3E/N1 IC , RK3E/KLF4-ER, RK3E/HA-KLF4, RK3E/pQCXIN, RK3E/MAML DN1 , RK3E/MAML DN2 and RK3E/CSL DN pools were derived by retroviral transduction of RK3E cells as described (Foster KW, et al. 1999; Louro ID, et al. 2002).
  • MCFlOA, ZR75-1 and BT474 cells were obtained from the American Type Culture Collection and cultured as recommended by the supplier.
  • Virus transduced cells were grown in the presence of 0.4-1.0 ⁇ g/ml puro (Sigma) or 400 ⁇ g/ml G41 8 (Mediatech). The focus assay was described previously (Foster KW, et al. 1999). The ⁇ SI L685, 458 (Sigma) was used at 10.0 ⁇ M in DMSO 5 and media were changed daily. siRNAs were synthesized by Therrno-Fisher/Dharmacon (see Table 5 for sequences), and transfected into RK3E or human tumor cell lines using LipofectamineTMRNAiMAX reagent (Invitrogen).
  • Hs KLF4-siR2 (AS) 3'UUCUCUCUGGCUCCUCAAGUU 5' SEQ ID NO:12
  • Bi-transgenic K14-rtTA; TRE-KLF4 mice were fed dox in drinking water as described previously (Foster KW, et al. 2005). Two mice were sacrificed at the indicated times (Fig.4A) and skin sections were obtained. Breast tumors were obtained from the University of Alabama hospital. Tissues were fixed and processed as described (Pandya AY, et al. 2004). Adjacent sections from 89 tumors were stained with KLF4 or N1-C20 antibodies, and statistical analysis was performed in consultation with the Biostatistics Shared Facility of the Comprehensive Cancer Center, University of Alabama at Birmingham. 12 of these 89 cases were also stained with Nl IC -Nter using heat-induced antigen retrieval. All experiments used normal rabbit immunoglobulin as a negative control. Human and animal studies conformed to NIH guidelines and were approved by the Institutional Review Board for Human Use and the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.
  • KLF4-ER cells were plated on poly-L-lysine coated coverslips at 30% confluence. 24 hours later, 4OHT or DMSO was added to the medium. After 10 hours, the cells were fixed and stained with N1-C20 antibody. Indirect immunofluorescence and digital imaging were performed as described (Pandya AY, et al. 2004). d. Reporter Assays
  • Fig. 3G 0.2 ⁇ g of pTK-/3-gal (internal reference), 0.5 ⁇ g Notchl promoter- reporter, and 0.2 ⁇ g pRK5-HA-KLF4 or control Vector (pcDNA3.1) were transfected into HEK293 cells at 50% confluence, using 1.8 ⁇ l TransIT-LTl (Minis) according to the manufacturer's protocol. Cells were extracted 48 hours later and assayed for luciferase and ⁇ -gal activity with the Dual Light System (Applied Biosystems). Luciferase activity was normalized to ⁇ -gal, and the fold activation (KLF4/Vector) was determined. Using similar methods (Fig.
  • MCFlOA cells at 95% confluence were washed twice with PBS.
  • Formaldehyde (37% solution; Fisher Scientific) was added to a final concentration of 1% in PBS, and the plates were incubated at room temperature for 10 min.
  • Cells were rinsed with ice-cold PBS and scraped into 6 ml of 100 mM Tris-HCl pH 9.4/10 mM DTT, transferred to a 15 ml tube and incubated at 30 0 C for 15 min.
  • the cells were centrifuged for 5 min at 3000 rpm, washed and centrifuged sequentially with 1.0 ml of buffer I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES-KOH pH 6.5) and 1.0 ml of buffer II (200 mM NaCl, 1.0 mM EDTA, 0.5 niM EGTA, 10 mM HEPES-KOH pH 6.5).
  • buffer I 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES-KOH pH 6.5
  • buffer II 200 mM NaCl, 1.0 mM EDTA, 0.5 niM EGTA, 10 mM HEPES-KOH pH 6.5.
  • Cells were re- suspended in 0.6 ml of lysis buffer with freshly added protease inhibitors (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1.0 mM PMSF, 1.0 mM benzamidine, 5.0 ⁇ g/ml leupeptin, 5.0 ⁇ g/ml aprotinin) and incubated on ice for 10 min. Samples were then sonicated with an Ultrasonic processor at 25% amplitude (twenty pulses for 10s each) on ice, then centrifuged at 4°C at 10,000 rpm for 10 min.
  • protease inhibitors 1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1.0 mM PMSF, 1.0 mM benzamidine, 5.0 ⁇ g/ml leupeptin, 5.0 ⁇ g/ml aprotinin
  • ChIP Dilution Buffer 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, with protease inhibitors as in lysis buffer above
  • the chromatin was pre-cleared with 12 ⁇ g of sheared sperm DNA, 10 ⁇ l normal mouse IgG (0.5 mg/ml, Santa Cruz) and protein A-Sepharose (lOO ⁇ l of 50% slurry in 10 mM Tris-HCl pH 8.0, 1.0 mM EDTA) with agitation for 2 hrs at 4°C.
  • Pre-cleared chromatin was incubated with 4.0 ⁇ g HA antibody, 9.0 ⁇ l of TFIIB antibody, or 9.0 ⁇ l normal IgG (Chip-IT Kit, Active Motif, Cat# 53001), and rocked at 4°C overnight. 100 ⁇ l of protein A-Sepharose (50% slurry) and 20 ⁇ g salmon sperm DNA were added to each sample, followed by agitation for a further 2.5 hrs at 4 0 C.
  • Beads were pelleted by centrifugation at 2000 x g for 3 min at 4°C and washed sequentially [10 min, in 1.0 ml with low salt buffer (0.1% SDS, 1% Triton X-100, 2.0 mM EDTA, 20 mM Tris- HCl pH 8.1, 150 mM NaCl ), TSE2 (0.1% SDS, 1% Triton X-100, 2.0 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl) and Buffer 3 (0.25% LiCl, 1% NP40, 1% sodium deoxycholate, 1.0 mM EDTA, 10 mM Tris-HCl pH 8.0)], followed by 3 washes in TE (10 mM Tris-HCl pH 9.0, 0.1 mM EDTA).
  • TE 10 mM Tris-HCl pH 9.0, 0.1 mM EDTA
  • the complexes were eluted in 250 ⁇ l of elution buffer (1% SDS, 0.1 M NaHCO 3 ) with agitation for 15 min. The beads were pelleted by centrifugation and the supernatants were transferred to clean tubes. The elution was repeated and the two eluates were combined. Crosslinks were removed by adding NaCl to 0.3 M with incubation at 65°C overnight. To reverse crosslinks in the input DNA, 100 ⁇ l inputs were heated without addition of NaCl. DNA was purified using Qiaquick columns (Qiagen) and eluted in 50 ⁇ l of 10 mM Tris-HCl pH 8.0. 2 ⁇ l of DNA was used in quantitative PCR reactions. PCR primers are listed in Table 6.
  • Oligonucleotides used for semiquantitative RT-PCR were used for semiquantitative RT-PCR.
  • Citron M. Strategies for disease modification in Alzheimer's disease. Nat.Rev.Neurosci., 5: 677-685, 2004.
  • ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc.Natl.Acad.Sci.U.S.A, 100: 8933-8938, 2003.
  • KLF4 and PCNA identify stages of tumor initiation in a conditional model of cutaneous squamous epithelial neoplasia. Cancer Biology and Therapy 4: 1401-1408, 2005.
  • Klf4 The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development, 129: 2619-2628, 2002.
  • Notch homologue mouse Notch 3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium. Mech.Dev., 46: 123-136, 1994. LaVoie, M. J. and Selkoe, D. J. The Notch ligands, Jagged and Delta, are sequentially processed by alpha-secretase and presenilin/gamma-secretase and release signaling fragments. J.Biol.Chem., 278:34427-34437, 2003.
  • KLF4 regulates Notchl transcription and signaling during epithelial transformation and tumorigenesis.
  • the Notchl receptor is cleaved constitutively by a furin-like convertase. Proc.NatLAcad.Sci.U.S.A, 95: 8108-8112, 1998.
  • TRAIL Tumor necrosis factor-related apoptosis-inducing ligand
  • KLF4 Dominant negative alleles of KLF4 associate with Sin3A and inhibit growth of mammary epithelial cells, hi preparation.
  • Prasad NB Biankin AV, Fukushima N, et al. Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells. Cancer Res 2005 ;65: 1619-26.
  • the KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nat.Cell Biol., 7: 1074-1082, 2005.
  • the zinc finger protein GLI transforms primary cells in cooperation with adenovirus El A. MoI Cell Biol 1991; 11:1724-8.
  • Widschwendter M., Berger, J., Daxenbichler, G., Muller-Holzner, E., Widschwendter, A., Mayr, A., Marth, C, and Zeimet, A. G. Loss of retinoic acid receptor beta expression in breast cancer and morphologically normal adjacent tissue but not in the normal breast tissue distant from the cancer. Cancer Res., 57: 4158-4161, 1997.

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Abstract

Disclosed herein are compositions and methods relating to Kriippel-Like Factor/Epithelial Zinc Finger (GKLF, or termed KLF4, Kriippel-like factor 4) and uses thereof in medical diagnosis and treatment.

Description

KRUPPEL-LIKE TRANSCRIPTION FACTOR KLF4/GKLF AND USES
THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. Provisional Application No. 61/026,276, filed February 5, 2008, which is hereby incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with government support under Grant ROl CA65686 and P50 CA89019 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
Cellular oncogenes have been isolated by characterization of transforming retroviruses from animal tumors, by examination of the breakpoints resulting from chromosomal translocation, and by expression cloning of tumor DNA molecules using mesenchymal cells such as NIH3T3. Several human tumor types exhibit loss-of-function mutations in a tumor suppressor gene that lead to activation of a specific oncogene in a large proportion of tumors. For example, c-MYC expression is regulated by the APC colorectal tumor suppressor; expression of GLI is activated by loss-of-function of PTC in human basal cell carcinoma and in animal models; E2F is activated by loss-of-function of the retinoblastoma susceptibility protein pi 05Rb; and RAS GTPase activity is regulated by the familial neurofibromatosis gene NFl. The comparative genomic hybridization assay and related methods have shown that numerous uncharacterized loci in tumors undergo gene amplification. These observations, and the infrequent genetic alteration of known oncogenes in certain tumor types, suggest that novel transforming oncogenes remain to be identified.
One limitation to the isolation of oncogenes has been the paucity of in vitro assays for functional expression cloning, as several oncogenes are known to exhibit cell-type specificity. For example, GLI, BCR-ABL, NOTCHl/TANl, and the G protein GIP2 have been found to transform immortalized rat cells, but not NIH3T3 or other cells, demonstrating the potential utility of alternate assays for oncogene expression cloning. While most studies have used NIH3T3 or other mesenchymal cells as host for analysis of oncogenes relevant to carcinoma, the potential utility of a host cell with epithelial characteristics has been discussed. Thus, there is a need for methods of identifying carcinoma oncogenes by utilizing a host cell with epithelial characteristics.
BRIEF SUMMARY
In an aspect, provided are methods and compositions related to Kruppel-Like Factor/Epithelial Zinc Finger (GKLF, or termed KLF4, Kruppel-like factor 4) and uses thereof in medical diagnosis and treatment.
Additional advantages of the disclosed methods and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or maybe learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and together with the description, serve to explain the principles of the disclosed methods and compositions.
Figure 1 shows Notch (Ntc) maturation and signaling. Figure IA shows structure of Ntc proteins. NtcEC: extracellular domain (-180 kDa) generated from full length by cleavage at site Sl in Golgi. Ntc™: transmembrane form (—120 kDa) generated by cleavage at S2 by TACE. NtcIC: intracellular form (~90 kDa), generated by cleavage at S3 by γ-secretase. LP, leader peptide; TM, transmembrane domain; ANK, 6 ankyrin repeat domain; NLS, nuclear localization signals; TAD, transcription transactivation domain. The binding sites for some Ntc interacting proteins are shown below. Figure IB shows Ntc signaling pathways. In the Classical Pathway, NtcIC displaces histone deacetylases (HDACl) and co-repressors (Hairless, CoR) from CSL. Then Mastermind- Like (MAML) proteins and histone acetylases (p300, CAF) can bind. The complex activates transcription through CSL binding sites in cellular promoters such as Hes (Hairy/Enhancer of split) genes. Alternate Pathways: signaling mediated by proteins other than CSL. In summary, KLF4 can activate Ntc expression, and can alter Ntc signaling by induction of the CSL- antagonist Hairless. This results in inhibition of Classical Pathway signaling as indicated by CSL activity and expression of Hesl (bottom left), and instead promotes Alternate Pathway signaling (bottom right).
Figure 2 shows Kaplan-Meier analysis of KLF4 and the Ntcl precursor form, Ntc ITM, in breast cancer. 153 breast cancer cases were stained and scored with rabbit polyclonal oKLF4 and with Ntcl (Santa Cruz, C-20). Cut-offs were selected to optimize the P value (logrank test). Median scores were: KLF4_Cyto=0.3, McI-CyIo=O-S, and KLF4_Nuc=0.45. Figure 2 A shows type 3 tumors, with high KLF4 cytoplasmic and low KLF4 nuclear staining, were at increased risk (P=0.031). Figure 2B shows tumors with high KLF4_Cyto and low Ntcl_Cyto were more likely to die from breast cancer (P=0.0052). Figure 2C shows three variables used in combination (P=0.0033). Kaplan- Meier methods were used to plot survival curves and log rank test was used to compare survival rates. Highest risk groups are indicated with an asterisk.
Figure 3 shows Notchl is a direct transcriptional target of KLF4 in breast epithelial MCFlOA cells. Figure 3 A shows KLF4-ER- or Vector (pBρuro)-transduced RK3E cells were treated with cycloheximide (CHX), or 4-hydroxytamoxifen (4OHT) as indicated above the lanes, and total RNA was isolated two hours later. Northern blot analysis used a labeled PCR fragment of rat Ntcl (upper panel). The membrane was stripped and reprobed with a cloned human /3-tubulin (/3-Tub) cDNA fragment as a loading control (below). Protein extracts were queried with V 1744 αNtclIC (Abeam) that recognizes the active form. Similar induction of the inactive precursor, NtclTM, was seen using oNtcl C20 (Santa Cruz). Figure 3B shows KLF4-ER- or Vectorexpressing RK3E cells were either not treated (left panels) or treated with 4OHT (right panels) for 10 hours. Indirect immunofluoresence staining used α-Ntcl C-20 antibody (Santa Cruz), Phalloidin and DAPI. Scale bar is 20 μm. Figure 3C shows a schematic of the human Ntcl promoter (- 9903 to +240 bp relative to the initiation codon, ATG). Vertical lines indicate consensus KLF4 binding sites. A broken arrow indicates the putative start site of transcription as determined by homology with the mouse gene. K-P indicate the positions and sizes in bp of PCR fragments scanned by CbJP. Figure 3D-F show ChIP. MCFlOA cells expressing HA-KLF4 were treated with formaldehyde. Sonicated, cross-linked chromatin was isolated and immunoprecipitated (IPd) with the indicated antibodies. The IPs were analyzed by PCR to amplify fragments K-P (panels F and D) from the Ntcl promoter or a fragment from the human GAPDH promoter (panel E). Lanes 2 and 6 show PCRs performed with 0.02% of the input material used for IP. In lane 1, template DNA was omitted (negative control). In Figure 3F, the efficiencies of IP of fragments K-N relative to that of fragment O are indicated below the panels. Figure 3G (left) is a schematic showing deletion mutants Δ1-Δ5 of the human Notchl promoter cloned in pGL3-Pro. These were transiently transfected with pTK-/3-gal (control) into HEK293 cells. The relative transcription efficiency of the mutants is shown in the histogram on the right. The experiment was done three times in triplicate and SE bars are shown. Figure 3H shows KLF4 induces Notchl ligands by an indirect (slow-onset) mechanism. Total RNA was isolated from Vector- (lanes 4, 5) or KLF4-ER- (lanes 1-3) expressing RK3E cells treated with 4OHT for 48 hrs. Semiquantitative RT-PCR analysis was done for the Notch ligands indicated to the left. For KLF4-ER, input was varied by 3-fold to ensure that PCR reactions were not saturated (lanes 2, 3).
Figure 4A shows rapid upregulation of Notchl following induction of KLF4 in the skin of 'tet-on! K14-rtTA; TREEXF4 transgenic mice. KLF4 was induced by addition of dox to the drinking water, and skin sections were obtained from two mice at each of the times shown. The sections were stained with rabbit oKLF4 or αNtcl-C20. The top and bottom panels represent nearly adjacent sections of one tissue sample. Scale bars, 50 μm. Figure 4B-D show expression of Notchl and KLF4 are strongly correlated in primary human breast tumors. Figure 4B shows staining of sub-adjacent sections of a representative breast tumor with the indicated antibodies. Scale bar, lOOμm. Figure 4C shows Pearson correlation analysis of KLF4 and Notchl in 89 cases of breast cancer. Scores were obtained using a 0.0-4.0 scale, with weighting based on the percentage of cells exhibiting each score. Cytoplasmic KLF4 was likewise correlated with Ntcl, with similar P and r values. Tumors with the same scores appear as one data point. Figure 4D shows contingency analysis of Notchl and KLF4 (Fisher's exact test, two-tailed).
Figure 5 shows transformation by KLF4 requires Ntcl signaling. Figure 5 A shows RK3E epithelial cells stably transduced with antisense (AS) Ntcl10 or Vector were superinfected with KLF4, Ras, c-Myc or GUI retroviruses and allowed to form foci for four weeks. The Wright-stained dishes shown are representative of three or more independent experiments. Transformation by RAS is expected to require Ntcl (see Miele L., Nat. Med. 2001). Figure 5B shows the results in A were quantitated by counting of foci. SE bars are shown. Figure 5C shows RK3E cells were transfected with increasing amounts of pcDNA3-Ntcl AS, and a constant amount of Ntcl , Hesluc reporter, and pTK-jS-Gal (a control for transfection efficiency). Luciferase activity was normalized to β- Gal. The experiment was performed in triplicate and SD bars are shown. Figure 5D shows RK3E cells were transduced by retroviruses endcoding NtclIC or KLF4-ER and allowed to form foci for two weeks in the presence of vehicle (DMSO) or 7SI (L-685,458). Transformation by the S3 cleavage product NtclIC is expected to be ^Si-independent (see Fig. IA).
Figure 6 shows neoplastic transformation of RK3E cells by full length Notch 1 (Ntc 1). Figure 6A shows FL Focus formation by RK3E cells infected with Vector, NtclIC or Ntc 1FL retroviral supernatants. Figure 6B shows immunoblot of extracts from cloned cell lines derived from individual Ntcl foci showing increased expression of NtclFL (lanes 3- 9) as compared with Vector cells (lanel) or NtclIC cells (lane 2). The same samples were probed for jS-actin (control; lower panel). Figure 6C shows NtclFL transformation correlates with increased Ntcl expression. RK3E cells were transduced, selected in puro, and either cultured at subconfluence (NtclFL*) or incubated for 2 weeks at confluence until a transformed morphology was observed (NtclFL). Whole cell extracts were analyzed by immunoblot with α-Ntcl C20 which recognizes all three species (top panel). The filter was stripped and reprobed with V 1744 which is specific for NtclIC (middle panel). Additional species (uncharacterized) that interact with C-20 appear in NtclFL and NtclIC transformed cells (asterisk). Amidoblack staining of spotted cell extract was used as a loading control (LC). NtclFL requires cleavage to NtclIC by γ-secretase for a transformed growth phenotype. NtclIC and NtclFL expressing cells were plated at low confluence in six well plates and allowed to form colonies in the presence of 7SI or vehicle (DMSO). This colony morphology assay was described previously (Foster, KW et al., Cell Growth Differ 1999). 7SI reduced the density of NtclFL but not NtclIC colonies. This strongly supports specificity since the product of the blocked enzymatic step, NtclIC, should be resistant to 7SI.
Figure 7 shows KLF4 blocks classical pathway activation by Notchl Figure 7A shows KLF4 suppresses classical Ntcl signaling. Hesl-luc, 8x wt CSL and 8x mutant CSL reporters were used in transient transfection assay of RK3E cells. The experiment was done in triplicate (bars, SD). Similar results were obtained in HEK293 cells. Figure 7B shows two mechanisms of transformation by Notchl . When KLF4 is low/absent (left panel), Notchl induces transformation by signaling through the classical pathway. Activated NtclIC relocates to the nucleus where it displaces Hairless (Hr) and Co- repressors (CoR) from CSL. MAML and Co-activators (CoA) are recruited to the complex which then activates targets such as the Hes genes. In the presence of KLF4 (right panel), Notchl induces transformation through an alternative pathway.
Figure 8 shows dominant negative (DN) MAML and DN CSL block transformation of RK3E by NtclIC but not by KLF4 or RAS. Figure 8 A shows to detect expression of DN proteins, lysates from RK3E cell lines stably expressing Vector or DN MAMLs were probed with α-MAMLl . The position of MAMLDN2 protein is indicated by an arrow (top panel). As MAMLDNl is not recognized by α-MAMLl, GFP antibody was used to detect MAMLDNI-GFP fusion protein (middle panel). /3-actin served as a control for protein loading (bottom panel). Figure 8B shows semi-quantitative RTPCR was done with total RNA isolated from the indicated cell lines using mouse-CSL specific primers. GAPDH served as a control. Figure 8C shows classical Notchl signaling is silenced in RK3E cells that stably expressing DN MAML or DN CSL. Hesl-luc reporter activity was measured in MAML DNl, MAML DN2, CSL DN or Vector (pQCXIN) cells 48 hrs. after co-transfection of reporter with Ntcl or Vector. Experiments were done twice in triplicate (bars, SE). Figure 8D shows focus formation assay. Cells stably carrying the retroviral vector indicated above the dishes were superinfected with NtclIC, KLF4, RAS or pBPuro retroviral supernatant as indicated on the left. Plates are representative of three independent experiments done in triplicate. Insets show KLF4 foci at higher magnification. Figure 8E shows quantification of foci. The histogram shows number of foci in DN cell lines normalized to Vector (bars, SD).
Figure 9 shows maturation of MCFlOA is blocked by KLF4 or Ntclic. MCFlOA cells were plated on a layer of Matrigel, and underwent morphogenesis over a period of 10-14 days to form smooth, round, hollow spheres of epithelium (Vector panels). Cells stably transduced with erbB2 formed extended, multi-lobular structures with smooth, round borders, consistent with hyperplasia and epithelial differentiation. In contrast, cells transduced with KLF4 or Ntcl retained the disorganized structure observed at 4-5 days, with rough, irregular borders, indicating an arrest of epithelial differentiation. The developmental arrest persisted until 19 days, when the experiment was terminated. Scale bars, lOOjLtm.
Figure 10 shows phenotypes of KLF4 proteins. Figure 1OA shows to detect expression of endogenous KLF4 transcripts, total RNA was isolated from the indicated cell lines and analyzed by Northern blot (left panel). A /3-tubulin probe served as a control for loading. To detect the endogenous protein, rabbit oKLF4 (upper panel) or normal rabbit Ig (lower panel) was applied to MCFlOA cells. Bound Ig was detected using a red- fluorescent secondary ab. Signal corresponding to KLF4 is shown superimposed on the cell nuclei (TRITC+DAPI). Scale bars, 50μm. Figure 1OB shows mammary epithelial cell lines (MCFlOA or MCFlOAT) were transduced using the indicated retrovirus supernatants (Vector, KLF4ala5, KLF4ala6) and selected in puromycin. Surviving colonies were stained with modified Wright's stain after 10 days in selection. MCF10A-KLF4DN colonies surviving puromycin were estimated as <1% of the Vector control. MCF10A-KLF4WT cells grew similarly, or formed even more dense colonies, as compared to the Vector control. Similar results were obtained in three independent experiments. Figure 1OC shows RK3E cells were transduced using the retrovirus supernatants Vector, KLF4ala5, or KLF4ala6 (Construct 1) and selected in puro. Surviving cells were transduced with c-MYC, ErbB2, GLIl, N-RAS, Notch 1 or KLF4 retroviruses (Construct 2) and incubated in puromycin at confluence for 3 weeks to allow the oncogene to induce transformation (there is no drug selection for the Construct 2 in focus assays). The background observed when Construct 2 is Vector is shown in the inset (CtI). Two independent experiments were performed in triplicate. Similar results were observed for KLF4-ala6. Figure 1OD shows Results of one experiment, including standard deviation bars, are shown. The ~10-fold suppression for ErbB2 shown in C is consistently observed (2 expts, 6 dishes total). These genetic studies indicate that endogenous EXF4 is required for proliferation/survival of MCFlOA cells, and for transformation by specific transforming oncogenes such as c-MYC and ErbB2. Further support for these conclusions was obtained using KLF4 WT-ER, which is DN in the absence of 4OHT.
Figure 11 shows RARγRXRα and NUR77 are direct transcriptional targets of KLF4. Figure 1 IA shows KLF4-ER or vector expressing cells were treated for two hours with 4OHT and CHX, RNA was isolated and assayed by microarray (Affimetrix chip RAE U230A). Two completely independent experiments were done. The histogram shows the induction of the indicated genes. p21 is a known target of KLF4. Figure HB shows RNAs prepared as in A were used as templates for RT-PCR using oligonucleotides specific to rat RARy and GAPDH (control). Figure 11C shows RK3E cells stably expressing KLF4-ER or Vector were induced with 4OHT for the times indicated above the lanes. RNA was isolated and analyzed by RTPCR using oligonucleotides specific for the genes indicated on the left. RXRo; was not detected by microarray. Figure 12 shows RXR-selective agonist (9cUAB30) specifically blocks KLF4 induced transformation. Figure 12A shows K14-rtTA;TRE-KLF4 transgenic mice were induced with dox for 2 weeks. Food was admixed with placebo or with 9cUAB30. Induction of dysplasia-SCC was scored by gross and microscopic exam. No abnormality was observed in 8/9 animals treated with 9ΛJAB30. 1/9 treated animals showed mild hyperplastic/dysplastic changes that were attenuated compared with controls, which exhibited the full dysplasia-SCC phenotype as published (Foster, KW et al., 2005). Figure 12B shows retroviral supernatants were generated using the expression vectors indicated on the left and applied to RK3E cells. Dishes were incubated for 3-4 weeks with addition of 9cUAB30 or DMSO to the culture media every two days. Transformed foci were identified by Wright stain. Similar results were obtained in 3 independent experiments, each performed in replicate.
Figure 13 shows efficacy of 9cUAB30 in therapy of established breast cancers. Breast cancers were induced in female rats using MNU and therapy was initiated at day 0, when the tumor size was -200 mm2 as described in the text. (Bars, SD).
Figure 14 shows KLF4-high tumor cells are sensitive to γSI or 9cUAB30. Figure 14A shows cells were plated at 5,000 cells/well in 24 well plates and treated with DMSO (0.2%) control, 1.0 M 9cUAB30, or 10 M 7SI in media/10% FBS. Drugs were added 24 hours after plating and drugs/medium was changed every other day. ATP levels were determined after 5 days of treatment. KLF4 expression levels (H, high; I, intermediate; L, low) were determined by Northern blot previously (Foster, KW, et al., 2000). NtclIC expression was shown by western blot (NT, not reported) (Stylianou S. et al., 2006). Figure 14B shows dose-response of MCF7 was determined at 5 days. Figure 14C shows in an experiment independent from the work shown in panel A, drugs were applied singly or in combination and MCF7 growth was determined after 5 days. For all data the mean of 4 replicates is shown. Bars, SD.
Figure 15 shows the genotype determines the tumor type following oral DMBA treatment of genetically altered mice. Figure 15A shows Kaplan-Meier analysis. Insets show the number of animals in each treatment group (N), the number of tumors of all types (T), the average multiplicity of tumors (T/N). The % of animals in each group that developed breast cancer or SCC is indicated (Tumor Incidence). Figure 15B shows characterization of Klf4 ab (Hl 80). SCC was induced by expressing human KLF4 in the skin of transgenic mice (Tet-on SCC). Hl 80 showed prominent nuclear and cytoplasmic staining, as published for this model (Foster KW et al., Oncogene 2005). Hl 80 also showed staining of endogenous Klf4 in normal mouse skin and adnexa (Normal skin). IgG was used as a negative control. Arrows indicate the dermoepidermal junction (DEJ). Figure 15C Staining of breast cancers from the MMTV-ErbB2;DMBA model. Klf4 and Ntc 1 were co-positive in Case #8 (see Table 3) but negative in Case #10. Scale bars, 100 μm.
Figure 16 shows KLF4 is expressed in primary human pancreatic cancers. Paraffin sections were stained with KLF4 antibody (Hl 80, Santa Cruz). Results were confirmed using an anti-KLF4 rabbit antibody (Foster KW et al., Oncogene 2005). DAB staining is indicated. As published for other tumor types, some tumors show mainly cytoplasmic staining (Case 3), others show nuclear staining (Case 5), and others appear mixed (Cases 2, 4). Normal rabbit antibody (Dako) was used as a negative control (upper right). Scale bar, 50μm.
Figure 17 shows KLF4 expression during pancreatic tumor progression in the PDX-Cre; LSLRasG12D; Smad41oxP/loxP mouse model. KLF4 immunostaining was low or absent in normal pancreatic ducts (Normal pancreas, arrows) or acini (arrowhead). A pancreatic islet is shown under the scale bar (50 μm). KLF4 was prominent in the early neoplastic ducts (PanlN) and was consistently detected where neoplastic ductal cells appeared more invasive (Pancreatic tumor).
Figure 18A shows Western blot analysis of human pancreatic cancer lines. A single filter was probed in sequence with each of the antibodies shown. Molecular weight markers are shown on the right. As a control, RK3E cells transduced with a KLF4 vector or an empty vector were analyzed in parallel (lanes 11-12). Figure 18B shows siRNA- mediated knockdown of KLF4. The KLF4-high cell line Pane 10.05 was tranfected with the indicated siRNA, and extracts were analyzed at 96h. A single filter was sequentially analyzed with the indicated antibodies. siRNA-Ctl is a commercially supplied control (Dharmacon) without similarity to human mRNAs. Ntcl knockdown was likewise observed when siRNA#2 or siRNA#3 were transfected alone. Figure 18C shows Growth effects of KLF4-Ntcl knockdown in pancreatic cancer cells. siRNAs were transfected in 6-well plates. Cell number at 96h posttransfection was estimated using the Bradford assay to quantitate total cellular protein. The results shown are the mean of two sequential, independent experiments performed by different operators (JMR and QL). Using a novel transfection protocol that we developed, transfection efficiencies for the two cell lines were estimated as >90% based upon a KIFl 1 control that gives mitotic arrest and cell death.
Figure 19 shows cytoxicity of KLF4 inhibitors in pancreatic cancer. Cells were plated at 1000 cells per well and allowed to equilibrate for 24h. Cells were then treated as indicated for 5 days. The SI was L685,458, which is a nontoxic, oncogene-specific antagonist of KLF4-induced transformation (RK3E cells). Some other γSIs tested were not compatible with long-term (3 weeks) cell culture assays. Cell number (normalized to DMSO control) was determined using the Pierce ATPLite assay (luciferase assay of ATP). Figures 19A-C show experiments performed once in triplicate. Figure 19D shows results of one triplicate experiment. Similar results were obtained in an independent experiment. Data for each cell line (4-5 conditions) were analyzed by 1 way ANOVA with post-Tukey test. Bars, SD.
Figure 20 shows subjacent sections of primary human breast tumor stained with the indicated antibodies. Within the section were areas representing either infiltrating ductal carcinoma (Tumor, upper row) or adjacent, uninvolved epithelium (lower row). Scale bar, 50 μm.
Figure 21 shows immunoblot analysis of Nl IC following siRNA-mediated suppression of KLF4 in BT474 and ZR75-1 human breast cancer cell lines. The upper and lower portions of a filter representing a single SDS-PAGE gel were queried in parallel with NlIC-Nter or KLF4 (Hl 80) antibodies, respectively. The lower portion was reprobed with β-tubulin antibody (loading control).
Figure 22 shows KLF4-ER cells transfected with Notchl -specific siRNAs, and Nl IC expression was determined by immunoblot 72 hours later. A portion of the filter was queried in parallel with -tubulin antibody (loading control).
Figure 23 shows quantification of transformed foci from the experiment shown in Fig. 24.
Figure 24 shows RK3E cells transfected with Notchl -specific siRNAs and then transduced with KLF4-ER retrovirus 24 hours later. Transformed foci were scored at 20 days. ErbB2 served as a control oncogene and showed no inhibition by siRNAs. Three independent experiments were performed in duplicate.
Figure 25 shows inhibition of canonical Notchl signaling by KLF4. Figure 25 A shows semi-quantitative RT-PCR used to analyze expression of Notchl and Notchl - regulated genes following activation of KLF4. Figure 25B shows immunblot analysis of extracts prepared in parallel with the RNAs used in panel A. The filter was queried with Hesl antibody, then stripped and reprobed with /3-actin antibody (loading control). Figure 25C shows RK3E cells transfected with the indicated luciferase reporter and an internal reference in combination with Nl IC, KLF4, and/or empty vector. Following normalization to adjust for differences in transfection efficiency or cell number, the basal activity of each reporter was assigned a value of 1.0 (lanes 1, 7, and 13). The data represent one experiment performed in duplicate. Similar results were obtained in independent experiments.
Figure 26 shows immunostaining of human breast cancers with antibodies to KLF4, and Notchl (N1-C20, and NlIC-Nter). Staining of tumor cells is indicated by a brown precipitate. Cases shown are representative of a total of 8 cases with positive staining and 4 cases with low/negative staining. No staining was observed when normal rabbit immunoglobulin was used as the primary antibody (not shown). Scale bar, 50μm.
DETAILED DESCRIPTION
The disclosed methods and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a peptide is disclosed and discussed and a number of modifications that can be made to a number of molecules including the peptide are discussed, each and every combination and permutation of peptide and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.
It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. A. Compositions
1. Oncogenes/ Tumor Suppressors i. GKLF/ KLF4
Disclosed herein are compositions and methods relating to the identification, diagnosis, and/or treatment of tumors based on expression of Gut-Enriched Kriippel-Like Factor (GKLF) protein/ Kruppel-like factor 4 (KLF4). US Patent No. 7,364,868 is incorporated by reference herein in its entirety for its teaching of detecting nuclear and cytosolic expression of KLF4 in cancer cells and uses thereof for pro gnosing for aggressive early-stage breast carcinoma.
A significant number of transcription factors use a conserved zinc finger domain to bind their target DNAs. In fact, the human genome encompasses over 700 genes that contain a particular C2H2-type of zinc finger, which employs two cysteine and two histidine amino acid residues to coordinate the single zinc atom in the finger-like structure. A further subgroup of the C2H2-zinc finger proteins exhibits homology to the Drosophila melanogaster segmentation gene product, Krύppel. Members of this subgroup are termed Kriippel-like factors (KLFs), and have been extensively studied for their roles in cell proliferation, differentiation and survival, especially in the context of cancer.
All KLF family members are characterized by their three Cys2His2 zinc fingers located at the C-terminus, separated by a highly conserved H/C link. DNA binding studies demonstrated that the KLFs have similar affinities for different GC -rich sites, or sites with CACCC homology, and can compete with each other for the occupation of such sites. KLFs also share a high degree of homology between the specificity protein (Sp) family of zinc-finger transcription factors and bind similar, if not the same sites, in a large number of genes.
KLF4, also known as Gut-enriched Kruppel-Like Factor (GKLF) acts as a transcriptional activator or a transcriptional repressor depending on the promoter context and/or cooperation with other transcription factors. For example, KLF4 transactivates the iNOS promoter in cooperation with p65 (ReIA), and the p21Cipl/Wafl promoter in cooperation with p53, but it directly suppresses the p53 promoter and inhibits ornithine decarboxylase promoter activity by competing with specificity protein- 1 (Sp-I). KLF4 also interacts with the p300/CBP transcription co-activators. KLF5, also known as Intestinal enriched Kruppel-Lke Factor (DCLF) or basic transcription element binding protein 2 (Bteb2) has been assigned purely transcriptional activation activity but, similar to KLF4, binds p300 which acetylates the first zinc finger conferring a trans-activating function. Importantly for KLF4 & KLF5, the amino acids that are predicted by the Klevit model to interact with DNA are identical and the two compete for the same CACCC element found in a wide variety of promoters. KLF4 & KLF5 can act antagonistically during cellular proliferation, differentiation, and promoter activation, either via direct competition or via alterations in their own gene expression. The expression of KLF4 in terminally-differentiated, post-mitotic intestinal epithelial cells as opposed to proliferating crypt cells which contain high levels of KLF5 is one example of such opposing effects. KLF4 inhibits proliferation through activation of p21Cipl/Wafl, and direct suppression of cyclin Dl and cyclin Bl gene expression. Both KLF4 & KLF5 proteins act on the klf4 promoter where KLF4 increases expression and KLF5 decreases expression of klf4 mRNA.
A full-length mouse cDNA clone encoding KLF4 was initially isolated from a NIH3T3 cDNA library by reduced-stringency screening with a DNA probe containing the zinc finger region of an immediate early gene product, Zif268 or Egrl. Mouse KLF4 contains 483 amino acids, has a predicted molecular weight of 53 kD, and is 90% identical to human KLF4. The carboxyl terminus of KLF4 contains three C2H2-zinc fingers. KLF4 is a nuclear protein whose cellular address depends on two nuclear localization signals. A survey of the tissue distribution in adult mice revealed that KLF4 is highly expressed in terminally differentiated, post-mitotic epithelial cells of the intestinal tract, a finding consistent with the antiproliferative effect of KLF4.
Gene sequences for Kruppel-like factors are listed in Table 1. Table 1. Kruppel-like factor Sequences
Symbol Aliases Chromosome Accession RefSeq IDs
Numbers
KLFl EKLF 19pl3.13-pl3.12 U37106 NM_006563
KLF2 LKLF 19pl3.13-pl3.11 AF123344
KLF3 BKLF 4pl6.1-pl5.2 AF285837
KLF4 EZF, GKLF 9q31 AF022184 NM_004235
KLF5 BTEB2, IKLF, CKLF 13q21.3 D14520
KLF6 BCDl, ST12, COPEB CPBP, 10pl5 U51869
GBF, Zf9, PACl
KLF7 UKLF 2q32 AB015132
KLF8 BKLF3, ZNF741, DXS741 Xpl l.21 U28282 NM 007250
KLF9 BTEBl 9q21.11 BC069431 NM_001206
KLFlO TIEG, EGRA 8q22.2 U21847
KLFI l TIEG2 Tieg3 2p25 AF028008 NM_003597
KLF12 AP-2rep 13q22 AJ243274
KLFl 3 RFLAT-I, BTEB3, NSLPl, 15 AF132599 NM_015995
FKLF-2
KLF14 BTEB5 7q32.3 AF490374 NM l 38693
KLFl 5 3 AB029254
KLF16 NSLP2, BTEB4, DREF 19pl3.3 AF327440
KLFl 7 ZNF393 Zfp393, FLJ40160 Ip34.1 BC049844 NM 173484
KLF4 is a member of the SpI -like family of transcription factors (Kaczynski, J., et al. 2003; Bieker, J. J. 2001). The 53kDa protein contains an N-terminal transactivation domain, a repression domain, a nuclear localization signal, and three C-terminal zinc fingers (ZFD) (Geiman, D. E., et al. 2000; Yet, S. F., et al. 1998; Ruppert, J. M., et al. 1988). The ZFD mediates binding to the consensus 5' RRGGYGY 3' (R^purine, Y=pyrimidine; SEQ ID NO:8) (Shields, J. M., et al. 1998). By use of expression cloning in RK3E cells KLF4 was identified as an oncogene (Foster, K. W., et al. 1999). KLF4 is expressed in post-mitotic cells of the skin and gut, and may function as a tumor suppressor in certain contexts (Garrett-Smha, L. A., et al. 1996; Shields, J. M., et al. 1996; Dang, D. T., et al. 2003; Zhao, W., et al. 2004). Klf4 functions in the suprabasal layers of the skin to form the water permeability barrier. Klf4-/- mice develop normally, but die soon after birth from dehydration (Segre, J. A., et al. 1999).
KLF4 mRNA and protein is up-regulated early in progression of breast cancer, oral squamous cell carcinoma (SCC), and cutaneous SCC (Foster, K. W., et al. 1999; Foster, K. W., et al. 2000; Foster, K. W., et al. 2005; Huang, C. C, et al. 2005). In contrast, KLF4 is down-regulated in several other tumor types (Foster, K. W., et al. 2000; Huang, C. C, et al. 2005). Based upon the increased KLF4 transcripts in oral SCCs and dysplasia, it was hypothesized that dysplasia results from increased KLF4 in proliferation-competent cells of the basal layer (Foster, K. W., et al. 1999). Breast and skin share a common embryologic origin, and tumors of these tissues show similar genetics (infrequent mutation of RAS, more frequent alterations of p53, pl6INK4A, c-MYC, ErbB family members) (Ruppert, J. M., et al. 1997; Forastiere, A., et al. 2001; Mao, L., et al. 2004). Indeed, several mouse models of cancer exhibit both breast and skin lesions (Jonkers, J., et al. 2001). Induction of KLF4 in mouse skin plays a role in tumor initiation (Foster, K. W., et al. 2005; Huang, C. C, et al. 2005). Within 48 hours of KLF4 induction, the proliferation- differentiation switch is inhibited. Low-level KLF4 induced epithelial turnover without cell accumulation, termed occult cell turnover (Huang, C. C, et al. 2005). Higher levels induced hyperplasia by one week. By 2-4 weeks hyperplasia, dysplasia, and tumor diffusely involve the skin.
By expressing candidate genes in differentiated fibroblasts, Klf4 and just three other genes (c-Myc, Oct3/4, Sox2) were found sufficient to induce the pluripotent embryonic stem cell phenotype (Takahashi, K., et al. 2006). These pluripotent cells contributed to embryonic development when injected into blastocysts, and when injected into athymic mice they formed tumors with tissues from all three germ layers. Consistent with this observation, KLF4 functions as an oncogene in several contexts (Foster, K. W., et al. 1999; Rowland, B. D., et al. 2005; Suzuki, T., et al. 2002; Li, Y., et al. 2005). Analysis of Klf4-deficient gut epithelium revealed a role of KLF4 in cell fate (Sancho, E., et al. 2003). These mice show selective loss of goblet cells, which derive from stem cells following several binary cell fate decisions (Sancho, E., et al. 2003). Just as KLF4 promotes the goblet cell fate, loss of CSL or Hesl or even induction of Ntcl induces goblet cells, linking the KLF4 and Ntcl pathways in cell fate specification (Sancho, E., et al. 2003; Zecchini, V., et al. 2005). As disclosed herein, KLF4 induces a profile (Ntcl high, Hesl low, c-MYC low, p21/Wafl high) that is the pattern expected of goblet cells, indicating that KLF4 functions as a regulator of cell fate in epithelia (Segre, J. A., et al. 1999; Sancho, E., et al. 2003; Katz, J. P., et al. 2005; Katz, J. P., et al. 2002). ii. Notchl (Ntcl)
Disclosed herein are compositions and methods relating to the identification, diagnosis, and/or treatment of tumors based on expression of Notchl (Ntcl).
The Notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms. Notch is present in all metazoans, and vertebrates possess four different notch receptors, referred to as Notchl to Notch4. The Notch receptor is a single-pass (i.e. it crosses the membrane once, in contrast to many other transmembrane proteins which loop back and forth between the extracellular and intracellular spaces) transmembrane receptor protein. It is a hetero-oligomer composed of a large extracellular portion which associates in a calcium dependent, non-covalent interaction with a smaller piece of the Notch protein composed of a short extracellular region, a single transmembrane-pass, and a small intracellular region.
The Notch protein sits like a trigger spanning the cell membrane, with part of it inside and part outside. Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to alter gene expression.
Notch signaling also has a role in neuronal function and development, stabilizing arterial endothelial fate and angiogenesis, regulating crucial cell communication events between endocardium and myocardium during both the formation of the valve primordial and ventricular development and differentiation. Notch signaling also has a role in cardiac valve homeostasis and has implications in other human disorders involving the cardiovascular system. Additionally, Notch signaling has a role in timely cell lineage specification of both endocrine and exocrine pancreas, influencing binary fate decisions of cells that must choose between the secretory and absorptive lineages in the gut, expanding the HSC compartment during bone development and participates in commitment to the osteoblastic lineage suggesting a potential therapeutic role for Notch in bone regeneration and osteoporosis. Notch signaling has a role in regulating cell-fate decision in mammary glands at several distinct development stages, and some non-nuclear mechanisms, such as controlling the actin cytoskeleton through the tyrosine kinase AbI. Notch signaling is often repressed in many cancers, and faulty Notch signaling is implicated in many diseases including T-ALL (T-cell acute lymphoblastic leukemia), CADASIL (Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy), MS (Multiple Sclerosis), Tetralogy of Fallot, Alagille syndrome, and myriad other disease states.
Maturation of the Notch receptor involves cleavage at the prospective extracellular side during intracellular trafficking in the Golgi complex. This results in a bipartite protein, composed of a large extracellular domain linked to the smaller transmembrane and intracellular domain. Binding of ligand promotes two proteolytic processing events; as a result of proteolysis, the intracellular domain is liberated and can enter the nucleus to engage other DNA-binding proteins and regulate gene expression.
Notch and most of its ligands are transmembrane proteins, so the cells expressing the ligands typically need to be adjacent to the Notch expressing cell for signaling to occur. The Notch ligands are also single-pass transmembrane proteins and are members of the DSL (Delta/Serrate/LAG-2) family of proteins.
Once the Notch extracellular domain interacts with a ligand, an ADAM-family metalloprotease called TACE (Tumor Necrosis Factor Alpha Converting Enzyme) cleaves the Notch protein just outside the membrane. This releases the extracellular portion of Notch, which continues to interact with the ligand. The ligand plus the Notch extracellular domain is then endocytosed by the ligand-expressing cell. There may be signaling effects in the ligand-expressing cell after endocytosis. After this first cleavage, an enzyme called γ-secretase cleaves the remaining part of the Notch protein just inside the inner leaflet of the cell membrane of the Notch-expressing cell. This releases the intracellular domain of the Notch protein, which then moves to the nucleus where it can regulate gene expression by activating the transcription factor CSL. Other proteins also participate in the intracellular portion of the Notch signaling cascade.
As disclosed herein, KLF4 is a regulator of Ntcl transcription and signaling. Ntcl, first identified as a translocation partner in T cell acute lymphoblastic leukemia (T-ALL) (Ellisen, L. W., et al. 1991), is a transmembrane (TM) receptor that interacts with membrane-anchored ligands (Nickoloff, B. J., et al. 2003; Weinmaster, G., et al. 1991; Weinmaster, G., et al. 1992; Reaume, A. G., et al. 1992; del Amo, F. F., et al. 1993; Lardelli, M., et al. 1994; Uyttendaele, H., et al. 1996). Human Ntcl is synthesized as a -270 kDa precursor, NtclFL (Fig. IA) (Nickoloff, B. J., et al. 2003; Selkoe, D., et al. 2003). Maturation involves removal of the leader peptide (LP) and cleavage by a furin-like convertase at the Sl site in the Golgi (Logeat, F., et al. 1998). In turn, cleavage at S2 yields the -120 kDa NtcTM, a substrate for cleavage at S3 by γ-secretase (Baron, M. 2003). Formation of the active intracellular form, NtcIC, is promoted by Ntcl ligands and blocked by -ySIs (Li, T., et al. 2003; Zhang, Z., et al. 2000).
The structural domains in Ntcl are shown in Fig. IA. Ntcl does not bind DNA directly, but interacts with other proteins such as CSL, also called CBFl (Lecourtois, M., et al. 1995; Baron, M., et al. 2002). In the absence of Ntc signaling, CSL exists in a repressor complex with histone deacetylases (HDACs) and co-repressors including Hairless (Morel, V., et al. 2001; Kao, H. Y., et al. 1998; Lai, E. C. 2002; Zhou, S., et al. 2000; Zhou, S., et al. 2001) (Fig. IB). Upon interaction with NtcIC, these molecules are displaced by histone acetyltransferases (HATs) such as CBP/P300 (Zhou, S., et al. 2000; Kitagawa, M., et al. 2001; Fryer, C. J., et al. 2002). The NtcIC-CSL complex activates transcription from CSL binding sites in the promoters of target genes such as Hesl (Iso, T., et al. 2003) (Fig. IB). NtclIC proteins (Fig. IA-B) are produced in T-ALL by translocation or by point mutation (>50%) (Weng, A. P., et al. 2004). In vitro, NtclIC was shown to transform El A-immortalized rat kidney cells (RK3E cells) similar to KLF4 (Foster, K. W., et al. 1999; Capobianco, A. J., et al. 1997; Ascano, J. M., et al. 2003). Mapping studies have failed to identify a single minimal transforming region of NtclIC (Fryer, C. J., et al. 2002; Aster, J. C, et al. 2000; Jeffries, S., et al. 2000; Dumont, E., et al. 2000). Both "Classical" and CSL-independent, Alternate NtclIC signaling pathways have been identified (Jeffries, S., et al. 2000; Brennan, K., et al. 2002; Shawber, C, et al. 1996; Nofziger, D., et al. 1999) (Fig. IB), but it has been unclear which pathway is important for transformation (Brennan, K., et al. 2003).
KLF4 and Ntcl are co-expressed in differentiating layers of the skin (Lin, M. H., et al. 2003). KLF4 and Ntcl are highly expressed in most breast cancers and head and neck SCC, but are low in other tumor types (Huang, C. C, et al. 2005; Nickoloff, B. J., et al. 2003; Brennan, K., et al. 2003; Lin, M. H., et al. 2003; Leethanakul, C, et al. 2000; Nicolas, M., et al. 2003). Indeed, KLF4 and Ntcl appear to function as oncogenes in breast cancer and cutaneous/oral SCC, but are candidate tumor suppressors in the cervix, gut, and hair follicle (Nicolas, M., et al. 2003; Maillard, L, et al. 2003; Rangarajan, A., et al. 2001; Talora, C, et al. 2002). Evidence supports a role for Ntcl signaling in breast cancer (Brennan, K., et al. 2003; Politi, K., et al. 2004; Kiaris, H., et al. 2004). Integration of MMTV into Ntcl results in synthesis of Ntcl and induces breast cancer (Brennan, K., et al. 2003; Callahan, R., et al. 2001; Dievart, A., et al. 1999). Likewise, transgenic mice expressing NtclIC develop mammary neoplasias (Kiaris, H., et al. 2004; Raafat, A., et al. 2004). At least some of the rearrangements (leukemia) or viral integrations (mouse breast cancer) appear to result in synthesis of constitutively active, NtclIC-like proteins (Fig. IA). Ntcl expression is prominent in human breast cancers (Brennan, K., et al. 2003; Weijzen, S., et al. 2002; Reedijk, M., et al. 2005). Unlike NtclIC, NtclFL (the form upregulated in human tumors) has not been found to act as an oncogene (Brennan, K., et al. 2003). As disclosed herein, KLF4 directly induces NtclFL transcription and NtclFL and Ntcl10 are both oncogenes.
2. Antibodies
Antibodies specific for Human KLF4 and KLF5 are commercially available from, for example, Abeam, Abnova Corporation, ABR- Affinity BioReagents, Aviva Systems Biology, CeMines, CHEMICON, GeneTex, IMGENEX, Lifespan Biosciences, Novus Biologicals, R&D Systems, Santa Cruz Biotechnology, Inc.
Also provided is a monoclonal antibody directed against residues 479-1197 of Kruppel-like factor 4 (SEQ ID NO: 6). Such antibody can be used to monitor a treatment, further evaluate effectiveness of the treatment in an individual. Specifically, the monoclonal antibody detects the localization and level of KLF4 protein, and wherein decreases of KLF4 protein level indicate effective response of the individual to the treatment. Still further provided in the present invention is a kit for monitoring a treatment thereby evaluating effectiveness of the treatment in an individual, comprising the monoclonal antibody disclosed herein and a suitable carrier.
Antibodies specific for Human Notchl are commercially available from, for example, Abeam, AbD Serotec, ABR- Affinity BioReagents, AlphaGenix, Inc., AnaSpec, BD Biosciences Pharmingen, BioLegend, Biomeda Corporation, Calbiochem, CHEMICON, Developmental Studies Hybridoma Bank, Epitomics, Inc., GeneTex, Lab Vision, Lifespan Biosciences, Novus Biologicals, Proteintech Group, Inc., and Sigma- Aldrich. i. Antibodies Generally
The term "antibodies" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term "antibodies" are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with human KLF4 or KLF5. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sd. USA, 81:6851-6855 (1984)).
The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro, e.g., using the HIV Env-CD4-co-receptor complexes described herein.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.
In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, MJ. Curr. Opin. Biotechnol. 3:348-354, 1992).
As used herein, the term "antibody" or "antibodies" can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response. ii. Human antibodies
The disclosed human antibodies can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole et al. {Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol., 147(l):86-95, 1991). Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J MoI. Biol, 227:381, 1991; Marks et al., J. MoI. Biol, 222:581, 1991).
The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Set USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol, 1:32> (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein. iii. Humanized antibodies
Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.
To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).
Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S. Patent No. 5,721,367 (Kay et al.), U.S. Patent No. 5,837,243 (Deo et al.), U.S. Patent No. 5, 939,598 (Kucherlapati et al.), U.S. Patent No. 6,130,364 (Jakobovits et al.), and U.S. Patent No. 6,180,377 (Morgan et al.). 3. Cancer Therapeutics i. Gamma Secretase Inhibitor
Disclosed herein are γ-secretase inhibitors (7SI) for use on tumors having the disclosed oncogene expression patterns.
Gamma secretase is a multi-subunit protease complex, itself an integral membrane protein, that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. The most well-known substrate of gamma secretase is amyloid precursor protein, a large integral membrane protein that, when cleaved by both gamma and beta secretase, produces a short 39-42 amino acid peptide called amyloid beta whose abnormally folded fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients. Gamma secretase is also critical in the related processing of the Notch protein.
The gamma secretase complex has not yet been fully characterized but minimally consists of four individual proteins: presenilin, nicastrin, APH-I (anterior pharynx- defective 1), and PEN-2 (presenilin enhancer 2). Presenilin, an aspartyl protease, is the catalytic subunit; mutations in the presenilin gene have been shown to be a major genetic risk factor for Alzheimer's disease.
Gamma secretase is an internal protease that cleaves within the membrane- spanning domain of its substrate proteins, including amyloid precursor protein (APP) and Notch. Substrate recognition occurs via nicastrin ectodomain binding to the N-terminus of the target, which is then passed via a poorly understood process between the two presenilin fragments to a water-containing active site at which the catalytic aspartate residue resides.
Numerous gamma secretase inhibitors (7SI) are known in the art and contemplated for use in the disclosed compositions and methods. An example of a γSIs undergoing clinical trials for the treatment of breast cancer is MK0752 (Merck, Whitehouse Station, NJ; ClinicalTrials.gov Identifier: NCT00106145). Additional examples of γSIs include MRK003-ONC (Merck), benzodiazepine, dibenzazepine, and Compound Z. U.S. Patent Nos. 7,208,488 (04/24/07), 7,208,602 (04/24/07), 7,232,820 (06/19/07), 7,253,158 (08/07/07), 7,253,195 (08/07/07), 7,253,306 (08/07/07), 7,256,186 (08/14/07), 7,262,208 (08/28/07), and 7,262,223 (08/28/07) are incorporated herein by reference in their entireties for the teaching of 7SIS.
Using cleavage products such as A/3 as the readout, 7-secretase has been intensively studied, and numerous small molecule inhibitors have been identified (Citron, M. 2004). Aspartyl protease transition state analogues such as pepstatin and L685458 inhibit γ-secretase. Other potent and specific γ-secretase inhibitors (γSIs) include sulfonamides and benzodiazepines (Tian, G., et al. 2002). The γ-secretase complex is an unusual protease that acts within the membrane to release intracellular fragments from Type I integral membrane proteins, a process termed regulated intramembrane proteolysis (RIP). 7-secretase is composed of the transmembrane proteins presenilin 1 (PSl) and presenilin 2 (PS2) as well as nicastrin and Aph-1 (Ye, Y., et al. 1999; Struhl, G., et al. 1999; De Strooper, B., et al. 1999; Song, W., et al. 1999; Shiraishi, H., et al. 2006; Fortini, M. E. 2002). In the late 1990's genetic studies in flies and mice showed that loss-of- function of PSl yielded similar phenotypes as for Ntcl, and it became clear that cleavage of APP and the RIP of Ntc were both mediated by γ-secretase (Fortini, M. E. 2002).
Since then several other substrates of γ-secretase-mediated RTP have been identified. These include ErbB4 (Ni, C. Y., et al. 2001), E-cadherin (Fortini, M. E. 2002), the Ntc ligands Jag2 and Dill (LaVoie, M. J., et al. 2003; Ikeuchi, T., et al. 2003), CD44 (Fortini, M. E. 2002), a protein tyrosine phosphatase (Anders, L., et al. 2006) and megalin (Biemesderfer, D. 2006). Unlike for Ntc, for none of these other substrates has the knockout phenotype been recapitulated by PSl knockout or 7SI treatment. In addition, there are no known Ntc-independent effects of γSIs in animals or humans, and these drugs are consistently described as well-tolerated at efficacious doses (Citron, M. 2004; Wong, G. T., et al. 2004; Siemers, E. R., et al. 2006). The observed selectivity for Ntc can be due to partial inhibition of γ-secretase or to functional redundancy within the other pathways regulated by γ-secretase. Alternatively there can be distinct forms of 7-secretase that preferentially cleave substrates such as Ntc versus other substrates, and these can be differentially affected by inhibitors (Fortini, M. E. 2002). γSIs inhibit the cleavage step that converts the inactive precursor Ntcl™ to NtclIC, the active signaling form that has transforming activity (Tian, G., et al. 2002). When administered to mice and rats these compounds rapidly suppress γ-secretase cleavage, induce differentiation of gut cells toward the goblet cell fate, and yield a modest diminution of thymic T cells and splenic B cells that was asymptomatic in a 15 day treatment (Wong, G. T., et al. 2004; Searfoss, G. H., et al. 2003; van Es, J. H., et al. 2005; Milano, J., et al. 2004).
Several reports of clinical trials indicate that γSIs are well tolerated in humans. LY450139 was administered to volunteers for 14 days (Siemers, E., et al. 2005). Subsequently a randomized, controlled, 6 week trial was performed in patients with AD (Siemers, E. R., et al. 2006). The drug was efficacious (38% reduction of plasma A/3) and well-tolerated, with the major side effect being mild and clinically inconsequential suppression of lymphocyte function. Gastrointestinal symptoms were modest. Current clinical trials include phase I analysis of the Merck γSI MK0752 in breast cancer and in T- ALL (Krop, I. E., et al. 2006; Pui, C. H., et al. 2006; Deangelo, D. J., et al. 2006). For T- ALL patients this drag was well tolerated below 300 mg/m2, and all doses tested were sufficient to inhibit γ-secretase (Deangelo, D. J., et al. 2006). When given to breast cancer patients at doses of 450 mg daily, 40% of patients required dose reduction secondary to Grade 2/3 fatigue, and alternate schedules are being explored (Krop, I. E., et al. 2006). ii. Retinoid X receptor (RXR) agonist (rexinoid)
Disclosed herein are retinoid X receptor (RXR) agonists (rexinoids) for use on tumors having the disclosed oncogene expression patterns. The retinoid X receptor (RXR) is a type of nuclear receptor which is activated by 9-cis retinoic acid. There are three retinoic acid receptors (RXR), RXR-alpha, RXR-beta, and RXR-gamma encoded by the RXRA, RXRB, RXRG genes respectively. RXR heterodimerizes with subfamily 1 nuclear receptors including CAR, FXR, LXR, PPAR, PXR, RAR, TR, and VDR. As with other type II nuclear receptors, the RXR heterodimer in the absence of ligand is bound to hormone response elements complexed with corepressor protein. Binding of agonist ligands to RXR results in dissociation of corepressor and recruitment of coactivator protein which in turn promotes transcription of the downstream target gene into mRNA and eventually protein.
Examples of RXR selective retinoids ("rexinoids"), such as 9-cis-retinoic acid (9cRA), are described in U.S. Pat. No. 5,780,676 and published international application WO 97/12853, which are incorporated herein by reference in their entirety for the teaching of these compounds.
The rexinoid can be 9-cis-UAB30 (9cUAB30), which is very effect in the chemoprevention of breast cancer and has very low toxicity. Atigadda et al, J. Med. Chem. 2003 is hereby incorporated by reference herein for the teaching of 9cUAB30 and methods of making and using same.
As disclosed herein, KLF4 activates transcription of nuclear hormone receptors (RARγ, RXRα, Nur77), and retinoid agonist can block KLF4 oncogenic activity. For chemoprevention of cancer, a retinoid agonist for RXRs, 9-cis-UAB30 (9cUAB30), was developed. This rexinoid is 50-fold less toxic than the pan-agonist 9-cis-retinoic acid (9cRA), 15 -fold less toxic than the RXR-agonist Targretin, and does not elevate triglycerides. In preclinical models of breast cancer, 9cUAB30 is a highly efficacious, nontoxic chemopreventive agent (Grubbs, C. J., et al. 2003; Atigadda, V. R., et al. 2003; Grubbs, C. J., et al. 2005). 9cUAB30 is also a candidate therapeutic, as indicated by its induction of apoptosis and inhibition of BrdU incorporation in established tumors (Grubbs, C. J., et al. 2005).
RARs and RXRs are members of the nuclear hormone receptor (NR) superfamily (Bastien, J., et al. 2004; Nagpal, S. et al. 2000). For both RAR and RXR, three isotypes (α, β, γ) are encoded by separate genes. All-trans retinoic acid (ATRA) and 13cRA are agonists for the RARs. 9cRA is an agonist for both RARs and RXRs. Retinoid receptors act through RA response elements (RAREs) within gene promoters to regulate transcription (Chambon, P. 1996; Giguere, V., et al. 1987; Petkovich, M., et al. 1987). In absence of ligand, NRs can localize to the nucleus, bind to RAREs, and recruit co- repressors (Bastien, J., et al. 2004; Dilworth, F. J., et al. 2001; Aranda, A., et al. 2001; Glass, C. K., et al. 2000). Liganded NRs can recruit a variety of co-activators, chromatin remodellers, and modifiers that recruit the RNA Polymerase II machinery (Dilworth, F. J., et al. 2001). In some cases growth suppression by retinoid agonists may be due to repression of AP-I (Karin, M., et al. 1997; Chen, C. F., et al. 2004). RARjS is lost in tumors such as breast cancer and SCC by gene deletion, mutation, or promoter methylation (Niles, R. M., et al. 2004; Hayashi, K., et al. 2001; Sirchia, S. M., et al. 2002; Nakayama, T., et al. 2001), and mice deficient in RXRα in the prostate develop high grade lesions (Huang, J., et al. 2002).
In addition to RARs, RXRs can heterodimerize with other partners including vitamin D3 receptors, PPARs, and orphan nuclear receptors including NUR77, Norl and Nurrl (Niles, R. M., et al. 2004; Winoto, A., et al. 2002; Sohn, S. J., et al. 2003). The RXR receptor of the heterodimers can be transcriptionally silent (e.g., RXR: VDR) or be permissively activated upon binding of either the RXR ligand or a ligand for the binding partner (e.g., RXR:PPARs). In contrast, RAR/RXR heterodimers are thought to require binding of an RAR ligand before the RXR ligand can enhance transcription. Upon binding of a ligand to both RAR and RXR and release of co-repressors, the two receptors synergize to activate transcription (Bastien, J., et al. 2004; Nettles, K. W., et al. 2005). RXR-Nurrl heterodimers are robustly activated by RXR ligands (Law, S. W., et al. 1992; Forman, B. M., et al. 1995; Perlmann, T., et al. 1995; Wallen-Mackenzie, A., et al. 2003) raising the possibility that the RXR-NUR77 heterodimers may be rexinoid-responsive. NUR77, a target of KLF4, mediates apoptosis by induction of apoptotic genes and by translocation of RXRα-NUR77 to mitochondria (Rajpal, A., et al. 2003; Cao, X., et al. 2004; Lee, K. W., et al. 2005). The binding of NUR77 to BCL2 converts the latter from a blocker of apoptosis to a promoter of cell death (Lin, B., et al. 2004). Natural ligands for NUR77 are unknown, but synthetic l,l-Bis(3'-indolyl)-l-(psubstituted phenyl)methanes function as NUR77 agonists and induce apoptosis in breast cancer cells (Chintharlapalli, S., et al. 2005). While 9cRA and SRl 1237 promote RXRα homodimerization, resulting in a block of NUR77-mediated apoptosis (Cao, X., et al. 2004), the effects of 9cUAB30 and its derivatives have not been determined. If these promote heterodimerization by binding to RXRα and/or NUR77 they may activate apoptosis. RA influences epithelial development and differentiation (Fisher, G. J., et al. 1996). In skin the most abundant RAR is RARγ (Fisher, G. J., et al. 1996; Darwiche, N., et al. 1995). The receptor heterodimer induced by KLF4, RARγ/RXRo; represents the major signal transducer in these cells (Chapellier, B., et al. 2002; Clifford, J. L., et al. 1999). Like KLF4 and Ntcl, these are most abundant in suprabasal keratinocytes (Huang, C. C, et al. 2005; Lin, M. H., et al. 2003). Furthermore, RA-induced growth inhibition of transformed keratinocytes is mediated by RARγ (Chen, C. F., et al. 2004; Goyette, P., et al. 2000). These and other retinoid receptors are likewise expressed in benign and malignant breast tissues (Pasquali, D., et al. 1997; Widschwendter, M., et al. 1997), and are thought to mediate the anti-tumor effects of retinoids in breast cancer (Toma, S., et al. 1997; Rao, G. N., et al. 1998; Wu, K., et al. 2002; Wu, K., et al. 2000). iii. Carriers
The disclosed cancer therapeutics can be combined, conjugated or coupled with or to carriers and other compositions to aid administration, delivery or other aspects of the inhibitors and their use. For convenience, such composition will be referred to herein as carriers. Carriers can, for example, be a small molecule, pharmaceutical drug, fatty acid, detectable marker, conjugating tag, nanoparticle, or enzyme.
The disclosed compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject, along with the composition, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically- acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds can be administered according to standard procedures used by those skilled in the art.
Pharmaceutical compositions can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
Some of the compositions can potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These can be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
The carrier molecule can be covalently linked to the disclosed cancer therapeutics. The carrier molecule can be linked to the amino terminal end of disclosed peptides. The carrier molecule can be linked to the carboxy terminal end of disclosed peptides. The carrier molecule can be linked to an amino acid within disclosed peptides. The herein provided compositions can further comprise a linker connecting the carrier molecule and disclosed cancer therapeutics. The disclosed inhibitors can also be conjugated to a coating molecule such as bovine serum albumin (BSA) (see Tkachenko et al., (2003) J Am Chem Soc, 125, 4700-4701) that can be used to coat microparticles, nanoparticles of nanoshells with the cancer therapeutics.
Protein crosslinkers that can be used to crosslink the carrier molecule to the cancer therapeutics, such as disclosed peptides, are known in the art and are defined based on utility and structure and include DSS (Disuccinimidylsuberate), DSP (Dithiobis(succinimidylpropionate)), DTSSP (3,3'-Dithiobis (sulfosuccinimidylpropionate)), SULFO BSOCOES (Bis[2- (sulfosuccinimdooxycarbonyloxy) ethyl] sulfone), BSOCOES (Bis[2- (succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST (Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene glycolbis(sulfosuccinimidylsuccinate)), DPDPB (l,2-Di[3'-(2'- pyridyldithio) propionamido]butane), BSSS (Bis(sulfosuccinimdyl) suberate), SMPB (Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB (Sulfosuccinimdyl-4-(p- maleimidophenyl) butyrate), MBS (3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS (3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SLAB (N- Succinimidyl(4-iodoacetyl) aminobenzoate), SULFO SLAB (N-Sulfosuccinimidyl(4- iodoacetyl)aminobenzoate), SMCC (Succinimidyl-4-(N-maleimidomethyl) cyclohexane- 1-carboxylate), SULFO SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane- 1-carboxylate), NHS LC SPDP (Succinimidyl-6-[3-(2-pyridyldithio) propionamido) hexanoate), SULFO NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio) propionamido) hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio) propionate), NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate), NHS IODOACETATE (N- Hydroxysuccinimidyliodoacetate), MPBH (4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH (4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid hydrazide hydrochloride), MBH (m-Maleimidobenzoic acid hydrazidehydrochloride), SULFO EMCS (N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide), EMCS (N-(epsilon- Maleimidocaproyloxy) succinimide), PMPI (N-(p-Maleimidophenyl) isocyanate), KMUH (N-(kappa-Maleimidoundecanoic acid) hydrazide), LC SMCC (Succinimidyl-4-(N- maleimidomethyl)-cyclohexane-l-carboxy(6-amidocaproate)), SULFO GMBS (N- (gamma-Maleimidobutryloxy) sulfosuccinimide ester), SMPH (Succinimidyl-6-(beta- maleimidopropionamidohexanoate)), SULFO KMUS (N-(kappa- Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS (N-(gamma- Maleimidobutyrloxy) succinimide), DMP (Dimethylpimelimidate hydrochloride), DMS (Dimethylsuberimidate hydrochloride), MHBH(Wood's Reagent) (Methyl-p- hydroxybenzimidate hydrochloride, 98%), DMA (Dimethyladipimidate hydrochloride). a. Nanoparticles, Microp articles, and Microbubbles
The term "nanoparticle" refers to a nanoscale particle with a size that is measured in nanometers, for example, a nanoscopic particle that has at least one dimension of less than about 100 ran. Examples of nanoparticles include paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohoms, nano-onions, nanorods, nanoropes and quantum dots. A nanoparticle can produce a detectable signal, for example, through absorption and/or emission of photons (including radio frequency and visible photons) and plasmon resonance.
Microspheres (or microbubbles) can also be used with the methods disclosed herein. Microspheres containing chromophores have been utilized in an extensive variety of applications, including photonic crystals, biological labeling, and flow visualization in microfluidic channels. See, for example, Y. Lin, et al., Appl. Phys Lett. 2002, 81, 3134; D. Wang, et al., Chem. Mater. 2003, 15, 2724; X. Gao, et al., J. Biomed. Opt. 2002, 7, 532; M. Han, et al., Nature Biotechnology. 2001, 19, 631; V. M. Pai, et al., Mag. & Magnetic Mater. 1999, 194, 262, each of which is incorporated by reference in its entirety. Both the photostability of the chromophores and the monodispersity of the microspheres can be important.
Nanoparticles, such as, for example, silica nanoparticles, metal nanoparticles, metal oxide nanoparticles, or semiconductor nanocrystals can be incorporated into microspheres. The optical, magnetic, and electronic properties of the nanoparticles can allow them to be observed while associated with the microspheres and can allow the microspheres to be identified and spatially monitored. For example, the high photostability, good fluorescence efficiency and wide emission tunability of colloidally synthesized semiconductor nanocrystals can make them an excellent choice of chromophore. Unlike organic dyes, nanocrystals that emit different colors (i.e. different wavelengths) can be excited simultaneously with a single light source. Colloidally synthesized semiconductor nanocrystals (such as, for example, core-shell CdSe/ZnS and CdS/ZnS nanocrystals) can be incorporated into microspheres. The microspheres can be monodisperse silica microspheres.
The nanoparticle can be a metal nanoparticle, a metal oxide nanoparticle, or a semiconductor nanocrystal. The metal of the metal nanoparticle or the metal oxide nanoparticle can include titanium, zirconium, hafnium, vanadium, niobium, tantalum, chromium, molybdenum, tungsten, manganese, technetium, rhenium, iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel, palladium, platinum, copper, silver, gold, zinc, cadmium, scandium, yttrium, lanthanum, a lanthanide series or actinide series element (e.g., cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium, and uranium), boron, aluminum, gallium, indium, thallium, silicon, germanium, tin, lead, antimony, bismuth, polonium, magnesium, calcium, strontium, and barium. In certain embodiments, the metal can be iron, ruthenium, cobalt, rhodium, nickel, palladium, platinum, silver, gold, cerium or samarium. The metal oxide can be an oxide of any of these materials or combination of materials. For example, the metal can be gold, or the metal oxide can be an iron oxide, a cobalt oxide, a zinc oxide, a cerium oxide, or a titanium oxide. Preparation of metal and metal oxide nanoparticles is described, for example, in U.S. Pat. Nos. 5,897,945 and 6,759,199, each of which is incorporated by reference in its entirety.
For example, cancer therapeutics can be immobilized on silica nanoparticles (SNPs). SNPs have been widely used for biosensing and catalytic applications owing to their favorable surface area-to-volume ratio, straightforward manufacture and the possibility of attaching fluorescent labels, magnetic nanoparticles (Yang, H.H. et al. 2005) and semiconducting nanocrystals (Lin, Y. W., et al. 2006).
The nanoparticle can also be, for example, a heat generating nanoshell. As used herein, "nanoshell" is a nanoparticle having a discrete dielectric or semi-conducting core section surrounded by one or more conducting shell layers. U.S. Patent No. 6,530,944 is hereby incorporated by reference herein in its entirety for its teaching of the methods of making and using metal nanoshells.
Targeting molecules can be attached to the disclosed compositions and/or carriers. For example, the targeting molecules can be antibodies or fragments thereof, ligands for specific receptors, or other proteins specifically binding to the surface of the cells to be targeted. b. Liposomes
"Liposome" as the term is used herein refers to a structure comprising an outer lipid bi- or multi-layer membrane surrounding an internal aqueous space. Liposomes can be used to package any biologically active agent for delivery to cells.
Materials and procedures for forming liposomes are well-known to those skilled in the art. Upon dispersion in an appropriate medium, a wide variety of phospholipids swell, hydrate and form multilamellar concentric bilayer vesicles with layers of aqueous media separating the lipid bilayers. These systems are referred to as multilamellar liposomes or multilamellar lipid vesicles ("MLVs") and have diameters within the range of 10 nm to 100 μm. These MLVs were first described by Bangham, et al., J MoI. Biol. 13:238-252 (1965). In general, lipids or lipophilic substances are dissolved in an organic solvent. When the solvent is removed, such as under vacuum by rotary evaporation, the lipid residue forms a film on the wall of the container. An aqueous solution that typically contains electrolytes or hydrophilic biologically active materials is then added to the film. Large MLVs are produced upon agitation. When smaller MLVs are desired, the larger vesicles are subjected to sonication, sequential filtration through filters with decreasing pore size or reduced by other forms of mechanical shearing. There are also techniques by which MLVs can be reduced both in size and in number of lamellae, for example, by pressurized extrusion (Barenholz, et al., FEBS Lett. 99:210-214 (1979)).
Liposomes can also take the form of unilamnellar vesicles, which are prepared by more extensive sonication of MLVs, and consist of a single spherical lipid bilayer surrounding an aqueous solution. Unilamellar vesicles ("ULVs") can be small, having diameters within the range of 20 to 200 nm, while larger ULVs can have diameters within the range of 200 nm to 2 μm. There are several well-known techniques for making unilamellar vesicles. In Papahadjopoulos, et al., Biochim et Biophys Acta 135:624-238 (1968), sonication of an aqueous dispersion of phospholipids produces small ULVs having a lipid bilayer surrounding an aqueous solution. Schneider, U.S. Pat. No. 4,089,801 describes the formation of liposome precursors by ultrasonication, followed by the addition of an aqueous medium containing amphiphilic compounds and centrifugation to form a biomolecular lipid layer system. Small ULVs can also be prepared by the ethanol injection technique described by Batzri, et al, Biochim et Biophys Acta 298:1015-1019 (1973) and the ether injection technique of Deamer, et al., Biochim et Biophys Acta 443:629-634 (1976). These methods involve the rapid injection of an organic solution of lipids into a buffer solution, which results in the rapid formation of unilamellar liposomes. Another technique for making ULVs is taught by Weder, et al. in "Liposome Technology", ed. G. Gregoriadis, CRC Press Inc., Boca Raton, FIa., Vol. I, Chapter 7, pg. 79-107 (1984). This detergent removal method involves solubilizing the lipids and additives with detergents by agitation or sonication to produce the desired vesicles.
Papahadjopoulos, et al., U.S. Pat. No. 4,235,871, describes the preparation of large ULVs by a reverse phase evaporation technique that involves the formation of a water-in- oil emulsion of lipids in an organic solvent and the drug to be encapsulated in an aqueous buffer solution. The organic solvent is removed under pressure to yield a mixture which, upon agitation or dispersion in an aqueous media, is converted to large ULVs. Suzuki et al., U.S. Pat. No. 4,016,100, describes another method of encapsulating agents in unilamellar vesicles by freezing/thawing an aqueous phospholipid dispersion of the agent and lipids.
In addition to the MLVs and ULVs, liposomes can also be multivesicular. Described in Kim, et al., Biochim et Biophys Acta 728:339-348 (1983), these multivesicular liposomes are spherical and contain internal granular structures. The outer membrane is a lipid bilayer and the internal region contains small compartments separated by bilayer septum. Still yet another type of liposomes are oligolamellar vesicles ("OLVs"), which have a large center compartment surrounded by several peripheral lipid layers. These vesicles, having a diameter of 2-15 μm, are described in Callo, et al., Cryobiology 22(3):251-267 (1985).
Mezei, et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also describe methods of preparing lipid vesicles. More recently, Hsu, U.S. Pat. No. 5,653,996 describes a method of preparing liposomes utilizing aerosolization and Yiournas, et al., U.S. Pat. No. 5,013,497 describes a method for preparing liposomes utilizing a high velocity-shear mixing chamber. Methods are also described that use specific starting materials to produce ULVs (Wallach, et al., U.S. Pat. No. 4,853,228) or OLVs (Wallach, U.S. Pat. Nos. 5,474,848 and 5,628,936). A comprehensive review of all the aforementioned lipid vesicles and methods for their preparation are described in "Liposome Technology", ed. G. Gregoriadis, CRC Press Inc., Boca Raton, FIa., Vol. I, II & III (1984). This and the aforementioned references describing various lipid vesicles suitable for use in the invention are incorporated herein by reference.
Fatty acids (i.e., lipids) that can be conjugated to the provided compositions include those that allow the efficient incorporation of the proprotein convertase inhibitors into liposomes. Generally, the fatty acid is a polar lipid. Thus, the fatty acid can be a phospholipid The provided compositions can comprise either natural or synthetic phospholipid. The phospholipids can be selected from phospholipids containing saturated or unsaturated mono or disubstituted fatty acids and combinations thereof. These phospholipids can be dioleoylphosphatidylcholine, dioleoylphosphatidylserine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, dioleoylphosphatidic acid, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylserine, palmitoyloleoylphosphatidylethanolamine, palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic acid, palmitelaidoyloleoylphosphatidylcholine, palmitelaidoyloleoylphosphatidylserine, palmitelaidoyloleoylphosphatidylethanolamine, palmitelaidoyloleoylphosphatidylglycerol, palmitelaidoyloleoylphosphatidic acid, myristoleoyloleoylphosphatidylcholine, myristoleoyloleoylphosphatidylserine, myristoleoyloleoylphosphatidylethanoamine, myristoleoyloleoylphosphatidylglycerol, myristoleoyloleoylphosphatidic acid, dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine, dilinoleoylphosphatidylethanolamine, dilinoleoylphosphatidylglycerol, dilinoleoylphosphatidic acid, palmiticlinoleoylphosphatidylcholine, palmiticlinoleoylphosphatidylserine, palmiticlinoleoylphosphatidylethanolamine, pamiiticlinoleoylphosphatidylglycerol, palmiticlinoleoylphosphatidic acid. These phospholipids may also be the monoacylated derivatives of phosphatidylcholine (lysophophatidylidylcholine), phosphatidylserine (lysophosphatidylserine), phosphatidylethanolamine (lysophosphatidylethanolamine), phophatidylglycerol (lysophosphatidylglycerol) and phosphatide acid (lysophosphatidic acid). The monoacyl chain in these lysophosphatidyl derivatives may be palimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoyl or myristoleoyl. The phospholipids can also be synthetic. Synthetic phospholipids are readily available commercially from various sources, such as AVANTI Polar Lipids (Albaster, Ala.); Sigma Chemical Company (St. Louis, Mo.). These synthetic compounds may be varied and may have variations in their fatty acid side chains not found in naturally occurring phospholipids. The fatty acid can have unsaturated fatty acid side chains with C14, C16, C18 or C20 chains length in either or both the PS or PC. Synthetic phospholipids can have dioleoyl (18:1)-PS; palmitoyl (16:0)-oleoyl (18:1)-PS, dimyristoyl (14:0)-PS; dipalmitoleoyl (16:1)-PC, dipalmitoyl (16:0)-PC, dioleoyl (18:1)- PC, palmitoyl (16:0)-oleoyl (18:1)-PC, and myristoyl (14:0)-oleoyl (18:1)-PC as constituents. Thus, as an example, the provided compositions can comprise palmitoyl 16:0. c. In vivofEx vivo
As described above, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject's cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).
If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject. B. Methods
1. Treatment
Provided herein is a method of treating or preventing a tumor in a subject comprising administering to the subject a therapeutically effective amount of a 7-secretase inhibitor CySI). Also provided herein is a method of treating or preventing a tumor in a subject comprising administering to the subject a therapeutically effective amount of a retinoid X receptor (RXR) agonist (rexinoid). Also provided herein is a method of treating or preventing a tumor in a subject comprising administering to the subject a therapeutically effective amount of a γ-secretase inhibitor CySI) and a retinoid X receptor (RXR) agonist (rexinoid). The 7SI can be any 7SI known in the art, including MK0752, MRK003-ONC, L- 685,458, or dibenzazepine (DBZ). The rexinoid can be any retinoid known in the art, including 9cUAB30.
In some aspects, the tumor of the disclosed method can be any tumor with a detectably higher level of Kruppel-like factor 4 (KLF4) as compared to normal reference cells. For example, the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of KLF4 in the tumor cells as compared to that of normal reference cells. m some aspects, the tumor of the disclosed method can be any tumor with predominantly cyotosolic expression of Kriippel-like factor 4 (KLF4) as compared to normal reference cells. For example, the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher cytosolic expression of KLF4 in the tumor cells as compared to that of normal reference cells. m some aspects, the tumor of the disclosed method can be any tumor with a detectably higher level of Notchl (Ntcl) as compared to normal reference cells. For example, the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of Ntclin the tumor cells as compared to that of normal reference cells.
In some aspects, the tumor of the disclosed method can be any tumor with predominantly nuclear expression of Ntcl as compared to normal reference cells. For example, the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher nuclear expression of Ntcl in the tumor cells as compared to that of normal reference cells.
The tumor of the disclosed method can be a breast cancer. Thus, the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget's disease of the breast. The breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2 -negative (triple-negative breast cancer). Thus, in some aspects of the disclosed method, the subject has been diagnosed with the breast tumor by mammogram or biopsy. In some aspects, the subject has undergone a lumpectomy or mastectomy, hi some aspects, the subject has been identified has having a genetic risk of developing a breast tumor.
The tumor of the disclosed method can be pancreatic cancer. Thus, the tumor can be an adenocarcinoma. The tumor of the disclosed method can be squamous cell carcinoma. Thus, the tumor can be in the skin, mouth, esophagus, prostate, lungs, or cervix.
2. Diagnostic/ Treatment Combination
Also provided herein is a method of treating or preventing a tumor in a subject comprising the steps of (a) detecting staining of Kriippel-like factor 4 (KLF4) in the tumor cells, (b) detecting a difference in the amount of KLF4 staining in the breast tumor cells as compared to that of normal reference cells, and (c) administering to the subject a therapeutically effective amount of a γ-secretase inhibitor (γSI). hi some aspects, the method comprises detecting a higher level of KLF4 in the tumor cells as compared to that of normal reference cells. For example, the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
Also provided herein is a method of treating or preventing a tumor in a subject comprising the steps of (a) detecting staining of Kriippel-like factor 4 (KLF4) in said breast tumor cells, (b) detecting a difference in the localization of KLF4 staining in the breast tumor cells as compared to that of normal reference cells, and (c) administering to the subject a therapeutically effective amount of a γ-secretase inhibitor (7SI). In some aspects, the method comprises detecting a predominantly cytosolic staining of KLF4 in the tumor cells, hi some aspects, the method comprises detecting a predominantly nuclear staining of KLF4 in the tumor cells.
The disclosed methods can further comprise administering to the subject a therapeutically effective amount of a retinoid X receptor (RXR) agonist (rexinoid).
The disclosed method can further comprise detecting a difference in the amount of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells. Thus, in some aspects the method comprises detecting a higher level of Ntcl in the tumor cells as compared to that of normal reference cells. The disclosed method can further comprise detecting a difference in the localization of Notch 1 (Ntcl) staining in the tumor cells as compared to that of normal reference cells. Thus, in some aspects, the method comprises detecting a predominantly nuclear staining of Ntcl in the tumor cells. In some aspects, the method comprises detecting a predominantly cytosolic staining of Ntcl in the tumor cells.
The tumor of the disclosed method can be a breast cancer. Thus, the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget' s disease of the breast. The breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative breast cancer).
Thus, in some aspects of the disclosed method, the subject has been diagnosed with the breast tumor by mammogram or biopsy. In some aspects, the subject has undergone a lumpectomy or mastectomy. In some aspects, the subject has been identified has having a genetic risk of developing a breast tumor.
The tumor of the disclosed method can be pancreatic cancer. Thus, the tumor can be an adenocarcinoma. The tumor of the disclosed method can be squamous cell carcinoma. Thus, the tumor can be in the skin, mouth, esophagus, prostate, lungs, or cervix.
The reference cells of the disclosed method can be from normal breast, such as from breast reduction or mammoplasty. The reference cells of the disclosed method can be from human tumor cell lines. The reference cells of the disclosed method can be from the subject. For example, the reference cells can be from normal breast tissue of the subject. 3. Treatment Selection
Also provided is a method of selecting a treatment for a subject diagnosed with a tumor, comprising examining the expression of Krϋppel-like factor 4 (KLF '4) in the tumor, wherein a difference in the amount of KLF4 in one or more tumor cells as compared to that of normal reference cells indicates that the selected treatment is γ-secretase inhibitor
(TSI).
In some aspects, the method comprises detecting a higher level of KLF4 in the tumor cells as compared to that of normal reference cells. For example, the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
The tumor of the disclosed method can be a breast cancer. Thus, the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (EDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget's disease of the breast. The breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative breast cancer).
In some aspects, a higher level of KLF4 in the tumor cells as compared to that of normal reference cells indicates that the selected treatment is 7SI.
The method can further comprise detecting a difference in the amount of Notch 1 (Ntc 1) staining in the tumor cells as compared to that of normal reference cells. In some aspects, a higher level of Ntc 1 in the tumor cells as compared to that of normal reference cells indicates that the selected treatment is -ySI.
The method can further comprise detecting a difference in the localization of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells. In some aspects, predominantly nuclear staining of Ntcl in the tumor cells indicates that the selected treatment is 7SI. hi some aspects, a predominantly cytosolic staining of Ntcl in the tumor cells indicates that the selected treatment is 7SI.
Also provided is a method of selecting a treatment for a subject diagnosed with a tumor, comprising examining the expression of Kriippel-like factor 4 (KLF4) in said breast tumor, wherein a difference in the localization of KLF4 in one or more breast tumor cells as compared to that of normal reference cells indicates that the selected treatment is 7-secretase inhibitor CySI).
In some aspects, a predominantly cytosolic staining of KLF4 in the tumor cells indicates that the selected treatment is 7SI. In some aspects, a predominantly nuclear staining of KLF4 in the tumor cells indicates that the selected treatment is 7SI.
The tumor of the disclosed method can be a breast cancer. Thus, the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget's disease of the breast. The breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2 -negative (triple-negative breast cancer).
The method can further comprise detecting a difference in the amount of Notchl (Ntc 1) staining in the tumor cells as compared to that of normal reference cells. In some aspects, a higher level of Ntc 1 in the tumor cells as compared to that of normal reference cells indicates that the selected treatment is ^SI.
The method can further comprise detecting a difference in the localization of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells. In some aspects, a predominantly nuclear staining of Ntcl in the tumor cells indicates that the selected treatment is 'ySI. In some aspects, a predominantly cytosolic staining of Ntcl in the tumor cells indicates that the selected treatment is 7SI. 4. Subject Identification
A method of identifying a subject diagnosed with a tumor as a candidate for treatment with a 7-secretase inhibitor (7SI), comprising examining the expression of Kriippel-like factor 4 (KLF4) in the tumor, wherein a difference in the amount of KLF4 in one or more breast tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI. In some aspects, the method comprises detecting a higher level of KLF4 in the tumor cells as compared to that of normal reference cells. For example, the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of EXF4 in the tumor cells as compared to that of normal reference cells.
The tumor of the disclosed method can be a breast cancer. Thus, the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (EBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget's disease of the breast. The breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2 -negative (triple-negative breast cancer). In some aspects, a higher level of KLF4 in the tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI.
The method can further comprise detecting a difference in the amount of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells. In some aspects, a higher level of Ntcl in the tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI.
The method can further comprise detecting a difference in the localization of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells. In some aspects, a predominantly nuclear staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 'ySI. In some aspects, a predominantly cytosolic staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 7SL
Also provided is a method of identifying a subject diagnosed with a tumor as a candidate for treatment with a γ-secretase inhibitor CySI), comprising examining the expression of Kruppel-like factor 4 (KLF4) in said breast tumor, wherein a difference in the localization of KLF4 in one or more tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI.
In some aspects, a predominantly cytosolic staining of KLF4 in the tumor cells identifies the subject as a candidate for treatment with a 'ySI. In some aspects, a predominantly nuclear staining of KLF4 in the tumor cells identifies the subject as a candidate for treatment with a 7SI.
The tumor of the disclosed method can be a breast cancer. Thus, the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget' s disease of the breast. The breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative breast cancer).
The method can further comprise detecting a difference in the amount of Notchl (Ntcl) staining in said tumor cells as compared to that of normal reference cells. In some aspects, a higher level of Ntcl in the tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI.
The method can further comprise detecting a difference in the localization of Notchl (Ntcl) staining in the tumor cells as compared to that of normal reference cells. In some aspects, a predominantly nuclear staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 7SI. In some aspects, a predominantly cytosolic staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 7SI.
5. Prognosis
A method of determining the prognosis of an individual diagnosed as having a tumor, comprising examining the expression of Kriippel-like factor 4 (KLF4) and Notchl (Ntcl) in one or more tumor cells, wherein a difference in the amount, localization, or both of KLF4 and a difference in the amount, localization, or both of Ntcl staining in the tumor cells as compared to that of normal reference cells indicates a lower likelihood of survival.
The tumor of the disclosed method can be a breast cancer. Thus, the breast tumor can be ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (ILC), medullary carcinoma, inflammatory breast cancer (IBC), tubular carcinoma (TC), colloid carcinoma, metaplastic carcinoma, papillary carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, or Paget' s disease of the breast. The breast tumor can be estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative breast cancer).
In some aspects, a higher level of KLF4 in the tumor cells as compared to that of normal reference cells indicates a lower likelihood of survival. For example, the method can comprise detecting an about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher level of KLF4 in the tumor cells as compared to that of normal reference cells.
In some aspects, a predominantly cytosolic staining of KLF4 in the tumor cells indicates a lower likelihood of survival. In some aspects, a predominantly nuclear staining of KLF4 in the tumor cells indicates a lower likelihood of survival. In some aspects, a higher level of Ntcl in the tumor cells as compared to that of normal reference cells indicates a lower likelihood of survival. In some aspects, a predominantly cytosolic staining of Ntcl in the tumor cells indicates a lower likelihood of survival. In some aspects, a predominantly nuclear staining of Ntcl in the tumor cells indicates a lower likelihood of survival. In some aspects, a predominantly cytosolic staining of KLF4 and a predominantly nuclear staining of Ntcl indicates a lower likelihood of survival.
6. Immunoassays
KLF4 and Notchl can be detected in cells using standard methods known in the art. For example, KLF4 and/or Notchl can be detected in cells using immunodetection methods. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding immunodetection methods. Immunoassays, in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Many types and formats of immunoassays are known and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmune precipitation assays (RIPA), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAP/ FLAP).
In general, immunoassays involve contacting a sample suspected of containing a molecule of interest (such as the disclosed biomarkers) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest (such as antibodies to the disclosed biomarkers) with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immunocomplexes. Contacting a sample with the antibody to the molecule of interest or with the molecule that can be bound by an antibody to the molecule of interest under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply bringing into contact the molecule or antibody and the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any molecules (e.g., antigens) present to which the antibodies can bind. In many forms of immunoassay, the sample- antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as the disclosed biomarkers or their antibodies) in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process. In general, the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label. See, for example, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference in its entirety and specifically for teachings regarding immunodetection methods and labels.
As used herein, a label can include a fluorescent dye, a member of a binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence. Substances suitable for detectably labeling proteins include fluorescent dyes (also known herein as fluorochromes and fluorophores) and enzymes that react with colorometric substrates (e.g., horseradish peroxidase). The use of fluorescent dyes is generally preferred in the practice of the invention as they can be detected at very low amounts. Furthermore, in the case where multiple antigens are reacted with a single array, each antigen can be labeled with a distinct fluorescent compound for simultaneous detection. Labeled spots on the array are detected using a fluorimeter, the presence of a signal indicating an antigen bound to a specific antibody.
Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5- Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-
Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4- methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4- 1 methylcoumarin; 9- Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis- BTC; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™-3; Bodipy492/515; Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO- PRO™ -3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green- 1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy- X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C- phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3τM. Cy5 i 8; cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3'DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4- ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DiIC 18(5)); DIDS; Dihydorhodamine 123 (DHR); DiI (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-
Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 1OGF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type' non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo- 1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-I; JO JO-I; JO-PRO-I; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-I; LO-PRO-I; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue- White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag- Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO- 1; POPO-3; PO-PRO-I; PO- 1 PRO-3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufm; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R- phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™ (super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-I; SNAFL-2; SNARF calcein; SNARFl; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red- X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-I; TO-PRO-3; TO-PRO-5; TOTO-I; TOTO-3; Tricolor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-I; YO- PRO 3; YOYO- l;Y0Y0-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof.
A modifier unit such as a radionuclide can be incorporated into or attached directly to any of the compounds described herein by halogenation. Examples of radionuclides useful in this embodiment include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, fluorine-18. In another aspect, the radionuclide can be attached to a linking group or bound by a chelating group, which is then attached to the compound directly or by means of a linker. Examples of radionuclides useful in the apset include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling techniques such as these are routinely used in the radiopharmaceutical industry.
The radiolabeled compounds are useful as imaging agents to diagnose neurological disease (e.g., a neurodegenerative disease) or a mental condition or to follow the progression or treatment of such a disease or condition in a mammal (e.g., a human). The radiolabeled compounds described herein can be conveniently used in conjunction with imaging techniques such as positron emission tomography (PET) or single photon emission computerized tomography (SPECT). Labeling can be either direct or indirect. In direct labeling, the detecting antibody (the antibody for the molecule of interest) or detecting molecule (the molecule that can be bound by an antibody to the molecule of interest) include a label. Detection of the label indicates the presence of the detecting antibody or detecting molecule, which in turn indicates the presence of the molecule of interest or of an antibody to the molecule of interest, respectively. In indirect labeling, an additional molecule or moiety is brought into contact with, or generated at the site of, the immunocomplex. For example, a signal- generating molecule or moiety such as an enzyme can be attached to or associated with the detecting antibody or detecting molecule. The signal-generating molecule can then generate a detectable signal at the site of the immunocomplex. For example, an enzyme, when supplied with suitable substrate, can produce a visible or detectable product at the site of the immunocomplex.
As another example of indirect labeling, an additional molecule (which can be referred to as a binding agent) that can bind to either the molecule of interest or to the antibody (primary antibody) to the molecule of interest, such as a second antibody to the primary antibody, can be contacted with the immunocomplex. The additional molecule can have a label or signal-generating molecule or moiety. The additional molecule can be an antibody, which can thus be termed a secondary antibody. Binding of a secondary antibody to the primary antibody can form a so-called sandwich with the first (or primary) antibody and the molecule of interest. The immune complexes can be contacted with the labeled, secondary antibody under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes can then be generally washed to remove any non-specifically bound labeled secondary antibodies, and the remaining label in the secondary immune complexes can then be detected. The additional molecule can also be or include one of a pair of molecules or moieties that can bind to each other, such as the biotin/avadin pair. In this mode, the detecting antibody or detecting molecule should include the other member of the pair.
Other modes of indirect labeling include the detection of primary immune complexes by a two step approach. For example, a molecule (which can be referred to as a first binding agent), such as an antibody, that has binding affinity for the molecule of interest or corresponding antibody can be used to form secondary immune complexes, as described above. After washing, the secondary immune complexes can be contacted with another molecule (which can be referred to as a second binding agent) that has binding affinity for the first binding agent, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (thus forming tertiary immune complexes). The second binding agent can be linked to a detectable label or signal-genrating molecule or moiety, allowing detection of the tertiary immune complexes thus formed. This system can provide for signal amplification.
Immunoassays that involve the detection of as substance, such as a protein or an antibody to a specific protein, include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are generally diagnostic means of determining the presence or absence of a specific protein, or an antibody to a specific protein, in a sample. Protein separation methods are additionally useful for evaluating physical properties of the protein, such as size or net charge. Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein, or antibody to a specific protein, in a sample. Finally, in vivo detection is useful for evaluating the spatial expression patterns of the substance, i.e., where the substance can be found in a subject, tissue or cell.
Provided that the concentrations are sufficient, the molecular complexes ([Ab- Ag] n) generated by antibody-antigen interaction are visible to the naked eye, but smaller amounts may also be detected and measured due to their ability to scatter a beam of light. The formation of complexes indicates that both reactants are present, and in immunoprecipitation assays a constant concentration of a reagent antibody is used to measure specific antigen ([Ab-AgJw), and reagent antigens are used to detect specific antibody ([Ab-AgJn). If the reagent species is previously coated onto cells (as in hemagglutination assay) or very small particles (as in latex agglutination assay), "clumping" of the coated particles is visible at much lower concentrations. A variety of assays based on these elementary principles are in common use, including Ouchterlony immunodiffusion assay, rocket Immunoelectrophoresis, and immunoturbidometric and nephelometric assays. The main limitations of such assays are restricted sensitivity (lower detection limits) in comparison to assays employing labels and, in some cases, the fact that very high concentrations of analyte can actually inhibit complex formation, necessitating safeguards that make the procedures more complex. Some of these Group 1 assays date right back to the discovery of antibodies and none of them have an actual "label" (e.g. Ag- enz). Other kinds of immunoassays that are label free depend on immunosensors, and a variety of instruments that can directly detect antibody— antigen interactions are now commercially available. Most depend on generating an evanescent wave on a sensor surface with immobilized ligand, which allows continuous monitoring of binding to the ligand. Immunosensors allow the easy investigation of kinetic interactions and, with the advent of lower-cost specialized instruments, may in the future find wide application in immunoanalysis.
The use of immunoassays to detect a specific protein can involve the separation of the proteins by electophoresis. Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique.
Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage. In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.
Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e., the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.
Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length. Further, it is usually necessary to reduce disulphide bridges in proteins (denature) before they adopt the random-coil configuration necessary for separation by size; this is done with 2-mercaptoethanol or dithiothreitol (DTT). In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight.
Determination of molecular weight is done by SDS-PAGE of proteins of known molecular weight along with the protein to be characterized. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye- front. A simple way of determining relative molecular weight by electrophoresis (Mx) is to plot a standard curve of distance migrated vs. loglOMW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel.
In two-dimensional electrophoresis, proteins are fractionated first on the basis of one physical property, and, in a second step, on the basis of another. For example, isoelectric focusing can be used for the first dimension, conveniently carried out in a tube gel, and SDS electrophoresis in a slab gel can be used for the second dimension. One example of a procedure is that of O'Farrell, P.H., High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), herein incorporated by reference in its entirety for its teaching regarding two-dimensional electrophoresis methods. Other examples include but are not limited to, those found in Anderson, L and Anderson, NG, High resolution two-dimensional electrophoresis of human plasma proteins, Proc. Natl. Acad. Sci. 74:5421-5425 (1977), Ornstein, L., Disc electrophoresis, L. Ann. N. Y. Acad. Sci. 121:321349 (1964), each of which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods.
Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680 (1970), which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods, discloses a discontinuous system for resolving proteins denatured with SDS. The leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine. Accordingly, the resolving gel and the stacking gel are made up in Tris-HCl buffers (of different concentration and pH), while the tank buffer is Tris-glycine. All buffers contain 0.1% SDS.
One example of an immunoassay that uses electrophoresis that is contemplated in the current methods is Western blot analysis. Western blotting or immunoblotting allows the determination of the molecular mass of a protein and the measurement of relative amounts of the protein present in different samples. Detection methods include chemiluminescence and chromagenic detection. Standard methods for Western blot analysis can be found in, for example, D.M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent 4,452,901, each of which is herein incorporated by reference in their entirety for teachings regarding Western blot methods. Generally, proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are transferred to a sheet of special blotting paper, e.g., nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel. The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein.
The attachment of specific antibodies to specific immobilized antigens can be readily visualized by indirect enzyme immunoassay techniques, usually using a chromogenic substrate (e.g. alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other possibilities for probing include the use of fluorescent or radioisotope labels (e.g., fluorescein, 125I). Probes for the detection of antibody binding can be conjugated antiimmunoglobulins, conjugated staphylococcal Protein A (binds IgG), or probes to biotinylated primary antibodies (e.g., conjugated avidin/ streptavidin).
The power of the technique lies in the simultaneous detection of a specific protein by means of its antigenicity, and its molecular mass. Proteins are first separated by mass in the SDS-PAGE, then specifically detected in the immunoassay step. Thus, protein standards (ladders) can be run simultaneously in order to approximate molecular mass of the protein of interest in a heterogeneous sample.
The herein disclosed oncogenes can be detected by cell separatation methods such as including fluorescence activated cell sorting (FACS). The use of separation techniques include, but are not limited to, those based on differences in physical (density gradient centrifugation and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rhol23 and DNA-binding dye Hoechst 33342).
Cells may be selected based on light-scatter properties as well as their expression of various cell surface antigens. The purified stem cells have low side scatter and low to medium forward scatter profiles by FACS analysis. Cytospin preparations show the enriched stem cells to have a size between mature lymphoid cells and mature granulocytes.
Various techniques can be employed to separate the cells by initially removing cells of dedicated lineage. Monoclonal antibodies are particularly useful. The antibodies can be attached to a solid support to allow for crude separation. The separation techniques employed should maximize the retention of viability of the fraction to be collected.
The separation techniques employed should maximize the retention of viability of the fraction to be collected. Various techniques of different efficacy may be employed to obtain "relatively crude" separations. Such separations are where up to 10%, usually not more than about 5%, preferably not more than about 1%, of the total cells present are undesired cells that remain with the cell population to be retained. The particular technique employed will depend upon efficiency of separation, associated cytotoxicity, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.
Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, or other convenient technique.
Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
Other techniques for positive selection may be employed, which permit accurate separation, such as affinity columns, and the like. The method should permit the removal to a residual amount of less than about 20%, preferably less than about 5%, of the non- target cell populations. The antibodies may be conjugated with markers, such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type. Any technique may be employed which is not unduly detrimental to the viability of the remaining cells.
Conveniently, after substantial enrichment of the cells lacking the DAZL marker, generally by at least about 50%, preferably at least about 70%, the cells may now be separated by a fluorescence activated cell sorter (FACS) or other methodology having high specificity. Multi-color analyses may be employed, with the FACS which is particularly convenient. The cells may be separated on the basis of the level of staining for the particular antigens.
Preferably, cells are initially separated by a coarse separation, followed by a fine separation, with positive selection of one or more markers associated with the stem cells and negative selection for markers associated with lineage committed cells. 7. Nucleic Acid Detection
As an alternative to immunodetection methods, KLF4 and Notch 1 mRNA can be detected in cells using standard methods known in the art. A number of widely used procedures exist for detecting and determining the abundance of a particular mRNA in a total or poly(A) RNA sample. For example, specific mRNAs can be detected using Northern blot analysis, nuclease protection assays (NPA), in situ hybridization, or reverse transcription-polymerase chain reaction (RT-PCR).
In theory, each of these techniques can be used to detect specific RNAs and to precisely determine their expression level. In general, Northern analysis is the only method that provides information about transcript size, whereas NPAs are the easiest way to simultaneously examine multiple messages. In situ hybridization is used to localize expression of a particular gene within a tissue or cell type, and RT-PCR is the most sensitive method for detecting and quantitating gene expression.
Northern analysis presents several advantages over the other techniques. The most compelling of these is that it is the easiest method for determining transcript size, and for identifying alternatively spliced transcripts and multigene family members. It can also be used to directly compare the relative abundance of a given message between all the samples on a blot. The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., intactness of the RNA sample and how efficiently it has transferred to the membrane). RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. Nonisotopic or high specific activity radiolabeled probes can be used including random-primed, nick- translated, or PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides. Additionally, sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an exon) may be used as probes.
The Nuclease Protection Assay (NPA) (including both ribonuclease protection assays and Sl nuclease assays) is an extremely sensitive method for the detection and quantitation of specific mRNAs. The basis of the NPA is solution hybridization of an antisense probe (radiolabeled or nonisotopic) to an RNA sample. After hybridization, single-stranded, unhybridized probe and RNA are degraded by nucleases. The remaining protected fragments are separated on an acrylamide gel. Solution hybridization is typically more efficient than membrane-based hybridization, and it can accommodate up to 100 μg of sample RNA, compared with the 20-30 μg maximum of blot hybridizations. NPAs are also less sensitive to RNA sample degradation than Northern analysis since cleavage is only detected in the region of overlap with the probe (probes are usually about 100-400 bases in length).
NPAs are the method of choice for the simultaneous detection of several RNA species. During solution hybridization and subsequent analysis, individual probe/target interactions are completely independent of one another. Thus, several RNA targets and appropriate controls can be assayed simultaneously (up to twelve have been used in the same reaction), provided that the individual probes are of different lengths. NPAs are also commonly used to precisely map mRNA termini and intron/exon junctions.
In situ hybridization (ISH) is a powerful and versatile tool for the localization of specific mRNAs in cells or tissues. Unlike Northern analysis and nuclease protection assays, ISH does not require the isolation or electrophoretic separation of RNA. Hybridization of the probe takes place within the cell or tissue. Since cellular structure is maintained throughout the procedure, ISH provides information about the location of mRNA within the tissue sample.
The procedure begins by fixing samples in neutral-buffered formalin, and embedding the tissue in paraffin. The samples are then sliced into thin sections and mounted onto microscope slides. (Alternatively, tissue can be sectioned frozen and post- fixed in paraformaldehyde.) After a series of washes to dewax and rehydrate the sections, a Proteinase K digestion is performed to increase probe accessibility, and a labeled probe is then hybridized to the sample sections. Radiolabeled probes are visualized with liquid film dried onto the slides, while nonisotopically labeled probes are conveniently detected with colorimetric or fluorescent reagents.
RT-PCR has revolutionized the study of gene expression. It is now theoretically possible to detect the RNA transcript of any gene, regardless of the scarcity of the starting material or relative abundance of the specific rnRNA. In RT-PCR, an RNA template is copied into a complementary DNA (cDNA) using a retroviral reverse transcriptase. The cDNA is then amplified exponentially by PCR. As with NPAs, RT-PCR is somewhat tolerant of degraded RNA. As long as the RNA is intact within the region spanned by the primers, the target will be amplified.
Relative quantitative RT-PCR involves amplifying an internal control simultaneously with the gene of interest. The internal control is used to normalize the samples. Once normalized, direct comparisons of relative abundance of a specific mRNA can be made across the samples. It is crucial to choose an internal control with a constant level of expression across all experimental samples (i.e., not affected by experimental treatment). Commonly used internal controls (e.g., GAPDH, β-actin, cyclophilin) often vary in expression and, therefore, may not be appropriate internal controls. Additionally, most common internal controls are expressed at much higher levels than the mRNA being studied. For relative RT-PCR results to be meaningful, all products of the PCR reaction must be analyzed in the linear range of amplification. This becomes difficult for transcripts of widely different levels of abundance.
Competitive RT-PCR is used for absolute quantitation. This technique involves designing, synthesizing, and accurately quantitating a competitor RNA that can be distinguished from the endogenous target by a small difference in size or sequence. Known amounts of the competitor RNA are added to experimental samples and RT-PCR is performed. Signals from the endogenous target are compared with signals from the competitor to determine the amount of target present in the sample. 8. Administration
The disclosed compounds and compositions can be administered in any suitable manner. The manner of administration can be chosen based on, for example, whether local or systemic treatment is desired, and on the area to be treated. For example, the compositions can be administered orally, parenterally (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection), , by inhalation, extracorporeally, topically (including transdermally, ophthalmically, vaginally, rectally, intranasally) or the like.
As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
The exact amount of the compositions required can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. Thus, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage can vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counter indications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
For example, a typical daily dosage of the γ-secretase inhibitor (7SI) used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
Following administration of a disclosed, the efficacy of the therapeutic 7-secretase inhibitor (7SI) can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a composition disclosed herein is efficacious in treating or inhibiting an tumor in a subject by observing that the composition reduces the tumor or prevents a further increase in tumor size. C. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds, reference to "the compound" is a reference to one or more compounds and equivalents thereof known to those skilled in the art, and so forth.
"Optional" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.
By "treatment" is meant the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder, hi addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
The term "therapeutically effective" means that the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. The term "carrier" means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. D. Examples
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in 0C or is at ambient temperature, and pressure is at or near atmospheric. 1. Example 1 i. Results a. Frequent deregulation of KLF4 in human breast cancer
To examine KLF4/GKLF RNA and protein in established breast cancers and DCIS several mRNA in situ hybridization and immunostaining assays of human KLF4 were developed (Foster, K. W., et al. 2000; Pandya, A. Y., et al. 2004; Foster, K. W., et al. 2005; Huang, C. C, et al. 2005). As compared to uninvolved epithelium, KLF4 mRNA and protein was shown to be upregulated in most cases of human breast cancer, unlike several other tumor types (Foster, K. W., et al. 2000). KLF4 is likewise upregulated in DCIS, identifying this as an early change. b. Analysis of KLF4 and Ntcl in breast cancer prognosis
A polyclonal rabbit α-KLF4 was developed (Foster, K. W., et al. 2005; Huang, C. C, et al. 2005) against a human peptide that is much superior at differentiating nuclear and cytoplasmic KLF4 as compared to the original monoclonal α-KLF4 (Foster, K. W., et al. 2000; Pandya, A. Y., et al. 2004). In addition, a polyclonal α-Ntcl was characterized (C20, Santa Cruz) which reacts well with cytoplasmic Ntcl (Ntcl™) and poorly with nuclear Ntcl (activated form, Ntcl ). Although Ntcl acts in the nucleus, western blot shows that the major species present in cells and tissues is the inactive precursor, Ntcl™ that must be cleaved by γ-secretase to yield NtclIC (Fig. 1). It is unknown how Ntcl™ in tissues correlates with the active Ntclic. Thus 3 variables were examined: KLF4 in the cytoplasm, KLF4 in the nucleus and Ntcl precursor (Ntcl™) in the cytoplasm.
These reagents were utilized to evaluate 153 UAB breast cancer cases of whom 68 recurred and died (44% overall mortality). As shown in Fig. 2 A, the category of high cytoplasmic KLF4, low nuclear KLF4 (i.e., Type 3 tumors) was a poor prognostic group of 18 patients with 12 deaths (67% mortality; P=0.031). hi the previous analysis, KLF4 was prognostic in a subgroup of patients with early stage disease (Pandya, A. Y., et al. 2004). However, in the current group of patients, the number of deaths in this subgroup (10 compared to 23 in the earlier study) limited the analysis of early stage disease. The 153 patients were next analyzed as to KLF4 and Ntcl. As shown in Fig. 2B, the category of high cytoplasmic KLF4 and low cytoplasmic Ntcl identified a group of poor prognosis patients with 14/22 deaths (64% mortality; P=0.0052).
Use of KLF4 and Ntcl in combination identified a higher risk group than when either was used alone. When used alone, low cytoplasmic Ntcl was associated with a poor outcome, but with a low hazard compared with Fig. 2A-B. For cytoplasmic NtcKO.4, the highest risk group identified using Ntcl alone, the mortality was 28/53 (53%; P=0.044). Using all 3 variables (Fig. 2C), the combination of high cytoplasmic KLF4, low nuclear KLF4 (i.e., Type 3 tumors), and low cytoplasmic Ntcl identified a very poor prognostic group with 9/11 deaths (82% mortality; P=0.0033). In addition to prognostic biomarker application, these findings indicate that KLF4 can promote Ntcl signaling as indicated by increased conversion of Ntcl™ precursor to NtclIC (thereby lowering cytoplasmic Ntcl), leading to an aggressive phenotype. This latter effect may be more prevalent than just the poor prognosis sub-group identified. c. KLF4 induces Ntcl and its ligands in epithelial cells To identify KLF4 target genes in RK3E cells (see methods in Fig. 3 legend), a conditional allele was generated by fusing KLF4 to a fragment of the estrogen receptor (i.e., KLF4-ER) (Foster, K. W., et al. 2005; Louro, I. D., et al. 2002). In response to 4- hydroxy tamoxifen (4OHT) the KLF4-ER fusion localizes to the nucleus and induces transformation (Foster, K. W., et al. 2005). Microarrays were used to identify transcripts induced by KLF4-ER in RK3E cells within 2-3 hours of induction in the presence of the protein translation inhibitor cycloheximide (CHX), which suppresses indirect target genes. Two sequential, independent experiments were performed (Table 2, Fig. 3). Northern analysis showed rapid (2hr) induction of Ntcl mRNA by 4OHT in KLF4-ER cells in the presence or absence of CHX (Fig. 3A, upper panels). Immunoblot using α-NtclIC (V1744, specific for the active form, see Fig. 1) showed increased active NtclIC following 4OHT addition to KLF4-ER cells (Fig. 3 A, bottom panels). α-Ntcl staining (C20, Santa Cruz) was increased by 4OHT in the cytoplasm of KLF4-ER cells, consistent with induction of Ntcl as an early, direct response to KLF4 (Fig. 3B). Table 2. Direct transcriptional targets of KLF4 (mean of two microarray expts).
Gene name Accession Fold Notes
Δ
Ntc Pathway
Ntc l BM390614 +7.5 MMTV Integration in breast ca.
Lunatic fringe AW915115 +2.0 Modifies Ntc
Hesl NM_024360 -2 Ntcl/CSL target gene
Ephrin pathway
Ephrin Al AW531877 +4.6 Vascular recruitment, contact inhibition,
EphA2 ligand
EphA2 AI602491 +2.8 Transforms MCFlOA breast cells
Ephexin AF254801.1 +3.9 Mediates Ephrin/EphA signaling
Retinoid pathway
RARγ BM386555 4.86 Retinoid receptor, tumor suppressor activity
NUR77 NM 024388 13.8 Breast cancer, apoptosis
Other pathways
Noggin AA859752 -7.0 BMP signaling, cell fate
Tgf-α NM 012671.1 +3.1 EGFR family ligand
ErbB3 BI284288 +3.7 ErbB2 coreceptor
WNT6 AI599019 +2.5 Breast development
WNT4 NM 053402.1 +4.4 Breast development c-MYC NM 012603.1 -11 Proliferation; cell fate
Prev. known targets
KLF4 NM_053713.1 +2.4 Epith. Differentiation p21wAFi/ciPi U24174.1 +6.4 Prognosis in breast cancer
Few or no proteins have been reported to regulate Ntcl transcription, and the human promoter has not been previously characterized (Fig. 3C). The promoter contains several potential KLF4 binding sites (5' RRGGYGY 3' (R=purine, Y=pyrimidine; SEQ ID NO:8)), some of which are evolutionarily conserved (see asterisks) and fall near the predicted Ntcl transcription start site (Tsuji, H., et al. 2003). Using mammary epithelial MCFlOA cells, ChIP analysis of the promoter indicated that the proximal fragments O and P are bound by HA-KLF4, while the promoter distal consensus site K was not bound (Fig. 3D-F). In promoter-reporter assays, fragments of the Ntcl promoter function as KLF4- regulated enhancers (Fig. 3G). Nearly all of the activity was contained within a 524 base, promoterproximal fragment (Δ5, Fig. 3G) that corresponds to ChIP fragment O (Fig. 3C).
KLF4 induced the Ntcl ligands Dill, D114, and Jagl (Fig. 3H). This induction was observed only late (i.e., 48hr), indicating that the ligands are indirectly induced. Jagl is a marker of poor outcome in breast cancer (Reedijk, M., et al. 2005). d. Concordance of KLF4 and Ntcl in mouse skin and human breast tumors
Induction of KLF4 in the skin by dox (Foster, K. W., et al. 2005) induced Ntcl by 48 hrs (Fig. 4A). At 14d staining was even more prominent, with Ntcl staining in the cytoplasm (Weijzen, S., et al. 2002) and KLF4 in the nucleus as reported (Foster, K. W., et al. 2005). Staining of KLF4 and Ntcl was performed in human breast cancer and scored slides using the semiquantitative approach described previously (Pandya, A. Y., et al. 2004). Pearson correlation revealed that KLF4 and Ntcl proteins are strongly associated (Fig. 4B-D and legend). Using median scores as cutoffs, 65 of 89 cases (73%) showed concordance of KLF4 and Ntcl (Fig. 4D, P-O.001). Ntcl is known to correlate with poor outcome in breast cancer (Pandya, A. Y., et al. 2004; Reedijk, M., et al. 2005; Parr, C, et al. 2004). In conclusion, KLF4 is the first transcription factor regulator of Ntcl, and probably the major determinant of Ntcl transcription in breast cancer. e. Ntcl antagonists, including 7SIs5 block transformation by KLF4 and NtclFL but not NtclIC
To block Ntcl in RK3E, NtclIcantisense (AS) RNA (Nickoloff, B. J., et al. 2003; Rangarajan, A., et al. 2001) was stably expressed by retroviral transduction (Fig. 5 A-B). These cells grew similarly to the Vector cells. NtclIC-AS efficiently blocked transformation by activated Ras, consistent with the report that NtclIC is required for transformation by RAS in other cells (Weijzen, S., et al. 2002). NtclIC-AS also efficiently blocked KLF4-induced, but not c-MYC- or GIi 1 -induced transformation. The Hesl-luc reporter assay assesses Ntcl activity in cells, and confirmed that NtclIC-AS specifically inhibits NtclIC in RK3E cells (Fig. 5C). Activity of the pTK-/3-gal control was unaffected. The KLF4-ER transformation assay (Foster, K. W., et al. 2005) was also used to examine the role of γ-secretase, required for S3 cleavage of Ntcl (Fig. IA). The γSI L-685,458 (Compound X; Sigma) permitted transformation of RK3E by NtclIC, but transformation by KLF4-ER was efficiently blocked (Fig. 4D).
Ntc 1IC, but not Ntc 1FL, can transform a cell line similar to RK3E (Capobianco, A. J., et al. 1997). As disclosed herein, NtclIC and NtclFL induced foci in RK3E, unlike a Vector control (Fig. 6A-C). The NtclFL transgene in these cells is WT in the PEST region (a motif conferring more rapid turnover) and heterodimerization domains that are mutated in T-ALL (Weng, A. P., et al. 2004). To further assess the specificity of γSIs as antagonists of KLF4, cells transformed by NtclFL or NtclIC were cultured in the presence of γSIs. Colonies formed by NtclFL cells appeared flat in the presence of 7SI, whereas colonies of Ntcl IC cells piled up and formed dense colonies of cells that were morphologically transformed, similar to the vehicle control (Fig. 6D). As these control oncogenes represent the substrate (Ntcl ) and the product (Ntcl ) of γ-secretase, these results strongly support the idea that ^SIs act specifically on 7-secretase to inhibit transformation. f. NtclIC induces transformation through a different downstream pathway in the presence of KLF4
An unresolved question is whether NtclIC transforms cells through CSL or by an alternate route (Brennan, K., et al. 2003) (Fig. IB). KLF4 potently inhibited both basal activity and NtclIC-induced activity of a Hesl-luc reporter in HEK293 cells (Fig. 7A, Hesl). This effect is likely due to inhibition of CSL activity, as Ntcl -dependent activity of an 8x wt CSL promoter-reporter was inhibited by KLF4 (Fig. 7A, 8x wt CSL). In addition to Ntcl, KLF4-ER cells treated with 4OHT +/- CHX also showed direct induction of the CSL-antagonist Hairless (Table 2, Fig. 7B). Inhibition of CSL reporter activity was due to induction of Hairless as shown using Hairless shRNAs (Fig. 75C). The effects of KLF4 on CSL are summarized in Fig. 7D. As shown in Fig. 8A-D, DN alleles of CSL and MAMLl block transformation by Ntc 1IC, but do not block transformation by KLF4, indicating that Ntcl can transform cells through two different routes (Fig. IB) and that KLF4 shunts Ntcl away from the classical pathway in favor of the alternate pathway. Since Ntcl acts very differently depending upon the presence/absence of KLF4, efficacy of 'ySIs in patients may depend upon levels of KLF4 rather than on levels of Ntcl alone.
Although KLF4 suppression of the classical Ntcl pathway has been demonstrated so far in RK3E and HEK293 cells, several in vivo observations suggest the effect is quite general. Reducing CSL-dependent pathway activity by deletion of the CSL or Hesl genes favors goblet cell formation in the gut, a known activity of KLF4 (Sancho, E., et al. 2003; Katz, J. P., et al. 2002; van Es, J. H., et al. 2005). Also, the early lesions induced by KLF4 in a transgenic mouse model of cutaneous SCC resemble lesions resulting from conditional knockout of CSL (Foster, K. W., et al. 2005; Yamamoto, N., et al. 2003), and SCC is similarly induced by either MAMLDNl or KLF4 transgenes in mouse skin (Proweller, A., et al. 2006). Potentially, loss of CSL or expression of MAML-DNl may mimic KLF4 by inducing Ntcl alternative pathway signaling. g. KLF4 blocks maturation of human MCFlOA breast epithelial cells and promotes tumor formation in MMTV-ErbB2 mice
Immortalized MCFlOA cells can be cultured in 2D monolayers on plastic, or else induced to recapitulate the polarized, acinar structures of breast epithelium by culture on a layer of basement membrane components (Matrigel) (Seton-Rogers, S. E., et al. 2004; Mills, K. R., et al. 2004; Seton-Rogers, S. E., et al. 2004; Debnath, J., et al. 2003a; Debnath, J., et al. 2003b; Debnath, J., et al. 2002; Muthuswamy, S. K., et al. 2001). ErbB2, but not ErbBl, reinitiates proliferation of preformed acini and induces a hyperplastic, multiacmar phenotype, with luminar repopulation, but with retention of other differentiated features such as apico-basal polarity of these epithelial cells (Muthuswamy, S. K., et al. 2001). ErbB2-induced hyperplastic lesions can be converted to fully transformed cells by TGF1S (Seton-Rogers, S. E., et al. 2004; Seton-Rogers, S. E., et al. 2004). hi 2D culture, MCFlOA cells transduced with KLF4 by retroviral transduction and then mass selected grew at the same rate and showed the same morphology as the Vector control. However in 3D culture KLF4 induced a block to differentiation/maturation (Fig. 9). When cultured on Matrigel, instead of the well-organized spheres and smooth, regular basal cell layer characteristic of untransformed cells, KLF4 induced grape-like clusters of cells with irregular basal layers. Clusters retained their roughened, dysplastic morphology throughout the period of observation of 19 days, suggesting that the maturation block is permanent. The similar maturation block by KLF4 and Ntcl demonstrates clearly that
KLF4 has a property of an oncogene in human breast cells because Ntcl was recently shown to induce soft agar growth and transformation of the same MCFlOA (Stylianou, S., et al. 2006).
To test the activity of KLF4 in vivo the latent tumor model, MMTV-ErbB2 was used . Bitransgenic MMTV-KLF4+/"; MMTV-ErbB2+/" virgin females (N= 12) were compared to mice transgenic for either transgene (N=9). KLF4 did not appreciably alter the latency of ErbB2-induced breast cancers (range 23-32 weeks) but bitransgenic animals showed more tumors per animal compared with MMTVErbB2 alone (2.50 vs. 1.44; p=0.0119, Mann Whitney test, two-tailed). No tumors were observed in WT or MMTV- KLF4+/~ mice as published (Foster, K. W., et al. 2005). These data suggest that KLF4 acts as an oncogene in breast epithelium. h. Characterization of KLF4DN alleles
Multiple KLF4 mutants, generated by site-directed mutation of conserved or charged residues to alanine, were screened for dominant negative (DN) activity. Two mutants, termed KLF4Ala5 and KLF4Ala6, were found to block transformation of RK3E cells by KLF4, but not by other oncogenes (e.g., RAS, GLIl). KLF4DNs was analyzed in MCFlOA cells and RAS-transformed MCFlOAT cells (Dawson, P. J., et al. 1996). MCFlOA cells express endogenous KLF4 (Foster, K. W., et al. 2000 ; Huang, C. C, et al. 2005) (Fig. 10A). Retroviral transduction with HA-KLF4WT vector yields cells that grow normally and stably express the exogenous protein. In contrast, in three independent experiments, KLF4Ala5 or KLF4Λla6 vectors induced only rare puroR colonies (Fig. 10B). Thus, KLF4DN is not tolerated by MCFlOA, consistent with an essential role of endogenous KLF4 in the growth of these cells. In contrast, KLF4DNs do not inhibit the Ras-transformed derivative, MCFlOAT (Fig. 10B). i. KLF4 is required for transformation by c-MYC and ErbB2
To identify pathways that might account for the KLF4 phenotype in MCFlOA, a screen for KLF4 dependence was used (Fig. 1 OC-D). Pooled populations of puroR RK3E- KLF4DN and RK3E- Vector cells were transduced with viruses for NtclIC, c-MYC, GIi 1, N-RAS or ErbB2, and foci were scored after 3 weeks. Independent experiments show that only c-MYC and ErbB2 are blocked by KLF4DN (Fig. 10D).
Activated ErbB2A648C induces transformation of RK3E (Li, X., et al. 2005). ErbB2 requires ErbB3 to drive the proliferation of breast cancer cells (Holbro, T., et al. 2003) and cooperates with ErbB3 in NIH3T3 transformation (Alimandi, M., et al. 1995). Transformation of RK3E by ErbB2 was blocked by either KLF4DN (experiments performed twice in triplicate for a total of 12 dishes - 2 expts x 2 alleles x 3 replicates) (Fig. 10C). KLF4DN cells showed >10 foldreduced transformation by ErbB2 retrovirus compared to control cells. Microarray data identified ErbB3, an essential ErbB2 signaling partner, as a direct transcriptional target of KLF4 (Table 2). Not only are these coexpressed in rumors, but also in normal skin ErbB2, ErbB3, and KLF4 are co-expressed in all cell layers, and increased in superficial, postmitotic cells (Huang, C. C, et al. 2005; Piepkorn, M., et al. 2003; Lebeau, S., et al. 2005). Like induction of Ntcl, KLF4-ErbB signaling links KLF4 to a pathway that is important in breast cancer. j. KLF4 induces nuclear receptors (NRs) in RK3E epithelial cells KLF4-ER directly induced transcription of RARγin each of the two microarray experiments (Fig. 1 IA; Table 2). Another NR, Nur77 (Lin, B., et al. 2004) was one of the most strongly induced transcripts on the array. Array results were confirmed by RT-PCR, using GAPDH as a control for input and Ntcl as a positive control (Fig. 1 IB-C). RXRα was induced by KLF4 in as little as 3 hrs, similarly to RARγ, Nur77, and Ntcl (Fig. HC). k. 9cUAB30 blocks skin tumor initiation by KLF4
Regulation of NRs by KLF4 prompted the testing of 9cUAB30 as an antagonist of KLF4. To determine whether 9cUAB30 can block KLF4 in vivo, the tet-on KLF4 model of SCC (Foster, K. W., et al. 2005) was used with addition of 9cUAB30 (1000 mg/kg diet) or placebo to the feed. This dose was shown to induce no side effects, such as weight loss, bone breakage, or any gross phenotype of the hair/skin. Following a 30 day treatment of dox + 9ΛJAB30, or dox + placebo, the skin phenotype of bitransgenic animals was examined grossly and microscopically. By gross and microscopic exam, induction of the skin phenotype was completely blocked in 8 of 9 animals treated with 9cUAB30 (Fig. 12A; P=0.0004, Fisher's exact test). Skin samples of the 9th mouse showed partial suppression of the phenotype. A control group fed placebo developed SCC-like lesions in 9/9 mice, as expected for this highly penetrant model (-100%) (Foster, K. W., et al. 2005). 1. 9cUAB30 is a specific inhibitor of KLF4
The above results demonstrated that 9ΛJAB30 is a highly effective antagonist of KLF4-induced transformation in vivo. To assess specificity of 9cUAB30 as an inhibitor of KLF4 in vitro transformation assays were performed in the presence/absence of 9cUAB30 (Fig. 12B). The RK3E assay where multiple carcinoma oncogenes can be analyzed in parallel was utilized. In this experiment 9cUAB30 inhibited transformation by KLF4, a result now demonstrated in three independent experiments, all performed in duplicate/triplicate. There was no effect on the size or number of foci induced by other oncogenes, including the breast cancer oncogenes Ntcl or ErbB2, and no effect on the BCC oncogene GUI. This striking result indicates that 9cUAB30 specifically targets the KLF4 pathway. m. Efficacy of 9cUAB30 in therapy of breast cancer
Rats were given a standard dose of MNU and followed over several months as described (Lubet, R. A., et al. 2006). When animals developed breast tumors of 200-300 mm2, they were randomly assigned to receive 9cUAB30 at 200 mg/kg diet or control diet. Caliper measurements of tumors were performed twice per week for 5-6 weeks. Tumor size at the start of therapy was assigned 100%. Figure 13 demonstrates the tumor growth curves for placebo-treated and 9cUAB30-treated mice. Using a linear mixed effects model, inhibition by 9cUAB30 was significant (p= 0.0068). To determine the effect on tumor doubling time, linear regression was applied to each animal individually. If a tumor did not double by the end of the study, the observation period of the study (42 days) was treated as the doubling time. The Kruskal-Wallis test was applied to compare the tumor doubling time between groups, indicating that 9cUAB30 significantly prolonged the doubling time with no observed side effects (mean 33.0 days vs. 18.4 days; p=0.0065). n. 9cUAB30 or 7SIS inhibit in vitro growth of human breast tumor cell lines with high KLF4 levels
KLF4 is highest in ZR75-1 and MCF7, lowest in MDA-MB-231 and MDA-MB- 453, and intermediate in other lines in a panel of breast tumor cell lines (Foster, K. W., et al. 2000). To examine whether KLF4 levels correlate with drug sensitivity the 5-day cytotoxicity of 1.0 μM 9cUAB30 or lOμM 7SI L-685,458 was determined (as used in Fig. 14D). Strikingly, the KLF4-high lines ZR75-1 and MCF7 were the most sensitive, and this was true for either drug (Fig. 14A). In ZR75-1 cells 9cUAB30 and 7SI showed similar cytotoxicity (-40%). MCF7 cells, which require KLF4 for rapid growth in culture (Rowland, B. D., et al. 2005), and Ntcl for growth in soft agar (Stylianou, S., et al. 2006), were more sensitive than any other line to 7SI (50% inhibition) and the second-most sensitive line to 9cUAB30 (22% inhibition). Effects were quite reproducible as shown in dose-response studies (Fig. 14B) and in the combined drug study (Fig. 14C). o. Development of a human-like model of breast cancer using oral DMBA in genetically-engineered mice
Therapy and prevention of breast cancer is hindered by absence of models with short latency, high penetrance, high throughput, and similarity to human cancer (Hursting, S. D., et al. 2005; Green, J. E., et al. , 2005; Ottewell, P. D., et al. 2006). Longer-latency models that are widely used are the rat DMBA model and the mouse MMTV-ErbB2 model (Li, B., et al. 1997; Zelazny, E., et al. 2001). By giving oral DMBA to MMTV- ErbB2+/" mice a rapid model was developed (-16 week latency, Fig. 15 A; collaboration with Project #1). Three groups of FVB/N females were treated with DMBA: 1) MMTV- ErbB2+/"; p53+/\ 2) MMTVErbB2+/-, and 3) p53+/\ Dosing was 1 mg/gavage, 1 dose/wk from age 50 days, treated for 8 weeks. No genotyping was needed because all breeders were homozygous (e.g., MMTV-ErbB2+/+) or else WT FVB. Kaplan-Meier analysis shows that the genotype of DMBA-treated animals determined the tumor type, with MMTV- ErbB2+/" promoting breast cancer (84% incidence) and p53+/" promoting SCC (75% incidence). The MMTV-ErbB2+/"; DMBA group showed only a 20% incidence of SCC. Nearly all breast tumors were adenocarcinoma, with tumor and stromal cells similar in appearance to human breast cancer (Fig. 15C).
For IHC α-Klf4 Hl 80 and Ntcl C20 were used (Santa Cruz). Hl 80 specifically detects the human KLF4 protein by western blot and stained the nuclei of SCC cells of tet- on KLF4 transgenic mice (Foster, K. W., et al. 2005) (Fig. 15B). H180 also detects endogenous mouse Klf4 (normal skin, Fig. 15B). Using this antibody approximately 50% of breast tumors in DMBA-treated mice were co-positive for Klf4 and Ntcl (Fig. 15C, Table 3). The association of Klf4 and Ntcl in these tumors is similar as in human breast cancer (Fig. 4). Breast tumors were also analyzed using antibodies that detect ER (MC-20, Santa Cruz) or p53 (CM5, Novacastra) (a subset is tabulated Table 3). Whereas MMTV- ErbB2-induced mouse tumors are typically ER-negative (Guy, C. T., et al. 1992; Wu, K., et al. 2002), it was found that overall 30% of MMTV-ErbB2+/-; DMBA tumors were ER+ (total tumors examined, N=30). A common concern over use of carcinogens is that Ras mutations will be frequent. Unlike for MNU, when oral DMBA is used to induce breast cancer in WT rats, the frequency of RAS activation is only -20% (El Sohemy, A., et al. 2000; Lu, J., et al. 1997). Taken together, the above data argues that the MMTV-ErbB2+A; DMBA model is similar to human breast cancer.
Table 3: Immunostaining analysis of the MMTV-ErbB2+/~; DMBA model.
Case# Klf4 Notchl P531 ER2"
1-6427T1 + + - -
2-6427T3 - - - +
3-6427T2 + + + -
4-6426T3 + - - +
5-6425T2 + + ++ -
6-6425T3 + + + -
7-6425T4 + +
8-6424T3 - -
9-6424T4 - . . .
Figure imgf000074_0001
p. KLF4 is required for in vitro growth of human pancreatic cancer cell lines with high KLF4 levels
Whole cell NP40 extracts of 10 pancreatic cancer cell lines were prepared and a western blot sequentially probed with antibody to KLF4 (Santa Cruz Hl 80), NtclIC (Abeam), and /3-actin (Fig. 18A). As for other tumor types, in these lines KLF4 migrates as several distinct species ranging in size from 50-65 kD, indicating posttranslational modification. This identification of EXF4 by Western was supported by positive/negative controls (Fig. 18A, lanes 11-12). While KLF4 is expressed in nearly all pancreatic cancer cell lines, the levels varied from line to line, hi contrast, antibody to NtclIC showed more consistent levels from cell line to cell line (Fig. 18A).
To test whether KLF4 is required for growth of these lines an efficient procedure was first developed for siRNA delivery to tumor cell lines. As a control for transfection efficiency, an siRNA to KTFl 1 that induces mitotic arrest and eventual cell death was used (Invitrogen). Cells were incubated with lipofectamine-siRNAKIFn complex or a control complex in a serum-free growth medium, followed by addition of normal growth medium. Growth was measured using the ATP assay (above) at 96h post-transfection. By this approach 90% of the growth of several human tumor cell lines was blocked over a 4 day period, indicating that >90% of the cells were successfully transfected. With this robust procedure in hand four different siRNAs to KLF4 were tested (Dharmacon). A mixture of two siRNAs (siRNA-KLF4#2/#3) was sufficient to block nearly all endogenous KLF4 expression by western (Fig. 18B). Cells transfected with the control siRNA (siRNA-Ctl; not similar to any human mRNA; Dharmacon) showed abundant KLF4. siRNA-KLF4#2/#3 was then transfected into Pane 08.13, Pane 10.05, or the KLF4- positive, 7S I- sensitive breast tumor cell line MCF7. Whole cell protein extracts were prepared at 96h post-transfection. Compared to controls, siRNA-KLF4#2/#3 appeared to suppress the final cell number. By microscopy there were many single cells instead of clusters of dividing cells. This observation was confirmed using a standard Bradford assay to quantify cellular protein in extracts of these wells, revealing a 50% reduction in cell mass (and by inference, cell number; Fig. 18C). MCF7 cells, which do not require KLF4 for cell growth, served as a control.
KLF4 knockdown cells showed reduced levels of active NtclIC by western blot, strongly supporting the notion that KLF4 regulates Ntcl in pancreatic cancer (Fig. 18B). Further, transfection of siRNA#2 or #3 alone OR as a mixture was sufficient to give the same growth defect, and consistently suppressed Ntcl compared to controls (siRNA#l, siRNA-Ctl). q. KLF4+ human pancreatic cancer cells are τSI-sensitive
To determine whether KLF4 protein levels correlate with drug sensitivity in pancreatic cancer, 1000 cells were plated per well in triplicate 96- wells, and the cell number determined after a 5 day treatment with vehicle (DMSO), 7SI (L-685,458), 9cUAB30, or both drugs. Cell counts were determined using an ATP-dependent luciferase reaction (Pierce ATPlite Assay) which in gave a strictly linear response from 100-100,000 cells per well. The day 5 cell counts in were always less than 25,000 cells, well within the linear range. In an initial screen of four lines, the KLF4-high lines Pane 10.05 and Pane 08.13 were sensitive to 7SI, while the KLF4-low lines Panel and S2013 were not. A titration study confirmed these results (Fig. 19A). Compared with DMSO, lOμM 'ySI blocked -50% of the growth of Pane 08.13 (pO.OOl) and Pane 10.05 (pO.Ol) but had little effect on Panel (p>0.05) (1 way ANOVA with Tukey's multiple comparison).
As a single agent, 9cUAB30 was less effective than "ySI, but showed statistically significant inhibition of Pane 10.05 or Pane 08.13 at 4.0-12.0 μM (p<0.05; Fig. 19B-C). In RK3E-KLF4-ER cells, 9cUAB30 inhibits transformation, but shows no cytoxicity in cell growth assays, indicating that it targets some other process besides cell proliferation. The combination of ^1+9CUABSO was also compared to 'ySI alone. In 2 of 3 experiments Pane 10.05 cells showed less growth in
Figure imgf000076_0001
than in 7SI alone (Fig. 19C-D). A screen of the remaining 6 cell lines showed that the KLF4-low lines were not usually sensitive to 7SI or to 9ΛJAB30 (Fig. 19D). Of these lines only Pane 02.03 was significantly inhibited by either drug (7SI, p<0.01). r. Frequentl upregulation of KLF4 in pancreatic cancer
To examine KLF4/GKLF protein in established cancers and intraepithelial neoplasia, several immunostaining assays of human KLF4 were developed. Paraffin sections of 37 cases of primary human pancreatic cancer were stained. As compared to uninvolved epithelium, KLF4 protein was upregulated in most cases of primary human pancreatic cancer (21 of 37 cases, 57%, Fig. 16), unlike in several other tumor types.
KLF4 was similarly analyzed in a genetically engineered mouse model of PanIN/PDAC. KLF4 was never detected in multiple sections of normal pancreatic ducts and acini (PDX-Cre control mice), but prominently upregulated in PanlN-like lesions (Fig. 17). This result identified KLF4 as an early change, similarly as shown in oral SCC, breast cancer, and cutaneous SCC. KLF4 was also expressed in mouse lesions that are histologically similar to invasive human PDAC, but was not detected in ductal epithelium entrapped within or else distant from invasive lesions. 2. Example 2 i. Results a. KLF4 upregulates Notchl in RK3E cells
To identify transcripts regulated by KLF4, a KLF4-estrogen receptor (KLF4-ER) fusion was used that is normally cytoplasmic but relocates to the nucleus and transforms RK3E cells in the presence of 4-hydroxytamoxifen (4OHT) (Foster KW, et al. 2005). RK3E cells stably expressing either KLF4-ER or Vector were treated for 2 hours with 4OHT, with 4OHT in combination with the protein synthesis inhibitor cycloheximide (CHX), or with vehicle alone. In two independent microarray experiments, Notchl mRNA was upregulated an average of 7.5 fold in KLF4-ER cells treated with 4OHT+CHX, but not in the control. The increased expression of Notchl mRNA was confirmed by Northern blot (Fig. 3A, top panel, lanes 4 and 5). In contrast, Notch 2, 3, 4 and Dll/Jag ligands were unchanged at 2 hours of induction.
Antibody Nl -C20, directed against the C-terminus of Notchl, showed strong immunofluorescent staining in KLF4-ER cells, but not Vector cells, treated with 4OHT for 10 hours (Fig. 3B). By immunoblot, antibody NlIC-Nter, specific for the amino- terminus of N1IC generated by -secretase cleavage, showed accumulation of N1IC with increasing time in 4OHT (Fig. 3A, middle panel). This accumulation was diminished by inclusion of a γ-secretase inhibitor (γSI) in the medium (Fig. 3A, bottom panel, lane 3). As cleavage of N1FL to N1IC is dependent upon the expression of Notch ligands and components of γ-secretase, these were examined for regulation by KLF4. Semi-quantitative RT-PCR showed that mRNAs for the Notch ligands Dill, D114 and Jagl increased by 48 hours of induction. As there was no detectable increase at 2 hours, these ligands can be indirect targets of KLF4. Immunoblotting showed upregulation of Dill and Jagl proteins but not D114, indicating that the latter can be regulated post-transcriptionally. The γ-secretase component, Presenillinl (PSl), showed little increase. These results indicate that Notchl is a transcriptional target of EXF4, and that it is processed to the active form, N1IC, in response to activation of KLF4. b. Induction of KLF4 in the skin of transgenic mice upregulates Notchl
It was next determined whether induction of human KLF4 by doxycycline (dox) in the cutaneous basal keratinocytes of KLF4 transgenic mice would be followed by upregulation of Notchl (Foster KW, et al. 2005). Formalin-fixed skin sections obtained at 0 hours, 48 hours and 14 days post-induction were stained with anti-human KLF4 or N1-C20 antibodies (Fig. 4A). At 0 hours, staining of the transgenic human KLF4 protein and endogenous Notchl was low, similar to that seen with control IgG (Fig. 4A, left). At 48 hours Notchl was uniformly positive across the skin while KLF4 was detected in a patchy pattern within the basal layer (middle). However, other markers of KLF4 activity, such as Keratin 1 , indicated that KLF4 was induced throughout the skin, with heterogeneity in the levels of expression (Foster KW, et al. 2005). Indeed, SCC-like lesions were apparent at 14 days and Notchl and KLF4 were uniformly positive (Fig. 4 A, right). KLF4 expression became increasingly nuclear with progression, as observed in human SCC (Foster KW, et al. 2005; Huang CC, et al. 2005). The close temporal correlation of KLF4 and Notchl expression supports regulation of Notch 1 by KLF4. c. Expression of KLF4 and Notchl in human breast carcinomas Compared with adjacent, uninvolved mammary epithelium, KLF4 mRNA and protein are upregulated in the majority of breast cancers, and KLF4 immunostaining identifies tumors with an aggressive phenotype (Pandya AY, et al. 2004). To correlate KLF4 and Notchl in human tumors, paraffin sections of primary breast cancers were immunostained. For Notchl, N1-C20 and NlIC-Nter antibodies were first characterized by staining subjacent sections representing 12 cases of infiltrating ductal carcinoma and adjacent uninvolved epithelium. 8 cases showed intense, uniform staining of tumor cells with each Notch 1 antibody, while the other 4 cases showed only low-level staining (Fig. 26). Concordant staining of these 12 cases was statistically significant as indicated by 2x2 contingency analysis (Table 4, P=O.002, Fisher's exact test).
Table 4: 2x2 contingency analysis of N1-C20 and NlIC-Nter immunostaining in 12
Figure imgf000078_0001
Total 8 4 12
P=0.002 (Fisher's exact test, two-tailed)
Although staining by the two Notch antibodies within and between sections was similar, N1-C20 predominantly stained the cytoplasm while NlIC-Nter mainly stained the nucleus. Consistent with these results, immunoblotting studies indicated that N1-C20 recognizes Nl , the transcriptionally active nuclear form, less efficiently than other forms of Notch 1, such as Nl ™.
For the cases above, KLF4 antibody gave uniform staining of the 8 Notchl-high tumors but showed little staining in 4 Notchl-low tumors (Fig. 26). To further test the correlation between Notchl and KLF4, a total of 89 tumors were stained with KLF4 and N1-C20 antibodies (Figs. 20 and 4C). KLF4-stained slides and Notchl -stained slides were scored independently. As described previously, intensity scores were determined using a 0.0-4.0 scale, and weighted according to the percentage of cells exhibiting that intensity (Pandya AY, et al. 2004). Median values were: KLF4 (nuclear) 0.5, Notchl (cytoplasmic) 0.75. Pearson analysis revealed that KLF4 and Notchl are correlated in these samples (r = 0.5, PO.0001; Fig. 4C). Using the median scores as cutoffs, 65 of 89 cases (73%) expressed KLF4 and Notchl concordantly (Fig 4C, P<0.001).
It was next tested whether Notch and KLF4 were coordinately expressed in human breast cancer cell lines. In BT474 and ZR75-1 cells, siRNA-mediated knockdown of KLF4 resulted in a corresponding decrease in endogenous Nl (Fig. 21). In these, and in other cell lines that were tested, there was no apparent effect on proliferation in response to KLF4 knockdown, as both cell confluence and the yield of extracted protein was similar to the control at 96 hours post-transfection. Thus, in these lines KLF4 appeared to regulate Notchl expression independently of cell proliferation. d. KLF4 binds to the Notchl promoter and activates transcription
Chromatin immunoprecipitation (ChIP) was used to determine if KLF4 could bind to the human Notchl promoter (Fig. 3C). MCFlOA cells were transduced with pBpuro-HA-KLF4 retrovirus and a puromycin (puro)-resistant pool selected. The Notchl promoter fragment P was specifically immunoprecipitated from these cells by HA antibodies but not by control IgG (Fig. 3F, lanes 1-4). In contrast, a GAPDH promoter fragment was brought down by TFIIB antibodies, but not by HA or control IgG (lanes 5-8). Further scanning of the Notchl promoter by ChIP showed that fragments L, M, N and O, within a ~2. 1 kb region encompassing the start of the Notchl open reading frame, were bound by KLF4. In contrast, fragment K was not bound (Fig. 3F, lanes 9- 12).
To map the KLF4-responsive region of the Notchl promoter, promoter-reporter assays were performed in HEK293 cells in which endogenous KLF4 is low. The 2.1 kb fragment Δl (-1866/+240) and a series of truncations 2 to 5 were cloned into the luciferase reporter vector, pGL3-Pro (Fig. 3G). Each construct was co-transfected with either KLF4 or Vector and a control for transfection efficiency. Relative to the Vector, Δl was activated 4-fold by KLF4 (Fig. 3G). Constructs that deleted two regions of the promoter, Δ2 (overlapping fragments L, M and N) and Δ4 (corresponding to part of fragment O), showed decreased activation. Thus, two regions of the Notchl promoter respond to activation by KLF4, consistent with interaction data obtained by ChIP (Fig. 3F). e. Notchl contributes to transformation by KLF4
To test whether Notchl played a role in transformation by KLF4, two strategies were used to suppress Nl in RK3E cells. Cells were transduced with pBpuroKLF4- ER or pBpuro-Nl retroviruses (Fig. 5D). As previously reported (Foster KW, et al. 2005), KLF4-ER cells formed foci in the presence of 4OHT (Fig. 5D). The addition of SI to the media inhibited formation of foci by KLF4-ER, but not by Nl (Fig. 5D). Although γ-secretase has multiple cellular substrates in addition to Notchl, these results are consistent with a role for Notchl in transformation by KLF4.
To determine if Notch 1 is a specific downstream effector of transformation by KLF4, endogenous Notchl was knocked down. Attempts to generate stable knockdown lines using lentiviral shRNA vectors were unsuccessful. For oncogene-transduced cells to establish transformed foci, they must continue to divide as the surrounding cells become confluent during the first 2-3 days following retroviral transduction. Thus, the suppression of oncogene transforming activity during the immediate post-transduction period should suffice to reduce the overall size and/or number of transformed foci. For example, transient expression of a dominant-negative allele of Snail 1 can block transformation by GIi 1 (Li X, et al. 2006). Transient transfection of RK3E cells with Notch 1 siRNAs was therefore used to reduce the expression of Nl IC relative to control (Fig. 22, compare lanes 1-3 with lane 4).
24 hours after siRNA transfection, RK3E cells were infected with KLF4-ER or ErbB2 retroviruses and allowed to form foci (Fig. 24). Following fixation and staining, foci larger than 0.5 mm were counted (Figs. 23-24). While focus formation by the control oncogene, ErbB2 was relatively unaffected, knockdown of Notch 1 diminished the efficiency of focus formation by KLF4 by -56-69% (Fig. 23). Thus, Notchl is an effector of transformation by KLF4. f. KLF4 suppresses activation of the Hesl promoter by Notchl
To examine Notchl transcriptional activity in KLF4-expressing cells, the expression of multiple known Notch 1 targets was analyzed at different times following induction of KLF4-ER cells with 4OHT. As expected, Notch 1 mRNA was rapidly induced within 3 hours (Fig.25 A, lane 8). The Notch target genes Nrarp and Cyclin Dl were upregulated by 12 hours (Fig. 25A), similar to the latency of N1IC, as shown in Fig. 3A. In contrast, several other targets of Notch signaling were not altered by 48 hours, including Hesl, Hes5, Herpl, Herp2, Herp3, Ascll and c-Myc (Fig. 25B). Therefore, in this context Notch 1 signaling appeared to be restricted to a subset of its target promoters.
Consistent with published data, the KLF4-expressing layers of normal skin did not immunostain for Hesl, nor was Hesl staining apparent in the SCC-like lesions in KLF4 transgenic skin (Moriyama M, et al. 2008). In the same sections, endothelial cells stained strongly for Hesl and served as a positive control. Thus, Hesl is not upregulated in response to KLF4-Notch signaling in these settings.
To further examine the transcriptional activity of Notch 1 in the presence of KLF4, a Hesl-luciferase reporter that is widely used to assay activity of the canonical Notchl pathway was used. RK3E cells were co-transfected with KLF4, Nl IC, or a combination of the two expression constructs, along with an internal control. Luciferase activity was measured 48 hours later (Fig. 25C). While N1IC activated the Hesl promoter as expected (Fig. 25C, lanes 3 and 5), KLF4 suppressed NlIC-dependent activation (compare lanes 3 and 4, and lanes 5 and 6). Similar results were obtained in HEK293 cells. The ability of Nl IC to activate a reporter construct containing 8 tandem CSL-binding sites was also blocked by KLF4 (Fig. 25C, lanes 9-12). A reporter containing 8 mutant CSLbinding sites served as a control (lanes 13-18). g. Notch acts through a noncanonical pathway in response to KLF4
To determine if canonical Notchl signaling plays a role in transformation by KLF4, dominant-negative inhibitors of this pathway were used . A point mutation in CSL (CSLDN) and truncated versions of MAMLl (MAMLDN1 and MAMLDN2) were previously shown to dominantly interfere with activation of the Hesl promoter by the N1IC-CSL- MAML complex (Maillard I, et al. 2004; Kato H, et al. 1997). Retroviral constructs and antibiotic selection were used to generate pools of RK3E cells that stably expressed Vector, MAMLDN1, MAMLDN2 or CSLDN. The expression of MAMLDN2 was detected by immunoblotting with a MAML-specific antibody (Fig. 8A). MAMLDN1, lacks the epitope recognized by this antibody, and was expressed as a GFP-fusion protein detectable by GFP antibodies (Fig. 8A). RT-PCR with species-specific primers was used to detect expression of CSLDN (Fig. 8B). The activation of the Hesl-luciferase reporter was next examined in the presence of exogenous N1IC by transient transfection into each of these pools of cells (Fig. 8C). Compared with Vector, stable expression of MAMLDN1, MAMLDN2, or CSLDN inhibited activation of the Hesl promoter by N1IC, indicating suppression of canonical Notchl signaling (Fig. 8C).
The transforming activities of retroviruses encoding KLF4, Nl IC, N-RAS, GIi 1 or c-MYC was then assayed in these cells. While focus formation by N1IC was suppressed in each of the three cell lines that expressed attenuators of the canonical pathway, transformation by KLF4, N-RAS, Glil or c-MYC was unaffected (Figs. 8D, 8E). These results indicate that the canonical Notchl signaling pathway is dispensable for transformation by KLF4. In contrast, in the absence of KLF4, Nl IC induces transformation via the canonical pathway. ii. Materials and Methods a. Antibodies
Preparation of cell extracts, isolation of RNA, RT-PCR, immunoblotting, microarray analysis and Northern blotting were as described (Foster KW, et al. 1999; Louro ID, et al. 2002). b. Cell lines and tissue culture
RK3E and HEK293 cells were grown in DMEM with 10% fetal bovine serum. RK3E/pBpuro, RK3E/N1IC, RK3E/KLF4-ER, RK3E/HA-KLF4, RK3E/pQCXIN, RK3E/MAMLDN1, RK3E/MAMLDN2 and RK3E/CSLDN pools were derived by retroviral transduction of RK3E cells as described (Foster KW, et al. 1999; Louro ID, et al. 2002). MCFlOA, ZR75-1 and BT474 cells were obtained from the American Type Culture Collection and cultured as recommended by the supplier. Virus transduced cells were grown in the presence of 0.4-1.0 μg/ml puro (Sigma) or 400 μg/ml G41 8 (Mediatech). The focus assay was described previously (Foster KW, et al. 1999). The γSI L685, 458 (Sigma) was used at 10.0 μM in DMSO5 and media were changed daily. siRNAs were synthesized by Therrno-Fisher/Dharmacon (see Table 5 for sequences), and transfected into RK3E or human tumor cell lines using Lipofectamine™RNAiMAX reagent (Invitrogen). Trypsinized cells were plated directly into wells containing lipid-siRNA complexes and incubated in serum- free medium (Optimem; Invitrogen) at 370C for 2 hours before adding complete medium. For in vitro transformation assays (Fig. 24), cells were exposed to retroviral supernatant 24 hours after transfection of siRNAs. For analysis of Notch 1 expression in KLF4 knockdown cells (Fig. 21), the siRNA-transfected breast cancer cells were analyzed at 96 hours posttransfection.
Table 5: Sequences of siRNAs used in this study
Name RNA sequence
Hs KLF4-siRl (S) 5ΑCCUCGCCUUACACAUGAAUU31 SEQ ID NO: 9
Hs KLF4-siRl (AS) 3'UUUGGAGCGGAAUGUGUACUU 5' SEQ ID NO: 10
Hs KLF4-siR2 (S) 51 GAGAGACCGAGGAGUUCAAUIB' SEQ ID NO: 11
Hs KLF4-siR2 (AS) 3'UUCUCUCUGGCUCCUCAAGUU 5' SEQ ID NO:12
Rn Nl -siRl (S) 5 'CUACAAGAUCGAAGCCGUA3 ' SEQ ID NO: 13
Rn Nl -siRl (AS) 5 'UACGGCUUCGAUCUUGUA3 ' SEQ ID NO: 14
Rn Nl -siR2 (S) 5 'CCAUGGAGCUUGCCGGGAU3 ' SEQ ID NO: 15
Rn Nl -siR2 (AS) 5 'AUCCCGGCAAGCUCCAUGGUU3 ' SEQ ID NO: 16
Rn Nl-siR3 (S) 5'GCGAGGAAGAGCUACGCAAS' SEQ ID NO:17
Rn Nl-siR3 (AS) 5'UUGCGUAGCUCUUCCUCG3' SEQ ID NO:18
Control (S) 5 'GAAUAUGGUUGUUUGAAGAUU3 ' SEQ ID NO: 19
Control (AS) 5 'UCUUCAAACAACCAUAUUCUU3 ' SEQ ID NO:20
c. Immunostaining
Bi-transgenic K14-rtTA; TRE-KLF4 mice were fed dox in drinking water as described previously (Foster KW, et al. 2005). Two mice were sacrificed at the indicated times (Fig.4A) and skin sections were obtained. Breast tumors were obtained from the University of Alabama hospital. Tissues were fixed and processed as described (Pandya AY, et al. 2004). Adjacent sections from 89 tumors were stained with KLF4 or N1-C20 antibodies, and statistical analysis was performed in consultation with the Biostatistics Shared Facility of the Comprehensive Cancer Center, University of Alabama at Birmingham. 12 of these 89 cases were also stained with NlIC-Nter using heat-induced antigen retrieval. All experiments used normal rabbit immunoglobulin as a negative control. Human and animal studies conformed to NIH guidelines and were approved by the Institutional Review Board for Human Use and the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.
For detection of Notchl in cultured cells (Fig. 3B), KLF4-ER cells were plated on poly-L-lysine coated coverslips at 30% confluence. 24 hours later, 4OHT or DMSO was added to the medium. After 10 hours, the cells were fixed and stained with N1-C20 antibody. Indirect immunofluorescence and digital imaging were performed as described (Pandya AY, et al. 2004). d. Reporter Assays
In Fig. 3G, 0.2 μg of pTK-/3-gal (internal reference), 0.5 μg Notchl promoter- reporter, and 0.2μg pRK5-HA-KLF4 or control Vector (pcDNA3.1) were transfected into HEK293 cells at 50% confluence, using 1.8 μl TransIT-LTl (Minis) according to the manufacturer's protocol. Cells were extracted 48 hours later and assayed for luciferase and β-gal activity with the Dual Light System (Applied Biosystems). Luciferase activity was normalized to β-gal, and the fold activation (KLF4/Vector) was determined. Using similar methods (Fig. 25C), 0.5 μg of the indicated luciferase reporter and 0.2 μg of pTK-gal were transiently transfected into RK3E cells with either no (-) or 0.5 g (+) of HA- KLF4 plasmid and 0.5 (+) or 1.0 (++) μg of pcDNA3.1-NlIc. The total DNA was kept constant by the inclusion of Vector. e. Chromatin Immunoprecipitation
MCFlOA cells at 95% confluence were washed twice with PBS. Formaldehyde (37% solution; Fisher Scientific) was added to a final concentration of 1% in PBS, and the plates were incubated at room temperature for 10 min. Cells were rinsed with ice-cold PBS and scraped into 6 ml of 100 mM Tris-HCl pH 9.4/10 mM DTT, transferred to a 15 ml tube and incubated at 300C for 15 min. The cells were centrifuged for 5 min at 3000 rpm, washed and centrifuged sequentially with 1.0 ml of buffer I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES-KOH pH 6.5) and 1.0 ml of buffer II (200 mM NaCl, 1.0 mM EDTA, 0.5 niM EGTA, 10 mM HEPES-KOH pH 6.5). Cells were re- suspended in 0.6 ml of lysis buffer with freshly added protease inhibitors (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1.0 mM PMSF, 1.0 mM benzamidine, 5.0 μg/ml leupeptin, 5.0 μg/ml aprotinin) and incubated on ice for 10 min. Samples were then sonicated with an Ultrasonic processor at 25% amplitude (twenty pulses for 10s each) on ice, then centrifuged at 4°C at 10,000 rpm for 10 min. Supernatants were collected and 4.5 ml of ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, with protease inhibitors as in lysis buffer above) was added. The chromatin was pre-cleared with 12 μg of sheared sperm DNA, 10 μl normal mouse IgG (0.5 mg/ml, Santa Cruz) and protein A-Sepharose (lOOμl of 50% slurry in 10 mM Tris-HCl pH 8.0, 1.0 mM EDTA) with agitation for 2 hrs at 4°C.
Pre-cleared chromatin was incubated with 4.0 μg HA antibody, 9.0 μl of TFIIB antibody, or 9.0 μl normal IgG (Chip-IT Kit, Active Motif, Cat# 53001), and rocked at 4°C overnight. 100 μl of protein A-Sepharose (50% slurry) and 20 μg salmon sperm DNA were added to each sample, followed by agitation for a further 2.5 hrs at 40C. Beads were pelleted by centrifugation at 2000 x g for 3 min at 4°C and washed sequentially [10 min, in 1.0 ml with low salt buffer (0.1% SDS, 1% Triton X-100, 2.0 mM EDTA, 20 mM Tris- HCl pH 8.1, 150 mM NaCl ), TSE2 (0.1% SDS, 1% Triton X-100, 2.0 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl) and Buffer 3 (0.25% LiCl, 1% NP40, 1% sodium deoxycholate, 1.0 mM EDTA, 10 mM Tris-HCl pH 8.0)], followed by 3 washes in TE (10 mM Tris-HCl pH 9.0, 0.1 mM EDTA). The complexes were eluted in 250 μl of elution buffer (1% SDS, 0.1 M NaHCO3) with agitation for 15 min. The beads were pelleted by centrifugation and the supernatants were transferred to clean tubes. The elution was repeated and the two eluates were combined. Crosslinks were removed by adding NaCl to 0.3 M with incubation at 65°C overnight. To reverse crosslinks in the input DNA, 100 μl inputs were heated without addition of NaCl. DNA was purified using Qiaquick columns (Qiagen) and eluted in 50 μl of 10 mM Tris-HCl pH 8.0. 2 μl of DNA was used in quantitative PCR reactions. PCR primers are listed in Table 6.
Table 6; Sequences of DNA oligonucleotides used in this study
Name Sequence SEQ ID NO Olignucleotides used for cloning
Rn Notchl promoter- AGAGATCTTCGCCAATGGAGGCACACAGGCAG SEQ ID NO: 21 BamHI (S)
Rn Notchl promoter- GGAATTCGAAGGTCGGTCCTCCCTGAT SEQ ID NO: 22 RΞcoRI (AS) hsnotchATG (S) CAGCCGGTGGGGAGGCTAACCGCCGCTCCTGGC SEQ ID NO: 23 hsnotchATG (AS) GCCAGGAGCGGCGGTTAGCCTCCCCACCGGCTG SEQ ID NO: 24 hsnotch740ATG (S) GCGGGGAGGCCAGCATCTAGAGGGAAAAGCG SEQ ID NO: 25 hsnotch740ATG (AS) CGCTTTTCCCTCTAGATGCTGGCCTCCCCGC SEQ ID NO: 26
Oligonucleotides used for ChIP analysis
K (S) GGCCCTGACACAGGCTGGCTTCC SEQ ID NO: 27
K (AS) GGTTCGTCTGGGTCACAAACGTCCC SEQ ID NO: 28
L (S) AGCCCCGCGCTTCCTTCTATGGA SEQ ID NO: 29
L (AS) CCTGGCACACCTCTTGCCAAATGC SEQ ID NO: 30
M (S) CCTGGCACACCTCTTGCCAAATGC SEQ ID NO: 31
M (AS) TTCTCCACCACGATGCCAGGCACG SEQ ID NO: 32
N (S) GGCACTGGTCCCGGCTGCTCCCT SEQ ID NO: 33
N (AS) GAGCAGCTGAGGCCACGTTGGGG SEQ ID NO: 34
O (S) TGGGCTCGGGACGCGCGGCTCAG SEQ ID NO: 35
O (AS) GCTCCGCGCCCGGCTCGTTCCTT SEQ ID NO: 36
P (S) GCTCAGCTCGGGGAGGCGCAAAG SEQ ID NO: 37
P (AS) GAAGGTCGGTCCTCCCTGAT SEQ ID NO: 38
Oligonucleotides used for semiquantitative RT-PCR
Rn DLLl (S) TGACAAGAGCAGCTTTAAGGCCCG SEQ ID NO: 39 Rn DLLl (AS) CTGTTTCTCAGCAGCAGTCCCTGG SEQ ID NO: 40 Rn DLL3 (S) GCTGACTCACAGCGCTTCCTTCT SEQ ID NO: 41 Rn DLL3 (AS) GTCGGTGTGGGGCAGGGATTGGA SEQ ID NO: 42 Rn DLL4 (S) TGTGGGCAGCCGCTGCGAGTTTC SEQ ID NO: 43 Rn DLL4 (AS) GTAACCGAAGTGGCACCTTCTCTCC SEQ ID NO: 44
Rn Jaggedl (S) CAGACAGTGCGTGCCGCCATAGGT SEQ ID NO: 45 Rn Jaggedl (AS) CAGCAACTGCTGACATCAAATCCCC SEQ ID NO: 46 Rn Jagged2 (S) TCTGCTCTGGAATCCGAGCCCTG SEQ ID NO: 47 Rn Jagged2 (AS) TTGTTGGCGCTCTCGTCCCGTGG SEQ ID NO: 48 Rn GAPDH (S) TACTAGCGGTTTTACGGGCG SEQ ID NO: 49 Rn GAPDH (AS) TCGAACAGGAGGAGCAGAGAGAGCGA SEQ ID NO: 50
f. Digital Imaging
For immunofluorescence studies, black and white images were collected using an Axio-Cam HRc digital camera (Zeiss), and the pseudo-colored images were merged using Axio vision software (version 3.1). Color images of stained slides were acquired similarly using brightfield microscopy. All images were exported from Axiovision as tiff files, and these were imported into CorelDraw (version 10). Within each Figure, minor adjustments were made to gamma, brightness, contrast, and intensity so as to more accurately portray the appearance of the actual samples. For the experimental and control panels within each figure or subfigure, all manipulations and adjustments were performed identically and in parallel. Within CorelDraw, color bitmaps were changed from RGB to CMYK and then the entire figure was exported from CorelDraw as a tiff file for submission. E. References
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F. Sequences
1. SEQ ID NOil tcagttactc agg
2. SEQ ID NO:2 cctgagtaac tgacaca
3. SEQ ID NO:3 cctcactcct tctctagctc
4. SEQ ID NO:4 aacaaattgg actaatcgat acg
5. SEQ ID NO:5 - cDNA sequences of GKLF/KFL4 tcgaggcgac cgcgacagtg gtgggggacg ctgctgagtg gaagagagcg 50 cagcccggcc accggaccta cttactcgcc ttgctgattg tctatttttg 100 cgtttacaac ttttctaaga acttttgtat acaaaggaac tttttaaaaa 150 agacgcttcc aagttatatt taatccaaag aagaaggatc tcggccaatt 200 tggggttttg ggttttggct tcgtttcttc tcttcgttga ctttggggtt 250 caggtgcccc agctgcttcg ggctgccgag gaccttctgg gcccccacat 300 taatgaggca gccacctggc gagtctgaca tggctgtcag cgacgcgctg 350 ctcccatctt tctccacgtt cgcgtctggc ccggcgggaa gggagaagac 400 actgcgtcaa gcaggtgccc cgaataaccg ctggcgggag gagctctccc 450 acatgaagcg acttccccca gtgcttcccg gccgccccta tgacctggcg 500 gcggcgaccg tggccacaga cctggagagc ggcggagccg gtgcggcttg 550 cggcggtagc aacctggcgc ccctacctcg gagagagacc gaggagttca 600 acgatctcct ggacctggac tttattctct ccaattcgct gacccatcct 650 ccggagtcag tggccgccac cgtgtcctcg tcagcgtcag cctcctcttc 700 gtcgtcgccg tcgagcagcg gccctgccag cgcgccctcc acctgcagct 750 tcacctatcc gatccgggcc gggaacgacc cgggcgtggc gccgggcggc 800 acgggcggag gcctcctcta tggcagggag tccgctcccc ctccgacggc 850 tcccttcaac ctggcggaca tcaacgacgt gagcccctcg ggcggcttcg 900 tggccgagct cctgcggcca gaattggacc cggtgtacat tccgccgcag 950 cagccgcagc cgccaggtgg cgggctgatg ggcaagttcg tgctgaaggc 1000 gtcgctgagc gcccctggca gcgagtacgg cagcccgtcg gtcatcagcg 1050 tcagcaaagg cagccctgac ggcagccacc cggtggtggt ggcgccctac 1100 aacggcgggc cgccgcgcac gtgccccaag atcaagcagg aggcggtctc 1150 ttcgtgcacc cacttgggcg ctggaccccc tctcagcaat ggccaccggc 1200 cggctgcaca cgacttcccc ctggggcggc agctccccag caggactacc 1250 ccgaccctgg gtcttgagga agtgctgagc agcagggact gtcaccctgc 1300 cctgccgctt cctcccggct tccatcccca cccggggccc aattacccat 1350 ccttcctgcc cgatcagatg cagccgcaag tcccgccgct ccattaccaa 1400 gagctcatgc cacccggttc ctgcatgcca gaggagccca agccaaagag 1450 gggaagacga tcgtggcccc ggaaaaggac cgccacccac acttgtgatt 1500 acgcgggctg cggcaaaacc tacacaaaga gttcccatct caaggcacac 1550 ctgcgaaccc acacaggtga gaaaccttac cactgtgact gggacggctg 1600 tggatggaaa ttcgcccgct cagatgaact gaccaggcac taccgtaaac 1650 acacggggca ccgcccgttc cagtgccaaa aatgcgaccg agcattttcc 1700 aggtcggacc acctcgcctt acacatgaag aggcattttt aaatcccaga 1750 cagtggatat gacccacact gccagaagag aattcagtat tttttacttt 1800 tcacactgtc ttcccgatga gggaaggagc ccagccagaa agcactacaa 1850 tcatggtcaa gttcccaact gagtcatctt gtgagtggat aatcaggaaa 1900 aatgaggaat ccaaaagaca aaaatcaaag aacagatggg gtctgtgact 1950 ggatcttcta tcattccaat tctaaatccg acttgaatat tcctggactt 2000 acaaaatgcc aagggggtga ctggaagttg tggatatcag ggtataaatt 2050 atatccgtga gttgggggag ggaagaccag aattcccttg aattgtgtat 2100 tgatgcaata taagcataaa agatcacctt gtattctctt taccttctaa 2150 aagccattat tatgatgtta gaagaagagg aagaaattca ggtacagaaa 2200 acatgtttaa atagcctaaa tgatggtgct tggtgagtct tggttctaaa 2250 ggtaccaaac aaggaagcca aagttttcaa actgctgcat actttgacaa 2300 ggaaaatcta tatttgtctt ccgatcaaca tttatgacct aagtcaggta 2350 atatacctgg tttacttctt tagcattttt atgcagacag tctgttatgc 2400 actgtggttt cagatgtgca ataatttgta caatggttta ttcccaagta 2450 tgccttaagc agaacaaatg tgtttttcta tatagttcct tgccttaata 2500 aatatgtaat ataaatttaa gcaaacgtct attttgtata tttgtaaact 2550 acaaagtaaa atgaacattt tgtggagttt gtattttgca tactcaaggt 2600 gagaattaag ttttaaataa acctataata ttttatctg 2639
6. SEQ ID NO:6 - amino acid sequences ofGKLF/KFL4
Met Ala VaI Ser Asp Ala Leu Leu Pro Ser Phe Ser Thr Phe Ala
5 10 15
Ser GIy Pro Ala GIy Arg GIu Lys Thr Leu Arg GIn Ala GIy Ala
20 25 30
Pro Asn Asn Arg Trp Arg GIu GIu Leu Ser His Met Lys Arg Leu
35 40 45
Pro Pro VaI Leu Pro GIy Arg Pro Tyr Asp Leu Ala Ala Ala Thr
50 55 60
VaI Ala Thr Asp Leu GIu Ser GIy GIy Ala GIy Ala Ala Cys GIy
65 70 75
GIy Ser Asn Leu Ala Pro Leu Pro Arg Arg GIu Thr GIu GIu Phe
80 85 90
Asn Asp Leu Leu Asp Leu Asp Phe lie Leu Ser Asn Ser Leu Thr
95 100 105
His Pro Pro GIu Ser VaI Ala Ala Thr VaI Ser Ser Ser Ala Ser
110 115 120
Ala Ser Ser Ser Ser Ser Pro Ser Ser Ser GIy Pro Ala Ser Ala
125 130 135
Pro Ser Thr Cys Ser Phe Thr Tyr Pro lie Arg Ala GIy Asn Asp
140 145 150
Pro GIy VaI Ala Pro GIy GIy Thr GIy GIy GIy Leu Leu Tyr GIy
155 160 165
Arg GIu Ser Ala Pro Pro Pro Thr Ala Pro Phe Asn Leu Ala Asp
170 175 180 lie Asn Asp VaI Ser Pro Ser GIy GIy Phe VaI Ala GIu Leu Leu
185 190 195
Arg Pro GIu Leu Asp Pro VaI Tyr lie Pro Pro GIn GIn Pro GIn
200 205 210
Pro Pro GIy GIy GIy Leu Met GIy Lys Phe VaI Leu Lys Ala Ser
215 220 225
Leu Ser Ala Pro GIy Ser GIu Tyr GIy Ser Pro Ser VaI He Ser
230 235 240
VaI Ser Lys GIy Ser Pro Asp GIy Ser His Pro VaI VaI VaI Ala
245 250 255
Pro Tyr Asn GIy GIy Pro Pro Arg Thr Cys Pro Lys He Lys Gin
260 265 270
GIu Ala VaI Ser Ser Cys Thr His Leu GIy Ala GIy Pro Pro Leu
275 280 285
Ser Asn GIy His Arg Pro Ala Ala His Asp Phe Pro Leu GIy Arg
290 295 300
GIn Leu Pro Ser Arg Thr Thr Pro Thr Leu GIy Leu GIu GIu VaI
305 310 315
Leu Ser Ser Arg Asp Cys His Pro Ala Leu Pro Leu Pro Pro GIy
320 325 330 Phe His Pro His Pro GIy Pro Asn Tyr Pro Ser Phe Leu Pro Asp
335 340 345
GIn Met GIn Pro GIn VaI Pro Pro Leu His Tyr GIn GIu Leu Met
350 355 360
Pro Pro GIy Ser Cys Met Pro GIu GIu Pro Lys Pro Lys Arg GIy
365 370 375
Arg Arg Ser Trp Pro Arg Lys Arg Thr Ala Thr His Thr Cys Asp
380 385 390
Tyr Ala GIy Cys GIy Lys Thr Tyr Thr Lys Ser Ser His Leu Lys
395 400 405
Ala His Leu Arg Thr His Thr GIy GIu Lys Pro Tyr His Cys Asp
410 415 420
Trp Asp GIy Cys GIy Trp Lys Phe Ala Arg Ser Asp GIu Leu Thr
425 430 435
Arg His Tyr Arg Lys His Thr GIy His Arg Pro Phe GIn Cys GIn
440 445 450
Lys Cys Asp Arg Ala Phe Ser Arg Ser Asp His Leu Ala Leu His
455 460 465 Met Lys Arg His Phe
470
7. SEQ ID NO:7 aauaaa
8. SEQ ID NO:8 rrggygy (r = purine, y = pyrimidine)
9. SEQ ID NO:9 - Hs KLF4-siRl (S)
Accucgccuuacacaugaauu
10. SEQ ID NO:10 - Hs KLF4-siRl (AS) uucauguguaaggcgagguuu
11. SEQ ID NO:11 - Hs KLF4-siR2 (S) gagagaccgaggaguucaauu
12. SEQ ID NO:12 - Hs KLF4-siR2 (AS) uugaacuccucggucucucuu
13. SEQ ID NO:13 - Rn Nl-siRl (S) cuacaagaucgaagccgua
14. SEQ ID NO:14 - Rn Nl-siRl (AS) auguucuagcuucggcau
15. SEQ ID NO:15 - Rn Nl-siR2 (S) ccauggagcuugccgggau
16. SEQ ID NO:16 - Rn Nl-siR2 (AS) uugguaccucgaacggcccua 17. SEQ ID NO:17 - Rn Nl-siR3 (S) gcgaggaagagcuacgcaa
18. SEQ ID NO:18 - Rn Nl-siR3 (AS) gcuccuucucgaugcguu
19. SEQIDNO:19 - Control (S) gaauaugguuguuugaagauu
20. SEQIDNO:20 -Control (AS) uucuuauaccaacaaacuucu
21. SEQIDNO:21 - rn notchl promoter- bamhi (s) agagatcttcgccaatggaggcacacaggcag
22. SEQ ID NO:22 - rn notchl promoter- recori (as) ggaattcgaaggtcggtcctccctgat
23. SEQIDNO:23 - hsnotchatg (s) cagccggtggggaggctaaccgccgctcctggc
24. SEQIDNO:24 - hsnotchatg (as) gccaggagcggcggttagcctccccaccggctg
25. SEQIDNO:25 - hsnotch740atg (s) gcggggaggccagcatctagagggaaaagcg
26. SEQ ID NO:26 - hsnotch740atg (as) cgcttttccctctagatgctggcctccccgc
27. SEQ ID NO:27 - k (s) ggccctgacacaggctggcttcc
28. SEQIDNO:28 -k(as) ggttcgtctgggtcacaaacgtccc
29. SEQIDNO:29 - 1 (s) agccccgcgcttccttctatgga
30. SEQIDNO:30 - 1 (as) cctggcacacctcttgccaaatgc
31. SEQIDNO:31 - m (s) cctggcacacctcttgccaaatgc
32. SEQIDNO:32 - m (as) ttctccaccacgatgccaggcacg 33. SEQ ID NO:33 - n (s) ggcactggtcccggctgctccct
34. SEQ ID NO:34 - n (as) gagcagctgaggccacgttgggg
35. SEQ ID NO:35 - o (s) tgggctcgggacgcgcggctcag
36. SEQ ID NO:36 - o (as) gctccgcgcccggctcgttcctt
37. SEQ ID NO:37 - p (s) gctcagctcggggaggcgcaaag
38. SEQIDNO:38 - p (as) gaaggtcggtcctccctgat
39. SEQ ID NO:39 - rn dill (s) tgacaagagcagctttaaggcccg
40. SEQ ID NO:40 - rn dill (as) ctgtttctcagcagcagtccctgg
41. SEQ ID NO:41 - rn dll3 (s) gctgactcacagcgcttccttct
42. SEQ ID NO:42 - rn dll3 (as) gtcggtgtggggcagggattgga
43. SEQ ID NO:43 - rn dll4 (s) tgtgggcagccgctgcgagtttc
44. SEQ ID NO:44 - rn dll4 (as) gtaaccgaagtggcaccttctctcc
45. SEQ ID NO:45 - rn jaggedl (s) cagacagtgcgtgccgccataggt
46. SEQ ID NO:46 - rn jaggedl (as) cagcaactgctgacatcaaatcccc
47. SEQ ID NO:47 - rn jagged2 (s) tctgctctggaatccgagccctg 48. SEQ ID NO:48 - rn jagged2 (as) ttgttggcgctctcgtcccgtgg
49. SEQ ID NO:49 - rn gapdh (s) tactagcggttttacgggcg
50. SEQ ID NO:50 - rn gapdh (as) tcgaacaggaggagcagagagagcga

Claims

UMS
What is claimed is:
1. A method of treating or preventing a tumor in a subject comprising administering to said subject a therapeutically effective amount of a γ-secretase inhibitor (^S I) and a therapeutically effective amount of a retinoid X receptor (RXR) agonist (rexinoid).
2. The method of claim 1 , wherein the rexinoid is 9cUAB30.
3. The method of claim 1, wherein the tumor has a high level of Kruppel-like factor 4 (KLF4) as compared to normal reference cells.
4. The method of claim 3, wherein the tumor is a breast cancer, pancreatic cancer, or squamous cell carcinoma.
5. The method of claim 4, wherein the breast tumor is estrogen receptor-negative, progesterone receptor-negative, and HER2 -negative (triple-negative breast cancer).
6. The method of claim 4, wherein the subject has been diagnosed with the breast tumor by mammogram or biopsy.
7. The method of claim 4, wherein the subject has undergone a lumpectomy or mastectomy.
8. The method of claim 4, wherein the subject has been identified has having a genetic risk of developing a breast tumor.
9. A method of treating breast cancer in a subject comprising the steps of:
(a) detecting increased staining of Krϋppel-like factor 4 (KLF4) staining in breast tumor cells as compared to that of normal reference cells, and
(b) administering to the subject a therapeutically effective amount of a γ- secretase inhibitor (7SI).
10. The method of claim 9, wherein step (a) further comprises detecting a predominantly cytosolic staining of Kruppel-like factor 4 (KLF4) staining in breast tumor cells as compared to that of normal reference cells.
11. The method of claim 9, wherein the tumor is a breast cancer, pancreatic cancer, or squamous cell carcinoma.
12. The method of claim 9, wherein step (b) further comprises administering to the subject a therapeutically effective amount of a retinoid X receptor (RXR) agonist (rexinoid).
13. The method of claim 9, wherein step (a) further comprises detecting a higher level of Ntc 1 in the breast tumor cells as compared to that of normal reference cells. (a) further comprises detecting a predominantly nuclear staining of Notchl (Ntcl) in the tumor cells.
15. A method of treating or preventing a tumor in a subject comprising the steps of:
(a) detecting a higher level of Kriippel-like factor 4 (KLF4) in the breast tumor cells as compared to that of normal reference cells, and
(b) administering to the subject a therapeutically effective amount of a γ- secretase inhibitor (7SI).
16. The method of claim 15, wherein the tumor is a breast cancer, pancreatic cancer, or squamous cell carcinoma.
17. The method of claim 15, further comprising administering to the subject a therapeutically effective amount of a retinoid X receptor (RXR) agonist (rexinoid).
18. The method of claim 15, wherein step (a) further comprises detecting a higher level of Notchl (Ntcl) in the tumor cells as compared to that of normal reference cells.
19. The method of claim 15, wherein step (a) further comprises detecting a predominantly nuclear staining of Notchl (Ntcl) in the tumor cells.
20. A method of selecting a treatment for a subject diagnosed with a tumor, comprising examining the expression of Kriippel-like factor 4 (KLF4) in the tumor, wherein a higher level of KLF4 in the tumor cells as compared to that of normal reference cells indicates that the selected treatment is 7-secretase inhibitor
CySi).
21. The method of claim 20, wherein the tumor is a breast cancer, pancreatic cancer, or squamous cell carcinoma.
22. The method of claim 20, further comprising examining the expression of Notchl (Ntcl) staining in the tumor cells, wherein a higher level of Ntcl in the tumor cells as compared to that of normal reference cells indicates that the selected treatment is -ySI.
23. The method of claim 20, further comprising examining the localization of Notchl (Ntcl) staining in the tumor cells, wherein a predominantly nuclear staining of Ntcl in the tumor cells indicates that the selected treatment is 7SI.
24. A method of selecting a treatment for a subject diagnosed with a tumor, comprising examining the expression of Kriippel-like factor 4 (KLF4) in said breast tumor, wherein a predominantly cytosolic staining of KLF4 in the tumor cells indicates that the selected treatment is γ-secretase inhibitor (7SI). tumor is a breast cancer, pancreatic cancer, or squamous cell carcinoma.
26. The method of claim 24, further comprising examining the expression of Notchl (Ntc 1) staining in the tumor cells, wherein a higher level of Ntcl in the tumor cells as compared to that of normal reference cells indicates that the selected treatment is -ySI.
27. The method of claim 24, further comprising examining the localization of Notchl (Ntcl) staining in the tumor cells, wherein a predominantly nuclear staining of Ntcl in the tumor cells indicates that the selected treatment is 7SI.
28. A method of identifying a subject diagnosed with a tumor as a candidate for treatment with a γ-secretase inhibitor (7SI), comprising examining the expression of Kriippel-like factor 4 (KLF4) in the tumor, wherein a higher level of KLF4 in the tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI.
29. The method of claim 28, wherein the tumor is a breast cancer, pancreatic cancer, or squamous cell carcinoma.
30. The method of claim 28, further comprising examining the expression of Notchl (Ntcl) staining in the tumor cells, wherein a higher level of Ntcl in the tumor cells as compared to that of normal reference cells identifies the subject as a candidate for treatment with a 7SI.
31. The method of claim 28, further comprising examining the localization of the localization of Notchl (Ntcl) staining in the tumor cells, wherein a predominantly nuclear staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 7SI.
32. A method of identifying a subject diagnosed with a tumor as a candidate for treatment with a 7-secretase inhibitor (7SI), comprising examining the expression of Kriippel-like factor 4 (KLF4) in said breast tumor, wherein a predominantly cytosolic staining of KLF4 in the tumor cells identifies the subject as a candidate for treatment with a 7SI.
33. The method of claim 32, wherein the tumor is a breast cancer, pancreatic cancer, or squamous cell carcinoma.
34. The method of claim 32, further comprising examining the expression of Notchl (Ntcl) staining in said tumor cells, wherein a higher level of Ntcl in the tumor ference cells identifies the subject as a candidate for treatment with a 7SI.
35. The method of claim 32, further comprising examining the localization of the localization of Notch 1 (Ntcl) staining in the tumor cells as compared to that of normal reference cells, wherein a predominantly nuclear staining of Ntcl in the tumor cells identifies the subject as a candidate for treatment with a 7SI.
36. A method of determining the prognosis of an individual diagnosed as having a tumor, comprising examining the expression of Kruppel-like factor 4 (KLF4) and Notch 1 (Ntcl) in one or more tumor cells,
(a) wherein a higher level of KLF4 in the tumor cells as compared to that of normal reference cells, a predominantly cytosolic staining of KLF4 in the tumor cells as compared to that of normal reference cells, or a combination thereof; and
(b) wherein a higher level of Ntcl in the tumor cells as compared to that of normal reference cells, a predominantly nuclear staining of Ntcl in the tumor cells as compared to that of normal reference cells, or a combination thereof; indicates a lower likelihood of survival.
37. The method of claim 36, wherein the tumor is a breast cancer, pancreatic cancer, or squamous cell carcinoma.
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CN112980799A (en) * 2021-02-10 2021-06-18 南京鼓楼医院 Method for constructing KLF12 high-expression mouse and application of method in construction of folate-independent neural tube defect mouse model
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CN112980799B (en) * 2021-02-10 2022-12-27 南京鼓楼医院 Method for constructing KLF12 high-expression mouse and application of method in construction of folate-independent neural tube defect mouse model
CN117866962A (en) * 2024-01-12 2024-04-12 山东第一医科大学附属省立医院(山东省立医院) Human telomerase reverse transcriptase promoter mutant and application thereof

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