WO2022271955A1 - Nouvelles nanoparticules de sharn ciblées pour la thérapie du cancer - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
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Definitions
- Colorectal cancer is the second leading cause of cancer-related deaths in Western countries including the USA, with incidences increasing by 2% annually (Siegel, R. L., et al., 2017, CA Cancer J Clin, 67:177-193; Siegel, R. L., et al., 2016, CA Cancer J Clin, 66:7-30).
- FOLFOX 5-fluorouracil [5-FU] + oxaliplatin
- Resistance to FOLFOX occurs through several mechanisms, including decreased influx of platinum, or it can also lead to the generation of reactive oxygen species that can directly induce apoptosis resistance (Masuda, H., et al., 1994, Biochem Biophys Res Commun, 203:1175-1180; Kruidering, M., et al., 1997, J Pharmacol Exp Ther, 280:638-649; Weng,
- the present invention provides a composition for treating chemoresistant cancer comprising one or more inhibitor of one or more chemoresi stance promoting molecule, wherein the one or more chemoresi stance promoting molecule comprises CD44 splice variant 6 (CD44v6).
- the one or more inhibitor is encapsulated in a nanoparticle.
- the one or more inhibitor comprises one or more nucleic acid molecules selected from the group consisting of: one or more shRNA, one or more miRNA, one or more siRNA, one or more antisense nucleic acid molecule, one or more ribozyme, one or more killer-tRNA, one or more sgRNA, one or more long non-coding RNA, one or more anti-miRNA oligonucleotide, and one or more plasmid.
- the one or more shRNA comprises a sense strand nucleotide sequence at least 95% identical to one or more nucleotide sequence selected from the group consisting of: SEQ ID NO:5 and SEQ ID NO:4; and an antisense strand nucleotide sequence at least 95% identical to one or more nucleotide sequence selected from the group consisting of: SEQ ID NO:7 and SEQ ID NO:6.
- the one or more shRNA comprises a sense strand nucleotide sequence of SEQ ID NO:5 and an antisense strand nucleotide sequence of SEQ ID NO:7.
- the one or more shRNA comprises a loop nucleotide sequence of uucaagaga (SEQ ID NO:8).
- the one or more shRNA comprises a nucleotide sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:9. In one embodiment, the one or more shRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:9.
- the one or more shRNA is encoded by a plasmid vector.
- the plasmid vector comprises a nucleotide sequence at least 90% identical to one or more selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 18, and SEQ ID NO:20.
- the composition further comprises a second plasmid vector encoding a Cre-recombinase.
- the recombinase is a Cre- recombinase comprising the amino acid sequence of SEQ ID NO: 15.
- the expression of the recombinase is conditional and requires the presence of a tissue-specific promoter.
- the tissue-specific promoter comprises an intestine/colon tissue-specific pFabpl promoter and comprises the nucleotide sequence of SEQ ID NO: 16.
- the second plasmid comprises a nucleotide sequence at least 90% identical to SEQ ID NO:22.
- the nanoparticle comprises: a) a polyalkylene glycol comprising one or more selected from the group consisting of: polyethylene glycol (PEG), polypropylene glycol (PPG), and polybutylene glycol (PBG); b) a polycation comprising one or more selected from the group consisting of: polyethylenimine (PEI), poly(allylanion hydrochloride) (PAH), polypropylenimine, poly-arginine, poly-lysine, poly-histidine, poly(trimethylenimine), poly(tetramethylenimine), aminoglycoside polyamine, poly(2-dimethylamino)ethyl methacrylate, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, putrescine, and cadaverine; and c) a targeting moiety.
- PEG polyethylene glycol
- PPG polypropylene glycol
- PBG polybutylene glycol
- the targeting moiety comprises transferrin (Tf), comprising the amino acid sequence of SEQ ID NO: 17.
- the nanoparticle comprises transferrin, comprising SEQ ID NO: 17, C-terminally conjugated to polyethylene glycol (PEG) which is in turn conjugated to polyethylenimine (PEI) in the order of Tf-PEG-PEI.
- the present invention provides a method of treating chemoresistant cancer in a subject in need thereof, comprising administering to the subject any one of the compositions described above.
- the chemoresistant cancer comprises colorectal cancer.
- the method further comprises administering a combination of folinic acid, fluorouracil, and oxaliplatin (FOLFOX) to the subject.
- the subject has been treated with a chemotherapeutic agent, such as FOLFOX.
- the present invention provides any one of the compositions described above for use in treating a subject.
- the present invention provides any one of the compositions described above for use as a medicament.
- the present invention provides any one of the compositions described above for use in treating chemoresistant cancer in a subject.
- the chemoresistant cancer comprises colorectal cancer.
- the subject has been treated with FOLFOX.
- the present invention provides any one of the compositions described above for use in a method of treating chemoresistant cancer in a subject.
- the chemoresistant cancer comprises colorectal cancer.
- the subject has been treated with FOLFOX.
- the method comprises administering FOLFOX to the subject.
- the present invention relates to a composition for treating chemoresistant cancer, comprising one or more inhibitor of one or more chemoresi stance promoting molecule, wherein said one or more chemoresi stance promoting molecule comprises one or more selected from the group consisting of: periostin (PN), Wnt family member 3A (WNT3A), Interleukin 17A (IL-17A) and CD44 splice variant 6 (CD44v6).
- PN periostin
- WNT3A Wnt family member 3A
- IL-17A Interleukin 17A
- CD44v6 CD44 splice variant 6
- the one or more inhibitor is encapsulated in a nanoparticle.
- said inhibitor comprises one or more selected from the group consisting of: an antibody, an antibody fragment, a peptide, a peptidomimetic, a small molecule, and a nucleic acid molecule.
- said nucleic acid molecule comprises one or more selected from the group consisting of: one or more siRNA, one or more microRNA, one or more shRNA, one or more antisense nucleic acid molecule, one or more ribozyme, one or more killer-tRNA, one or more guide RNA (part of the CRISPR/CAS system), one or more long non-coding RNA, one or more anti-miRNA oligonucleotide, one or more mRNA molecule, and one or more plasmid vector.
- said one or more shRNA comprises a sense strand nucleotide sequence encoded by a nucleotide sequence at least 95% identical to one or more selected from the group consisting of: SEQ ID NO:4 and SEQ ID NO:5; and an antisense strand nucleotide sequence encoded by a nucleotide sequence at least 95% identical to one or more selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7.
- said one or more shRNA comprises a loop nucleotide sequence at least 95% identical to SEQ ID NO: 8.
- said one or more shRNA comprises a nucleotide sequence at least 95% identical to a nucleotide sequence selected from the group consisting of: SEQ ID NO:9 and SEQ ID NO: 10.
- said shRNA is encoded by a first plasmid vector.
- the expression of said shRNA encoded by said first plasmid vector is conditional and controlled by the presence or absence of a recombinase.
- said first plasmid vector comprises a nucleotide sequence at least 90% identical to one or more selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO:21.
- the composition of the invention further comprises a second plasmid vector encoding a recombinase.
- said recombinase is Cre-recombinase comprising the amino acid sequence of SEQ ID NO: 15.
- said expression of said recombinase by said second plasmid vector is conditional and requires the presence of a tissue-specific promoter.
- said tissue-specific promoter comprises an intestine/colon tissue-specific promoter.
- said intestine/colon tissue-specific promoter is pFabpl comprising the nucleotide sequence of SEQ ID NO: 16.
- said second plasmid vector comprises a nucleotide sequence at least 90% identical to SEQ ID NO:22.
- said nanoparticle of the composition comprises one or more selected from the group consisting of: a) a polyalkylene glycol; b) a polycation; and c) a targeting moiety.
- said polyalkylene glycol of the nanoparticle is one or more selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), and polybutylene glycol (PBG).
- said polycation of the nanoparticle is one or more selected from the group consisting of: polyethylenimine (PEI), poly(allylanion hydrochloride) (PAH), putrescine, cadaverine, polylysine, poly-arginine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside polyamine, dideoxy-diamino-b-cyclodextrin, Spermine, spermidine, cadaverine, poly(2- dimethylamino)ethyl methacrylate, and poly (histidine).
- PEI polyethylenimine
- PAH poly(allylanion hydrochloride)
- putrescine putrescine
- cadaverine polylysine
- poly-arginine poly(trimethylenimine)
- polypropylenimine aminoglycoside polyamine
- said targeting moiety of the nanoparticle comprises transferrin (SEQ ID NO: 17).
- said nanoparticle comprises transferrin (Tf; SEQ ID NO: 17) C-terminally conjugated to polyethylene glycol (PEG) which in turn is conjugated to polyethylenimine (PEI) in the order Tf-PEG-PEI.
- Tf transferrin
- PEG polyethylene glycol
- PEI polyethylenimine
- the present invention relates to a method of administering a therapeutic composition comprising an inhibitor of one or more chemoresi stance promoting molecule to a subject in need thereof, wherein said therapeutic composition comprises an inhibitor of one or more chemoresi stance promoting molecule encapsulated in a nanoparticle, wherein said one or more chemoresi stance promoting molecule comprises one or more selected from the group consisting of: PN, WNT3A, IL-17A and CD44v6.
- the present invention relates to a method of treating or preventing one or more disease or disorder associated with chemotherapeutic resistance in a subject in need thereof, comprising administering to the subject a composition comprising an inhibitor of one or more chemoresistance promoting molecule encapsulated in a nanoparticle, wherein said one or more chemoresistance promoting molecule comprises one or more selected from the group consisting of: PN, WNT3A, IL- 17A and CD44v6.
- said one or more disease or disorder associated with chemotherapeutic resistance comprises colorectal cancer.
- the method of treating or preventing one or more disease or disorder further comprises administering to the subject one or more additional therapeutic selected from the group consisting of: one or more inhibitor of one or more chemoresistance promoting molecule, one or more activator of one or more chemosensitivity promoting molecule, one or more chemotherapeutic agent, and one or more cancer immunotherapeutic agent.
- said one or more additional therapeutic comprises one or more inhibitor of one or more chemoresistance promoting molecule selected from the group consisting of: WNT3A, Periostin, IL-17A, b-catenin, LRP6, Frizzled, DVL, TCF4 and MDR1.
- said one or more additional therapeutic comprises one or more activator of one or more chemosensitivity promoting molecule selected from the group consisting of: GSK3 , DKK1, and DAB2.
- Figure 1 depicts exemplary results of CD44v6 expression induced by FOLFOX and ICso analyses of 5-Fluorouracil (5FUR), oxaliplatin (OXA) and FOLFOX in SW480 cell growth.
- Figure 1 A depicts exemplary results of mRNA expression levels shown for CD44v6 in 6 CRC cell lines. mRNA levels were measured by Real-time PCR (QPCR). Real-time PCR data for each target gene at 2-DDO ⁇ are presented as the fold change in gene expression normalized to an endogenous reference gene GAPDH, and data are expressed as fold change relative to pre-neoplastic Ape 10.1 cells as controls.
- QPCR Real-time PCR
- Figure IB depicts exemplary results of FOLFOX-resistant cells (FR) of parental SW480, WIDR, LOVO and HT29 sensitive (S) cells generated by repeated exposure to increasing concentrations (l-4x) of FOLFOX as described in Materials and Methods of Example 1 below.
- the 50% inhibitory concentration (ICso) was determined as a concentration pg/ml of chemotherapeutic drugs (5-Fluorouracil [5-FU] and oxaliplatin [OXA] required to achieve a 50% growth inhibition relative to untreated controls using the ATP Glo-based growth assay as described in Methods.
- FOLFOX-resistant cells (FR) of parental SW480, WIDR, LOVO and HT29 sensitive (S) cells were generated by repeated exposure to increasing concentrations (l-4x) of FOLFOX as described in Methods.
- Figure 1C depicts exemplary results of the ICso 5-FU
- Figure ID depicts exemplary results of the ICso of oxaliplatin (OXA)
- Figure IE depicts exemplary results of the ICso of FOLFOX for sensitive and corresponding resistant SW480 cells.
- Figure IB depicts the ICso for 5-Fluorouracil (5- FU) and oxaliplatin (OXA) of three selected CRC cells.
- Figure IF depicts the constant and variable exons shown for the PCR primers used to amplify CD44 variable (v) and standard (s) isoforms in the human CD44v6 gene.
- Figure 2 depicts exemplary results demonstrating that FOLFOX induces CD44v6 expression and establishes FOLFOX (FR) resistant colorectal cancer (CRC) cells.
- Figure 2A depicts exemplary results demonstrating that colon tumor cells derived from SW480 that are resistant to 5- Fluorouracil (5FUR), oxaliplatin (OXAR), and FOLFOX (FR) selectively overexpressed CD44v6 mRNAs (by QPCR) compared to sensitive (S) pairs of cells.
- 5FUR 5- Fluorouracil
- OXAR oxaliplatin
- FR FOLFOX
- Figure 2B depicts exemplary results of serum-starved SW480-S cells stimulated with 1 x FOLFOX (1 x FOLFOX: 50 pg/ml 5-flurouracil + 10 pM oxaliplatin + 1 pM leucovorin) as described in Materials and Methods of Example 1 and collected at the indicated time points.
- the mRNA expressions were analyzed by semiquantitative RT-PCR from total RNA (GAPDH as internal control). Agarose gels of the PCR products are shown. The PCR products are separated in 2% agarose gel. The primers for v6 isoforms generate v6 and v6-v8 PCR products.
- FIG. 2C depicts exemplary RT-PCR results for the CD44v isoforms using the different primers in the tumor cells derived from colorectal patients (PD) who are resistant to 5FU, OXA and FOLFOX (PD-5-FU, PD-OXA and PD-FR).
- Figure 2D depicts exemplary RT-PCR results for the CD44v isoforms using the different primers (C5v6v8v9C7, C5v6v8C7 and C5v6C7) in the PD-FR, PD-OXA and PD-5-FU cells.
- Figure 2E depicts exemplary Western blots for antibodies that recognize either CD44v6, the active hypo phosphorylated b-catenin (Active b-catenin, [ABC]), b-catenin, MDRl or b-tubulin in sensitive (S) and FOLFOX resistant (FR) clones of SW480 cells following stimulation with lx FOLFOX for 4-48 hours.
- the expressions of indicated proteins in FR and S cells are representative of three different experiments.
- Figure 2F depicts exemplary semi-quantitative RT-PCR analyses for CD44 variants in SW480-S and SW480-FR cells transfected with pSicoR-non targeted shRNAl (NT shl) or pSicoR-v6 shRNAl (v6 shl) for 72 hours followed by FOLFOX stimulation for 12 hours.
- Figure 2F, lower Panel depicts exemplary Western blot analyses for p-LRP6 (S1490), active hypo-phosphorylated b-catenin (ABC), or total b-catenin, or MDRl or tubulin following FOLFOX (lx) stimulation for 12 hours in cells transfected with NT shl or v6 shl.
- Figure 3 depicts a table of primers used for CD44 exon specific PCR as described in the Materials and Methods of Example 1.
- Figure 4 depicts exemplary results of CD44v6 expression induced by FOLFOX.
- Figure 4A depicts exemplary semi quantitative RT-PCR analyses for CD44 variants in SW480-S and SW480-FR cells transfected with pSicoR-non targeted shRNA2 (NT sh2) or pSicoR-v6 shRNAl (v6 sh2) for 72 hours followed by FOLFOX stimulation for 12 hours.
- NT sh2 pSicoR-non targeted shRNA2
- v6 sh2 pSicoR-v6 shRNAl
- Figure 4B depicts exemplary Western blot analyses from experiments shown in D for p-LRP6 (S1490), active hypo-phosphorylated b-catenin (ABC), or total b-catenin, or MDR1 or tubulin following FOLFOX (lx) stimulation for 12 hours in cells transfected with NT sh2 or v6 sh2.
- Figure 4C depicts exemplary results demonstrating that silencing CD44v6, and FOLFOX induced WNT3A attenuates FOLFOX resistant cell survival.
- Figures 4D-E depict exemplary validations of CD44v6 shRNAs (v6 shl, and v6 sh2; Figure 4D) and WNT3A shRNAs (WNT3A shl and WNT3 A sh2; Figure 4E) by the indicated shRNA mediated knockdown and the corresponding knock-in (KI) gene transfections as described in Materials and Methods of Example 1.
- Target proteins were analyzed by WB analysis (b-tubulin, internal control).
- Figure 4F depicts exemplary validation of expression vectors CD44v6 cDNA (v6 cDNA) by WB analysis of target proteins (b-tubulin, internal control).
- Figure 5 depicts exemplary results demonstrating that FOLFOX induces CD44v6 expression and establishes FOLFOX (FR) resistant colorectal cancer (CRC) cells.
- Figure 5A depicts exemplary results demonstrating that silencing CD44v6 and FOLFOX induced WNT3A attenuates FOLFOX resistant cell survival.
- SW480-FR cells grown in serum free medium were treated with various concentrations of FOLFOX in the presence or absence of vector, NT shl, v6 shl, pSicoR-WNT3A shRNA 1 (WNT3A shl), or WNT3A shl + v6 cDNA. 48 hours after transfections, cells were incubated in complete medium for 72 hours.
- FIG. 5B depicts exemplary results of anchorage-independent growth in soft agar for SW480-FR, WIDR-FR and LOVO-FR cells and compared with their sensitive (S) pairs. Scale bars, 100 pm.
- Figure 5C depicts exemplary tumor-sphere formation assays for the SW480-FR, WIDR-FR and LOVO-FR cells and compared with their sensitive (S) pairs. Scale bars, 100 pm.
- FIG. 5D depicts exemplary Tumor formation in nude mice injected with either 5 x 10 4 SW480-FR cells, or 5 x 10 4 SW480-S or 1 x 10 6 SW480-S cells.
- SW480-FR cells formed tumor nodules in all injected mice (7/7). Starting at week 3, whereas SW480-R (5 x 10 4 ) cells induce tumor nodules whereas SW480-S (5 x 10 6 ) cells induced much smaller tumor nodules starting a week later than the SW480-FR cells (7/7 mice).
- SW480-S (5 x 10 4 ) cells were unable to induce tumors. Growth curves are shown for these xenograft tumors in immunocompromised mice.
- Figure 6 depicts exemplary results demonstrating that CD44v6 defines CRC cancer initiating cells (CICs).
- Figure 6A depicts exemplary experimental setup wherein single-cell suspensions from dissociated patient derived (PD) biopsy tissues collected from 5-FU (PD-5FUR), Oxaliplatin (PD-OXAR) and FOLFOX (PD-FR) resistant tumor tissues, and from FOLFOX-resistant SW480-FR tumor cells from subcutaneously (SQ) implanted tumors were sorted for high expressions of CD44v6-PE. Highly CD44v6 positive (+) populations were gated between 6-11%.
- PD dissociated patient derived
- PD-OXAR Oxaliplatin
- Highly CD44v6 positive (+) populations were gated between 6-11%.
- Representative analyses show the distributions of CD44v6+/EpCAM+ cells assessed by flow cytometry on freshly purified CRC cells from a PD-FR tumor either unstained or stained with antibodies to EpCAM-FITC and CD44v6-PE.
- Purified CD44v6+/EpCAM+ and CD44v6-/EpCAM+ cells from various tumors were cultured separately and grown in fresh medium for 2 weeks.
- Figure 6B depicts exemplary CD44v6+/EpCAM+ cells from various tumors that were cultured separately and grown in fresh medium for 2 weeks.
- the cells were then subjected to flow cytometric analysis for isolation of CD44v6+EpCAM+ALDHl+CD133+ (CICs), and CD44v6-EpC AM+ ALDH 1 +CD 133+ (Non-CICs).
- CICs andNon-CICs were isolated from SW480-FR tumors and other CRC tumors and grown for 2 weeks for other experiments.
- FIG. 6C depicts exemplary quantitative RT-PCR analyses of CIC-stemness markers (ALDH1, SOX2, OCT4, Nanog, c-Myc and CD44v6) on CICs from 3 independent experiments with tumor cells from PD-FR tumor specimens, SW480-FR SQ and SW480-S SQ xenograft tumor samples.
- ADH1, SOX2, OCT4, Nanog, c-Myc and CD44v6 CIC-stemness markers
- Figure 6D depicts exemplary percentages of tumor sphere formation that were measured in a sphere-formation assay.
- CICs black circles
- non-CICs open circles
- Data are presented as a percentage of wells containing colon tumor spheres compared to the total number of wells.
- Figure 6E depicts exemplary results demonstrating that 5 x 10 3 CICs from SW480-FR xenograft (SQ) tumors when resuspended in Matrigel were tumorigenic while 100-fold more unsorted cells (Bulk) resuspended in Matrigel were required to generate tumors in four independent implantations. Five mice per group were used.
- Figure 7 depicts exemplary results showing that CICs isolated from patient-derived (PD) tumor specimens demonstrate resistance to FOLFOX-treatment.
- Figure 7A depicts a graphical presentation of the CD44v6+/EpCAM+ and CD44v6-/EpCAM+ populations as a percentage of unsorted cells for 3 independent experiments for the PD-FR tumor cells.
- Figures 7B-C depict exemplary percentages of ALDH1+ cells in CD44v6+EpCAM+ and CD44v6- EpCAM+ sorted cells ( Figure 7B) and the percentages of CD 133+ cells in CD44v6+EpC AM+ALDH 1 + and CD44v6-EpCAM+ALDHl+ cells from PD-FR tumor tissues ( Figure 7C).
- CD44v6+EpCAM+ALDHl+CD133+ are identified as CICs
- CD44v6-EpCAM+ALDHl+CD133+ cells as Non-CICs (details in Materials and Methods of Example 1).
- Figure 7D depicts exemplary results demonstrating that PD- FR, PD-OXAR and PD-5FUR cells selectively overexpressed CD44v6 mRNAs (by QPCR) when treated with 1 x FOLFOX. Data are presented as fold change of CD44v6 mRNA expression relative to adjacent control cells from colon tissue.
- Figures 7E-F depict exemplary cell growth of CICs and non-CICs from three independent PD-FR ( Figure 7E) and PD-5-FUR ( Figure 7F) human specimens that were assessed by an ATP based assay (CellTiter-Glo) following 1 x FOLFOX treatment.
- Figure 8 depicts a table of primers used for real-time PCR (QPCR) of various genes associated with the sternness functions of CICs as described in the Materials and Methods of Example 1.
- QPCR real-time PCR
- Figure 9 depicts exemplary results demonstrating that CICs isolated from patient-derived (PD) tumor specimens demonstrate resistance to FOLFOX-treatment.
- Figure 9A-B depicts exemplary results of apoptosis of CICs and non-CICs from three independent PD-FR ( Figure 9A) and PD-5- FUR ( Figure 9B) cultures following FOLFOX treatment as assessed by Caspase 3 ELISA assay.
- Figure 9C depicts exemplary results demonstrating that xenograft CICs from the PD-FR cells were more tumorigenic than unsorted cells (Bulk) at two different injected cell numbers. Five mice per group were used.
- FIG. 9D depicts representative images of tumors initiated from CICs from PD-FR human specimens subcutaneously implanted into immunocompromised mice.
- Figure 9E depicts representative images of tumors initiated from CICs (resuspended in Matrigel) FACS sorted from SW480-FR xenografts and from PD-FR tumor specimen in immunocompromised mice.
- Figure 10 depicts exemplary results demonstrating that CD44v6 defines CRC cancer initiating cells (CICs).
- Figure 10A depicts exemplary results demonstrating that FACS sorted 2 x 10 3 CICs (resuspended in Matrigel) were tumorigenic while 250-fold more unsorted Bulk cells (resuspended in Matrigel) were unable to generate the same capacity of tumorigenesis in four independent specimens. Six mice per group were used.
- Figure 10B depicts exemplary enrichment of CICs in bulk cells from three sources - 1) patient derived specimens, 2) from SQ developed tumor samples of in vitro developed FR-cells, and 3) the corresponding sensitive pairs, that were assessed by FACS analysis for CD44v6 after FOLFOX treatment. Data are representative of four independent human specimens, and three independent tumor samples from sensitive and FR cells.
- Figure IOC depicts exemplary FACS sorted CD44v6 (+) CICs (2 x 10 3 ), CD44v6 (-) Non-CICs (5 x 10 5 ), and the unfractionated bulk tumor cells (5 x 10 5 ), from indicated tumor samples that were resuspended in Matrigel and implanted in immunocompromised mice.
- Figure 11 depicts exemplary results demonstrating that CICs isolated from resistant cells demonstrate resistance to FOLFOX treatment through WNT3 A/b-catenin signaling.
- Figure 11 A depicts exemplay results demonstrating that CICs isolated from SW480-FR SQ, SW480-OXA SQ and SW- 5-FU SQ tumor cells selectively overexpressed CD44v6 mRNAs (by QPCR) compared to sensitive (S) pairs of cells, and FOLFOX-treatment further stimulated this induction at different levels in these CICs.
- Figure 1 IB demonstrates exemplary results of secretion of WNT3A measured (by ELISA) in sensitive and FOLFOX resistant SW480 cells after treatment with FOLFOX for the indicated times.
- Figure 11C depicts exemplary results of Sensitive and FR cells of SW480 transfected with TOPFlash and TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors.
- the TOP/FOPFlash promoter was activated by treatment with FOLFOX (lx) for 1 hour. Cells were lysed and subjected to luciferase measurements. Reporter gene assays are described in Methods.
- Figure 1 ID depicts exemplay results of CD44v6 negative PD-FR/NON-CICs transfected with 50 ng TOPFlash, and control with 50 ng TK-Renilla vectors, or with 50 ng FOPFlash and 50 ng TK-Renilla vectors together with increasing time of incubation with 50 ng of CD44v6 cDNAs.
- 48 hours after co-transfection the cells were stimulated with WNT3 A (20 ng/ml). After 12 hours, cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis with the indicated proteins.
- TOPFlash : FOPFlash ratios are presented in the luciferase data.
- Figures 11E-F depict exemplary results of PD-FR CICs transfected with 100 ng control shRNA (non-targeted shRNA [NT shl]) or with CD44v6 shRNAl (CD44v6 shl) ( Figure 1 IE), or with NT sh2 or CD44v6 sh2 ( Figure 1 IF).
- NT shl non-targeted shRNA
- Figure 1 IE CD44v6 shRNAl
- Figure 1 IF Figure 1 IF.
- cells were transfected together with TOPFlash and TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors for 48 hours.
- 48 hours after reporter vector transfection the cells were stimulated with 20 ng/ml WNT3A for 12 hours.
- Cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis with the indicated proteins.
- Data represent results from 3 independent experiments performed in triplicates. Values in Figure 11 A-F represent means ⁇
- Figure 12 depicts exemplary results demonstrating that CICs isolated from resistant cells demonstrate resistance to FOLFOX treatment through WNT3 A/b-catenin signaling.
- Figures 12A-B depict exemplary results of SW480-FR CICs transfected with 100 ng NT shRNA (NT sh), or CD44v6 shRNA (v6 shl), or b-catenin shRNA (b-catenin shl). 48 hours after the transfections, cell growth was assessed by counting colonies in a clonogenic growth assay (left panel of Figure 12 A), and apoptosis was assessed by the Annexin V positive stain assay (left panel of Figure 12B).
- SW480-FR/(Non-CICs) were transfected with either vector, or CD44v6 cDNA or constitutively active (CA) b-catenin cDNA. 48 hours after transfections, cell growth was assessed by counting colonies in a clonogenic growth assay (right panel of Figure 12 A), and apoptosis was assessed by the Annexin V positive stain assay (right panel of Figure 12B).
- Figures 12C-D depict exemplary validations of CD44v6 shRNAs (b-catenin shl and b-catenin sh2) ( Figure 12C) and validations of constitutively active b-actin ( Figure 12D) done by the indicated shRNA mediated knockdown and the corresponding knock-in (KI) gene transfections as described in Materials and Methods of Example 1.
- Target proteins were analyzed by WB analysis (b- tubulin, internal control).
- Figure 13 depicts a table of shRNA sequences used in pSico and pSicoR vectors as described in the Materials and Methods of Example 1.
- Figure 14 depicts exemplary results demonstrating that CD44v6 regulated b-catenin signaling establishes FOLFOX resistance in CRC-CICs.
- Figure 14A depicts exemplary results of CD44 negative COS7 cells stably transfected with vector control or with CD44v6 cDNA.
- Nuclear (N) and cytosolic (C) fractions were prepared from COS-7/vector and COS-7/Flag-CD44v6 stable transfectants and immunoprecipitated by anti-Flag antibody followed by Western blotting with the indicated proteins.
- Figure 14B-E depict exemplary results of CICs from SW480- S ( Figures 14B and 14D) and SW480-FR ( Figures 14C and 14E) cells transfected with 100 ng NT shRNAl or CD44v6 shRNAl. After 48 hours, cells were transfected together with TOPFlash and TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors. 48 hours after reporter vector transfection, the cells were stimulated with or without 1 x FOLFOX. After 12 hours, cells were lysed and subjected to luciferase measurements ( Figures 14B and 14C) or, in parallel, to WB analysis with the indicated proteins ( Figures 14D and 14E).
- Figure 15 depicts exemplary results demonstrating that CD44v6 regulated b-catenin signaling establishes FOLFOX resistance in CRC-CICs.
- Figures 15A-C depict exemplary results of COS-7/vector and COS-7/Flag-CD44v6 stable clones further transfected with TOPFlash and TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors for 48 hours.
- Figure 15A depicts exemplary results of PD-FR CICs transfected with 100 ng NT sh2 or CD44v6 shl.
- Figure 15E depicts exemplary COS7-Vector and COS7-CD44v6 clones transfected with TOPFlash and TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors for 48 hours.
- the reporter was stimulated with 20 ng/ml WNT3 A for 12 hour or by further transfection with LRP6 (50 ng), DVL2 (50 ng) or constitutively active (CA) b-catenin (25 ng).
- LRP6 50 ng
- DVL2 50 ng
- CA constitutively active
- b-catenin 25 ng
- Figure 16 depicts exemplary results demonstrating that Caveolin-mediated endocytosis is essential for CD44-LRP6 ⁇ - catenin signaling to maintain FOLFOX resistance.
- Figure 16A depicts exemplary results of detergent-resistant membranes, Triton X-100 (1%) insoluble fractions of FR cells, that were separated in the OptiPrep linear gradient, and distributions of protein and cholesterol across the gradient are shown (details in Materials and Methods of Example 1).
- WB analyses show the presence of caviolinl (CAV1) and clathrin in different Optiprep fractions.
- Figure 16B depicts exemplary results demonstrating that CD44v6 and activated LRPs affiliate with the lipid raft.
- FIG. 16C depicts exemplary results of SW480-S cells treated with/without 5 mM methyl ⁇ -cyclodextrin (Mbq ⁇ ) for 1 hour, and the R and NR fractions were analyzed by WB for the indicated proteins.
- Figure 16D depicts exemplary results of dominant negative dynamin (DN Dyn [DN K44A]) used to inhibit both caveolin-1 and clathrin mediated endocytosis.
- SW480- FR and SW480-S cells transfected with dominant negative dynamin were co transfected with 50 ng TOPFlash and 50 ng TK-Renilla vectors, or with 50 ng FOPFlash and 50 ng TK-Renilla vectors. After 48 hours, cells were treated with WNT3 A (20 ng/ml) for 1 hour, and the cell lysates were subjected to luciferase activity assays and quantitated.
- Figure 16E depicts exemplary results of SW480-S and SW480-FR cells transfected with TOPFlash or FOPFlash luciferase reporter constructs.
- Transfected cells were treated for 1 hour with the indicated concentrations of nystatin, known to block caveolin-1 -mediated endocytosis, or with monodansylcadaverine (MDC), known to block clathrin-mediated endocytosis followed by stimulation with WNT3 A 20 ng/ml for 1 hour.
- Figure 16F depicts exemplary results of SW480-S cells
- Figure 16G depicts exemplary results of SW480-FR cells pre-treated for 1 hour with Nystatin (150 pg/ml) or MDC (150 pg/ml), and the cells were stimulated with WNT3A for 0.5 or 1.0 hours.
- the cell lysates were analyzed by WB for the indicated proteins.
- Figure 17 depicts exemplary results demonstrating that DAB2 favorably sequesters a CD44v6-LRP6 complex in the direction of clathrin-dependent endocytosis to retain FOLFOX sensitivity.
- Figure 17A depicts exemplary expressions of disheveled protein 2 (DVL-2) and DAB2 protein in sensitive and resistant pairs of cells as shown by QPCR.
- Figure 17B depicts exemplary results of Vector and DAB2 cDNA transfected SW480-FR cells treated with or without WNT3 A for 1 hour, and whole cell lysates (WCL) were analyzed by WB for the indicated proteins.
- WCL whole cell lysates
- Figure 18A depicts schematic representations of individual CD44v6 mutants; ED, extracellular domain; TM, transmembrane domain; ICD, intracellular domain.
- Figure 18B depicts exemplary results of CD44v6 negative SW480- FR/(NON-CICs) transfected with individual CD44v6 mutants as depicted in ( Figure 18 A).
- Individual CD44v6 cell clones were either untreated (control) or challenged with FOLFOX (lx) for 30 minutes.
- Raft (R) and non-raft (NR) fractions were prepared as described in Materials and Methods of Example 1.
- Figure 18C depicts exemplary results demonstrating that blocking endocytosis with potassium (K+) depletion reduces CD44v6 receptor internalization in SW480-S and SW480-R cells.
- Indicated cells were incubated with biotin conjugated anti-CD44v6 antibody at 4° C separately followed by further incubation at 37° C for 10, 20 and 30 minutes as indicated. Internalization was measured by flow cytometry after staining with fluorescein conjugated anti-biotin antibody. The percentage of internalization was calculated by setting the mean fluorescence intensity of cells after biotin labeling but without glutathione incubation as 100%.
- Figure 18D depicts exemplary results of SW480-FR cells cultured in complete media with and without K+ depletion at 37° C for 1 hour followed by further incubation in the presence of WNT3A for 30 minutes. Total cell lysates and endosomes purified by sucrose gradient centrifugation were analyzed by western blotting.
- Figures 18E-F depict exemplary results of SW480-FR cells surface labeled with biotinylating agent (non-cleavable Sulfo-NHS- LC-Biotin). Cells were stimulated with WNT3A at 37° C for the times indicated and placed at 4° C for 1 hour of labelling with the biotinylating agent.
- biotinylating agent non-cleavable Sulfo-NHS- LC-Biotin
- FIGS 18G-I depict exemplary results of sensitive and FR SW480 cells stably transfected with vector and a DAB2 construct. These stable clones were co-transfected with non-targeted (NT), or caveolinl (CAV1) shRNAl or clathrin shRNAl. After 48 hours, cells were then transfected with TOP/FOPFlash luciferase reporter constructs prior to WNT3A (20 ng/ml) stimulation for 1 hour, and cell lysates were subjected to luciferase activity determination (Figure 18G) and processed for WB analysis with the indicated proteins ( Figures 18H-I).
- NT non-targeted
- CAV1 caveolinl
- Figures 18J-K depict exemplary results of validations of CAVlshRNAs (CAV1 shl and CAV1 sh2) and Clathrin shRNAs (Clathrin shl and Clathrin sh2) done by the indicated shRNA mediated knockdown and the corresponding knock-in (KI) gene transfections as described in Materials and Methods of Example 1.
- Target proteins were analyzed by WB analysis (b-tubulin, internal control).
- FACS data in Figure 18C represent mean +/- SD, *p ⁇ 0.05 from at least 3 independent experiments performed in triplicates.
- Data in Figure 18G represent results from 3 independent experiments performed in triplicates. All WBs are representative of 3 independent experiments.
- Figure 19 depicts exemplary results demonstrating that Nuclear localization (NLS) site in the ICD domain of CD44v6 is required for nuclear translocation of CD44v6 through endosomal sorting, and its subsequent association with TCF4 contributes to enrichment of TCF4/TOPFlash transcription.
- Figure 19A depicts exemplary results of the association of CD44v6 with LRP6 and actin examined in SW480-FRNON-CICs/CD44v6 cell clones expressing the indicated CD44v6 mutants. After stimulation with WNT3A for 12 hours, the individual CD44v6-expressing SW480-FRNON-CIC clones were lysed.
- FIG. 19B depicts exemplary WB analyses of endosomal and nuclear fractions in individual SW480-FRNON-CICs/CD44v6 cell clones expressing the nuclear localization signal mutant D67 (NLC), the mutant (generated by removing intracellular domain (ICD) sequences containing the NLS binding site and one palmitoyl binding site, see Figure 18 A). All WB data are representative of 4 independent experiments.
- Figre 19C depicts exemplary results of SW480-FRNON-CICs/CD44v6 cells incubated with biotin- conjugated CD44v6 at 4° C for 1 hour.
- FIG. 19D depicts exemplary results demonstrating that CD44v6A67 (devoid of ICD) is expressed at the membrane. Lysates from indicated SW480-FRNON-CIC/CD44v6 cell clones expressing indicated CD44v6 mutants were subjected to cytosol and membrane fractionation. Cell fractions were analyzed by WBs. The relative purities of the membrane and cytosolic fractions were confirmed by probing for the cytoplasmic protein HSP90 and the membrane protein transferrin receptor (Tf-R).
- Tf-R membrane protein transferrin receptor
- FIGS 19E-F depict exemplary results demonstrating that mutation of the NLS, or CD44v6 devoid of ICD (CD44v6 D67) reduces CD44 mediated enrichment of WNT signaling.
- SW480-FRNON-CIC/CD44v6 cell clones expressing the indicated CD44v6 A67mutants ( Figure 19E), and the CD44v6 NLS mutants ( Figure 19F) were transfected with TOPFlash and control TK-Renilla vectors, or FopFlash and TK- Renilla vectors. 48 hours after reporter vector transfection, cells were lysed and subjected to luciferase measurements and in parallel to WB analysis. All luciferase data represent at least 3 independent experiments done in triplicates. All WBs are representative of 4 independent experiments.
- Figure 20 depicts exemplary results demonstrating that Nuclear CD44 associates with TCF4 and functions to modulate MDR1 transcription in FOLFOX resistant cells.
- Figure 20A depicts exemplary results demonstrating that nuclear CD44v6 associates with TCF4 and MDR1.
- Nuclear (N) and cytosolic (C) fractions were immunoprecipitated with TCF4 or IgG (Control) followed by Western blotting for the CD44v6, MDR1 and TCF4 proteins in the SW480-FR cells, and in the COS-7 CD44v6 clones expressing the indicated mutants and vector controls.
- Figure 20B depicts exemplary QPCR analyses of CD44v6, b-catenin and MDR1 levels in SW480-S, SW480-FR, SW480-OXAR and SW480-5FUR cells.
- Figure 20C depicts exemplary QPCR analyses of CD44v6, b-catenin and MDR1 levels in SW480-S , SW480-5FUR and SW480-OXAR cells treated with or without 1 x FOLFOX or 20 ng/ml WNT3A for 12 hours.
- Figures 20D-E depict exemplary QPCR analyses of TCF4, b- catenin, CD44v6 and MDR1 levels in SW480-S cells overexpressing constitutively active (CA) TCF4 cDNA ( Figure 20D) or CA b-catenin ( Figure 20E).
- Figures 20F-G depict exemplary results of transcription activities of the MDR1 promoter with TCF4 binding sites measured using indicated pGL3 reporters. In Figure 20F, the scheme shows the constructs with TCF binding sites in the MDR1 promoter.
- FIG 20G MDR1 Luciferase activity reporter assays are shown for SW480-FR cells overexpressing NT shRNA (Control), or b-catenin shRNA, or CD44v6 shRNA, or a dominant negative TCF4-DN construct for 48 hours.
- Figures 20H-J depict exemplary results of MDR1 gene expressions regulated by TCF4 in SW480-FR cells.
- the sketch map shows the predicted TCF4 binding sites (CTTTGA) within the indicated MDR1 promoter. The transcriptional start site was at +1, and ATG is at the translation start site.
- the putative TCF4 binding sites (MDR1 [A], MDR1 [B] and MDR1 [C]) are shown, and their locations are labeled.
- ChIP assays were done using either anti-TCF4, anti-CD44v6, anti b- catenin or irrelevant IgG antibody as negative control in SW480-FR cells overexpressing specific shRNAs against CD44v6 or Non-targeted shRNA (NT-sh), or CD44v6 WT or CD44v6NLS mutant constructs.
- Quantitative ChIP-QPCR data representing the PCR products in immunoprecipitated DNA versus 10% input DNA of ChIP primers for designated MDR1 (A) site are shown.
- QPCR data in Figures 20B-E represent mean +/- SD, *p ⁇ 0.05 from at least 3 independent experiments performed in triplicates.
- Luciferase data in Figure 20G and ChIP QPCR Data in Figures 20I-J represent results from 3 independent experiments performed in triplicates. WB and semi-quantitative data are representative of 3 independent experiments.
- Figure 21, comprising Figure 21 A through Figure 21E, depicts exemplary results demonstrating that Nuclear TCF4 modulates CD44v6 transcription in resistant cells, and ICD domain of CD44v6 is required for oxaliplatin (a component of FOLFOX) efflux.
- Figures 21A-D depicts exemplary results demonstrating that CD44v6 was transcriptionally regulated by TCF4 in SW480-FR cells.
- Figure 21 A the sketch map of predicted TCF4 binding sites (CTTTGA) within the CD44v6 promoter (CD44v6 [a], and CD44v6 [b]) is shown.
- IB CD44v6 luciferase (Luc) activity reporter assays are shown for SW480-FR cells overexpressing dominant negative (DN) TCF4, or b- catenin shRNA or NT-shRNA (Control) for 48 hours.
- DN dominant negative
- b- catenin shRNA or NT-shRNA Control
- Figure 21C semiquantitative PCR products using ChIP PCR primers (CD44v6 [A] as depicted in the schematic diagram in upper panel of Figure 21C) are shown for designated TCF4 binding sites of the CD44v6 promoter.
- FIG. 2 ID ChIP-QPCR using PCR primers for designated CD44v6 (A) sites (as shown in the schematic diagram in upper panel of Figure 21C) was used for amplification of the CD44v6 gene of untreated SW480-FR cells and of cells overexpressing the indicated vector and TCF4-DN cDNA.
- Figure 2 IE depicts exemplary results demonstrating that the intracellular domain (ICD) of CD44v6 induces 14 C Oxaliplatin Efflux/Retention in SW480-S and SW480-FR cells by FOLFOX and WNT3 A treatment.
- tumor cells were transfected for 48 hours with CD44v6shRNA, or with a CD44A67 construct (which is devoid of the ICD region of CD44v6).
- Figure 22 depicts a table of cis-sequences bound by CD44v6.
- ChIP assay was performed with chromatin from the S2480-FR CICs using CD44v6 antibody.
- the immunoprecipitated DNA-chromatin complex was amplified by PCR and subcloned. A total of 11 clones were sequenced. Blast analysis revealed the presence of various cis- binding sites for stemness/drug resistance related transcription factors in these DNA sequences.
- Figure 23 depicts a table of ChIP PCR primers for MDR1 and CD44v6 promoters as described in Materials and Methods of Example 1.
- Figure 24 depicts a proposed model for a positive feedback loop that couples P-catenin/TCF4 activation and CD44 alternate splicing, and CD44v6 then sustains cancer initiating cell proliferation and drug resistance.
- Figure 24A depicts a model showing the shunting of CD44v6 and LRP6 towards the caveolin endocytic pathway in the absence pf DAB2 in the FOLFOX resistant cells.
- elevated WNT3 A and CD44v6 through its nuclear localization site (NLS) recruits LRP6 in a lipid raft complex in resistant cells.
- NLS nuclear localization site
- the NLS site of CD44v6 coincides with the ezrin-radixin-moesin (ERM) site of the intracellular domain (ICD) of CD44v6.
- ERP ezrin-radixin-moesin
- ICD intracellular domain
- CD44v6 acts as a regulator of the FOLFOX-induced WNT3 A/b-catenin signaling and the binding and activation of LRP6.
- the consequent b-catenin transcription requires the NLS binding domain of CD44v6.
- the nuclear accumulation of a CD44v6- b-catenin complex activates MDR1 and CD44v6 promoters, which sustains drug resistance and CD44v6 rich cancer initiating cells (CICs).
- CICs CD44v6 rich cancer initiating cells
- Figure 24B depicts, by contrast, a model wherein sensitive cells in the presence of DAB2, the CD44v6-LRP6 complex is internalized through the clathrin- mediated endocytosis pathway and fails to inhibit the b-oh ⁇ eh ⁇ h-0044n6 complex accumulation in the nucleus by promoting a b-catenin destruction complex.
- Figure 25 depicts exemplary results demonstrating that cancer associated fibroblasts (CAFs) account for tumor resistance to FOLFOX.
- Figure 25A-B depicts single cell suspensions from a dissociated FOLFOX resistant (FR) patient colorectal tumor (PD-FR) that were sorted by FACS using PDGFR-a-PE and EpCAM-FITC.
- Figure 25C depicts the quantified percentages of EpCAM (-)/PDGFR-a (+) (CAFs) and EpCAM (+)/PDGFR-a (+) (Non-CAFs) in total unsorted cell populations.
- Figure 25D depicts the integrities of EpCAM-/PDGFRa + gated CAFs from the dissociated CRC cells from the patient colorectal tumor (PD-5FUR, PD-OXAR, PD-FR) and from the sensitive and FOLFOX resistant SW480/subcutaneous (SQ) tumor tissues as confirmed by QPCR analyses for CAF-associated markers FSP1, FAP, PDGFR-a and epithelial cell marker EpCAM (negative control).
- Figure 25E depicts EpCAM (-) /PDGFR-a (+) cells from the PD-5FUR, PD-OXAR, PD-FR, SW480-S and SW489-FR subcutaneous (SQ) tumor tissues that were cultured in DMEM with 10%
- FIG. 25F-G depicts the influence of CAFs on the viability of cancer initiating cells (CICs) that were evaluated by co-culture of CICs with CAFs ( Figure 25F), and by using CAFs derived conditioned media (CM) treatment with CICs ( Figure 25G).
- CICs cancer initiating cells
- CM CAFs derived conditioned media
- Figure 25H- I depicts the ability of the CAFs to affect the tumor growth of CICs from three different batches of SW480-S ( Figure 25H) and of SW480-FR ( Figure 251) tumor samples that were tested in vivo (see Methods of Example 2 below).
- Indicated cell numbers of SW480-S/SQ CAFs and SW480-FR/SQ CAFs that were pre-treated with vehicle (DMSO) or with FOLFOX for 72 hours were subcutaneously co-implanted in immunocompromised mice with the indicated SW480-S/SQ CICs and SW480-FR/SQ CICs. Tumors were harvested every week to evaluate the latency, and weights were measured to evaluate their development.
- Figure 26 depicts exemplary results demonstrating that FOLFOX induces cytokine secretion in CAFs.
- Figure 26A-B depicts basal mRNA expression levels of PN and the indicated growth factors, and of growth promoting cytokines (Figure 26A) and related proteins, receptors and transcription factors (Figure 26B) that were analyzed by semi quantitative RT-PCR in eight indicated CAFs isolated from drug resistant patient tissues and from three normal fibroblasts (Fb). GAPDH was the reference gene.
- Figure 26C demonstrates via Venn diagram that, although there were several molecules unique to each CAF, several cytokines were common to PD-5FUR, PD-OXAR and PD-FR chemo-therapy resistant patient tissues.
- Figure 26D-H depicts CAFs derived from three human surgical tissues and one commercial normal intestinal fibroblast line that were analyzed for their indicated five major cytokine and PN profiles after FOLFOX therapy treatment.
- Figure 261 depicts QPCR analysis of periostin (PN) and 4 major cytokines (WNT3A, IL-17A, IL6 and TGFpi) in patient tumor CAFs treated with DMSO or FOLFOX for 72 hours. GAPDH was the reference gene.
- Figure 26J depicts autocrine expression of two dominant cytokines (IL17A and WNT3A) and PN that were assessed by using an ELISA assay on PD-FR CICs treated with vehicle (DMSO) or with FOLFOX for 72 hours.
- Figure 26K depicts the effects of PN, IL17A and WNT3A on cell viability as assessed by using ATP Glo assay on PD-FR CICs and SW480-FR CICs. The effects were assessed in these different specimens by culturing 10 4 CICs with either vehicle, or 20 ng/ml of PN, IL-17A or WNT 3 A.
- Figure 26L depicts the effects of the autocrine PN, IL-17A and WNT3A production in CICs examined by studying the effects of blockade of PN, or IL- 17A or WNT3A on the tumor-initiating capacity of 1 x 10 4 SW480-FR/CICs in the absence of SW480-FR/CAFs.
- FIG. 26M depicts a confirmation of involvement of CD44v6 expression in regulating WNT3 A production as assessed in PD-FR CAFs by examining the effects of blockade of CD44v6 using specific shRNAs on the 10 4 SW480-FR/CAFs in the absence and presence of FOLFOX.
- Figure 27 depicts exemplary results demonstrating that CD44v6 expression is critically regulated by the WNT pathway stimulated by PN and/or IL17A.
- Figure 27A-B depicts results showing that selective shRNA- knockdown of CD44v6, but not CD44v8 (upper panels), greatly inhibits the ability of 50 ng/ml PN ( Figure 27A) or 50 ng/ml IL17A ( Figure 27B) to activate b-catenin and p-LRP6, and to upregulate MDR1 (lower panels) in serum starved SW480-OXAR tumor cells.
- Figure 27C depicts results showing that CD44v6 shRNA and WNT3 A shRNA (upper panels) greatly inhibit the ability of 50 ng/ml WNT3 A or of 1 x FOLFOX to activate b-catenin and to upregulate MDR1 (lower panels) in serum starved SW480- OXAR/SQ tumor cells.
- FIG. 27D-F depicts WNT reporter (TOPFLASH/FOPFLASH) activities determined in SW480-S ( Figure 27D), SW480- OXAR ( Figure 27E), and SW480-FR ( Figure 27F) SQ CICs in response to culturing with vehicle (Control), or with PN, or IL-17A, or WNT3A alone (20 ng/ml), or in combination PN + WNT3A, or IL-17A + WNT3A.
- Figure 27G depicts results showing that dominant negative TCF4 mediated down regulation of TCF4 inhibits CD44v6 expression in nuclear lysates of SW480-FR SQ tumor cells.
- Figure 27H depicts flow cytometry analyses of percent enrichment of CD44v6+/EpCAM+ CICs shown for unsorted cells from SW480- 5FUR/SQ, SW480-OXAR/SQ and SW480-FR/SQ cells overexpressing WNT3A or vector control.
- Figure 271 depicts percentages of colon tumor sphere formation as measured in a sphere-formation assay. CICs and Non-CICs in SW480-OXAR SQ tumor cells overexpressing WNT3 A or vector control were plated in 96-well plates for 14 days.
- Figure 28 depicts exemplary results demonstrating that Dickkopf (DKK) and its receptor (Kremen) subfamily expression regulated the WNT/p-catenin signaling in CRC cells.
- Figure 28A-B depicts Dickkopf (DKK) and Kremen subfamily gene expressions done using cDNA from the FR cells and sensitive cells (SW480, HT29, WIDR and LOVO).
- Figure 28C depicts autocrine expression of DKK1 as assessed by using an ELISA assay on S and FR cells of SW480, HT29, WIDR and LOVO.
- Figure 28D depicts the effects of PN and IL-17A on DKK1 secretion as measured by ELISA in sensitive and FR cells of SW480 transfected with PN or IL17A vectors for 36 hours.
- Figure 28E depicts results showing that Kremens cooperate with DKK1 in WNT/p-catenin signaling inhibition assessed by measuring luciferase WNT reporter (TOPFlash) assays with the indicated genes transfected in SW480-FR CICs. (See details of TOPFlash/FOP Flash assay in Methods of Example 2 below and in Example 1 above). Plasmids used were DKK1, FZ (frizzled), LRP6, KRM1 and KRM2.
- Figure 28F-I depicts pull downs of Western blots showing that over expressed Flag-tagged PN and Flag-tagged IL17A interact with WNT3A (Figure 28F, G) but not with DKK1 ( Figure 28H, I).
- Figure 28 J depicts the assessment of depletion of cell surface LRP6 by DKK1.
- SW480-FR CICs were transfected with Flag-LRP6, and with or without KRM2 as indicated. After 36 hours, cells were treated with DKK1 for 2 hours at 37° C and followed by cell surface biotinylation.
- FIG. 28K depicts SW480-FR CICs that were transfected with Flag-LRP6, KRM2 or GFP (Transfection control) and treated with DKK1 for the indicated times. Subsequently, cells were surface-biotinylated and analyzed as above.
- Figure 29, depicts exemplary results demonstrating that redistribution of CD44v6 and LRP6 in response to DKK1.
- Figure 29A depicts CD44v6 promoter luciferase activities after treating the SW480-S cells with either DKK1, WNT3A, WNT3A + DKK1, PN, PN + DKK1, IL17A, IL17A + DKK1 or vehicle control.
- Figure 29B depicts lysates of SW480-S and SW480-FR cells that were fractionated by OptiPrep gradient centrifugation, and lipid raft fractions (3-5) were immunoprecipitated with caveolin-1 (marker of lipid raft) and probed with anti- CD44v6 or anti-LRP6, or anti-CK 1g and anti-Caveolin antibody. Endogenous Caveolin-1 indicates the positions of the lipid raft fractions.
- Figure 29C-G depicts SW480-FR cells that were preincubated with or without the clathrin-microdomain inhibitor monodansylcadaverine (MDC) for 30 minutes, and then the cells were treated for 1 hour with DKK1, PN or IL17A (20 ng/ml each).
- MDC clathrin-microdomain inhibitor monodansylcadaverine
- Endogenous caveolin-1 and clathrin indicate the positions of the lipid raft (R) and non lipid raft (NR) fractions
- the band intensities of LRP6 and CD44v6 in the lipid raft and nonlipid raft fractions were quantified using NIH image, and the percentages of LRP6 and CD44v6 in the lipid raft fractions were calculated as (lipid raft/[lipid raft + nonlipid raft]) x 100 for only DKK1 (Figure 29E), DKK1 with PN ( Figure 29F), and DKK1 with IL17A (Figure 29G).
- Figure 30 depicts exemplary results demonstrating that PN and IL17A induce association of nuclear P-catenin/TCF4 with CD44v6 and MDR1 to modulate drug resistance.
- Figure 30A-B depicts PN or IL17A induced MDR1 promoter luciferase activities as measured in SW480-FR cells using the indicated pGL3-mdrl (a) reporter containing TCF4 binding sites (CTTTGA).
- CTTTGA pGL3-mdrl
- the scheme shows the pGL3-mdrl (a) reporter constructs with TCF binding sites.
- FIG. 30B MDR1 Luciferase activity reporter assays are shown for SW480-FR cells overexpressing a dominant negative TCF4-DN construct for 48 hours followed by co-transfection with or without PN, or IL17A expression vectors.
- Figure 30C-D depicts results showing that PN or IL17A induced MDR1 gene expression regulated by TCF4 in SW480-FR cells.
- the sketch map shows the predicted TCF4 binding sites (CTTTGA) within the indicated MDR1 promoter. The transcriptional start site was at “+1”, and ATG is at the translation start site. The putative TCF4 binding sites (MDR1 [A], MDR1 [B] and MDR1 [C]) are shown, and their locations are labeled.
- PCR primers designated for MDR1 (A) as shown in (C) were used for amplification of the potential TCF4 binding sites of the MDR1 gene.
- ChIP assays were done using either anti-CD44v6, anti b-catenin or irrelevant IgG antibody as negative control in SW480-FR cells overexpressing specific shRNAs against CD44v6 or Non-targeted shRNA (NT-sh) with or without cotransfection with PN, or IL- 17A constructs.
- NT-sh Non-targeted shRNA
- a representative quantitative QPCR representing the PCR product in immunoprecipitated DNA versus 10% input DNA of ChIP primers for designated MDR1 (A) site is shown.
- Input total genomic DNA was used as control for the PCR.
- FIG. 30E depicts the sketch map of predicted TCF4 binding sites (CTTTGA) within the CD44v6 promoter luciferase construct (CD44v6 [a]) is shown.
- Figure 30F depicts CD44v6 luciferase activity reporter assays shown for SW480-FR cells overexpressing dominant negative (DN) TCF4, for 48 hours followed by co-transfection with or without PN or IL17A expression vectors.
- FIG. 30G-H depicts results showing that PN or IL17A induced CD44v6 gene expression regulated by TCF4 in SW480-FR cells is shown.
- the sketch map shows the predicted TCF4 binding sites (CTTTGA) within the indicated CD44v6 promoter.
- the transcriptional start site was at “+1”, and ATG is at the translation start site.
- the putative TCF4 binding sites (MDR1 [A], MDR1 [B] and MDR1 [C]) are shown, and their locations are labeled.
- PCR primers, designated for CD44v6 (A) as shown in Figure 30G were used for amplification of the potential TCF4 binding sites of the CD44v6 gene.
- Figure 31 depicts exemplary results demonstrating that FOLFOX induced PN, WNT3A and IL17A signaling regulates CIC growth in vivo.
- Figure 31 A depicts a timeline for antibody treatments with and without FOLFOX in xenograft tumors implanted by a mixture of CICs plus CAFs from PD-FR human tissues ( Figure 3 IB, D) and from SW480-FR ( Figure 31C, E) xenograft tumor cells.
- the dependence of CICs on IL17A, WNT3A, PN and CD44v6 were evaluated in vivo. 2 xlO 4 CICs and 6 x 10 4 CAFs were injected into mice.
- FIG. 31B-C depicts representative tumors from three experiments from mice treated as indicated.
- Figure 32 depicts exemplary results demonstrating that tissue specific knockdown of CD44v6 by pFabpl-Cre inhibits tumor growth of implanted SW480-FR/SQ/CICs plus SW480-FR/SQ/CAFs in immune compromised mice.
- Figure 32A-E depicts SW480-FR cells that were transfected with pSico-CD44v6shRNA/Tf-PEG-PEI (pSico v6 shRNA/Nano) with Fabpl-Cre/Tf-PEG- PEI (Fabpl-Cre/Nano, or pSicoR v6shRNA/Nano with Fabpl-Cre/Nano.
- FIG. 32D Schematic representations of pSico ( Figure 32D) and pSicoR ( Figure 32E) after Cre-mediated recombination to synthesize active shRNA are shown.
- Figure 32A cells were analyzed by epifluorescence microscopy to detect GFP. Similar cell densities and identical exposure times were used for all images.
- Figure 32B-C total RNAs extracted from the transfected cells were analyzed for CD44v6 and GAPDH mRNA by semi-quantitative PCR.
- Figure 32F depicts PCR detection for Cre-mediated recombination of pSico-CD44v6 shRNA in tumor DNAs extracted from mice. Genomic DNA was extracted 4 days after shRNA/Nano plus indicated Cre/Nano treatments and subjected to PCR.
- Figure 33 depicts exemplary results demonstrating that CD44v6 signaling regulates colorectal CIC growth and self renewal.
- Figure 33 A depicts a timeline for pSico-v6-sh/Nano treatment with and without pFabpl-Cre/Nano in xenograft tumors derived from CICs plus CAFs from SW480-FR/SQ tumors and PD-FR/SQ tumors.
- Figure 33B depicts the dependence of CICs and CAFs on CD44v6 as evaluated in vivo. 2 x 10 4 CICs and 6 x 10 4 CAFs were injected into mice. When tumors reached approximately 0.3 cm 3 in volume, treatment was initiated.
- Figure 33C-D depicts the kinetics of relative tumor weights with time during in vivo SQ tumor growth at the indicated weeks generated by CICs plus CAFs from SW480-FR/SQ tumors ( Figure 33C) and from PD-FR/SQ tumors ( Figure 33D) injected (i.p.) with pSico-NT sh/Nano, pSico- v6 sh/Nano ⁇ pFabpl-Cre/Nano.
- Figure 33E depicts Western blot analyses using CD44v6, MDR1, and b-catenin antibodies in extracts from the various treated xenograft tumors derived from CICs plus CAFs from SW480-FR/SQ tumors collected at 6 weeks from the experiment in Figure 33B.
- Figure 33F depicts bars which represent densitometric ratios of CD44v6/p-actin, and MDRl/p-actin in the Western blot analyses using CD44v6, MDR1, and b-catenin antibodies in extracts from the various treated xenograft tumors derived from CICs plus CAFs from PD-FR/SQ tumors collected at 6 weeks from the experiment in Figure 33B.
- the results shown are means ⁇ SE from four independent experiments.
- Figure 33G depicts QPCR analyses of the indicated sternness related transcription factors from the total RNA from the various treated tumors collected at 6 weeks (Figure 33B).
- Figure 34 depicts a schematic model demonstrating that colon cancer associated fibroblast derived periostin and IL17A promote WNT3A induced CD44v6 expression in tumor cells and Cancer Initiating Cells (CICs).
- CD44v6 positive CICs induce drug resistance and colorectal tumorigenesis by enhancing CD44v6 ⁇ catenin nuclear localization and MDR1 gene expression in tumor cells and CICs.
- CD44v6 regulated IL17A, PN and WNT3A derived from chemotherapy induced CAFs contribute to CIC maintenance through induction of the CD44v6 receptor.
- Figure 35 depicts exemplary results demonstrating that FOLFOX-stimulated PN and IL17A secretion in CAFs promote WNT3A secretion and tumorigenic function of CICs.
- Figure 35A-C depicts CAFs derived from SW480-S and SW480-FR xenograft (SQ) tumor tissues that were analyzed for their indicated five major cytokine and PN profiles with and without FOLFOX treatment.
- Figure 35D depicts PN and IL17A stimulated WNT3A production in CAFs isolated from sensitive and FOLFOX resistant (FR) tumors of SW480 as assessed by transfecting these freshly isolated CAFs by vector control, PN, and IL-17A expression plasmids for 72 hours. Secretion of WNT3A was measured by an ELISA assay.
- Figure 35E depicts results showing the involvement of CD44v6 in regulating WNT3A production as assessed in PD-FR CAFs by examining the effects of blockade of CD44v6 using specific shRNAs in the absence and presence of PN and IL17A. The effects were evaluated by measuring secreted WNT3A in cultures by ELISA.
- Figure 35F-G depicts the percentages of colon tumor sphere formation as measured in a sphere-formation assay in the presence and absence of 50 ng/ml of either PN, or IL17A or WNT3A proteins (Figure 35F) or in presence and absence of 100 ng/ml of either PN-blocking antibody, or IL17A- blocking antibody or WNT3A- blocking antibody ( Figure 35G) in CICs isolated from the SW480-FR SQ tumors and SW480-S SQ tumors.
- Figure 36 depicts semi quantitative RT-PCR primers for cytokines, growth factors, PN and related receptors.
- Figure 37 depicts real-time PCR (QPCR) primers for various genes used in Example 2.
- Figure 38 depicts real-time PCR (QPCR) primers for various genes associated with CICs sternness functions.
- Figure 39 depicts shRNA sequences in pSico and pSicoR vectors used in Examples 1 and 2.
- Figure 40 depicts exemplary results demonstrating that pSicoCD44v6 shRNAl New and pSicoCD44v6 shRNA2 New are better silencing agents for tumor growth suppression by inhibiting MDR1 activation compared to that of CD44v6 shRNA Old (see Misra S et al., J Biol Chem, 2009,
- Figure 40A depicts a timeline shown for pSico-v6-sh/Nano treatment with and without pFabpl-Cre/Nano in xenograft tumors derived from CICs plus CAFs from SW480-FR/SQ tumors.
- Figure 40B depicts exemplary results demonstrating the dependence of CICs and CAFs on CD44v6 evaluated in vivo. 2 x 10 4 CICs and 6 x 10 4 CAFs were injected into mice. When tumors reached approximately 0.3 cm 3 in volume, treatment was initiated. The shRNA-nanoparticles were delivered by i.p. thrice a week. Seven SCID mice per group were used.
- FIG. 40C depicts exemplary results of relative tumor weights during in vivo SQ tumor growth at the indicated weeks generated by CICs plus CAFs from SW480-FR/SQ tumors treated with various sets of SW480-FR/SQ tumors injected (i.p.) with various groups of pSico-NT sh/Nano, pSico-v6 sh/Nano ⁇ pFabpl-Cre/Nano.
- Figures 40D-F depict exemplary Western blot analyses using CD44v6, MDR1, and b- tubulin antibodies in extracts from pSico-CD44v6 shRNA Old- ( Figure 40D), that pSico- CD44v6 shRNAl New- ( Figure 40E), and pSico-CD44v6 shRNA2 ( Figure 40F)-treated xenograft tumors derived from CICs plus CAFs from SW480-FR/SQ tumors collected at 6 weeks from the experiment in Figure 40B.
- Figure 41 depicts exemplary results demonstrating that EGF, and HGF stimulation increases pAkt-pGSK3P-ABC-pSTAT3 signaling.
- SW480-S cells were treated with 20ng/ml of EGF, and HGF for the indicated time and cell lysates were analyzed for the indicated proteins.
- Figure 42 depicts exemplary results demonstrating that CD44v6 shRNAl New and CD44v6 shRNA2 New are better silencing agents as compared CD44v6shRNA Old with respect to reduction of active-Pcatenin, pSTAT3, and MDRl expression in FOLFOX resistant SW480-FR cells.
- FOLFOX resistant SW480-FR cells were transfected with indicated shRNA constructs. After 72 hour the lysates were processed for western blotting to analyze the indicated proteins.
- Figure 43 depicts exemplary results demonstrating that CD44v6 shRNAl New and CD44v6 shRNA2 New efficiently reduce the expression of active-Pcatenin, pSTAT3, and MDR1 expression in HGF-, EGF-, and FOLFOX-stimulated FOLFOX resistant SW480-FR cells.
- Sensitive SW480-S cells were transfected with indicated shRNA constructs. After 72 hour the cells were treated with 20ng EGF, or HGF, or lx FOLFOX for 8 hours. Then the lysates were processed for western blotting to analyze the indicated proteins.
- Figure 44 depicts the differences in the size +/- standard deviation of Tf- PEG-PEI nanoparticles for pSico-CD44v6 shRNA Old + pFabpl-Cre and pSico-CD44v6 shRNAl/2 New and pFabpl-Cre individually.
- an element means one element or more than one element.
- the term “activate,” as used herein, means to induce or increase a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein’s expression, stability, function or activity by a measurable amount relative to a control comparator.
- Activators are compounds that, e.g., bind to, partially or totally induce stimulation, increase, promote, induce activation, activate, sensitize, or up regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., agonists.
- Antisense refers particularly to the nucleic acid sequence of the non coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double-stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
- anti-tumor effect refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
- An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.
- binding refers to the specific association or other specific interaction between two molecular species, such as, but not limited to, protein-DNA/RNA interactions and protein-protein interactions, for example, the specific association between proteins and their DNA/RNA targets, receptors and their ligands, enzymes and their substrates, etc.
- binding may be specific or non-specific, and can involve various noncovalent interactions, such as including hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, pi-pi interactions, and/or electrostatic effects.
- cancer as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.
- cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, oral cancer and the like.
- the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base pairing rules.
- sequence “A-G-T,” is complementary to the sequence “T- C-A.”
- Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
- the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
- a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
- a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
- an “effective amount” as used herein means an amount which provides a therapeutic or prophylactic benefit.
- Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
- a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
- Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
- expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
- “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
- An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
- Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
- fragment refers to a subsequence of a larger nucleic acid.
- a “fragment” of a nucleic acid can be at least about 36 nucleotides in length; for example, at least about 40 nucleotides to about 50 nucleotides; at least about 50 to about 60 nucleotides, at least about 60 to about 70 nucleotides; at least about 70 nucleotides to about 80 nucleotides; about 80 nucleotides to about 90 nucleotides; or about 100 nucleotides (and any integer value in between).
- fragment refers to a subsequence of a larger protein or peptide.
- a “fragment” of a protein or peptide can be at least about 12 amino acids in length.
- inhibitor means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely.
- Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and mRNA stability, expression, function and activity, e.g., antagonists.
- isolated means altered or removed from the natural state.
- a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
- An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
- modulate refers to the activation or inhibition of molecule, as described in the respective definitions herein.
- nucleic acid refers to a polynucleotide and includes poly ribonucleotides and poly-deoxyribonucleotides.
- Nucleic acids according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, for example, cytosine, thymine, and uracil, and adenine and guanine, respectively. (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982), which is herein incorporated in its entirety for all purposes).
- the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like.
- the polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
- the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
- overexpressed or overexpression is intended to indicate an abnormal level of expression of a particular gene in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ.
- Patients having solid tumors or a hematological malignancy characterized by overexpression of a particular gene can be determined by standard assays known in the art.
- patient refers to any animal, or cells thereof whether in vitro or in situ , amenable to the methods described herein.
- the patient, subject, or individual is a human.
- peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
- a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
- Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
- the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
- Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
- the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
- pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and/or in humans.
- polynucleotide includes cDNA, RNA, DNA/RNA hybrid, antisense RNA, ribozyme, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural or derivatized, synthetic, or semi -synthetic nucleotide bases. Also, contemplated are alterations of a wild type or synthetic gene, including, but not limited to deletion, insertion, substitution of one or more nucleotides, or fusion to other polynucleotide sequences.
- prevent refers to ability to block or delay the onset, or otherwise mitigate the severity of symptoms in a subject at risk of developing said disease or disorder.
- the terms “therapy” or “therapeutic regimen” refer to those activities taken to alleviate or alter a disorder or disease state, e.g., a course of treatment intended to reduce or eliminate at least one sign or symptom of a disease or disorder using pharmacological, surgical, dietary and/or other techniques.
- a therapeutic regimen may include a prescribed dosage of one or more drugs or surgery. Therapies will most often be beneficial and reduce or eliminate at least one sign or symptom of the disorder or disease state, but in some instances the effect of a therapy will have non-desirable or side- effects. The effect of therapy will also be impacted by the physiological state of the subject, e.g., age, gender, genetics, weight, other disease or disorder conditions, etc.
- therapeutically effective amount refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician.
- therapeutically effective amount includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.
- the therapeutically effective amount will vary depending on the compound, the disease or disorder and its severity and the age, weight, etc., of the subject to be treated.
- To “treat” a disease or disorder as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
- ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
- a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
- vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
- the term “vector” includes an autonomously replicating plasmid or a virus.
- the term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
- examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
- the present invention generally relates to compositions and methods for the treatment, prevention, or combination thereof, of one or more disease or disorder associated with chemotherapeutic resistance.
- the composition comprises one or more modulator of one or more chemoresi stance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof.
- the chemoresi stance promoting molecule is one or more selected from the group consisting of: periostin (PN), Wnt family member 3 A (WNT3A), Interleukin 17A (IL-17A) and CD44 splice variant 6 (CD44v6).
- the inhibitor is a nucleic acid molecule encoding shRNA directed against said one or more chemoresi stance promoting molecule.
- the one or more chemoresi stance promoting molecule inhibitor is encapsulated in a nanoparticle for delivery to a one or more cell or a tissue of a subject in need thereof.
- the method comprises administering a composition comprising an inhibitor one or more chemoresi stance promoting molecule to subject in need thereof.
- the subject has, or is at risk of developing, one or more disease or disorder associated with chemotherapeutic resistance.
- the method comprises treating or preventing one or more disease or disorder associated with chemotherapeutic resistance, such as cancer.
- the present invention comprises a method of diagnosing a cancer as chemotherapeutic resistant and treating said cancer with one or more inhibitor of said one or more chemoresistance promoting molecule.
- the indicator of chemotherapeutic resistance is overexpression of CD44v6 and the treatment is a composition comprising an inhibitor of CD44v6.
- the inhibitor of CD44v6 comprises an shRNA molecule specific for binding to CD44v6.
- the present invention relates to a composition
- a composition comprising one or more modulator of one or more chemoresistance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof.
- said modulator mediates chemotherapeutic agent efflux.
- the modulator is an inhibitor of a chemoresistance promoting molecule.
- the chemoresistance promoting molecule is one or more selected from the group consisting of: WNT3A, PN, IL-17A, b-catenin, low-density lipoprotein receptor-related protein 6 (LRP6), Frizzled, disheveled protein (DVL), T-cell factor 4 (TCF4) and multidrug resistance protein 1 (MDRl).
- the inhibitor comprises one or more selected from the group consisting of: an antibody, an antibody fragment, a peptide, a peptidomimetic, a small molecule, and a nucleic acid molecule.
- the modulator of the invention serves as an inhibitor of the expression or activity of a gene or gene product.
- the modulator is an inhibitor of one or more chemoresistance promoting molecule. It will be understood by one skilled in the art, based upon the disclosure provided herein, that inhibition of one or more chemoresistance promoting molecule encompasses the decrease in the expression, including transcription, translation, or both. The skilled artisan will also appreciate, once armed with the teachings of the present invention, that inhibition of one or more chemoresistance promoting molecule includes a decrease in the activity of the one or more chemoresistance promoting molecule.
- inhibition of one or more chemoresistance promoting molecule includes, but is not limited to, decreasing the amount of polypeptide of the one or more chemoresistance promoting molecule, decreasing transcription, translation, or both, of a nucleic acid encoding the one or more chemoresistance promoting molecule; and decreasing any activity of the one or more chemoresistance promoting molecule.
- the modulator of the invention serves as an activator of the expression or activity of a gene or gene product.
- the modulator is an activator of a chemosensitivity promoting molecule.
- the chemosensitivity promoting molecule is one or more selected from the group consisting of: glycogen synthase kinase 3 beta (GSK3 ), Dickkopf-related protein 1 (DKK1), and disabled homolog 2 (DAB2).
- the activator comprises one or more selected from the group consisting of: an antibody, an antibody fragment, a peptide, a peptidomimetic, a small molecule, and a nucleic acid molecule.
- activation of one or more chemosensitivity promoting molecule encompasses the increase in the expression, including transcription, translation, or both.
- activation of one or more chemosensitivity promoting molecule includes an increase in the activity of the one or more chemosensitivity promoting molecule.
- activation of one or more chemosensitivity promoting molecule includes, but is not limited to, increasing the amount of polypeptide of the one or more chemosensitivity promoting molecule, increasing transcription, translation, or both, of a nucleic acid encoding the one or more chemosensitivity promoting molecule; and increasing any activity of the one or more chemosensitivity promoting molecule.
- the modulator of the invention is a small molecule
- Such methods include chemical organic synthesis or biological means.
- Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.
- Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries.
- the method may use a variety of techniques well- known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.
- an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles.
- the shape and rigidity of the core determines the orientation of the building blocks in shape space.
- the libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores.
- the modulator of the one or more chemoresi stance promoting molecule, the one or more chemosensitivity promoting molecule, or combination thereof comprises one or more antibody or antibody fragment.
- the antibody may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“PR”) se t w hich provide support to the CDRs and define the spatial relationship of the CDRs relative to each other.
- the CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3,” respectively.
- An antigen binding site therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
- the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site.
- the enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)2 fragment, which comprises both antigen-binding sites.
- the antibody can be the Fab or F(ab’)2.
- the Fab can include the heavy chain polypeptide and the light chain polypeptide.
- the heavy chain polypeptide of the Fab can include the VH region and the CHI region.
- the light chain of the Fab can include the VL region and CL region.
- the antibody can be an immunoglobulin (Ig).
- the Ig can be, for example, IgA, IgM, IgD, IgE, and IgG.
- the immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide.
- the heavy chain polypeptide of the immunoglobulin can include a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region.
- the light chain polypeptide of the immunoglobulin can include a VL region and CL region.
- the antibody can be a polyclonal or monoclonal antibody.
- the antibody can be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody.
- the humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human
- polyclonal antibodies useful in the present invention are generated by immunizing rabbits according to standard immunological techniques well-known in the art (see, e.g., Harlow et ah, 1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY).
- Such techniques include immunizing an animal with a chimeric protein comprising a portion of another protein such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and/or a moiety such that the antigenic protein of interest is rendered immunogenic (e.g, an antigen of interest conjugated with keyhole limpet hemocyanin, KLH) and a portion comprising the respective antigenic protein amino acid residues.
- the chimeric proteins are produced by cloning the appropriate nucleic acids encoding the marker protein into a plasmid vector suitable for this purpose, such as but not limited to, pMAL-2 or pCMX.
- Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well-known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynski et al. (1988, Blood, 72: 109-115). Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.
- the modulator of the one or more chemoresistance promoting molecule, the one or more chemosensitivity promoting molecule, or combination thereof comprises a nucleic acid molecule.
- said nucleic acid molecule comprises one or more selected from the group consisting of: one or more siRNA, one or more microRNA, one or more shRNA, one or more antisense nucleic acid molecule, one or more ribozyme, one or more killer-tRNA, one or more guide RNA (part of the CRISPR/CAS system), one or more long non-coding RNA, one or more anti-miRNA oligonucleotide, one or more mRNA molecule, and one or more plasmid vector.
- RNA interference is normally triggered by double-stranded RNA (dsRNA) or endogenous microRNA precursors (pri-miRNAs/pre-miRNAs). Since its discovery, RNAi has emerged as a powerful genetic tool for suppressing gene expression in mammalian cells.
- dsRNA double-stranded RNA
- pri-miRNAs/pre-miRNAs endogenous microRNA precursors
- the nucleic acid molecule inhibits the expression or activity of a gene using a stable gene knockdown strategy.
- Stable gene knockdown can be achieved by expression of synthetic short hairpin RNAs (shRNAs).
- shRNAs consist of a stem-loop structure that can be transcribed in cells from an RNA polymerase II or RNA polymerase III promoter on a plasmid construct. It has been shown that expression of shRNA from a plasmid can be stably integrated for constitutive expression, which may provide certain advantages over synthetic siRNA.
- shRNAs as opposed to siRNAs, are synthesized in the nucleus of cells, further processed and transported to the cytoplasm, and then incorporated into the RNA-induced silencing complex (RISC) for activity.
- RISC RNA-induced silencing complex
- said one or more shRNA comprises a sense strand and an antisense strand connected via a loop.
- the antisense strand is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% complementary to mRNA encoding one or more chemoresistance promoting molecule (e.g. CD44v6), thereby leading to RISC- mediated repression of translation of mRNA encoding the one or more chemoresistance promoting molecule (e.g. CD44v6).
- chemoresistance promoting molecule e.g. CD44v6
- the sense strand is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to mRNA encoding one or more chemoresistance promoting molecule (e.g. CD44v6) and complementary to the antisense strand, thereby Watson-Crick base pairing to form the stem of the shRNA stem-loop.
- the antisense strand is 100% complementary to mRNA encoding one or more chemoresi stance promoting molecule (e.g.
- the sense strand is 100% identical to mRNA encoding one or more chemoresi stance promoting molecule (e.g. CD44v6) and complementary to the antisense strand, thereby Watson-Crick base pairing to form the stem of the shRNA stem-loop.
- the sense strand of the shRNA comprises a nucleotide sequence at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length.
- the sense strand comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5.
- the sense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5.
- the antisense strand of the shRNA comprises a nucleotide sequence at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length.
- the antisense strand comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7.
- the antisense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7.
- the shRNA loop comprises a nucleotide sequence at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides in length.
- the shRNA loop comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:8.
- the shRNA loop comprises the nucleotide sequence of SEQ ID NO: 8.
- the shRNA molecule comprises a nucleotide sequence at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, or at least 70 nucleotides in length.
- the shRNA molecule comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:9 or SEQ ID NO: 10. In one embodiment, the shRNA molecule comprises a nucleotide sequence of SEQ ID NO:9 or SEQ ID NO: 10.
- the mRNA encoding one or more chemoresistance promoting molecule comprises mRNA encoding human CD44v6.
- said mRNA encoding human CD44v6 comprises the sequence of SEQ ID NO:2.
- said mRNA encoding human CD44v6 is transcribed from DNA comprising the nucleotide sequence of SEQ ID NO:l.
- said mRNA encoding human CD44v6 encodes a protein comprising the amino acid sequence of SEQ ID NO:3.
- siRNA polynucleotide is an RNA nucleic acid molecule that interferes with RNA activity that is generally considered to occur via a post-transcriptional gene silencing mechanism.
- the siRNA polynucleotide comprises a double- stranded RNA (dsRNA), but is not intended to be so limited and may comprise a single- stranded RNA (see, e.g., Martinez etal ., 2002 Cell 110:563-74).
- siRNA polynucleotide included in the invention may comprise other naturally occurring, recombinant, or synthetic single-stranded or double-stranded polymers of nucleotides (ribonucleotides or deoxyribonucleotides or a combination of both) and/or nucleotide analogues as provided herein (e.g., an oligonucleotide or polynucleotide or the like, typically in 5’ to 3’ phosphodiester linkage).
- nucleotides ribonucleotides or deoxyribonucleotides or a combination of both
- nucleotide analogues as provided herein (e.g., an oligonucleotide or polynucleotide or the like, typically in 5’ to 3’ phosphodiester linkage).
- siRNA polynucleotides comprise double-stranded polynucleotides of about 18-30 nucleotide base pairs.
- siRNA polynucleotides comprise double-stranded polynucleotides of about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, or about 27 base pairs, and in other embodiments about 19, about 20, about 21, about 22 or about 23 base pairs, or about 27 base pairs, whereby the use of “about” indicates that in certain embodiments and under certain conditions the processive cleavage steps that may give rise to functional siRNA polynucleotides that are capable of interfering with expression of a selected polypeptide may not be absolutely efficient.
- siRNA polynucleotides may include one or more siRNA polynucleotide molecules that may differ (e.g., by nucleotide insertion or deletion) in length by one, two, three, four or more base pairs as a consequence of the variability in processing, in biosynthesis, or in artificial synthesis of the siRNA.
- the siRNA polynucleotide of the present invention may also comprise a polynucleotide sequence that exhibits variability by differing (e.g., by nucleotide substitution, including transition or transversion) at one, two, three or four nucleotides from a particular sequence.
- siRNA polynucleotide sequence can occur at any of the nucleotide positions of a particular siRNA polynucleotide sequence, depending on the length of the molecule, whether situated in a sense or in an antisense strand of the double-stranded polynucleotide.
- the nucleotide difference may be found on one strand of a double- stranded polynucleotide, where the complementary nucleotide with which the substitute nucleotide would typically form hydrogen bond base pairing, may not necessarily be correspondingly substituted.
- the siRNA polynucleotides are homogeneous with respect to a specific nucleotide sequence.
- the sense strand of the siRNA comprises a nucleotide sequence at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length.
- the sense strand comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:4 or SEQ ID NO:5.
- the sense strand comprises a nucleotide sequence of SEQ ID NO:4 or SEQ ID NO:5.
- the antisense strand of the siRNA comprises a nucleotide sequence at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length.
- the antisense strand comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:6 or SEQ ID NO:7.
- the antisense strand comprises a nucleotide of SEQ ID NO:6 or SEQ ID NO:7.
- shRNAs or siRNAs of the present invention may affect silencing of the target polypeptide expression to different degrees.
- the shRNAs or siRNAs thus, must first be tested for their effectiveness. Selection of shRNAs or siRNAs are made therefrom based on the ability of a given shRNAs or siRNAs to interfere with or modulate the expression of the target gene. Accordingly, identification of specific shRNA or siRNA polynucleotide sequences that are capable of interfering with expression of a desired target gene requires production and testing of each shRNA or siRNA.
- the methods for testing each shRNA or siRNA and selection of suitable shRNAs or siRNAs for use in the present invention are fully set forth herein the Examples. Since not all siRNAs that interfere with protein expression will have a physiologically important effect, the present disclosure also sets forth various physiologically relevant assays for determining whether the levels of interference with target protein expression using the shRNAs or siRNAs of the invention have clinically relevant significance.
- the nucleic acid molecule is an antisense nucleic acid sequence which is expressed by a plasmid vector.
- the antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of a desired regulator in the cell.
- the invention should not be construed to be limited to inhibiting expression of a regulator by transfection of cells with antisense molecules. Rather, the invention encompasses other methods known in the art for inhibiting expression or activity of a protein in the cell including, but not limited to, the use of a ribozyme, the expression of a non-functional regulator (i.e. transdominant negative mutant) and use of an intracellular antibody.
- Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press).
- Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule thereby inhibiting the translation of genes.
- antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289).
- Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5,190,931.
- antisense molecules of the invention may be made synthetically and then provided to the cell.
- Antisense oligomers of between about 10 to about 30, and in some instances about 15 nucleotides, are easily synthesized and introduced into a target cell.
- Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).
- Ribozymes and their use for inhibiting gene expression are also well known in the art (see, e.g., Cech et al., 1992, J. Biol. Chem. 267:17479-17482; Hampel et ah, 1989, Biochemistry 28:4929-4933; Eckstein et al., International Publication No. WO 92/07065; Altman et al., U.S. Patent No. 5,168,053).
- Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences encoding these RNAs, molecules can be engineered to recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J. Amer. Med.
- ribozymes There are two basic types of ribozymes, namely, tetrahymena-type (Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while hammerhead-type ribozymes recognize base sequences 11-18 bases in length. The longer the sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating specific mRNA species, and 18-base recognition sequences are preferable to shorter recognition sequences, which may occur randomly within various unrelated mRNA molecules.
- Ribozymes useful for inhibiting the expression of a regulator may be designed by incorporating target sequences into the basic ribozyme structure, which are complementary to the mRNA sequence of the desired target of the present invention. Ribozymes targeting the desired regulator may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.
- a miRNA or a synthetic miRNA is used as a therapeutic agent to regulate gene expression.
- the miRNA may contain one or more design elements. These design elements include, but are not limited to: i) a replacement group for the phosphate or hydroxyl of the nucleotide at the 5' terminus of the complementary region; ii) one or more sugar modifications in the first or last 1 to 6 residues of the complementary region; or, iii) noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3' end of the complementary region and the corresponding nucleotides of the miRNA region.
- a synthetic miRNA has a nucleotide at its 5' end of the complementary region in which the phosphate and/or hydroxyl group has been replaced with another chemical group (referred to as the “replacement design”).
- the phosphate group is replaced, while in others, the hydroxyl group has been replaced.
- the replacement group is biotin, an amine group, a lower alkylamine group, an acetyl group, 2'0-Me (2' oxygen-methyl), DMTO (4,4'- dimethoxytrityl with oxygen), fluoroscein, a thiol, or acridine, though other replacement groups are well known to those of skill in the art and can be used as well.
- Additional embodiments concern a synthetic miRNA having one or more sugar modifications in the first or last 1 to 6 residues of the complementary region (referred to as the “sugar replacement design”).
- sugar modifications there are one or more sugar modifications in the first 1, 2, 3, 4, 5, 6 or more residues of the complementary region, or any range derivable therein.
- first and “last” are with respect to the order of residues from the 5' end to the 3' end of the region.
- the sugar modification is a 2'0-Me modification.
- noncomplementarity design there is a synthetic miRNA in which one or more nucleotides in the last 1 to 5 residues at the 3' end of the complementary region are not complementary to the corresponding nucleotides of the miRNA region.
- the noncomplementarity may be in the last 1, 2, 3, 4, and/or 5 residues of the complementary miRNA.
- the miRNA region and the complementary region may be on the same or separate polynucleotides. In cases in which they are contained on or in the same polynucleotide, the miRNA molecule will be considered a single polynucleotide. In embodiments in which the different regions are on separate polynucleotides, the synthetic miRNA will be considered to be comprised of two polynucleotides.
- the RNA molecule is a single polynucleotide
- the single polynucleotide is capable of forming a hairpin loop structure as a result of bonding between the miRNA region and the complementary region.
- the linker constitutes the hairpin loop. It is contemplated that in some embodiments, the linker region is, is at least, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
- the linker is between 3 and 30 residues (inclusive) in length.
- flanking sequences as well at either the 5' or 3' end of the region.
- the nucleic acid molecule comprises one or more guide RNA specific to one or more target sequence (i.e. nucleic acid sequence) to be inhibited by one or more CAS enzyme via the CRISPR/CAS system.
- said target sequence comprises a nucleic acid sequence encoding one or more chemoresi stance promoting molecule (e.g. CD44v6).
- a “target sequence” refers to a sequence to which a CRISPR RNA (crRNA) sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
- a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
- a target sequence is located in the nucleus or cytoplasm of a cell.
- the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast.
- the ability of a crRNA to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay.
- the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence.
- cleavage of a target nucleotide sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
- Other assays are possible, and will occur to those skilled in the art.
- a crRNA sequence, and hence a nucleic acid-targeting crRNA may be selected to target any target nucleic acid sequence.
- the nucleic acid molecule comprises a DNA, RNA, cDNA molecule, or the like which encodes one or more modulator of one or more chemoresi stance promoting molecule (e.g. CD44v6), one or more chemosensitivity promoting molecule, or combination thereof.
- the nucleic acid molecule may be transcribed and/or translated into one or more modulator of one or more chemoresi stance promoting molecule (e.g. CD44v6), one or more chemosensitivity promoting molecule, or combination thereof.
- the nucleic acid molecule may be transcribed into a shRNA specific for inhibiting CD44v6.
- the present invention relates to a composition
- a composition comprising one or more nucleic acid molecule encoding one or more modulator of one or more chemoresi stance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof.
- said one or more nucleic acid molecule comprises one or more expression vector.
- the one or more vectors can contain an origin of replication.
- the one or more vectors can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
- the one or more vectors can be either a self-replication extra chromosomal vector, or a vector which integrates into a host genome.
- Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA” vector, and the like.
- a “vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
- the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
- Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
- Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and include both the expression and non-expression plasmids.
- the vector includes linear DNA, enzymatic DNA or synthetic DNA.
- a recombinant microorganism or cell culture is described as hosting an "expression vector" this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s).
- the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
- the one or more vectors can be a plasmid.
- the plasmid may be useful for transfecting cells with the recombinant nucleic acid sequence construct.
- the plasmid may be useful for introducing the recombinant nucleic acid sequence construct into the subject.
- the plasmid may also comprise a regulatory sequence, which may be well suited for gene expression in a cell into which the plasmid is administered.
- the plasmid may also comprise a mammalian origin of replication in order to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in a cell.
- the plasmid may be pVAXl, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may comprise the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region, which may produce high copy episomal replication without integration.
- the backbone of the plasmid may be pAV0242.
- the plasmid may be a replication defective adenovirus type 5 (Ad5) plasmid.
- the plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may be used for protein production in Escherichia coli (E.coli).
- the plasmid may also be pYES2 (Invitrogen, San Diego, Calif.), which may be used for protein production in Saccharomyces cerevisiae strains of yeast.
- the plasmid may also be of the MAXBACTM complete baculovirus expression system (Invitrogen, San Diego, Calif.), which may be used for protein production in insect cells.
- the plasmid may also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which may be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.
- the one or more vectors may be circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).
- the vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
- said one or more expression vector comprises one or more plasmid vector. In one embodiment, said one or more expression vector comprises at least two plasmid vectors. In one embodiment, said at least two plasmid vectors comprises a first plasmid vector and a second plasmid vector.
- said first plasmid vector comprises a conditionally expressed vector.
- said conditionally expressed vector comprises at least two recombinase target sequences.
- said at least two recombinase target sequences comprise at least two Lox sequences.
- said at least two Lox sequence are present in the same orientation.
- said at least two Lox sequences flank either side of a sequence encoding a stop codon, thereby forming a stop cassette.
- said stop cassette is positioned upstream of a sequence encoding one or more shRNA of the present invention.
- the stop cassette is positioned downstream of an RNA polymerase III (RNA pol III) promoter.
- the RNA pol III promoter comprises a type 1, type 2 or type 3 RNA pol III promoter. In one embodiment, the RNA pol III promoter comprises a type 3 RNA pol III promoter. In one embodiment, the type 3 RNA pol III promoter comprises a U6 promoter. In one embodiment, the components off the vector are arranged in the following order, from 5’ to 3’: U6-Lox-Stop-Lox-shRNA. In the presence of a recombinase, recombination of the Lox sequences in the same orientation excises the stop cassette, allowing the U6 promoter to driver expression of shRNA.
- the first vector comprises a pSico (plasmid for Stable RNA interference, conditional) vector.
- the first vector comprises a pSicoR (plasmid for Stable RNA interference, conditional, reverse) vector.
- pSico allows for conditional (Cre-Lox-dependent), stable expression of shRNAs for RNA interference in cells and transgenic mice. Addition of Cre TURNS ON shRNA expression.
- pSicoR allows for conditional (Cre-Lox-dependent) repression of shRNAs for RNA interference in cells and transgenic mice. Addition of Cre TURNS OFF shRNA expression (Ventura A, et al., Proc Natl Acad Sci USA, 2004, 101(28): 10380-5).
- the sequence encoding one or more shRNA of the present invention comprises a forward oligonucleotide and reverse oligonucleotide that are annealed and then inserted into the first vector via restriction digestion and ligation.
- said forward oligonucleotide comprises a nucleotide at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11 or SEQ ID NO: 13. In one embodiment, said forward oligonucleotide comprises SEQ ID NO: 11 or SEQ ID NO: 13.
- said reverse oligonucleotide comprises a nucleotide sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12 or SEQ ID NO: 14. In one embodiment, said reverse oligonucleotide comprises SEQ ID NO: 12 or SEQ ID NO: 14.
- the first vector comprises a nucleotide sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.
- the first vector comprises a nucleotide sequence of SEQ ID NO: 18,
- SEQ ID NO: 19 SEQ ID NO:20 or SEQ ID NO:21.
- said second plasmid vector comprises a tissue-specific expression vector. In one embodiment, the second plasmid vector comprises a tissue- specific promoter. In one embodiment, the tissue-specific promoter limits expression to the epithelium of the intestine, colon or a combination thereof. In one embodiment, the tissue-specific promoter is a pFabpl promoter. In one embodiment, said pFabpl promoter comprises a nucleotide sequence of SEQ ID NO: 16.
- said second plasmid vector comprises a nucleotide sequence encoding a recombinase.
- the nucleotide sequence encoding the recombinase is downstream of the tissue-specific promoter.
- the recombinase is Cre-recombinase.
- said Cre-recombinase comprises an amino acid sequence of SEQ ID NO: 15.
- the tissue specific promoter of the second plasmid is operably linked to the sequence encoding the recombinase.
- the presence of the tissue-specific promoter limits expression of the recombinase to the tissue type(s) in which the promoter is activated.
- the second vector comprises a nucleotide sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of SEQ ID NO:22.
- the second vector comprises a nucleotide sequence of SEQ ID NO:22.
- the composition of the present invention comprises said first plasmid vector encapsulated by a nanoparticle and said second plasmid vector encapsulated by a nanoparticle.
- Co-delivery of the vectors to a subject in need thereof ensures that the recombinase encoded by the second plasmid vector is only expressed in target tissues limited by the promoter, which in turn limits expression of the shRNA encoded by the first plasmid vector to said target tissues.
- one or more nucleic acid molecule may be provided as a linear nucleic acid, or linear expression cassette (“LEC”), that is capable of being efficiently delivered to a subject (for example via electroporation) and expressing the inhibitor encoded by the nucleic acid sequence.
- LEC linear expression cassette
- the LEC may be any linear DNA devoid of any phosphate backbone.
- the LEC may not contain any antibiotic resistance genes and/or a phosphate backbone.
- the LEC may not contain other nucleic acid sequences unrelated to the desired gene expression.
- the expression vector is a viral vector.
- Viral vectors are provided herein which are capable of delivering a nucleic acid of the invention to a cell.
- the expression vector may be provided to a cell in the form of a viral vector.
- Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), and in Ausubel et al. (1997), and in other virology and molecular biology manuals.
- Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
- a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.
- a promoter sequence for example, WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193.
- Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
- Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
- the one or more vectors can be formulated or manufactured using a combination of known devices and techniques. In some embodiments, they are manufactured using a plasmid manufacturing technique that is described in a licensed, co-pending U.S. provisional application U.S. Serial No. 60/939,792, which was filed on May 23, 2007. In some examples, the DNA plasmids described herein can be formulated at concentrations greater than or equal to 10 mg/mL.
- the manufacturing techniques also include or incorporate various devices and protocols that are commonly known to those of ordinary skill in the art, in addition to those described in U.S. Serial No. 60/939792, including those described in a licensed patent, US Patent No. 7,238,522, which issued on July 3, 2007.
- the above-referenced application and patent, US Serial No. 60/939,792 and US Patent No. 7,238,522, respectively, are hereby incorporated in their entirety.
- the present invention relates to a composition comprising one or more modulator of one or more chemoresistance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof, encapsulated within a nanoparticle for delivery to a cell or tissue of a subject in need thereof.
- the present invention relates to a composition comprising a nucleic acid encoding one or more modulator of one or more chemoresistance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof, encapsulated within a nanoparticle.
- any nanoparticles known in the art as suitable for the delivery of one or more modulator of one or more chemoresistance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof, or a nucleic acid molecule encoding one or more modulator of one or more chemoresi stance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof, can be used in the compositions and methods of the present invention.
- nanoparticle refers to a particle having at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500, 1000 nm, and all integers and fractional integers in between).
- the nanoparticle has at least one dimension, e.g., a diameter, of about 100 nm.
- the nanoparticle has a diameter of about 200 nm, a diameter of about 500 nm, or a diameter of about 1000 nm (1 pm).
- Nanoparticles having a diameter of at least 1000 nm also may be referred to as a “microparticle.”
- microparticle includes particles having at least one dimension in the range of about one micrometer (pm), i.e., 1 xlO 6 meters, to about 1000 pm.
- pm micrometer
- particle as used herein is meant to include nanoparticles and microparticles.
- Nanoparticles suitable for use in the presently disclosed compositions and methods may exist in a variety of shapes, including, but not limited to, spheroids, rods, disks, pyramids, cubes, cylinders, nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles, arrow-shaped nanoparticles, teardrop-shaped nanoparticles, tetrapod- shaped nanoparticles, prism-shaped nanoparticles, and a plurality of other geometric and non-geometric shapes.
- the disclosed nanoparticles have a spherical shape.
- the nanoparticles of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition (2005), Lippincott Williams & Wilkins, Philadelphia, Pa.).
- the compound (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier.
- the carrier can be a solid or a liquid, or both.
- the carrier is formulated with the compound as a unit- dose formulation, for example, a tablet, which can contain from 0.01 or 0.5% to 95% or 99% by weight of the compound.
- One or more compounds can be incorporated in the formulations of the invention, which can be prepared by any of the well-known techniques of pharmacy.
- the nanoparticle comprises one or more selected from the group consisting of: a polyalkylene glycol, a polycation and a targeting moiety. In one embodiment, the nanoparticle comprises a polyalkylene glycol, a polycation and a targeting moiety.
- Polyalkylene glycol means straight or branched polyalkylene glycol polymers including, but not limited to, polyethylene glycol (PEG), polypropylene glycol (PPG), and polybutylene glycol (PBG), as well as co-polymers of PEG, PPG and PBG in any combination, and includes the monoalkylether of the polyalkylene glycol.
- the polyalkylene glycol in the nanoparticles of this invention can be, but is not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and any combination thereof.
- the polyalkylene glycol of the nanoparticle is polyethylene glycol or “PEG.”
- PEG subunit refers to a single polyethylene glycol unit, i.e., — (CH2CH20) — .
- PEG of any suitable molecular weight may be employed in the nanoparticle, and is available over a wide range of molecular weights.
- the PEG molecular weight may be, for example, between about 300 g/mol to about 10,000,000 g/mol (e.g., about 600, 1,000, 5,000, 10,000 g/mol, or a range defined by any two of the foregoing values).
- the nanoparticle of the present invention comprises a polycation.
- polycation refers to a compound having a positive charge, for example, at least 2 positive charges, at a selected pH, for example, at physiological pH.
- Poly cationic moieties have between about 2 to about 15 positive charges, for example, between about 2 to about 12 positive charges.
- the polycation has between about 2 to about 8 positive charges at selected pH values.
- Suitable constituents of polycations include basic amino acids and their derivatives such as arginine, asparagine, glutamine, lysine and histidine; cationic dendrimers; and amino polysaccharides.
- Suitable polycations can be linear, such as linear tetralysine, branched or dendrimeric in structure.
- the polycation can be, but is not limited to polyethyleneimine, polyethylenimine, poly(allylanion hydrochloride; PAH), putrescine, cadaverine, polylysine, poly-arginine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy- diamino-b-cyclodextrin, spermine, spermidine, cadaverine, poly(2-dimethylamino)ethyl methacrylate, poly(histidine), cationized gelatin, dendrimers, chitosan, and any combination thereof.
- PAH putrescine
- Exemplary polycations also include, but are not limited to, synthetic polycations based on acrylamide and 2-acrylamido-2-methylpropanetrimethylamine, poly(N-ethyl-4-vinylpyridine) or similar quartemized polypyridine, diethylaminoethyl polymers and dextran conjugates, polymyxin B sulfate, lipopoly amines, poly(allylamines) such as the strong polycation poly(dimethyldiallylammonium chloride), polyethyleneimine, polybrene, and polypeptides such as protamine, the histone polypeptides, polylysine, polyarginine and polyomithine.
- synthetic polycations based on acrylamide and 2-acrylamido-2-methylpropanetrimethylamine
- poly(N-ethyl-4-vinylpyridine) or similar quartemized polypyridine diethylaminoethyl polymers and dextran conjugates
- the polycation is polyethylenimine (PEI).
- PEI polyethylenimine
- the amine-rich cationic polymer polyethylenimine (PEI) is an efficient nucleotide carrier that binds to the negatively-charged phosphate backbone of nucleotides and negatively-charged elements of cell membranes, facilitating endocytotic uptake of PEI-nucleotide complexes into cells (Bieber, T et. al. (2002) J. Control Release V82.441-454.; Boussif, O et. al. (1995) Proc. Natl. Acad. Sci. U.S.A V92. 7297-7301.; Godbey, W T et. al.
- the polycation e.g., PEI
- an active agent of this invention e.g., a polynucleotide, an oligonucleotide, an anionic protein, an anionic drug, a polynucleotide or oligonucleotide covalently bonded to a peptide or protein, as well as any combination thereof
- physical electrostatic force e.g., wherein the negative charges in the active agent(s) bind with the positive charges in the polycation.
- the nanoparticle is functionalized for enhanced delivery to target tissues and enhanced cellular uptake of the one or more modulator of one or more chemoresi stance promoting molecule, the one or more chemosensitivity promoting molecule, or a combination thereof.
- Addition of functional groups to the nanoparticle can improve stability, improve biodistribution, improve tissue delivery of the cargo (i.e. the one or more inhibitor of the one or more chemoresi stance promoting molecule), improve nanoparticle and/or cellular uptake, and any combinations thereof.
- Non-limiting examples of functional groups are gold nanoparticles (GNP), TAT-PTD and derivatives thereof, ApoE, albumin, antibody, antibody fragment, magnetic nanoparticle, iron oxide, transferrin, AAV tropism fragment, and combinations thereof.
- the functional group is a targeting moiety.
- the targeting moiety is transferrin.
- Transferrin is the protein that transports iron in human and animal plasma, in which its concentration is approximately 2.5 g/1. This major function of transferrin derives from its ability to specifically bind trivalent iron. Once iron is resorbed into the small intestine or picked up by the iron-storage protein ferritin, it is transported in the trivalent form to other tissues. (U.S. Pat. No. 5,041,537). Transferrin receptors are highly expressed in colon tumors. Thus, transferrin promotes targeting to and uptake of functionalized nanoparticles in colorectal cancer cells.
- transferrin comprises an amino acid sequence of SEQ ID NO: 17.
- the nanoparticle comprises a targeting moiety comprising SEQ ID NO: 17 or a fragment or variant thereof.
- the nanoparticle comprises a targeting moiety comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17.
- the nanoparticle comprises transferrin (Tf; SEQ ID NO: 17) C-terminally conjugated to polyethylene glycol (PEG) which in turn is conjugated to polyethylenimine (PEI) in the order Tf-PEG-PEI (see Bellocq NC, et al., 2003, Bioconjug Chem, 14(6): 1122-32; Misra, S., et al., 2009, J Biol Chem, 284: 12432- 12446; Ghatak, S., et al., 2008, Connect Tissue Res, 49: 265-269; Ghatak S, et al., J Biol Chem, 2017, 292(25): 10490-10519, each of which are incorporated herein by reference in their entireties)
- Tf transferrin
- PEG polyethylene glycol
- PEI polyethylenimine
- the present invention generally relates to methods of administering one or more modulator of one or more chemoresi stance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof, to a cell or tissue of a subject in need thereof.
- the chemoresi stance promoting molecule is one or more selected from the group consisting of: PN, WNT3A, IL-17A and CD44v6.
- the chemosensitivity promoting molecule comprises one or more selected from the group consisting of: GSK3 , DKK1, and DAB2.
- the modulator comprises one or more selected from the group consisting of: an antibody, an antibody fragment, a peptide, a peptidomimetic, a small molecule, and a nucleic acid molecule.
- the nucleic acid molecule comprises an shRNA capable of reducing expression of one or more chemoresi stance promoting molecule.
- the nucleic acid molecule comprises a nucleic acid molecule encoding one or more inhibitor of one or more chemoresi stance promoting molecule.
- the nucleic acid molecule comprises a nucleic acid molecule encoding one or more shRNA capable of reducing expression of one or more chemoresi stance promoting molecule.
- the one or more modulator of one or more chemoresi stance promoting molecule, the one or more chemosensitivity promoting molecule, or combination thereof, of the invention may be prepared as pharmaceutical compositions containing an effective amount of the modulator as an active ingredient in a pharmaceutically acceptable carrier.
- Carrier refers to a diluent, adjuvant, excipient, or vehicle with which the antibody of the invention is administered.
- Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
- 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter.
- compositions may be sterilized by conventional, well-known sterilization techniques (e.g., filtration).
- the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc.
- concentration of the one or more modulator of the invention in such pharmaceutical formulation may vary, from less than about 0.5%, usually to at least about 1% to as much as 15 or 20% by weight and may be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the mode of administration selected.
- Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia,
- the mode of administration of the one or more modulator may be any suitable route such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intratumoral, intravenous or subcutaneous, transmucosal (oral, intranasal, intravaginal, rectal) or other means appreciated by the skilled artisan, as well known in the art.
- parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intratumoral, intravenous or subcutaneous
- transmucosal oral, intranasal, intravaginal, rectal
- the one or more modulator is administered during surgical resection of a tumor.
- a pharmaceutical composition of the invention for intramuscular injection may be prepared to contain 1 ml sterile buffered water, and between about 1 ng to about 100 mg/kg, e.g. about 50 ng to about 30 mg/kg of the one or more modulator of one or more chemoresi stance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof.
- the pharmaceutical composition comprises about 5 mg to about 25 mg/kg of the one or more modulator.
- a pharmaceutically acceptable carrier can be an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to the subject.
- a pharmaceutically acceptable carrier can include a buffer, excipient, stabilizer, or preservative.
- pharmaceutically acceptable carriers are solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, such as salts, buffers, antioxidants, saccharides, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifying agents, or combinations thereof.
- the amounts of pharmaceutically acceptable carrier(s) in the pharmaceutical compositions may be determined experimentally based on the activities of the carrier(s) and the desired characteristics of the formulation, such as stability and/or minimal oxidation.
- compositions may comprise buffers such as acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffers, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); antibacterial and antifungal agents; and preservatives.
- buffers such as acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffers, HEPPSO, HEPES, neutral buffered saline, phosphate buffered s
- compositions of the present invention can be formulated for a variety of means of parenteral or non-parenteral administration.
- the compositions can be formulated for infusion or intravenous administration.
- Pharmaceutical compositions disclosed herein can be provided, for example, as sterile liquid preparations, e.g., isotonic aqueous solutions, emulsions, suspensions, dispersions, or viscous compositions, which may be buffered to a desirable pH.
- Formulations suitable for oral administration can include liquid solutions, capsules, sachets, tablets, lozenges, and troches, powders liquid suspensions in an appropriate liquid and emulsions.
- the present invention generally relates to methods of diagnosing cancer in a subject as chemotherapeutic resistant and treating said cancer with a composition to target mechanisms of resistance.
- the method comprises: a) obtaining a sample from the subject, b) measuring the expression level one or more chemoresistance promoting molecule, c) detecting an elevated level of said chemoresi stance promoting molecule as compared to a control sample (i.e. a comparator), and d) diagnosing said subject with chemotherapeutic resistant cancer.
- the method further comprises administering to the subject one or more inhibitor of one or more chemoresistance promoting molecule in said cancer.
- the method further comprises administering to the subject one or more activator of one or more chemosensitivity promoting molecule in said cancer.
- the method comprises: a) obtaining a sample from the subject, b) measuring the expression level one or more chemosensitivity promoting molecule, c) detecting an depressed level of said chemosensitivity promoting molecule as compared to a control sample (i.e. a comparator), and d) diagnosing said subject with chemotherapeutic resistant cancer.
- the method further comprises administering to the subject one or more inhibitor of one or more chemoresistance promoting molecule in said cancer.
- the method further comprises administering to the subject one or more activator of one or more chemosensitivity promoting molecule in said cancer.
- the present invention generally relates to methods of assessing the risk of developing chemotherapeutic resistant cancer in a subject and preventing progression of said chemotherapeutic resistant cancer with a composition to target mechanisms of resistance.
- the method comprises: a) obtaining a sample from the subject, b) measuring the expression level one or more chemoresistance promoting molecule, c) detecting an elevated level of said chemoresistance promoting molecule as compared to a control sample (i.e. a comparator), and d) determining that said subject is at risk of developing chemotherapeutic resistant cancer.
- the method further comprises administering to the subject one or more inhibitor of one or more chemoresistance promoting molecule in said cancer.
- the method further comprises administering to the subject one or more activator of one or more chemosensitivity promoting molecule in said cancer.
- the method comprises: a) obtaining a sample from the subject, b) measuring the expression level one or more chemosensitivity promoting molecule, c) detecting a depressed level of said chemosensitivity promoting molecule as compared to a control sample (i.e. a comparator), and d) determining that said subject is at risk of developing chemotherapeutic resistant cancer.
- the method further comprises administering to the subject one or more inhibitor of one or more chemoresi stance promoting molecule in said cancer.
- the method further comprises administering to the subject one or more activator of one or more chemosensitivity promoting molecule in said cancer.
- said measuring of the expression level of one or more chemoresi stance promoting molecule or chemosensitivity promoting molecule includes measuring the level of RNA or messenger RNA (mRNA).
- mRNA messenger RNA
- any methods known in the art for measuring the level of RNA in a sample can be used in the methods of the present invention.
- mRNA can be detected by methods including but not limited to mass spectroscopy, PCR microarray, thermal sequencing, capillary array sequencing, solid phase sequencing, and the like.
- said measuring of the expression level of one or more chemoresi stance promoting molecule or chemosensitivity promoting molecule includes measuring the level of protein. It should be recognized that any methods known in the art for measuring the level of protein in a sample can be used in the methods of the present invention.
- the protein may be detected by methods including but not limited to ELISA, Western blot, flow cytometry, immunofluorescence, immunohistochemistry, mass spectroscopy, and the like.
- the one or more chemoresi stance promoting molecule comprises CD44v6. In one embodiment, the one or more composition comprises an inhibitor of CD44v6. In one embodiment, the one or more composition comprises a nucleic acid molecule encoding an inhibitor of CD44v6. In one embodiment, the inhibitor is one or more shRNA molecule targeting CD44v6 mRNA.
- Non-limiting examples of comparators include, but are not limited to, a negative control, a positive control, standard control, standard value, an expected normal background value of the subject, a historical normal background value of the subject, a reference standard, a reference level, an expected normal background value of a population that the subject is a member of, or a historical normal background value of a population that the subject is a member of.
- the comparator is a level of one or more chemoresistance promoting molecule or chemosensitivity promoting molecule in a sample obtained from a subject not having a disease or disorder.
- the comparator is a level of one or more chemoresistance promoting molecule or chemosensitivity promoting molecule in a sample obtained from a subject known not to have a disease or disorder.
- the level of the one or more chemoresistance promoting molecule is determined to be elevated when the level of the one or more chemoresistance promoting molecule in the sample is increased by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, by at least 1000%, by at least 1500%, by at least 2000%, by at least 2500%, by at least 3000%, by at least 4000%, or by at least 5000%, when compared with a comparator.
- the level of the one or more chemoresistance promoting molecule is determined to be elevated when the level of the one or more chemoresistance promoting molecule in the sample is increased by at least 1 fold, at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 1 fold, at least 1.1 fold, at
- the level of the one or more chemosensitivity promoting molecule is determined to be depressed when the level of the one or more chemosensitivity promoting molecule in the sample is decreased by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, by at least 1000%, by at least 1500%, by at least
- the level of the one or more chemosensitivity promoting molecule is determined to be depressed when the level of the one or more chemoresistance promoting molecule in the sample is decreased by at least 1 fold, at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 1 fold, at least 1.1 fold, at
- the present invention generally relates to methods of treating or preventing one or more disease or disorder associated with chemotherapeutic resistance.
- said method comprises administering to the subject one or more modulator of one or more chemoresistance promoting molecule, one or more chemosensitivity promoting molecule, or a combination thereof.
- the method comprises administering one or more inhibitor of CD44v6.
- the method comprises administering one or more nucleic acid molecule encoding one or more inhibitor of CD44v6.
- the one or more inhibitor comprises shRNA directed against CD44v6 mRNA.
- said disease or disorder is cancer.
- the cancer is a chemoresistant cancer.
- types of cancers that can be treated or prevented by the methods and compositions of the disclosure include solid tumor cancers, liquid cancers, blood cancers, teratomas, sarcomas, and carcinomas.
- cancers that can be treated or prevented by the methods and compositions of the invention: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumor, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cerebral astrocytotna/malignant glioma, cervical cancer, childhood visual pathway tumor, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngio
- said cancer is colorectal cancer. In one embodiment, the cancer is chemoresistant colorectal cancer.
- the precise amount of the one or more modulator of one or more chemoresi stance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof, of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, and condition of the subject.
- Delivery systems useful in the context of the one or more modulator of one or more chemoresistance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof, of the invention may include time-released, delayed release, and sustained release delivery systems such that the delivery of the one or more modulator occurs prior to, and with sufficient time to cause, sensitization of the site to be treated.
- the composition can be used in conjunction with other therapeutic agents or therapies. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain composition embodiments of the invention.
- release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide- glycolide), copolyoxalates, polyesteramides, polyorthoesters, polycaprolactones, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109.
- Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di- and tri glycerides; sylastic systems; peptide based systems; hydrogel release systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
- lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di- and tri glycerides
- sylastic systems such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di- and tri glycerides
- sylastic systems such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di- and tri glycerides
- peptide based systems such as fatty acids or neutral fats such as mono-di- and tri glycerides
- hydrogel release systems such as those described
- the administration of the one or more modulator of one or more chemoresistance promoting molecule, one or more chemosensitivity promoting molecule, a combination thereof, and compositions thereof may be carried out in any manner, e.g., by parenteral or nonparenteral administration, including by aerosol inhalation, injection, infusions, ingestion, transfusion, implantation or transplantation.
- the one or more modulator and compositions thereof described herein may be administered to a patient trans-arterially, intradermally, subcutaneously, intratumorally, intramedullary, intranodally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
- the compositions of the present disclosure are administered by i.v.
- compositions of the present disclosure are administered to a subject by intradermal or subcutaneous injection.
- the compositions of one or more modulator may be injected, for instance, directly into a tumor, lymph node, tissue, organ, or site of infection.
- the compositions of the present disclosure are administered during surgical resection or debulking of a tumor.
- administration may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer.
- Repeated courses of treatment are also possible, as is chronic administration.
- the repeated administration may be at the same dose or at a different dose.
- the one or more modulator of one or more chemoresi stance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof, of the invention may be administered in combination with at least one additional therapeutic.
- the one or more modulator of the disclosure may be administered in combination with one or more other therapies useful for the prevention, management, treatment or amelioration of a disease or disorder or one or more symptoms thereof to a subject in need thereof to prevent, manage, treat or ameliorate a disease or disorder or one or more symptoms thereof.
- the at least one additional therapeutic comprises one or more inhibitor of one or more chemoresi stance promoting molecule.
- the one or more inhibitor can be one or more selected from the group consisting of: an antibody, an antibody fragment, a peptide, a peptidomimetic, a small molecule, and a nucleic acid molecule.
- the one or more chemoresi stance promoting molecule comprises one or more selected from the group consisting of: WNT3A, Periostin, IL-17A, b-catenin, LRP6, Frizzled, DVL, TCF4 and MDR1.
- the one or more inhibitor of one or more chemoresi stance promoting molecule comprises one or more selected from the group consisting of: a blocking antibody, an shRNA, and a nucleic acid encoding an shRNA.
- the at least one additional therapeutic comprises one or more activator of one or more chemosensitivity promoting molecule.
- the one or more activator can be one or more selected from the group consisting of: an antibody, an antibody fragment, a peptide, a peptidomimetic, a small molecule, and a nucleic acid molecule.
- the one or more chemosensitivity promoting molecule comprises one or more selected from the group consisting of: GSK3 , DKK1 and DAB2.
- the one or more activator of one or more chemosensitivity promoting molecule comprises one or more selected from the group consisting of: a recombinant protein of said one or more chemosensitivity promoting molecule, and a nucleic acid encoding a recombinant protein of said one or more chemosensitivity promoting molecule.
- the at least one additional therapeutic comprises one or more chemotherapeutic agent.
- the at least one additional therapeutic comprises one or more cancer immunotherapeutic agent.
- the cancer immunotherapeutic agent comprises an agent that opsonizes a target cell.
- An agent that opsonizes a target cell is any agent that can bind to a target cell (e.g., a cancer cell) and opsonize the target cell (e.g., mark the target cell for phagocytosis and/or for antibody-dependent cell mediated cytotoxicity (ADCC)).
- ADCC antibody-dependent cell mediated cytotoxicity
- any antibody that can bind to a target cell e.g., a cancer cell such as a tumor cell
- the antibody has an FC region
- the agent that opsonizes a target cell is an antibody that binds to a target cell (e.g., an anti -turn or antibody, an anti-cancer antibody, and the like).
- that agent that opsonizes the target cell is Rituximab.
- Rituximab is a chimeric unconjugated monoclonal antibody directed at the CD20 antigen.
- CD20 has an important functional role in B cell activation, proliferation, and differentiation.
- that agent that opsonizes the target cell is Cetuximab. Cetuximab binds to the EGF receptor (EGFR), and has been used in the treatment of solid tumors including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).
- EGFR EGF receptor
- the cancer immunotherapeutic agent comprises a specific antibody.
- exemplary antibodies selective for tumor cell markers, radiation, surgery, and/or hormone deprivation seeKwon et al. , Proc. Natl. Acad. Sci U.S.A., 96: 15074-9, 1999.
- Angiogenesis inhibitors can also be combined with the methods of the invention.
- a number of antibodies are currently in clinical use for the treatment of cancer, and others are in varying stages of clinical development. For example, there are a number of antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies.
- the CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for treatment of chronic lymphocytic leukemia.
- CD22 is targeted by a number of antibodies, and has recently demonstrated efficacy combined with toxin in chemotherapy-resistant hairy cell leukemia.
- Alemtuzumab (Campath) is used in the treatment of chronic lymphocytic leukemia;
- Gemtuzumab Mylotarg finds use in the treatment of acute myelogenous leukemia;
- Ibritumomab (Zevalin) finds use in the treatment of non-Hodgkin's lymphoma;
- Panitumumab (Vectibix) finds use in the treatment of colon cancer.
- Monoclonal antibodies useful in the methods of the disclosure that have been used in solid tumors include, without limitation, edrecolomab and trastuzumab (herceptin).
- Edrecolomab targets the 17-1A antigen seen in colon and rectal cancer, and has been approved for use in Europe for these indications.
- trastuzumab targets the HER- 2/neu antigen.
- the cancer immunotherapeutic agent is one or more selected from the group consisting of: cetuximab (binds EGFR), panitumumab (binds EGFR), rituximab (binds CD20), trastuzumab (binds HER2), pertuzumab (binds HER2), alemtuzumab (binds CD52), brentuximab (binds CD30), tositumomab, ibritumomab, gemtuzumab, ibritumomab, and edrecolomab (binds 17-1A), and a combination thereof.
- the cancer immunotherapeutic agent comprises an antigen binding region that targets one or more selected from the group consisting of:
- CD 19 CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD 134, CD 138, CD 152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7- H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1 A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FG
- the cancer immunotherapeutic agent comprises an antigen binding region that targets one or more selected from the group consisting of:
- CD 19 CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, SIRPA, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR, 17- 1 A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).
- the cancer immunotherapeutic agent comprises an immunomodulatory agent.
- the immunomodulatory agent includes, but not limited to, an anti-CTLA4 antibody, an anti -PD-1 antibody, an anti-PD-Ll antibody, a TIGIT antibody, a TIM3 antibody, a LAG3 antibody, a VISTA antibody, a B7H3 antibody, a B7H4 antibody, a CD40 agonist, a 4-1BB modulator (e.g., a 41BB- agonist), an OX-40 modulator (e.g., an OX-40 agonist), a GITR modulator (e.g., a GITR agonist), a CD47 binding agent such as an anti-CD47 antibody or a high affinity CD47 binding agent, a SIRPA binding agent such as an anti-SIRPA antibody or high affinity SIRPA binding agent, and the like), a TGFbeta antagonist such as an anti-TGFbeta antibody, a cytokine or a
- the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”.
- the delivery of one treatment ends before the delivery of the other treatment begins.
- the treatment is more effective because of combined administration.
- the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.
- delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
- the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
- the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
- other therapeutic agents may be administered before, after, or at the same time (simultaneous with) as the one or more modulator.
- the one or more modulator of one or more chemoresi stance promoting molecule, one or more chemosensitivity promoting molecule, or combination thereof, and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially.
- the one or more modulator described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
- Example 1 A positive feedback loop between b-catenin/MDRl signaling and CD44v6 splicing sustains chemoresi stance of cancer initiating cells in colorectal cancer
- WNT/p-catenin signaling pathway remains important throughout life as it has crucial roles in self-renewal for adult stem and progenitor cells (Lee, M. A., et ah, 2014, BMC Cancer, 14:125; Clevers, H., 2006, Cell, 127:469-480; Clevers, H., et ah, 2012, Cell, 149: 1192-1205).
- WNTs are lipid-modified glycoprotein ligands that bind to both Frizzled and low-density lipoprotein receptor-related protein 6 (LRP6) (Clevers, H., et ah, 2012, Cell, 149:1192-1205).
- b-catenin is phosphorylated and degraded by a complex composed of glycogen synthase kinase 3b (GSK3 b), Axin, adenomatous polyposis coli (APC), and casein kinase 1 (CK1).
- GSK3 b glycogen synthase kinase 3b
- Axin adenomatous polyposis coli
- CK1 casein kinase 1
- the WNT-Frizzled-LRP5/6 complex is phosphorylated and activates disheveled protein (DYL) (Malbon, C. C., et ah, 2006, Curr Top Dev Biol, 72:153-166).
- DVL activation inhibits GSK3p, which subsequently decreases b-catenin degradation and allows for its stabilization and translocation to the nucleus, where it binds to the T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors and activates gene transcription (Logan, C. Y., et al., 2004, Annu Rev Cell Dev Biol, 20:781-810).
- Endocytic adaptor protein DAB2 is a tumor suppressor protein (Sheng, Z., et al., 2000, Genomics, 70:381-386; Sheng, Z., et al., 2000, Oncogene, 19:4847-4854) involved in several receptor-mediated pathways (Xu, X. X., et al., 1995, J Biol Chem, 270:14184- 14191; Hocevar, B. A., et al., 2001, EMBO J, 20:2789-2801; Hocevar, B.
- Oncogenic CD44 is a downstream target gene of the WNT/p-catenin signaling pathway (Wielenga, V. J., et al., 1999, Am J Pathol, 154:515-523).
- WNT/p-catenin signaling pathway a downstream target gene of the WNT/p-catenin signaling pathway (Wielenga, V. J., et al., 1999, Am J Pathol, 154:515-523).
- induction of this pathway with a chemotherapy induced WNT ligand was not tested.
- Recent investigations have also suggested that the regulatory effects of the WNT/b- catenin signaling pathway respond to chemotherapeutic agents in CRC, and since b- catenin can be activated by several pathways to maintain chemotherapy induced resistance to apoptosis, the requirement of CD44v6 for the FOLFOX induced b-catenin activation remains to be addressed.
- CD44 is a multi-structural and multi-functional transmembrane glycoprotein that acts as a receptor for hyaluronan (also called hyaluronic acid).
- CD44 is encoded by a single gene containing 20 exons, ten of which are alternatively spliced to generate the numerous CD44 splice variants (CD44v) (Screaton, G. R., et al., 1992, ProcNatl Acad Sci USA, 89:12160-12164; Zoller, M., 2011, Nat Rev Cancer, 11 :254-267).
- CD44s The standard isoform of CD44 (CD44s) has no variant exons, is small, and is nearly ubiquitous in vertebrate cells (Naor, D., et al., 2008, Semin Cancer Biol, 18:260-267).
- Variant 6 of CD44 (CD44v6) participates in tumor development and progression in many ways that are restricted to stem cell subpopulations and cancer development.
- CD44v6 but no other of the variants, promotes generation of gut adenomas in mouse models of familial adenomatous polyposis (Zeilstra, J., et al., 2014, Oncogene, 33:665-670).
- CD44 isoforms are strongly overexpressed in dysplastic crypts and adenomas in humans, and in mice with mutated APC (Wielenga, V. J., et al., 1999, Am J Pathol, 154:515-523) suggesting that crosstalk between WNT and CD44/CD44v6 signaling pathways is important in CRC tumorigenesis. Additionally, a study showed that CD44 regulates P-catenin-COX2 signaling in colon tumor cells (Misra, S., et al., 2008, Connect Tissue Res, 49:219-224; Misra, S., et al., 2008, J Biol Chem, 283:14335-14344).
- a WNT canonical pathway (WNT/Frizzled/LRP6-GSK3p-p- catenin/TCF4) induces drug resistance (Katoh, M., et al., 2007, Clin Cancer Res, 13:4042-4045; MacDonald, B. T., et al., 2009, Dev Cell, 17:9-26).
- WNT also relies on the simultaneous activation of different intracellular transducers to induce non-canonical WNT pathways, such as the non-canonical WNT/RhoA pathway that controls MDR1 expression and activity in brain cancer (Allen, C., et al., 2010, Stroke, 41:2056-2063; Pinzon-Daza, M., et al., 2012, Br J Pharmacol, 167:1431-1447).
- Canonical and non- canonical WNT pathways are often reciprocally modulated, with either cooperative or antagonistic effects (Rossol-Allison, J., et al., 2009, Cell Signal, 21:1559-1568; Chang,
- CRC cells expressing CD166 (Vermeulen, L., et al., 2008, ProcNatl Acad Sci USA, 105:13427-13432), CD44 (Ohata, H., et al., 2012, Cancer Res, 72:5101-5110), CD44v6 (Todaro, M., 2014, Cell Stem Cell, 14:342-356), CD66c (Vermeulen, L., et al., 2008, ProcNatl Acad Sci USA, 105:13427-13432) and aldehyde dehydrogenase (ALDHl) (Huang, E.
- CD44v6 positive (+)/CICs have been associated with increased metastatic behavior in both pancreatic cancer (Zoller, M., 2011, Nat Rev Cancer, 11:254-267; Zoller, M., 2009, Nat Rev Cancer, 9:40-55; Gherardi, E., et al.,
- CD44v isoforms but not the CD44s isoform, promote adenoma formation in Ape (Min/+) mice (Zeilstra, J., et al., 2014, Oncogene, 33:665-670).
- CD44v6 predicts poor prognosis and is a marker of constitutive and reprogrammed CICs driving CRC metastasis (Todaro, M., 2014, Cell Stem Cell, 14:342- 356).
- CD44v8-10 is a specific marker for gastric CICs (Lau, W.
- CD44v3 is a specific CIC marker of head and neck cancers (Bourguignon, L. Y., et al., 2012, J Biol Chem, 287:32800-32824). These studies provide supporting examples that various CD44v isoforms are specially expressed on cancer cells and are required for tumorigenesis and progression. Thus, the CD44v6 isoform is likely to be a better CIC marker than the CD44s isoform in CRCs.
- CICs are the parental source for which all other malignant cells within a given tumor arise and are responsible for CRC tumor growth/maintenance, metastatic spread, resistance to conventional chemotherapies and relapse after cancer therapy (Valent, P., et al., 2012, Nat Rev Cancer, 12:767-775; Brabletz, T., et al., 2005, Nat Rev Cancer, 5:744- 749). Therefore, CRC/CICs must be effectively targeted to inhibit tumor growth and improve survival of patients with CRC.
- CAVl caveolin-1
- CD44 also regulates WNT signaling in the developing brain of Xenopus laevis embryos by association with LRP6 in the membrane (Schmitt, M., et al., 2015, Cell Death Differ, 22:677-689).
- Many of the oncogenic activities that have been previously attributed to CD44, in particular those relevant to ligand induced translocation of receptors into discrete microdomains (caveolin or clathrin) in the plasma membrane that strengthen signaling pathways, could be ascribed in part to CD44v6-signaling interactions in CRC.
- signaling is further modulated within lipid rafts through endocytosis, resulting in internalization of receptor complexes (Sabharanjak, S., et al., 2002, Dev Cell, 2:411-423).
- Endosomes containing internalized receptor complexes and shuttling of signaling molecules to the nucleus that concentrate essential components of the signaling pathway together with transcription factor transactivation represents a previously unappreciated step in signal transduction.
- CD44v6+CICs are involved in the development of drug-resistance, treatment failure, and tumor relapse in cancer (Ghatak, S., et al., 2005, J Biol Chem 280:8875-8883; Sherman, L., et al., 1998, Genes Dev, 12:1058-1071). This hypothesis was based on observations in this study that CICs acquire high levels of CD44v6-P-catenin mediated transactivation of TOPFlash promoter.
- the CD44v6 and LRP6 are translocated to the membrane micro-domains, where LRP6 is phosphorylated at serine residues (notably, serine 1490) through interaction with CD44v6 to form highly ordered CD44v6-LRP6-signaling through caveolin-1 endocytic pathway.
- LRP6/phospho-LRP6 (S1490)-CD44v6 distributions were significantly impacted by the presence of DAB2. Caveolin-containing endosomes, once internalized, appear to couple with the b-catenin destruction complex in that LRP6 phosphorylation at S1490 is observed.
- the CD44v6-LRP6- signalosomes in FOLFOX resistant cells are recruited to the endosome for sorting of CD44v6-P-catenin/TCF4-complex vesicles, which are then destined to the nucleus.
- TCF4 maintains distinctive transcriptional programs via interactions with MDR1 and CD44v6 gene promoters and sustains CD44v6-mediated auto-resistance in CICs.
- b- catenin/TCF4 signaling promotes CD44v6 splicing
- CD44v6 then sustains b- catenin/TCF4/MDRl signaling, which is important for maintaining the FOLFOX resistance.
- Nonidet P-40, EGTA, sodium orthovanadate, glycerol, phenylmethyl sulphonyl fluoride, leupeptin, pepstatin A, aprotinin and HEPES were from Sigma.
- Recombinant human WNT3 A protein was from R&D Systems (Minneapolis, MN).
- CD44v6, p-LRP6 Serine 1490
- LRP6, b-catenin Anti-Active-P-catenin (anti-ABC) antibody
- clone 8E7 TCF4, MDRl
- b actin b-tubulin
- horseradish peroxidase-linked anti-rabbit and anti-mouse antibodies Luminol reagent
- Blocking antibodies for CD44v6 (2F10), and isotype control were from R&D Systems.
- Blocking antibody for WNT3A (1H12L14) protein was from Thermo Fisher.
- Radiocarbon-labeled oxaliplatin [14C]oxaliplatin was purchased from Amersham Biosciences, Piscataway, NJ.
- HCT116 Human colorectal adenocarcinoma cell lines: 1) WIDR (CCL-218) was maintained in Eagle's Minimum Essential Medium (EMEM) +10% FBS; 2) LOVO (CCL-229) was maintained in F-12K Medium 2 mM L-glutamine and 1500 mg/L sodium bicarbonate; 3) HCT116 was provided by Dr. B.
- HCA-7 colony 29 was purchased from European Collection of Authenticated Cell Cultures and maintained in DMEM + 10% FBS + 2 mM L-Glutamine + 110 mg/L sodium pyruvate. Cell growth survival or apoptosis assays.
- the Caspase-Glo® 3/7 assay depends on the formation of free amino luciferin after adding caspase-3/7 DEVD-amino luciferin substrate to cell lysates and measuring amino luciferin by the luciferase present in the substrate reagent.
- the luminescent signal is proportional to caspase 3/7 activity and measured using a luminometer (PerkinElmer).
- IC50 values were determined for 5-Flourouracil (5-FU) and for oxaliplatin (OXA) ( Figure IB), because these molecules are the components of FOLFOX.
- 5-FU 5-Flourouracil
- OXA oxaliplatin
- To determine these IC50 values cells were separately pretreated with various concentrations of 5-FU, or OXA, or vehicle. After a 24-hour incubation at 37° C, growth assays were analyzed as described above. The 50% inhibitory concentration (IC50) was identified as a concentration of drug required to achieve a 50% growth inhibition relative to untreated controls.
- the average IC50 values for HT29, SW480 and SW948 cells for 5-FU is 50 mM and for OXA is 10 pM.
- FOLFOX resistance cells were generated by incubating the sensitive parental (HT29-S, SW480-S and SW948-S) cells with increasing concentrations from lx FOLFOX (50 pM 5-FU + 10 pM OXA + 1 pM leucovorin) to 5 x FOLFOX over 3 days. This exposure and withdrawal cycle was repeated five times for each dose of FOLFOX. The surviving cells were cultured in normal medium for 5 days. The resistance of these resistant clones was compared to sensitive pairs by determining the number of colonies in soft agar growth with lx FOLFOX - 5x FOLFOX therapy.
- Human CRC tissues were obtained from drug resistant patients (age range, 55-80 years) undergoing colorectal resection, in agreement with the ethical standards of the institutional review board.
- Patient tumor tissues, and cells from the generated FOLFOX resistant (FR) clones were maintained through subcutaneous xenografts in the flanks of immunocompromised [NOD-SCID/IL2Rynull (NSG)] mice and SCID mice, respectively.
- Fresh normal colonic tissue and colorectal tumors were rinsed in DMEM (Life Technologies) supplemented with 200 units/mL of penicillin, 200 pg/mL of streptomycin, and 4 units/mL of amphotericin B and minced, followed by incubation with 300 units/mL of collagenase (Worthington Biochemical) at 37o C for 3 h. A single cell suspension was obtained by filtration through a 40 pm filter.
- FACS fluorescence- activated cell sorting
- Flow cytometry was performed using a FACS Cell Sorter.
- PE phycoerythrin
- CD44v6 Single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5- isothiocyanate (FITC)-conjugated monoclonal antibody against EpCAM (R&D Systems).
- FITC Fluorescein-5- isothiocyanate
- CD44v6+/EpCAM+ and CD44v6-/EpCAM+ cells were subjected to flow cytometric analysis for isolation of CD44v6+EpCAM+ALDHl+, and CD44v6- EpCAM+/ALDHl+ cells using FITC-conjugated monoclonal antibody against ALDHl and grown in fresh medium for 2 weeks.
- CD44v6+EpCAM+ALDHl+ and CD44v6-EpCAM+/ALDHl+ cells were subjected to flow cytometric analysis for isolation of CD44v6+EpCAM+ALDHl+CD133+ (designated as CICs), and CD44v6- EpCAM+ALDHl+CD133+(designated as Non-CICs) using FITC-conjugated monoclonal antibody against CD 133.
- CICs were cultured in serum-free media with basic fibroblast growth factor (bFGF, 10 ng/ml; R&D Systems) and epidermal growth factor (EGF, 10 ng/ml; R&D Systems).
- bFGF basic fibroblast growth factor
- EGF epidermal growth factor
- CICs were cultured in serum-free media with basic fibroblast growth factor (bFGF, 10 ng/ml; R&D Systems) and epidermal growth factor (EGF, 10 ng/ml; R&D Systems).
- bFGF basic fibroblast growth factor
- EGF epidermal growth factor
- tumor formation medium 500 ml Dulbecco’s Modified Eagle Medium/F12
- 20 ng/ml epidermal growth factor 10 ng/ml basic fibroblast growth factor
- 5 pg/ml insulin 0.4% bovine serum albumin.
- 200 live cells / 200 m ⁇ of tumor sphere medium were suspended in ice. This suspension was kept on ice and mixed well for plating.
- PBS was added to the first and last columns (column 1 and 12) of the 96-well plate to help minimize medium evaporation. This leaves 10 wells available for each row.
- 200 pL of the cells were suspended in tumor sphere medium into each well (200 cells per well).
- cells were seeded into the wells of 2 rows for a total of 20 wells.
- the upper and lower edges of the 96-well plate were sealed with laboratory tape to avoid evaporation of medium, and cells were placed in an incubator set to 37° C and cultured in 5% C02 for 10 -14 days with change of media every 48 hours.
- tumor sphere numbers were counted under a phase-contrast microscope using the 40X magnification lens. Data are presented as a percentage of wells containing tumor spheres compared to the total number of wells. Cell lysis and immunoblotting.
- Cells were cultured until they were 75% confluent. Cells were washed twice at 4° C with phosphate-buffered saline (PBS), harvested with 0.05% Versene, and then washed in cold PBS again. The cells were pelleted by centrifugation at 5,000 x g for 2 minutes at 4° C.
- PBS phosphate-buffered saline
- the pellets were treated with the lysis buffer containing 1% Nonidet P-40, 0.5 mM EGTA, 5 mM sodium orthovanadate, 10% (v/v) glycerol, 100 pg/ml phenylmethyl sulphonyl fluoride, 1 pg/ml leupeptin, 1 pg/ml pepstatin A, 1 pg/ml aprotinin, and 50 mM HEPES, pH 7.5.
- the lysates were clarified by centrifugation at 12,000 x g for 20 minutes at 4° C and then stored at -80° C.
- the proteins on the blots were analyzed with antibodies from commercial sources for CD44v6, CD44s, p-LRP6, LRP6, MDR1, b-actin, and Active-b- actin (b-tubulin and b-actin were used as internal standards). They were detected by treatment with horseradish peroxidase-linked anti-rabbit or anti-mouse antibodies as secondary antibodies followed by treatment with luminol reagent (Santa Cruz Biotechnology). Each protein was analyzed in samples from at least three independent experiments from each set of tumor cells, CICs and CAFs.
- Lipid rafts from cultured cells were fractionated by using Opti prep gradient (Graham, J. M., 2002, ScientificWorldJournal, 2:1440-1443; Macdonald, J. L., et ah, 2005, J Lipid Res, 46:1061-1067). All procedures were done at 4o C.
- Cells from four 150 mm plates were washed to free from growth medium and scraped into base buffer [Tris- Sucrose buffer (20 mM Tris-HCl, pH 7.8, 250 mM sucrose) containing 1 mM CaC12 and 1 mM MgC12] Cells were pelleted by centrifugation at 4o C for 2 minutes at 250 g and suspended in 1 ml of base buffer with a cocktail of protease inhibitors at final concentrations of 0.2 mM aminoethyl-benzene sulfonyl fluoride, 1 ug/ml aprotinin, 10 uM pepstatin, 3 uM E-64, 10 ug/ml leupeptin, 2 uM pepstatin, and 50 ug/ml calpain inhibitor I.
- base buffer [Tris- Sucrose buffer (20 mM Tris-HCl, pH 7.8, 250 mM sucrose) containing 1 mM CaC12 and 1
- the cells were then lysed by passage through a 22 g needle 30 times. Nuclei were removed from the lysates by centrifuging at 4o C at 1,000 g for 10 minutes. The post-nuclear supernatant #1 was collected and saved. The pellet was further lysed by the addition of 1 ml base buffer plus divalent cations and protease inhibitors and sheered 30 times through a 22G needle and syringe. The second lysate was centrifuged for 10 minutes at 1,000 g, the post-nuclear supernatant #2 was combined with #1.
- OptiPrep Fifty percent OptiPrep was prepared in 2 ml of Tris-sucrose buffer containing protease inhibitors and was added to the combined 2 ml post nuclear supernatants and placed in the bottom of a 12 ml centrifuge tube. An 8 ml gradient of 0% to 20% OptiPrep in Tris-Sucrose buffer with protease inhibitors was poured on top of the lysate, which was now 25% in OptiPrep. Gradients were centrifuged for 90 min at 52,000 g using an SW-41 rotor in a Beckman ultracentrifuge. After centrifugation, cloudiness could be seen throughout the gradient.
- a diffuse band was observed about one-third of the way down the gradient, and a distinct band was apparent at the interface of the 20% end of the gradient and the 25% OptiPrep bottom layer.
- Gradients were fractionated into 0.250 ml fractions, and protein and cholesterol contents of each fraction were determined. The distributions of various proteins were assessed by Western blotting.
- Endosomes from SW480-FR-NON-CICs/CD44v6 cell clones expressing acting binding NLS mutant (nuclear localization signal mutant) and D67 mutant cells were isolated by sucrose density gradient (Walker, L. R., et ak, 2016, Cell Biosci, 6: 1). The cells were grown in plates and washed with PBS three times to remove growth medium. All operations were done at 4o C. Homogenization buffer 0.5 ml (250 mM sucrose, 1 mM EDTA, 1 mM phenylmethyl sulphonyl fluoride (PMSF)) was added and the cells detached from the surface.
- PMSF phenylmethyl sulphonyl fluoride
- the cells were lysed by passing through 22G needle and syringe.
- the lysed cells were centrifuged at lOOOxg for 10 minutes.
- One ml supernatant was adjusted to 25% sucrose /l mM EDTA.
- Step gradient in four layers was set up in a SW41Ti tube: bottom layer 2.4 ml of 45 % sucrose, overlaid with 5.2 ml of 35 % sucrose, 3.9 ml of 25 % sucrose, and 1 ml of post nuclear supernatant in 25 % sucrose.
- the tubes were centrifuged at 100,000xg for one hour. Fractions (2 ml) were collected from top to bottom. The densities of the fractions were measured by refractometry. The fractions were analyzed by western blotting.
- pcDNA-Wnt3A-V5 was a gift from Marian Waterman (Addgene plasmid # 35927; http://n2t.net/addgene:35927; RRID:Addgene #3 5927), human beta-catenin pcDNA3 was a gift from Eric Fearon (Addgene plasmid # 16828; http://n2t.net/addgene: 16828; RRID: Addgene # 16828).
- CD44v6 specific PCR amplification products were isolated with polyadenylated RNA from the HT29 cell line.
- PCR product was cloned in pcDNA3.1 vector and used as previously described (Ghatak, S., et ak, 2017, J Biol Chem, 292:10465-10489).
- Myc-tagged human full length TCF4E pcDNA3 was a gift from Frank McCormick (Addgene plasmid # 32738; http ://n2t. net/ addgene : 32738 ; RRID: Addgene # 32738).
- pDONR223_DKKl_WT was a gift from Jesse Boehm & William Hahn & David Root (Addgene plasmid # 82250; http://n2t.net/addgene: 82250; RRID: Addgene # 82250).
- the MDR1 and CD44v6 reporter constructs were synthesized by Bio basic (US) and cloned into the firefly pGL3 -basic vector (Promega) upstream of the Luciferase reporter gene.
- CD44v6 contains one TCF binding site (- 1700/500); and 2) CD44v6 (b) contains basal promoter and two TCF binding sites (- 2100/500).
- the M50 Super 8x TOPFlash vector (plasmid 12456) with a luciferase gene under the control of seven TCF/LEF -binding sites and the corresponding M51 Super 8x FOPFlash vector (plasmid 12457) with mutated TCF/LEF -binding sites were obtained from Addgene (Cambridge, MA, USA).
- the normalization vector pRL-TK renilla with a HSV-TK promotor driving Renilla luciferase was purchased from Promega.
- Transient transfection and luciferase reporter assay For the transient assays, 1.0 x 105 cells from both cell lines were transfected using Lipofectamine LTX 2000 (Invitrogen) with 1 pg of each Luciferase construct and 100 ng of pRL-SV40 vector (Promega), according to the manufacturer’s instructions. Firefly and Renilla Luciferase activities were measured in cell lysates 48 hours after transfection using the DualGlo Luciferase Assay System (Promega) on a Veritas TM Microplate Luminometer (PerkinElmer) following the manufacturer’s protocol. All experiments were done in triplicate.
- Ratios of Renilla luciferase readings to firefly luciferase readings were taken for each experiment, and triplicates were averaged. The average values of the tested constructs were normalized to the activity of the empty pGL3 -basic vector, which was arbitrarily set at value 1.
- b-catenin/TCF Reporter assays All reporter gene assays were done in 96-well plates.
- PD-FR/CICs or CD44v6 overexpressing SW480-FR/SQ/Non-CICs (1.0 x 104/well) were transfected with Super TOPFlash reporter (25 ng) and TK-Renilla (5 ng), and with the respective plasmid DNA as indicated using LipofectamineTM 3000 transfection reagent according to the manufacturer’s protocol. Each transfection was adjusted to 150 ng DNA/transfection with pcDNA3.1 empty vector. Where indicated, cells were transfected at 50-70% confluency with shRNA constructs using LipofectamineTM 3000 transfection Reagent in 6 cm petri dishes according to the manufacturer’s protocol 24 h before seeding the cells for the reporter assays.
- RNA extraction and cDNA synthesis (Andoorfar, S., et al., 2019, Mol Biol Rep, 46:5057-5062).
- Harvested cells were transferred to 1.5 ml Eppendorf tubes with very little amount of lx PBS, and 1 ml of TRizol (Invitrogen) was added and vortexed. The tube was kept in room temperature for 5 minutes. Then, 200 pi chloroform was added and left at room temperature for 10 minutes. The tube was centrifuged at 1300 rpm for 15 minutes at 4o C. The upper phase containing RNA was transferred to a new tube, and 600 pi ice cold isopropanol was added.
- the tube was inverted several times, kept in ambient temperature for 10 min, and centrifuged at 1200 rpm for 13 minutes at 4o C. After removing the supernatant, the white RNA pellet was washed with 1 ml of alcohol 75% and left air-dried. The sample was dissolved in 50 m ⁇ DEPC-treated water. The quality and quantity of extracted RNA was checked by a spectrophotometer. The extract was electrophoresed on 1% agarose gel. DNA contamination was removed from all RNA samples by treating the samples with DNase- 1. 500 ng of RNA was used for cDNA synthesis.
- the reaction contained 1 m ⁇ of each cDNA sample, 0.5 m ⁇ of each primer, 5 m ⁇ Taq DNA Polymerase 2x Master Mix Red (Amplicon Co.) and 3 m ⁇ dd water in a final volume of 10 m ⁇ .
- the PCR conditions including thermal conditions and the number of cycles and the cDNA concentrations, were optimized.
- temperature conditions included one initial denaturation cycle (3 min at 95° C) followed by 35 cycles with a denaturation step for 5 sec at 95 °C and a combined annealing and extension step for 35 sec at 61° C.
- the PCR products were electrophoresed on agarose 2.5%, stained with Ethidium bromide and photographed. The analysis of band intensity was done by ImageJ software.
- the PCR mixture (25 m ⁇ ) contained 12.5 m ⁇ of 2 SYBR Green PCR Master Mix (Bio-Rad), 5 m ⁇ of diluted RT product (1:20), and 0.5 mM sense and antisense primer sets.
- the QPCR primers used in this study in analyses of various genes associated with CIC sternness function were presented in Figure 8.
- the real-time PCR assays were done in three individual experiments with duplicate samples using standard conditions in a CFX96 real-time PCR detection machine. After incubations at 95° C for 3 minutes, the amplification protocol consisted of 50 cycles of denaturing at 95° C for 10 sec, annealing, and extension at 60° C for 30 sec.
- the standard curve was made from a series dilution of template cDNA. Expression levels of tested genes were calculated after normalization with the housekeeping gene GAPDH or b-actin.
- shRNA sequences used in this study, 1) coding nucleotide sequences of the genes were obtained from the NCBI, National Institutes of Health, website (www.ncbi.nlm.nih.gov); 2) hairpin shRNAs were designed to target a transcript sequence using the Broad Institute GPP Web Portal
- pSicoR-CD44v6 shRNAl (CD44v6 shl), pSicoR-CD44v6 shRNA2 (CD44v6 sh2), pSicoR-WNT3A shRNAl (WNT3A shl), pSicoR-WNT3A shRNA2 (WNT3A sh2), pSicoR-p-catenin shRNAl (b- catenin shl), pSicoR-P-catenin shRNA2 (b-catenin sh2) transfectants constitutively silence respective CD44v6, WNT 3 A and b-catenin genes in the cells.
- pSicoR-Non targeted shRNA (NT sh) transfectants were used as control to the above shRNA transfectants (see Figure 13 for shRNA sequences used in this study).
- shRNA knockdown experiments were also confirmed, comparing the knockdown effects of shRNAs for CDS either with those of NCDS (as proper negative controls) or with rescue of the observed shRNA-mediated knockdown phenotype by expression of a resistant form of the targeted mRNA. This is done by (Ghatak, S., et ak, 2017, J Biol Chem, 292:10465-10489): 1) transfecting the cells with specific shRNAs for the CDS of the target gene, or 2) co-transfecting the shRNA (CDS) for the target gene with or without corresponding cDNA transfection or 3) by the indicated shRNA-mediated knockdown and corresponding KI gene transfection.
- ChIP chromatin immunoprecipitation
- Nuclear b-catenin-associated chromatins were immunoprecipitated with b-catenin or CD44v6 antibodies for 3 hours. Chromosomal DNAs were purified and analyzed using QPCR with primers for TCF4 sites of MDRl to detect the MDRl promoter regions. Similarly, SW480-FR cells were transfected with or without NT sh, or b-catenin shl, or dominant negative TCF4 (TCF4-DN) constructs for 48 hours. Nuclear TCF4-associated chromatins were immunoprecipitated with b-catenin, or CD44v6 antibodies for 3 hours.
- Chromosomal DNAs were purified and analyzed using QPCR with primers for TCF4 sites of CD44v6 to detect the CD44v6 promoter regions. Control IgGs were used as negative controls for immunoprecipitation. Chromatin inputs were used as loading controls for PCR. The primers used for ChIP PCR studies are presented in Figure 23. In vivo tumorigenic potential of CICs.
- cells were treated in the presence or absence of FOLFOX or WNT3 A conditioned media at 370C for the times indicated and washed three times with ice-cold phosphate-buffered saline (PBS; pH 8.0) to remove any contaminating proteins.
- PBS ice-cold phosphate-buffered saline
- Cells 70-80% confluence (2.5 x 10 7 cells/ml) were resuspended in PBS and 50 ml of 20mM Sulfo-NHS-SS-Biotin per milliliter of reaction volume with gentle agitation for 60 min at 4°C.
- Cells were washed with Sulfo-NHS-SS- Biotin blocking reagent (50mM NH4C1 in PBS containing ImM MgC12; 0. ImM CaC12) to quench free Sulfo-NHS-SS-Biotin, followed by several ice-cold PBS washes.
- Cell lysates were prepared in lysis buffer and biotinylated proteins were precipitated using streptavidin beads from equal amounts of cell lysates. Precipitates were washed three times with cell lysis buffer and analyzed by SDS-PAGE and immunoblotting with appropriate antibodies.
- cell surface proteins were biotin-labelled as described above at room temperature for 1 h, followed by treatment with or without WNT3 A for the indicated times at 37C. Following stimulation, cells were incubated with 0.1 M glycine in PBS for 30 min at 4°C to quench the unreacted biotin. Surface-retained biotin was removed using reduced glutathione (60mM glutathione, 0.83M NaCl, with 0.83M NaOH and 1% bovine serum albumin (BSA) added before use) for two 30-min incubations, followed by ice-cold PBS washes four times. Cells were collected and lysed and biotinylated proteins precipitated using streptavidin beads from equal amounts of cell lysates. The amounts of receptor bound to beads were determined by followed by SDS- PAGE and immunoblot analysis.
- the cells were then washed three times with PBS and incubated for 24 hours with 0.2 mM oxaliplatin containing 300 dpm (2.16 pmole) [14C] oxaliplatin (77.6 pCi/mmole).
- the cells were then washed to remove free radioactive oxaliplatin and incubated in drug-free medium containing 1 x FOLFOX or WNT3A (20 pg/ml) or no FOLFOX, or no WNT3A, or CD44v6shRNA, or CD44A57 construct transfection for 48 hours prior to treatment with and without FOLFOX or WNT3 A for 2 hours.
- a two-tailed Student’s t-Test was used to compare mean values between sensitive and resistant cells using the following parameters: mean DDOT values for QPCR; mean colony number for soft agar growth assays; mean densitometry values for QPCR and WB; mean percentage of cell viability assay (CellTiter-Glo) and FACS analysis; mean luminescence for ATP activity in cell growth, Caspase Glow assays in Apoptosis measurements; and mean tumor weight in xenograft studies. Chi-squared analysis was performed to compare incidences between sensitive and resistant cells for the following assays: number of positive wells containing tumor spheres in sphere formation assays; and number of mice developing tumors in xenograft studies.
- Figure 1C- 1E shows the IC50 values of sensitive SW480-S cells compared with resistant SW480- 5FUR, SW480-OXAR or SW480-FOLFOX cells.
- the resistant cell lines have 3-5 fold higher IC50 values.
- ex vivo cultures were derived from biopsies (PD) collected from 5-FU resistant (PD-5FUR), Oxaliplatin (PD-OXAR), and FOLFOX (PD-FR) tumors and from subcutaneous xenograft (SQ) tumor samples derived from FOLFOX resistant (FR) cell lines (WIDR-FR, LOVO-FR, HT29-FR and SW480-FR) generated from corresponding sensitive (S) pairs of cells.
- CD44v6 induction in SW480-S cells was evaluated upon exposure to lx FOLFOX.
- serum depleted SW948-S cells were stimulated by addition of lx FOLFOX in media.
- the expression profiles of CD44 variants in SW480 cells were examined after stimulation with FOLFOX by exon-specific reverse transcription-PCR (RT-PCR) analysis.
- RT-PCR reverse transcription-PCR
- the expression levels of CD44 variants were monitored by quantitative RT-PCR using distinct sets of primers (See the schematic diagram of CD44 gene in Figure IF).
- Exon v6 was expressed together with exons v6-v8 and as an independent isoform (Figure 2B).
- the v6-v8 variants were detected using a 3' primer from c7 (reverse primer) of CD44 and two distinct 5 '-primers (forward primers) complementing to v6 and v8 exons of CD44, respectively.
- the v6 primers and CD44s primers each principally amplified a single product ( Figure 2B).
- the v8 primer gave rise to two alternately spliced variants of CD44 containing (1) variant exons v6, v7, and v8 (illustrated as v6-v8); and (2) variant exon v8 (shown as v8), all joined to the 3'- constitutive exon 7 (Figure IF). All products were confirmed by DNA sequencing as described [58] Following 24 hours of serum starvation, the relative expressions of CD44 variants were low, while stimulation with FOLFOX upregulated v6 mRNA expression that peaked between 4 and 16 hours and returned to basal levels at 24-36 hours likely due to the exhaustion of FOLFOX within the media ( Figure 2B).
- RT-PCR results showed that PD-FR, PD-OXAR and PD-5FUR expressed similar CD44 isoforms as shown in FOLFOX stimulated SW480-S cells ( Figure 2B), patient-derived specimens also express low-(Figure 2C) molecular-weight isoforms detected by the RT-PCR analysis with primers v3, v5, v9 and v6 ( Figure 2C).
- the focus of this Example is CD44v6.
- RT-PCR analysis was done using a forward primer that base pairs with both the v6 and C5 exons, and a reverse primer that base pairs with the C7 exon.
- FOLFOX resistant SW480-FR tumor cell viability was assayed using various doses of FOLFOX in the presence or absence of two different sets of shRNAs targeted to CD44v6 and WNT3A (CD44v6 v6shRNAl/2, or WNT3A shRNAl/2, or WNT3A shRNAl/2 plus CD44v6 cDNA) ( Figure 5A).
- Figure 5A A similar experiment was done with a second set of shRNAs for CD44v6 and WNT3 A ( Figure 4C) to confirm that the effects of CD44v6 and WNT3 A are specific for FOLFOX stimulated colon tumor resistance.
- WNT3 A shRNA alone inhibited tumor cell proliferation to the same extent as v6 shRNA ( Figure 5A, and Figure 4C).
- WNT3 A shRNA combined with v6 cDNA nearly eliminated WNT3 A shRNA-mediated inhibition of FOLFOX resistance in SW480-FR cells ( Figure 5A, and Figure 4C).
- chemotherapeutic drugs such as 5-FU, OXA and their combination in FOLFOX in CRC cells ( Figures 2, 5, and Figures 4A-C).
- Figures 4D-F show the validation of WNT3A shRNA, CD44v6 shRNA, and v6 cDNA expression vectors.
- CD44v6 has key roles for FOLFOX-induced WNT3 A/b-catenin/MDR 1 activation that is clearly inhibited by CD44v6 shRNA, confirming that FOLFOX might induce WNT ligands to mediate CD44v6-dependent WNT/p-catenin signaling, and 2) constitutive activation of CD44v6 and WNT3A are necessary for maintaining FOLFOX resistance in CRC cells through a WNT3 A-CD44v6-P-catenin-MDRl pathway.
- FR cells were able to form increased anchorage- independent growth assessed by formation of large numbers of soft agar colonies ( Figure 5C).
- Sphere-forming activity of both parental and FR cells was next investigated. Compared with parental sensitive cells, FR cells were able to form significantly greater numbers of tumor-spheres in serum free medium (Figure 5B). Additionally, to evaluate, whether FOLFOX resistant cells increased in vivo tumor growth compared to the corresponding sensitive cells, 5 c 10 4 FR cells, 5 xlO 4 S cells and 5 x 10 6 S SW480 cells, were each implanted into 7 immunocompromised mice in 3 separate experiments.
- CD44v6 expression defines highly tumorigenic colorectal cancer-initiating cells (CICs).
- CICs Cancer initiating cells
- sternness and resistance to conventional chemotherapies are considered to remain after chemotherapy and to initiate metastasis.
- CD44v6 has important roles in the sternness of CICs (Zoller, M.,
- CD44v6 defines CRC/CIC subpopulations with drug resistance and tumorigenic properties was investigated in clinical samples (PD-FR, PD-5FUR and PD-OXAR) isolated from patients who were resistant to several chemotherapeutic drugs as well as in the previously described FOLFOX resistant WIDR, HT29 and SW480 cells.
- CICs were isolated from these samples by FACS sorting using several of the previously reported candidates (CD44v6, CD133, EpCAM and ALDHl) (Ohata, H., et ak, 2012, Cancer Res, 72:5101-5110; Todaro, M., 2014, Cell Stem Cell, 14:342-356; Dalerba, P., et ak, 2007, Annu Rev Med, 58:267-284; Vermeulen, L., et ak, 2008, Cell Death Differ, 15:947-958; Horst, D., et ak, 2009, Cancer Invest, 27:844-850).
- FIG. 6 A shows that CD44v6+/EpCAM+ sorted cells (10% of unsorted PD- FR fresh tumor cells, Figure 7A) overlapped with CIC markers ALDHl and CD 133 antigen expressions in PD-FR patient tissues (Figure 6B).
- the data in Figure 7B and 7C show the percentages of CD44v6+EpCAM+ALDHl+, and CD44v6+EpC AM+ALDH 1 +CD 133 +cell s (hence forth
- CD44v6+EpCAM+ALDHl+CD133+cells will be referred to as CICs) compared to CD44v6(-)/ EpCAM+ALDHl+, and CD44v6(-)EpCAM+ALDHl+CD133+ cells (hence forth CD44v6(-)EpCAM+ALDHl+/CD133+ cells will be referred to as Non-CICs) respectively.
- Results in Figure 6C showed increased CIC-stemness related gene expressions (primers are shown in Figure 8) in CICs isolated from both PD-FR patient tissues and SW480-FR/SQ tumors compared to SW480-S/SQ tumors. Overall, the data in Figures 6A-C validate that the CICs overexpressing CD44v6 were originated from epithelial and stem cells.
- CD44v6 and b-catenin activation are CRC-CIC markers (Todaro, M., 2014, Cell Stem Cell, 14:342-356; Vermeulen, L., et al., 2010, Nat Cell Biol, 12:468-476) and FOLFOX therapy induces CD44v6 associated b-catenin- MDR1 signaling ( Figure 2E, and 2F), whether CD44v6- b-catenin signaling can classify CRC/CICs as a FOLFOX-resistant phenotype was examined.
- PD-5FUR CICs from SQ tumors displayed partial sensitivity to FOLFOX (40%) compared to no sensitivity in PD-FR CICs indicating that the PD-FR CICs are indeed resistant to FOLFOX, whereas PD-5FUR CICs are only partially resistant to FOLFOX ( Figures 7E-F and 9A-B).
- the CICs increased tumor incidence and reduced the latency of tumor formation by these cells with increased tumor size in mice implanted with CICs compared to unsorted (Bulk) tumor cells, which required 100-fold more cells compared to CICs ( Figure 6E, Figure 9C).
- the transplant ability and tumor growth between unsorted tumor cells and sorted CD44v6+CICs were compared.
- CD44v6+/CICs were injected in immune-compromised mice, 80-100% develop tumors compared to 50- 65% of mice injected with unsorted (Bulk) cells ( Figure 10A).
- CD44v6 (+) CICs 2 x 10 3 CD44v6 (+) CICs, 5 x 10 5 CD44v6 (-) Non-CICs, and 5 x 10 5 unfractionated tumor cells from primary tumor xenografts were transplanted into secondary xenograft model. It was found that implanted CD44v6 (+) CICs increased tumor incidence, grew rapidly and reduced the latency of tumor formation by CICs with increased tumor size ( Figure 9C). Furthermore, CD44v6 (+) CICs obtained from similar CD44v6 (+) CICs derived secondary xenografts were subsequently transplanted into third generation of xenografts in mice.
- CD44v6 (+) CICs did not lose their tumorigenic potential but instead increased their long-lasting faster tumor growth (Figure 10D).
- Figure 10E the tumorigenic potentiality of the CD44v6 (-) Non-CICs was entirely lost in secondary recipients ( Figure 10E), suggesting that CD44v6 (-) Non-CICs include more differentiated nontumorigenic cells whereas tumorigenic colorectal CICs are restricted to the small population of the CICs expressing CD44v6 ( Figures 10C-E).
- FOLFOX-induced WNT3A and CD44v6 signaling establishes cell autonomous resistance to conventional FOLFOX chemotherapies in colorectal CICs.
- FOLFOX stimulation had a small stimulatory effect on CD44v6 mRNA expression in FR cells (only -0.3-0.4 fold), whereas the stimulatory effect for CD44v6 expression is 2.5, 2.2 and 4-fold respectively in OXAR, 5-FUR and SW480S-CICs with FOLFOX treatment ( Figure 11 A).
- SW480-FR cells are already secreting WNT3 A at a much higher rate before treatment, and additional FOLFOX treatment increases WNT3A (Figure 1 IB).
- This relative higher WNT3 A secretion in response to FOLFOX treatment suggests that WNT3 A-induced TOPFlash transactivation may be enriched after FOLFOX treatment.
- a luciferase construct containing four native TCF/LEF binding sites (TOPFlash) or its negative-control counterpart (FOPFlash) containing four mutated LEF/TCF binding sites were overexpressed along with a Renilla construct. At 24 hours post-transfection, luciferase activity was measured using the dual- luciferase system.
- the TCF/LEF responsive reporter TOPFlash transactivation increased significantly higher in SW480-FR cells compared to SW480-S cells in response to 1 x FOLFOX treatment for 1 hour ( Figure 11C), indicating that FOLFOX stimulates WNT3 A pathway activation.
- Non-CICs show sensitivity to FOLFOX as determined by reduced colony formation in clonogenic growth assays and increased apoptosis determined by apoptosis assay (right panel of Figure 12A- B).
- CD44v6 was overexpressed using CD44v6 cDNA in CD44v6 (-)
- Co-IPs showed that WNT3 A stimulated active b-catenin and TCF4 were in a nuclear complex with CD44v6 in CD44v6 overexpressing COS-7 cells ( Figure 14A).
- WNT3A induced a mature glycosylated membrane bound form of LRP6 (upper band of LRP6) (Hsieh, J. C., et al., 2003, Cell, 112:355-367) that is reduced by knocking down CD44v6 in SW480-FR-CICs ( Figure 14G), whereas in sensitive cells, knocking down CD44v6 represses both the immature endoplasmic reticulum bound (ER) form of LRP6 (faster migrating band) and the mature membrane bound form of LRP6 (slower moving band) ( Figures 14F).
- ER immature endoplasmic reticulum bound
- CD44v6 regulates multiple receptor tyrosine kinase and non- tyrosine kinase signaling pathways
- casein kinase 1 (CK1) family members are known to phosphorylate LRP6 (Liang, J., et ak, 2011, Mol Cell Biol, 31 :2577-2590).
- CK1 casein kinase 1
- these pathway inhibitors were used and whether these pathways affect CD44v6-LRP6/WNT signaling was examined. The results indicate that MEK or PI3K pathways did not impact CD44v6 regulated WNT3 A-induced TOPFlash transactivation in a COS-7-CD44v6 stable transfectant clone ( Figures 15A-B).
- TOPFlash promoter activation occurs by membrane constituents WNT3 A or LRP6, but not by cytoplasmic molecules such as DVL-2 or CA-P-catenin, providing evidence that WNT3 A-induced TOPFlash transactivation occurs only in the membrane associated LRP6 activated by CD44v6 presumably in association with CK1 ( Figures 15C-E).
- Endocytosis of transmembrane signaling receptors is an important regulatory event in signal transduction including CD44/CD44v6 (Ghatak, S., et ak, 2005, J Biol Chem 280:8875-8883; Neame, S. I, et ak, 1995, J Cell Sci, 108:3127-3135; van Meer, G., 2005, EMBO J, 24:3159-3165) and WNT/p-catenin signaling (Conner, S. D., et ak, 2003, Nature, 422:37-44; Sorkin, A., et ak, 2002, Nat Rev Mol Cell Biol, 3:600-614).
- Clathrin-mediated endocytosis has a crucial role in terminating cell signaling by inhibiting association of cell surface receptors (Hanover, J. A., et ak, 1984, Cell, 39:283- 293; Beguinot, L., et ak, 1984, Proc Natl Acad Sci USA, 81:2384-2388).
- Clathrin- independent internalization/endocytosis occurs mostly at lipid rafts, which are microdomains of the plasma membrane enriched in cholesterol-sphingolipids-caveolin-1 (CAV1) (Gong, Q., et ak, 2008, J Cell Mol Med, 12:126-144).
- CAV1 cholesterol-sphingolipids-caveolin-1
- a C AVI -endocytosis pathway has been shown to function as a platform for receptor mediated signaling by accelerating the sequestering of receptors and signaling molecules within caveolae (Le Roy, C., et ak, 2005, Nat Rev Mol Cell Biol, 6: 112-126; Razani, B., et ak, 2002, Pharmacol Rev, 54:431-467).
- CD44v6 regulates WNT signaling at the level of association with mature LRP6 at the membrane (Figure 15D-E)
- Optiprep gradient layer and depicted as NR.
- increased levels of CD44v6 and LRP6 (S1490) localized in raft fractions of FR cells, which was absent in non-raft fractions expressing Clathrin in sensitive cells.
- Methyl -b-cyclodextrin a cholesterol depleting agent, abolished recruitment of CD44v6 and LRP6 to lipid raft ( Figure 16C).
- the FR and sensitive cells of SW480 were treated with nystatin, which disrupts caveolin dependent endocytosis and with monodansylcadaverine (MDC), which blocks clathrin-mediated endocytosis (Yamamoto, FL, et al., 2008, Dev Cell, 15:37-48), prior to stimulation with WNT3A.
- MDC monodansylcadaverine
- LRP6 and CD44v6 distributions to a clathrin domain in sensitive cells may be linked to DAB2 in response to brief treatment with FOLFOX or WNT3 A.
- DAB2-mediated internalization of LRP6 through the clathrin pathway may be the likely mechanism for failing to couple with the destruction complex and subsequent attenuation of the b- catenin/TCF4 promoter TOPFlash activation as seen in sensitive SW480-S cells and in SW480-S CICs ( Figure 14B and Figure 11C) compared to resistant SW480-FR cells and in SW480-FR/CICs ( Figure 14C, and Figure 11C).
- CD44v6 and LRP6 (S1490) co-immunoprecipitated with the lighter caveolin-containing R fractions, and WNT3 A stimulation appears to promote this association into a caveolin-compartment.
- phospho-LRP6 (S1490), indicative of activated WNT3 A/b-catenin signaling, is significantly increased with caveolin-containing fractions in WNT3 A- stimulated control (vector transfected) resistant cells ( Figure 17C) and is not present in DAB2-transfected cells following WNT3A treatment ( Figure 17D).
- Figure 17C the relative associations and distributions of other b-catenin modulator proteins was determined.
- axin and OdK3b in caveolin-immunoprecipitates depended on WNT3A ( Figures 17C-D).
- CD44v6 is also associated with lipid rafts via NLS sequence of CD44v6, a CD44v6NLS Mutant was generated by changing the putative NLS sequence 360RRRCGQKKK368 to 360 AAACGQ A AA368.
- the CD44v6-NLS-site (360RRR362CGQ366KKK368) is the site where ezrin, radixin, and moesin bind (CD44v6ERM) (Legg, J.
- CD44v6A67Mut is devoid of NLS sites of CD44v6.
- overexpression of the CD44v6A67PALM Mut and the CD44v6A61 PALM-NLS Mut completely block the association of CD44v6 to lipid rafts.
- the CD44v6NLS Mut did not completely block association of this mutant to lipid rafts.
- the engagement of the CD44v6A67PALM Mut and CD44v6A61PALM-NLS Mut that were defective in PALM and NLS sites fail to induce lipid raft redistribution/reorganization of CD44v6.
- both caveolin and clathrin were silenced in SW480-FR-Vec and SW480-FR-DAB2 cells by targeted shRNA for caveolin (CAV1) and clathrin.
- Figures 18G-I show that caveolin knockdown blocks WNT3 A/b-catenin transcriptional activation, active b-catenin (ABC) and MDR1 expressions in vector transfectant cells, whereas clathrin knockdown overturned the inhibitory effect of DAB 2 on ⁇ nNT3A/b- catenin mediated TOPFlash transactivation (Figure 18G) as well as on ABC and MDR1 expressions in DAB2 transfected FR cells ( Figures 18H-I).
- Validations of CAV1 and clathrin shRNAs are shown in Figures 18J-K) following previously published methods (Ghatak, S., et al., 2017, J Biol Chem, 292:10465-10489).
- Co-immunoprecipitation assays as shown in Figure 19A indicate that mutation of the PALM motif in the NLS-deleted-CD44v6A67 Mut (CD44v6A67PALM Mut), or by mutation of this NLS motif in CD44v6A61PALM Mut (CD44v6A61PALM-NLS Mut) disrupted the association of CD44v6 with LRP6 and actin protein predominantly in WNT3A stimulated cells.
- CD44v6 proteins containing an intact NLS motif in CD44v6A61 PALM Mut and CD44v6PALM Mut were constantly associated with actin and LRP6, and engagement of CD44v6 strongly enhanced the formation of the CD44v6- LRP6-actin signalosome complex in response to WNT3A stimulation (Figure 19A).
- CD44v6NLS mutant internalized efficiently but failed to enter the nucleus, indicating that CD44v6 is internalized through endosomal sorting and imported to the nucleus through the nuclear pore complex ( Figure 19B).
- SW48-FR-Non-CIC/CD44v6 clones were incubated with biotin-labeled CD44v6 in an endocytosis assay, the internalized CD44v6 formed a complex with TCF4 in both the cytosol and in the nucleus, whereas the CD44v6NLS Mutant only formed a complex with TCF4 in the cytoplasm, and CD44A67 was not internalized (Figure 19C).
- CD44v6NLS mutant does not allow the CD44v6-TCF4 complex to migrate to the nucleus ( Figure 19C)
- the CD44v6NLS mutant failed to increase WNT/p-catenin signaling activation ( Figure 19F) demonstrating that the effect of CD44v6 on WNT3 A/b-catenin signaling is mediated through the CD44v6-LRP6 binding, which requires the CD44v6/NLS site to be translocated to the nucleus with TCF4 for augmentation of WNT/p-catenin signaling in FOLFOX resistant cells.
- Nuclear b-oh ⁇ 6h ⁇ h/TER4 associates with CD44v6 and functions to modulate transcription of MDR1 and CD44v6
- CD44 expression is downstream of WNT3A ⁇ -catenin signaling (Wielenga, V. J., et al., 1999, Am J Pathol, 154:515-523; Misra, S., et al., 2008, Connect Tissue Res, 49:219-224; Misra, S., et al., 2008, J Biol Chem, 283:14335-14344).
- WNT3A ⁇ -catenin signaling Wielenga, V. J., et al., 1999, Am J Pathol, 154:515-523; Misra, S., et al., 2008, Connect Tissue Res, 49:219-224; Misra, S., et al., 2008, J Biol Chem, 283:14335-14344.
- CD44v6 a similar regulation of CD44v6 by WNT ⁇ -catenin in response to FOLFOX stimulation has yet to be identified.
- CD44v6-LRP6 is internalized in the presence of FOLFOX-stimulated WNT3A, and after internalization the CD44v6 is translocated to the nucleus by the stimulation of WNT3A.
- the full-length CD44v6 complexes with TCF4 and MDRl because CD44v6 and MDR1 promoters have TCF4 binding sites (see Figures 20H, and 21C).
- chromatin immunoprecipitation ChIP was done to identify DNA sequences bound by nuclear CD44v6 complexes. DNA fragments were pulled down by anti-CD44v6 antibody from a total of 11 clones.
- the MDR1 promoter luciferase constructs negatively responded to co transfection with dominant-negative TCF4-DN, b-catenin shl and CD44v6 shl. These inhibitory constructs reduced the responsiveness in PGL3-mdrl (a) and PGL3-mdrl (b) cells ( Figure 20G). These reductions provide evidence that TCF promoter binding and activation of MDR1 is mediated through both CD44v6 and b-catenin in the nucleus.
- pGL3-CD44v6 (a) and pGL3-CD44v6 (b) contain TCF4 binding sites.
- Luciferase assay was used to directly examine the interaction between P-catenin/TCF4 and the CD44v6 promoter.
- the luciferase activities in SW480-FR cells transfected with dominant negative TCF4-DN and b-catenin shRNAl (shl) were significantly lower than in the vector group ( Figure 2 IB), while b-catenin and TCF4 overexpression significantly increased the luciferase activity (data not shown). This provides evidence that b- catenin/TCF4 increases CD44v6 transcription activity.
- Figure 21E shows that the efflux of 14C- OXA (a component of FOLFOX) elevated, leaving low levels of intracellular drug retention after the addition of 1 x FOLFOX, or 20 ng/ml WNT3 A for 2 hours. Elevation of efflux of 14C-OXA in these cells increases in a time-dependent manner reaching a plateau level 2-2.5 h after FOLFOX, or WNT3A treatments (data not shown). The present results clearly show that the efflux of oxaliplatin was elevated in control FR- tumor cells compared to sensitive SW480 cells (Figure 2 IE). This high level of FOLFOX-mediated drug efflux causes low levels of intracellular OXA retention in FR cells compared to sensitive cells ( Figure 2 IE).
- WNT3A shRNAl blocked WNT3A in FOLFOX treated cells sensitizes the cells to FOLFOX ( Figure 5A, and Figure 4C) and fails to stimulate the efflux of OXA by increasing drug retention (data not shown).
- this Example indicates that FOLFOX treatment induces both WNT3A and CD44v6, which through its NLS site recruitment of LRP6 to CAVl-raft and activates LRP6 (S1490) to promote WNT3 A/p-catenin/TCF4 signaling induced CD44v6 expression.
- This functions through a positive feed-back loop between CD44v6 and b- catenin/TCF4 that activates MDR1 gene expression and CD44v6 splicing that sustains FOLFOX resistance.
- the failure to recruit MDR1 into a complex with CD44v6 using overexpression of CD44v6A67 or silencing CD44v6 abolishes FOLFOX-induced active multidrug efflux and increases drug retention.
- CIC heterogeneity develops through time as CICs evolve genetic or epigenetic alterations that allow them to differentiate into multiple tumor cell types (Rich, J. N., 2016, Medicine (Baltimore), 95:S2-7).
- CICs are most often defined as being multipotent, long-lived, slow cycling/quiescent, self-renewable, and asymmetrically dividing cells within tumors that have tumorigenic potential when transplanted into immune-deficient mice (Vermeulen, L., et al., 2008, Proc Natl Acad Sci USA, 105:13427-13432; Valent, P., et al., 2012, Nat Rev Cancer, 12:767-775; Vermeulen, L., et al., 2008, Cell Death Differ, 15:947-958; Visvader, J. E., 2011, Nature, 469:314-322; Visvader, J.
- CICs can be serially transplanted through multiple generations to relapse the tumor displaying strong drug-resistance and metastatic traits (Zeilstra, J., et al., 2014, Oncogene, 33:665-670; Valent, P., et al., 2012, Nat Rev Cancer, 12:767-775; Moitra, K., et al., 2011, Clin Pharmacol Ther, 89:491-502; Maugeri-Sacca, M., et al., 2011, Clin Cancer Res, 17:4942- 4947; Calcagno, A.
- chemotherapeutic stress may offer a selective advantage to cells with a high degree of sternness function of CICs.
- 5-fluorouracil (5- FU) alone or in combination with other chemotherapeutic agents, such as folinic acid/leucovorin plus oxaliplatin (FOLFOX) have been used as the standard therapy for advanced CRC (Sinicrope, F.
- These functional tests include WNT ⁇ -catenin/TCF4 mediated TOPFlash promoter activity, lipid raft localization assay, internalization/endosomal sorting and nuclear trafficking analysis, Cell viability, Annexin V positive cells expressing cell apoptosis, tumor sphere formation, xenograft tumor growth, MDR1 and CD44v6 gene transcription through nuclear CD44v6-TCF4 complex.
- FOLFOX induces alternative splicing of CD44 to generate the v6 isoforms, which is important for activation of the WNT3 A/b- catenin/MDRl signaling ( Figures 2A-F, and Figure 14A). It was demonstrated herein for the first time that FOLFOX induces WNT3 A secretion ( Figure 1 IB) that mediates b- catenin activation upon engagement of LRP6 with the cell surface receptor CD44v6 ( Figures 14B-G).
- the FOLFOX induced CD44v6 isoform regulates WNT/b- catenin TOPFlash reporter activity and FOLFOX resistance in CICs indicating that the induction of CD44v6 is a positive regulator of WNT ⁇ -catenin mediated CIC’s autonomous resistance ( Figures 1, 2, 4, 5, 6, 7, 9, 10-12, and 14-15).
- Targeting WNT or CD44v6 induced FOLFOX sensitivity and completely blocked the WNT3A mediated transactivation of the CICs Figures 4C and 5A, indicating that the CIC-chemoresi stance was generated by WNT ⁇ -catenin via CD44v6.
- CD44 (NLS) mutant sequesters TCF4 in the cytosol.
- the TCF4 remains associated with CD44v6 and binds to the TCF4, MDR1, and CD44v6 promoter, leading to increased MDR1 activity and drug efflux.
- WNT3 A-mediated phosphorylation of S1490 of LRP6 by CD44v6 is required for its association with DVL-2 and Caveolin, whereas DAB2 attenuates the WNT signaling by shifting the CD44v6- LRP6 complex to clathrin mediated endocytosis in sensitive cells.
- CD44v6-WNT3 A-mediated therapeutic drug resistance in CICs of colon tumor cells providing evidence that targeting CD44v6-WNT3 A mediated b- catenin/TCF4-MDRl signaling pathways and increased MDR1 efflux function may represent a novel approach to overcome chemotherapy resistance in colon tumor CICs.
- tumorigenic potential of isolated colon CD44v6+CICs was evaluated. Specifically, the ability of tumor-derived CD44v6+CICs and CD44v6 (-) Non- CICs was investigated using FACS sorting and implantation into immunocompromised mice for the formation of colon tumors. In this xenograft model of CRC, as many as 5 x 10 5 patient-derived and SW480-FR/SQ-derived CD44v6 (-) Non-CICs did not induce tumor formation. In contrast, 5 x 10 5 unfractionated bulk tumor cells or as few as 2 x 10 3 CD44v6+CICs generated visible tumors after 2-3 weeks ( Figures 7F, 9A-C, 10C-D, and 10F).
- CD44v6+CICs obtained from secondary xenografts were then transplanted into third- generation mice. Even though the higher number of CD44v6+ cells present in 5 x 10 5 unfractionated bulk tumor cells, tumor formation capacity of sorted CD44v6+CIC cells was faster and more efficient than tumor formation obtained with the unfractionated bulk tumor cells (Figure IOC). During the in vivo passaging, CD44v6+CICs did not lose their tumorigenic potential but instead increased their faster tumor growth (Figure 10D).
- CD44v6+CICs form tumor spheres in primary, second- and third generation of mice whereas tumorigenic potential of CD44v6 (-) Non-CICs were completely lost in secondary and tertiary recipients of xenografts.
- the CD44v6+CICs confined to a small cell population resident in the colon tumor and has the ability to generate reproduce long-term tumorigenic potential in serial recipient xenografts with unlimited tumorigenic potential.
- CD44v6 appears as a biomarker and therapeutic target for CRC patients.
- LRP6 and CD44v6 are subsequently internalized through the caveolin pathway and is sorted in endosomes that results in a b- catenin-CD44v6 complex accumulation in the nucleus, whereas in sensitive cells in the presence of DAB2, a CD44v6-LRP6 complex is internalized through the clathrin pathway and fails to inhibit the P-catenin-CD44v6 complex accumulation in the nucleus by promoting a b-catenin destruction complex. Moreover, CD44v6 acts as a regulator of the FOLFOX-induced WNT3A ⁇ -catenin signaling, and the binding and activation of LRP6 with consequent b-catenin transcription requires the NLS binding domain of CD44v6.
- CD44v6-TCF4 causes transcriptional activation and the expression of its target genes, including sternness associated genes.
- CD44v6-TCF4 also induces TCF4-specific transcriptional activation leading to activation of TOPFlash promoter, MDRl and the CD44v6 promoter, which sustains drug resistance in CICs expressing high CD44v6.
- FOLFOX- induced WNT3 A binding promotes MDRl association with CD44v6. This CD44v6- MDRl complex formation results in an increased efflux of the chemotherapeutic drug oxaliplatin.
- the coordinated FOLFOX/WNT3A-mediated CD44v6-WNT3A ⁇ - catenin/TCF4 signaling and the FOLFOX/WNT3A-mediated increased multidrug efflux (as diagrammed in Figure 24) is believed to be a potential mechanism underlying various tumor stem cell-specific behaviors, including transcriptional activation, tumor cell growth, and CIC autonomous multidrug resistance during CRC tumor progression.
- Example 2 Interplay between chemotherapy-activated cancer associated fibroblasts and cancer initiating cells expressing CD44v6 promote colon cancer resistance
- CICs Cancer-initiating cells
- CAFs cancer-associated fibroblasts
- the data in Example 1 shows that CICs expressing CD44v6 display cell-autonomous resistance to FOLF OX-therapy.
- FOLFOX-therapy stimulated a significant increase in CAFs that boosted CIC-chemoresi stance by secreting paracrine-factors (periostin, IL17A and WNT3A) as well as expressing CD44v6.
- CAFs-derived periostin and IL17A recruit WNT3A by inhibiting Dickkopfl (DKK1) and consequently increased CD44v6 expression in CICs by activating the WNT3 A/b-catenin pathway. Blocking either of these paracrine-factors or CD44v6 reduced colon tumor growth by disrupting the interplay between CICs and CAFs.
- DKK1 Dickkopfl
- this approach reduced xenograft-tumor growth more effectively than using CD44v6- blocking antibodies because this tissue-specific conditionally-silencing of CD44v6 resulted in longer-lasting changes maintained by sustained CD44v6/signaling resulting from a positive-feedback loop linking WNT3 A signaling-dependent alternative-splicing of CD44 to tumor cellular hierarchy through CAFs-secreted paracrine-factors.
- CRC is ranked second in women after breast cancer and third in men after prostate and lung cancers (Ferlay, J., et al., 2015, Int J Cancer, 136:E359-386). Recent studies show that the number of people age ⁇ 50 diagnosed with CRC is significantly increasing. Moreover, five-year survival in CRC ranges from 90% in early stages to less than 10% in advanced, metastatic cases (Coppede, F., et al., 2014, World J Gastroenterol, 20:943-956).
- Solid tumors display the heterogeneity that originated from a small subset of cancer cells known as cancer initiating cells (CICs), which are the unique source of all tumor cells and display cellular hierarchies from which tumor clones originate with self-renewing, extensive proliferating capacity to differentiate into multi-direction and cancer stemlike cells, or into cancer-initiating cells (CICs) at the apex (de Sousa e Melo, F., et al., 2017, Nature ,543:676-680; Bu, Y., et al., 2012, Front Biosci (Schol Ed), 4:819-830; Valent, P., et al., 2012, Nat Rev Cancer, 12:767-775).
- CICs cancer initiating cells
- CD44 is a multi -structural and multi-functional transmembrane glycoprotein that is encoded by a single gene containing 20 exons, ten of which are alternatively spliced to generate the numerous CD44 splice variants (CD44v) (Screaton, G. R., et al., 1992, ProcNatl Acad Sci USA, 89:12160-12164; Zoller, M., 2011, Nat Rev Cancer, 11 :254-267).
- the standard isoform of CD44 (CD44s) has no variant exons, is small and is nearly ubiquitous in vertebrate cells (Naor, D., et al., 2008, Semin Cancer Biol, 18:260-267).
- CD44s, CD44v6 and CD44v4-10 are detected in the human gut epithelium (Zeilstra, J., et al., 2014, Oncogene, 33:665-670)
- experiments using knock-in mice that express either CD44v4-10 or CD44s isoforms have demonstrated that CD44v isoforms, but not the CD44s isoform, promote adenoma formation in Apc/Min/+ mice (Zeilstra, J., et al., 2014, Oncogene, 33:665-670).
- CD44v6 (+) CICs have been identified that have self-renewal capacity and are able to differentiate into non tumorigenic cells.
- CICs have an important role in tumor niche generation by recruiting and activating tumor micro-environment cells through different signaling pathways (Paltridge, J. L., et al., 2013, Biochim Biophys Acta, 1834:2233-2241). Many of these pathways result in a communication loop between CICs with interactive niches that are specialized microenvironments comprised of various stromal cell types including fibroblasts (Medema, J.
- Mechanisms driving CIC maintenance and resistance are controlled by cell-cell interactions mediated through various mechanisms that involve extracellular matrix components and networks of growth factors, cytokines and chemokines (Todaro, M., et al., 2014, Cell Stem Cell, 14:342-356; Korkaya, H., et al., 2011, J Clin Invest, 121:3804- 3809; Vermeulen, L., et al., 2010, Nat Cell Biol, 12:468-476 Li, H. J., et al., 2012, Cancer Discov, 2:840-855).
- cytokines and chemokines Todaro, M., et al., 2014, Cell Stem Cell, 14:342-356; Korkaya, H., et al., 2011, J Clin Invest, 121:3804- 3809; Vermeulen, L., et al., 2010, Nat Cell Biol, 12:468-476 Li, H. J., et al., 2012
- the CD44v6-ectodomain can bind to growth factors/cytokines, such as HGF (Orian-Rousseau, V., et al., 2002, Genes Dev, 16:3074-3086), VEGF (Tremmel, M., et al., 2009, Blood, 114:5236-5244), EGF (Cheng, C., et al., 2006, Genes Dev, 20:1715- 1720) and TGFpl (Ghatak, S., et al., 2014, J Biol Chem, 289:7856-7872; Ghatak, S., et ak, 2017B, J Biol Chem, 292:10465-10489), and can potentiate receptor tyrosine kinase- associated signaling pathways where CD44v6 acts as a coreceptor.
- HGF Human-Rousseau, V., et al., 2002, Genes Dev, 16:3074-3086
- CD44v6 functions as a signaling platform that responds to microenvironmental paracrine cues through growth-factor/cytokines, and transduces these signals to membrane-associated proteins that induce a variety of genes related to apoptosis resistance
- Cytokines and chemokines function as both paracrine and autocrine factors, supporting tumor-microenvironment secreted molecules as ideal mediators of interactions between the CICs and other tumor cellular components.
- CD44v6 increases pathologically in the gut of the APC/Min+ mouse model (Zeilstra, J., et ak, 2014, Oncogene, 33:665-670).
- One of the stromal factors is periostin (PN), a 93.3 kDa matricellular protein (Morra, L., et ak, 2011, Virchows Arch, 45:465-475).
- Periostin mediates cell activation by promoting hyaluronan-CD44 mediated signaling through binding to integrins in other cell types (Ghatak, S., et ak, 2014, J Biol Chem, 289:8545- 8561).
- PN is observed mainly in tumor stroma and at a minor level in the cytoplasm of cancer cells, and stromal PN has a key role in regulating CIC maintenance and expansion during metastatic colonization by increasing WNT signaling (Malanchi, k, et ak, 2011, Nature, 481:85-89).
- WNT activity defines CRC stem cells and is regulated by the microenvironment (Vermeulen, L., et al., 2010, Nat Cell Biol, 12:468-476; Zeilstra, J., et al., 2008, Cancer Res, 68:3655-3661).
- CD44v6 is involved in regulating T-cell activity in vivo (Arch, R., et al., 1992, Science, 257:682-685).
- Cancer-associated fibroblasts (CAFs) create a chemo resistant niche in CRC by releasing cytokines, including IL17A, as a colorectal CIC maintenance factor (Lotti, F., et al., 2013, J Exp Med, 210:2851-2872).
- IL17A cytokines are also produced by immune cells within the gut mucosa and colorectal tissue, and CD44v6 is involved in regulating T-cell activity in vivo (Arch, R., et al., 1992, Science, 257:682-685).
- the WNT signaling involves either a canonical or a noncanonical pathway, and only the canonical pathway is well studied (Clevers, H., et al., 2012, Cell, 149:1192-1205; Gougelet, A., et al., 2012, Int J Hepatol, 2012:816125).
- 1-catenin is the main effector of the canonical signaling.
- the cytosolic b- catenin is continuously ubiquitinylated for degradation through phosphorylation by the casein kinase 1 (CK1) and the subsequent phosphorylation of the glycogen synthase kinase 3 (GSK3).
- Frizzled and scaffolding protein Dvl are required for the process of the phosphorylation of LRP6 and the recruitment of Axin Zeng, X., et al., 2008, Development, 135:367-375; Bilic, J., et ak, 2007, Science, 316:1619-1622).
- the Dickkopf (DKK) family comprises four members (DKK1 - DKK4), which antagonize WNT signaling (Niehrs, C., 2006, Oncogene, 25:7469-7481).
- DKK1 functions through two mechanisms: 1) DKK1 prevents Fz-LRP6 complex formation and inhibits b-catenin signaling (Semenov, M.
- DKK1 in addition to LRP6, DKK1 interacts with transmembrane receptor proteins Kremenl (KRMl) and KRM2 (Niehrs, C., 2006, Oncogene, 25:7469-7481), and association of DKK1/LRP6 complex with KRM1 and KRM2 modulates endocytosis and attenuates WNT signaling.
- FOLFOX stimulation increased the frequency of CAFs measured by stimulating the relative proportion of fibroblasts to the epithelial component (aSMA versus EpCAM). Subsequently CAFs create a chemo resistant niche by releasing pre-dominantly periostin, WNT3A and IL17A, which sustain a WNT3A-CD44v6 induced CIC-maintenance shared by WNT ligands through inhibition of DKK1.
- a common denominator is the cell- surface marker CD44v6-signaling that involves tumor-stroma interaction through periostin/WNT3A- and/or IL17A/WNT3A-induced augmentation of CD44v6-signaling, and that blocking the CD44v6-signaling will disrupt the CIC-autonomous resistance mechanism.
- Nonidet P-40, EGTA, sodium orthovanadate, glycerol, phenylmethyl sulphonyl fluoride, leupeptin, pepstatin A, aprotinin, and HEPES were from Sigma.
- Recombinant human periostin, WNT3A and IL17A proteins were from R&D Systems (Minneapolis, MN).
- the antibodies against CD44v6, b-catenin, Anti-Active-P-catenin (anti-ABC) antibody, clone 8E7, TCF4, MDRl, b-actin, b-tubulin, horseradish peroxidase-linked anti -rabbit and anti-mouse antibodies, and Luminol reagent were purchased from commercial sources (R&D, Santa Cruz Biotechnology Inc., Abeam, Ebioscience, Thermo Fisher, and Cell Signaling Technology).
- Blocking antibodies for CD44v6 (2F10), IL17A and isotype control were from R&D Systems.
- Blocking antibody for periostin (OC-20) was from Adipogen Life Sciences.
- Blocking antibody for WNT3A (1H12L14) was from Thermo Fisher.
- pDONR223_DKKl_WT was a gift from Jesse Boehm & William Hahn & David Root (Addgene plasmid # 82250; http://n2t.net/addgene:82250; RRID: Addgene_82250).
- IL17 (IL17A) cDNA (Myc-DDK-tagged) in expression vector pCMV6-entry (Cat# RC 18057) was from Origene.
- pcDNA-Wnt3A-V5 was a gift from Marian Waterman (Addgene plasmid # 35927; http://n2t.net/addgene:35927; RRID:Addgene #3 5927).
- 3XFlag DVL2 SPY
- LRP6-pCS2-VSVG was a gift from Xi He (Addgene plasmid # 27282; http://n2t.net/ addgene:27282; RRID:Addgene_27282).
- KREMEN2 transcript variant 2 Myc-DDK-tagged
- KREMENl transcript variant 3 Myc-DDK-tagged
- WIDR CCL-218 was maintained in Eagle's Minimum Essential Medium (EMEM) +10% FBS; 2) LOVO (CCL-229) was maintained in F-12K Medium 2 mM L-glutamine and 1500 mg/L sodium bicarbonate; 3) HCT116 was maintained in McCoy's 5 A medium; 4) HT29 (HTB-38) was maintained in McCoy's 5 A medium; 5) SW480 (CCL-228) was maintained in Leibovitz's L-15 Medium that was purchased from ATCC, Manassas, Virginia.
- HCA-7 colony 29 was purchased from European Collection of Authenticated Cell Cultures and maintained in DMEM + 10% FBS + 2 mM L-Glutamine + 110 mg/L sodium pyruvate.
- Pre-neoplastic Ape 10.1 cells isolated from Ape min/+ mice were cultured in Dulbecco’s modified Eagle medium supplemented with 20% FBS and harvested by a 15-30 min treatment with trypsin-EDTA solution (De Giovanni, C., et al., 2004, Int J Cancer, 109:200-206).
- Drug resistant human CRC resected tissues from patients (age range, 55- 80 years) was procured from a tissue bank.
- Cells were isolated from the resected human tumor tissues (designated as PD), and FOLFOX resistant (FR) clones were generated that were maintained through subcutaneous xenografts in the flanks of immunocompromised [NOD-SCID/IL2Rynull (NSG)] mice and SCID mice respectively.
- Fresh normal colonic tissue and colorectal tumors were rinsed in DMEM (Life Technologies) supplemented with 200 units/mL of penicillin, 200 pg/rnL of streptomycin, and 4 units/mL of amphotericin B and minced, followed by incubation with 300 units/mL of collagenase (Worthington Biochemical) at 37° C for 3 hours. A single cell suspension was obtained by filtration through a 40 pm filter.
- FACS fluorescence-activated cell sorting
- Flow cytometry was done in a cell sorter.
- single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC)-conjugated monoclonal antibody against EpCAM (R&D Systems).
- FITC Fluorescein-5-isothiocyanate
- Purified CD44v6+/EpCAM+ and CD44v6-/EpCAM+ cells from various tumors were cultured separately and grown in fresh CIC growth medium.
- CICs were cultured in serum-free media with basic fibroblast growth factor (bFGF, 10 ng/ml; R&D Systems) and epidermal growth factor (EGF, 10 ng/ml; R&D Systems) for 2 weeks. Then, the cultured CD44v6+/EpCAM+ and CD44v6-/EpCAM+ cells were subjected to flow cytometric analysis for isolation of D44v6+EpCAM+ALDHl+CD133+ (designated as CICs), and CD44v6-EpCAM+ALDHl+CD133+ (designated as Non-CICs) using appropriate fluorescence-conjugated antibodies as outlined in the Method section of Example 1. CICs were cultured in serum-free media with basic fibroblast growth factor (bFGF, 10 ng/ml; R&D Systems) and epidermal growth factor (EGF, 10 ng/ml; R&D Systems).
- bFGF basic fibroblast growth factor
- EGF epidermal growth factor
- Single cell suspensions from a dissociated FOLFOX resistant (FR) patient colorectal tumor (PD-FR) were sorted by FACS using PDGFR-a-PE and EpCAM-FITC. Percentages of EpCAM (-)/PDGFR-a (+) (CAFs) and EpCAM (+)/PDGFR-a (+) (Non- CAFs) in total unsorted cell populations were quantified.
- EpCAM (- )/PDGFRa (+) gated CAFs from the dissociated CRC cells from the patient colorectal tumors (PD-5FUR, PD-OXAR, PD-FR) and from the sensitive and FOLFOX resistant SW480/subcutaneous (SQ) tumor tissues were confirmed by QPCR analyses for CAF- associated markers FSP1, FAP, PDGFR-a, and epithelial cell marker EpCAM (negative control).
- EpCAM (-)/PDGFR-a (+) cells referred to as ‘CAFs’ were isolated from the indicated tumor tissues.
- the high PDGFRa positive population was gated in a range of 4- 20% (depending on the percentage of the total positive cell population of each sample) of the tail of the positive cells.
- EpCAM (-)/PDGFR-a (+) cell populations were cultured in DMEM with 10% BSA for 12 days, and were sorted by FACS using a- SMA-PE and EpCAM-FITC antibodies. The percentages of the sorted a-SMA (+)/PDGFR-a (+) cells in total unsorted CAFs are designated as enrichment of active CAFs. Dead cells were eliminated by using the viability dye DAPI. Isotype controls were used to establish proper gates.
- CICs Cell growth survival and apoptosis assays.
- the roles of paracrine factors on tumor sphere formation of CICs were determined by plating five thousand cells in triplicate into 96-well plates containing appropriate growth media. After 16 hours growth, cultures were incubated in media containing no serum for 16 hours at 37°C in 5% CO2, 95% air. Vehicle or 20 ng/ml of WNT3A, or PN, or IL17A, or chemotherapeutic drug was added to the plate and grown for 48 hours. In each experiment, a total of five plates (6 wells/treatment) were used. Experiments were repeated 3 times. The growth of these cells was determined by measuring increases in readings of ATP levels for viability (CellTiter-Glo, Promega).
- the luminescent signal is proportional to cell viability and measured using a luminometer (PerkinElmer).
- the Caspase-Glo ® 3/7 assay depends on the formation of free amino luciferin after adding caspase-3/7 DEVD-amino luciferin substrate to cell lysates and measuring amino luciferin by the luciferase present in the substrate reagent.
- the luminescent signal is proportional to caspase 3/7 activity and measured using a luminometer (PerkinElmer).
- cells were treated in the presence or absence of FOLFOX or WNT3A conditioned media at 37°C for the times indicated and washed three times with ice-cold phosphate-buffered saline (PBS; pH 8.0) to remove any contaminating proteins, and Biotinylation followed by immunoprecipitation and western blotting was done as described in Example 1.
- PBS ice-cold phosphate-buffered saline
- cell surface proteins were biotin-labelled as described in Example 1. The amounts of receptor bound to beads were determined by followed by SDS-PAGE and immunoblot analysis.
- tumor formation medium 500 ml Dulbecco’s Modified Eagle Medium/F12
- 20 ng/ml epidermal growth factor 10 ng/ml basic fibroblast growth factor
- 5 pg/ml insulin 0.4% bovine serum albumin.
- 200 live cells / 200 m ⁇ of tumor sphere medium were suspended in ice. This suspension was kept on ice and mixed well for plating.
- PBS was added to the first and last columns (column 1 and 12) of the 96-well plate to help minimize medium evaporation. This leaves 10 wells available for each row.
- Lipid rafts from cultured cells that were treated or transfected with indicated reagents or constructs for a particular experiment indicated in the brief description of the figures were fractionated by using Opti prep gradient (Graham, J. M.,2002, ScientificWorldJournal, 2:1440-1443; Macdonald, J. L., et ah, 2005, J Lipid Res, 46:1061-1067).
- the experimental method for isolation of lipid raft and non-raft fractions was described in Example 1.
- pcDNA-Wnt3A-V5 was procured (Addgene plasmid # 35927; http://n2t.net/addgene:35927; RRID:Addgene #3 5927), human beta-catenin pcDNA3 was a gift from Eric Fearon (Addgene plasmid # 16828; http://n2t.net/addgene: 16828; RRID: Addgene # 16828).
- CD44v6 specific PCR amplification products were isolated with polyadenylated RNA from the HT29 cell line.
- PCR product was cloned in pcDNA3.1 vector and used as described previously (Ghatak, S., et ak, 2017, J Biol Chem, 292:10465-10489).
- Myc-tagged human full length TCF4E pcDNA3 was procured (Addgene plasmid # 32738; http://n2t.net/addgene:32738; RRID: Addgene # 32738).
- pDONR223_DKKl_WT was procured (Addgene plasmid # 82250; http://n2t.net/addgene: 82250; RRID: Addgene # 82250).
- Reporter vectors and lucif erase reporter assays The MDR1, CD44v6 reporter PGL3 constructs, TOPFlash and FOPFlash were used as described in Example 1.
- the normalization vector pRL-TK renilla with a HSV-TK promotor driving Renilla luciferase was purchased from Promega.
- Transient transfection and luciferase reporter assay for PGL3 mdrl and PGL3 cd44v6 and b-catenin/TCF Reporter assays were done as described in Example 1.
- RNA extraction and cDNA synthesis were done as described in Example 1.
- the semi-quantitative PCR primer sequences used for CD44 exon specific PCR are given in Figure 3.
- the semi-quantitative PCR primer sequences used for proteins and cytokines of CAFs are presented in Figure 36.
- the QPCR primers used in this study in analyses of various genes associated with fibroblast specific markers as well as for PN, IL17A, and WNT3A are presented in Figure 37.
- the QPCR primers used in this study in analyses of various genes associated with CIC sternness factors are presented in Figure 38.
- shRNA sequences For determining shRNA sequences, coding nucleotide sequences of the genes were used, obtained from the National Institutes of Health database, website (www.ncbi.nlm.nih.gov). The hairpin shRNAs to target a transcript sequence were designed using the Broad Institute GPP Web Portal
- NT sh The pSicoR-Non targeted shRNA (NT sh) was used as control to the above shRNA transfectants (see Figure 13 for shRNA sequences used in this study).
- the specificities of prepared shRNAs were confirmed (Ghatak, S., et al., 2017B, J Biol Chem, 292:10465-10489; Ghatak, S., et ah, 2017, JBiol Chem, 292:10465-10489) as described in Example 1.
- the primers for various shRNAs used in this study are given in Figure 39.
- ChIP chromatin immunoprecipitation
- CICs and CAFs were isolated from subcutaneous SW480-FR/SQ xenografts in SCID mice (using approved IACUC protocol).
- Tumorigenic potential of CICs alone or in combination with CAFs was determined by subcutaneous implantation in the flanks of six-week-old SCID female mice from the Jackson Laboratory.
- the CAFs were treated with DMSO or FOLFOX (50 mM 5-FU + 10 pM OXA + 1 pM Leucovorin) for 3 days.
- CAFs (4 x 10 4 ) pre-treated with FOLFOX were determined by SQ implantation as mentioned above. Twenty-five mice per cell types were used. The appearance of tumors were monitored, and five mice were sacrificed every 2 weeks. Tumors were removed and weighed to evaluate the tumor development (Figure 33).
- shRNA vectors are inherently inactive due to the presence of a Lox-Stop-Lox cassette prior to the start codon. When injected intraperitoneally (i.p.), there will be ubiquitous cellular uptake of these vectors. However, only cells expressing FABPL (i.e. intestinal epithelium) will express the Cre protein and activate the shRNAs.
- FABPL i.e. intestinal epithelium
- Tf-PEG-PEI nanoparticles were prepared and both plasmids (floxed pSico-CD44v6shRNA + p Fabpl- Cre ) were encapsulated into them as previously described (Misra, S., et al., 2009, J Biol Chem, 284:12432-12446).
- the pSico-CD44v6shRNA was validated in SW480-FR cells ( Figure 33A, B, D).
- transferrin (Tfi-coated PEG-PEI) The transferrin (Tf)- PEG-PEI/Nanoparticle (Nano) was prepared as validated in previous studies (Ghatak, S., et al., 2017B, J Biol Chem, 292: 10465-10489; Misra, S., et al., 2009, J Biol Chem, 284:12432-12446).
- transferrin was linked with N-hydroxy succinimide/ PEG/maleimide and then allowed to react with a mercaptopropionate-modified branched PEI to form Tf-PEG-PEI (Ghatak, S., et ak, 2017B, J Biol Chem, 292:10465-10489; Misra, S., et ak, 2009, J Biol Chem, 284:12432-12446).
- the pSico-CD44v6 shRNA, or pFabpl-Cre plasmid was encapsulated in the purified Tf-PEG-PEI conjugate separately (size: ⁇ 80 ⁇ 31 nm).
- PCR was done to amplify the recombined and unrecombined genomic plasmid DNA from SW480-FR cells.
- Group 3 1 x FOLFOX [(3 x per Wk. for 4 Wks.)] with pSico NT shRNA-Nano (3 x per Wk. for 4 Wks.)]; Group 4: 1 x FOLFOX [(3 x per Wk.) for 4 Wks.] with Fabpl Cre -Nano (3 x per Wk. for 4 Wks.); Groups 5. 7. and 9: pSico v6 shRNA-Nano [(20 pg, Group 5): (50 pg, Group 7): (100 pg, Group 9) (3 x per Wk. for 4 Wks.)]; Group 6. 8.
- a two-tailed Student’s t-Test was used to compare mean value between sensitive and resistant cells using the following parameters: mean AACT values for QPCR; mean colony number for soft agar growth assays; mean densitometry values for QPCR and WB; mean percentage of cell viability assay (CellTiter-Glo) and FACS analysis; mean luminescence for ATP activity in cell growth, Caspase Glow assays in Apoptosis measurements; and mean tumor weight in xenograft studies. Chi-squared analysis was performed to compare incidences between sensitive and resistant cells for the following assays: number of positive wells containing tumor spheres in the sphere formation assay; and numbers of mice developing tumors in xenograft studies.
- Example 1 revealed that the CIC immunophenotype (O ⁇ 44n6 M ⁇ 1i+ ) within a tumor is responsible for tumor formation, progression and resistance to FOLFOX therapies.
- the interaction between CICs and their tumor niches (microenvironment) is strongly linked to the CIC survival/self-renewal (Peitzsch, C., et al., 2017, Semin Cancer Biol, 44:10-24).
- CICs can preserve the tumor heterogeneity that underlies the important malignant behaviors and therapy resistance (Ben-Porath, T, et al., 2008, Nat Genet, 40:499-507).
- FOLFOX-stimulated CAFs promote CIC growth.
- CAFs are known to support tumorigenesis (Vermeulen, L., et al., 2010, Nat Cell Biol, 12:468-476), viable CAFs were isolated from freshly resected patient colon tumor tissues and from SQ tumor samples by FACS using platelet-derived growth factor receptor-a (PDGFRa) and EpCAM antibodies ( Figures 25A-B). Data in Figure 25C shows that unsorted PD-FR cells contain about 20% amounts of EpCAM (-) PDGFRa (+) (referred to CAFs) and slightly more than 20% EpCAM (+) PDGFRa (+) cells (referred to as Non-CAFs).
- the patient-derived and xenograft (SQ) tumor-derived CAF cultures expressed high levels of fibroblast mRNA markers, including aSMA, PDGFRa, FAP and FSP1 via QPCR analyses, but very little or no EPCAM, an epithelial cell marker (Figure 25D).
- the data in Figure 25D also demonstrated that fibroblast markers, including aSMA and PDGFRa, increased after FOLFOX (FR) treatment.
- Figure 25E shows quantitative measures of fibroblast component a-SMA versus epithelial component EpCAM with or without FOLFOX treatment (measured by FACS sorting), and the results show that CAFs derived from FR tumor cells increase the proportion of fibroblasts to the epithelial component (aSMA versus EpCAM), compared to 5-FUR, OXAR and sensitive (S) tumor cells.
- the aSMA versus EpCAM ratio is further increased with FOLFOX treated CAFS in all the tumor types ( Figure 25E), confirming that CAFs are enriched in post-cytotoxic therapy on tumors.
- CAFs under both experimental conditions demonstrated a paracrine effect through secreted factors from CAFs pre-treated with FOLFOX to promote CIC growth (Figures 25F, G).
- normal fibroblasts which are not CAFs, also increased CIC viability upon FOLFOX treatment relative to untreated cells, but at much reduced effects compared to FOLFOX-treated CAFs ( Figure 25F).
- FOLFOX-treated CAFs enhanced the ability of CICs to initiate tumors and increase tumor growth rates in immunocompromised mice (Figure 25H-K).
- xenografts generated by CICs co-implanted with FOLFOX-treated CAFs displayed increased xenograft tumor incidence with increased tumor size (Figure 25H-K) and reduced the latency of tumor formation by CICs (Figure 25J-K).
- Figure 25 demonstrate that FOLFOX-therapy activates CAFs to secrete biological components in the microenvironment that stimulate CIC growth and maintenance.
- the response to FOLFOX by these CAFs appears to be preconditioned by the tumor microenvironment since normal fibroblasts treated with FOLFOX could not induce CIC viability to the same degree ( Figure 25F, and G).
- FOLFOX stimulates CAF cytokine secretion.
- Figures 25F-K provide evidence for the presence of biologically important components in the CAF secretome.
- comparative analyses was done of CAF associated periostin, cytokines, and their related receptors and transcription factor gene expressions at baseline in PD- 5FUR/CAFs, PD-OXAR/CAFs and PD-FR/CAFs, as well as in normal fibroblasts (Normal-Fb) by semi-quantitative RT-PCR analysis ( Figures 26A,B).
- FIG. 26A shows several molecules that are exclusive to each CAF, and there were only 12 cytokines and matricellular protein molecules common to all three patient-tumor derived (PD) CAFs shown in the Ven diagram ( Figure 26C).
- PD patient-tumor derived
- Figure 26C conditioned no-serum media
- CAFs-derived factors have a central role for different inflammatory pathways that can enhance CD44v6 regulated b-catenin signaling (as seen in Example 1) in the absence of inflammatory immune cells.
- chemotherapy treated CAFs directly enhanced CIC growth in the absence of inflammatory cells ( Figures 25F, and G), and the three stromal-secreted factors (PN, WNT3A and IL17A) were largely induced in FOLFOX stimulated CAFs ( Figures 26D- F).
- each of these three stromal-secreted factors directly augmented CIC growth in the absence of CAFs (ATP activity, Figure 26K) with reduced apoptosis (apoptosis data not shown).
- these three CAF-secreted factors stimulated CIC’s tumorigenic potential as measured by tumor sphere formation in CICs from sensitive and FR tumors of SW480 (Figure 35F).
- Example 1 shows that FOLFOX induced CD44v6 regulated WNT3 A/b-catenin signaling in CICs.
- CAFs derived chemo resistant tumor cells PD-5FUR, PD-OXAR and PD-FR CAFs
- PD-5FUR, PD-OXAR and PD-FR CAFs also express significant levels of CD44v6 compared to normal-fibroblasts
- WNT3 A expression was analyzed in vehicle or FOLFOX treated PD-FR CAFs in which CD44v6 was knocked down, and the results show that CD44v6 regulates WNT3A production in FOLFOX treated PD-FR/CAFs (Figure 26M). Furthermore, this regulation of CD44v6 on WNT3A is through IL17A and PN in FR-tumor cell derived CAFs but not in sensitive tumor cell derived CAFs ( Figure 35E), confirming that FOLFOX induce IL17A and PN production in CAFs-derived from FOLFOX resistant tumors which is nearly absent in CAFs-derived from sensitive tumor cells that were not exposed to FOLFOX (as seen in Figure 35A-B).
- PN and IL17A can contribute to CIC maintenance through WNT3A- CD44v6 signaling.
- IL17A and WNT3A in CAFs (Figure 26A-H) that matched with the levels of aSMA/EpCAM ratios in human patient samples-derived CAFs before and after FOLFOX treatment ( Figure 25E).
- PN and IL17A have been reported to be required for CIC maintenance (Malanchi, F, et ak, 2011, Nature, 481:85-89; Bie, Q., et ak, 2016, Sci Rep, 6:25447).
- PN, WNT3A and IL17A act as a CIC niche component that can promote CIC maintenance and expansion by augmenting WNT-CD44v6-P-catenin-MDRl -signaling.
- PN and IL17A recruit WNT ligand through inhibiting DKK1
- Periostin and IL17A have been reported to be required for colorectal CIC maintenance (Malanchi, L, et al., 2011, Nature, 481:85-89; Bie, Q., et al., 2016, Sci Rep, 6:25447).
- a prior report also showed that PN stimulated WNT3 A through inhibition of tumor suppressor DKK1 (Malanchi, L, et al., 2011, Nature, 481:85-89).
- DKK1 inhibits LRP6-mediated WNT3 A/b-catenin signaling and promotes b-catenin degradation (Mao, B., et al., 2002, Nature, 417:664-667; Mao, B., et al., 2001, Nature, 411:321-325).
- PN and IL17A have a functional role in the molecular events within the tumor environment that coordinates with inhibition of DKK1 for WNT-CD44v6- MDRl signaling activation for CIC maintenance is virtually unknown.
- CICs isolated from FR-SQ tumors were used, which display no DKK1 (Figure 28A) and less KRM receptors ( Figure 28A), and these CICs were transfected with the luciferase reporter construct TOPFLASH carrying TCF-binding sites (Korinek, V., et al., 1997, Science, 275:1784-1787).
- CD44v6 forms a complex with LRP6 for endocytosis (see Example 1) by extension DKK1 also forms a complex with LRP6-KRM2-CD44V6 and eliminates WNT3 A/LRP6 ⁇ -catenin signaling by removing the LRP6-CD44V6 from the plasma membrane as seen in Figure 28J and K and perturbing their interaction.
- PD-FR/SQ/CICs transfected with the TOPFLASH and FOPFlash were used.
- PN, IL17A or WNT3A treatment in PD- FR/CICs induces the TOPFLASH reporter activity ( Figure 28L).
- co transfection of PD-FR/CICs with DKK1 and KRM2 leads to strong synergistic -80% inhibition even following WNT3 A treatment.
- DKK1/KRM2 has similar effects on PN or IL17A induced TOPFLASH activity in PD-FR/CICs ( Figure 28L).
- SW480-FR cells were transfected with DKKland KRM2 followed by transfection with or without a WNT3A expression vector.
- SW480-FR cells were transfected with DKKland KRM2 expression vectors because the FR cells contain low levels of KRM2 and no expression of DKK1 as seen in Figure 28B, and 28A.
- Results in Figure 29A indicate that DKK1 inhibits WNT3 A target CD44v6 gene expression.
- DKK1 caused a similar reduction of LRP6 in the lipid raft fraction in clathrin-inhibitor monodansylcadaverine (MDC)-treated cells ( Figures 29C, 29D, and 29E-G). This result indicates that DKK1 may remove CD44v6 and LRP6 from the lipid raft fraction independently of clathrin-mediated endocytosis.
- PN or IL17A did not affect the distribution of CD44v6 and LRP6 between the lipid raft (R) and nonlipid raft (NR) fractions in the absence of DKK1, but they reversed the reduction of CD44v6 and LRP6 in the ‘R’ fraction induced by DKK1 when clathrin-dependent endocytosis was blocked ( Figures 29D, and 29F-G). Overall, these results indicate that DKK1 induces the internalization of LRP6 with clathrin, thereby suppressing the b-catenin signaling.
- PN and IL17A induce association of nuclear with CD44v6 and MDR1 to modulate drug resistance.
- a WNT3A ⁇ -catenin pathway regulates CD44v6 expression and vice versa ( Figures 27A-C) and enriches CD44v6 in CICs ( Figures 27H, and I).
- the data demonstrated that WNT3A ⁇ -catenin also regulate MDR1 expression through CD44v6 in CICs and in CD44v6 expressing COS-7 cells.
- data demonstrated that in the presence of FOLFOX or WNT3A, a CD44v6-LRP6 complex is recruited to lipid raft, which is necessary to activate WNT ⁇ -catenin promoter TOPFlash activation.
- CD44v6-LRP6 complex is then internalized through endosomal sorting (see Example 1) and subsequently only CD44v6 through its nuclear localization site (NLS) enters the nucleus (see Example 1) where it binds to TCF4 and MDR1 (see Example 1). Furthermore, the data (see Example 1) indicate that WNT3A/p- catenin/TCF4 drive CD44v6 and MDR1 gene expression.
- ChIP assays were done using CD44v6- immunoprecipitated (IP’d) DNA, and TCF4-IP’d DNA and input DNA were amplified using primers covering the indicated TCF4 binding sites (scheme in Figure 30C).
- ChIP assays (Figure 30D) showed robust binding of P-catenin/TCF4 to MDR1 sites in the SW480-FR cells. Results also indicated that CD44v6 bound to this site and was associated with binding of TCF4 and b-catenin ( Figure 30D).
- CD44v6 Luciferase assay was used to directly examine the interaction between PN and IL17A induced b- catenin/TCF4 and the CD44v6 promoter.
- the luciferase activities in SW480-FR cells transfected with dominant negative TCF4-DN were significantly lower than vector group (Figure 3 OF), while PN and IL17A overexpression significantly increased the luciferase activity ( Figure 30F).
- the results ( Figure 30F) also indicate that PN and IL17A induced CD44v6 promoter activity requires WNT/TCF binding to the CD44v6 promoter.
- PN and IL17A inhibited DKK1 to promote the WNT3A ⁇ -catenin promoter activity (Figure 28F-28H, and 28L). Furthermore, WNT3A induced by PN and IL17A internalizes LRP6 and CD44v6 through caveolinl ( Figure 29C-G); and 6) PN and IL17A regulated CD44v6 expression through a WNT3A/TCF4 pathway ( Figure 27).
- Stromal secreted factors can maintain CIC growth and tumorigenic function.
- Tissue specific inhibition of CD44v6 in vivo inhibits MDR1 expression and CRC progression by retaining FOLFOX sensitivity.
- CD44v6 there is no small molecule inhibitor for CD44v6 commercially available.
- experiments were conducted to test the effect of pSico- CD44v6shRNA plus pFabpl-Cre by intraperitoneal treatment with engineered nanoparticle delivery systems to express CD44V6 shRNA in the SW480-FR/CICs plus PD-FR/CAFS implanted xenograft tumors in immunocompromised mice.
- the idea was to transactivate a conditionally silenced pSico- plasmid with CD44v6 shRNA oligonucleotide (pSico-CD44v6shRNA) by Cre-recombinase produced in response to an intestine/colon tissue-specific pFabpl promoter.
- the principle is as follows.
- the recombinase produced under the influence of a tissue-specific promoter in the cells, will eliminate the (CMV-EGFP)-cassette from U6-(CMV-EGFP) f/f -CD44vd shRNA from the pSico-CD44v6 shRNA, and the U6 promoter will induce synthesis of CD44v6 shRNA.
- Normal cells in intestine/colon will not be affected because they rely mostly on the standard CD44s expression, which does not have any variant exons of CD44. Even if cells in other organs express CD44v6 shRNA, knockdown of CD44v6 will not be produced due to lack of response to the tissue-specific promoter.
- pSico-CD44v6 shRNA plus pFabpl-Cre plasmids were generated and encapsulated in a Tf-polyethylene glycol (PEG)-polyethylene imine (PEI) nanoparticle following previous studies (Misra, S., et al., 2009, J Biol Chem, 284:12432- 12446).
- PEG polyethylene glycol
- PEI polyethylene imine
- CD44v6shRNA sequence was cloned in a pSicoR vector ( Figure 32E schematic representation of pSicoR) and was used to constitutively silence endogenous CD44v6 in the absence of Fabpl-Cre in in vitro experiments.
- NT non targeted shRNA was also cloned in these vectors.
- Cre- regulated v6-shRNAl/nanoparticles were validated in SW480-FR cells, which were transfected with either pSico-CD44v6 shRNAl ⁇ Fabpl-Cre containing nanoparticles, or pSiocoR-CD44v6 shRNAl alone.
- a pFabpl-Cre plasmid was validated in conditional silencing of CD44v6 in vivo tumors derived from CICs plus CAFs from SW480-FR tumor cells.
- shRNA knockdown of CD44v6 in CICs plus CAFs implanted SW480-FR/SQ tumors depended on the inclusion of a pFabpl-Cre plasmid and induced nearly complete recombination due to loss of GFP, whereas a pARR2-Probasin- Cre plasmid, which is specific for prostate tissue, could not induce a recombination of the pSico-CD44v6-shRNA plasmid and did not show any effect in colon tissue.
- the inset shows that pARR2 -Probasin Cre releases b-galactosidase from a conditional b-gal plasmid in the mouse prostate, further validating tissue specific delivery of the plasmid if tissue specific Cre is in right place.
- tissue specificity of pFabpl-Cre dependent knockdown of another gene was validated.
- Figure 32G shows the in vivo transfection and pFabpl-Cre dependent knockdown of the firefly luciferase gene was specific to colorectal xenograft tumor tissue, as well as to small and large intestine indicating that the nanoparticles carrying plasmids activate only in specific tissues that are dependent on promoter driven Cre expression.
- xenograft tumors were established by injecting SW480-FR/CICs in combination with SW480-FR/CAFs, and PD-FR/CICs in combination with PD-FR/CAFs (Figure 33B), subcutaneously into immunocompromised mice (experimental timeline is presented in Figure 33A).
- Cre- mediated conditional silencing of endogenous CD44v6 was used in combination with FOLFOX therapy.
- Administration of Cre-mediated CD44v6-shRNA nanoparticles prevented tumor growth ( Figure 33B-33D) and inhibited CD44v6 and MDR1 expression ( Figure 33E-33F) in a dose-dependent manner.
- Figures 24-33 indicate the central role of CD44v6- WNT signaling in tumor growth and resistance to chemotherapy.
- the data assessed the mechanism of induction of WNT-CD44v6-MDRl signaling in CICs through the FOLFOX-induced CAFs-secreted factors (PN, IL17A and WNT3A).
- PN, IL17A and WNT3A FOLFOX-induced CAFs-secreted factors
- colon tumorigenic potential resides in a rare population of undifferentiated cells that express CD44v6, which defines CIC resistance to cytotoxic therapy.
- CD44v6 defines CIC resistance to cytotoxic therapy.
- the data in Example 1 demonstrated that as few as 2 x 10 3 CICs expressing CD44v6 were capable of inducing xenograft tumor formation in immunocompromised mice, whereas, as many as 5 x 10 5 Non-CICs did not result in any tumor formation.
- Example 1 demonstrated that CICs from FOLFOX resistant cells had higher nuclear b- catenin accumulation and WNT/b catenin activation, and greater resistance with a higher number of CICs expressing CD44v6.
- CD44v6 regulated WNT3A ⁇ -catenin activation sustained FOLFOX resistance appears to be associated with poor outcome on the survival of colon tumor growth in xenograft models derived from CICs plus CAFs from preclinical tumors (as seen in Figures 31-33).
- CICs may be accountable for tumor resistance to conventional chemotherapy (Valent, P., et ak, 2012, Nat Rev Cancer, 12:767-775; Gottesman, M. M., et ak, 1993, Annu Rev Biochem, 62:385-427).
- intrinsic and extrinsic pathways act through two ways for tumor progression in CICs: intrinsic and extrinsic pathways.
- the intrinsic mechanisms involve gene mutation, while the extrinsic mechanisms incorporate the production of distinct growth factors and cytokines by the tumor microenvironment leading to the activation of specific signaling pathways (Plaks, V., et ak, 2015, Cell Stem Cell, 16:225-238).
- TGFpi and IL6 may be involved in CD44v6- regulation of WNT3A via regulation of TGFpi and IL6/STAT3 mediated PN and/or IL17A.
- IL17A was shown to be induced by TGFpi in TH17 cells in CRC (Wu, S., et al., 2009, Nat Med, 15:1016-1022; Schafer, M., et al., 2008, Nat Rev Mol Cell Biol, 9:628-638), and PN is a signaling intermediate in TGFpi induced EMT in glioblastoma cancer (Ouanouki, A., et al., 2018, Oncotarget, 9:2.2023-22037).
- PN is a signaling intermediate in TGFpi induced EMT in glioblastoma cancer
- CD44v6 regulated a WNT/p-catenin axis, which in turn further transactivates MDRl and CD44v6 gene expression.
- Activation of WNT-CD44v6 signaling has a key role in maintaining the CIC pool in the gut and in promoting self-renewal of CR-CSCs (Todaro, M., et al., 2014, Cell Stem Cell, 14:342-356; Todaro, M., et al., 2007, Cell Stem Cell, 1:389-402; Leedham, S. J., et al., 2013, Gut, 62:83-93).
- Stromal elements contribute significantly to maintain the undifferentiated status and clonogenic activity of the tumorigenic cells (Medema, J.
- the drug sensitivity in the parental cells which are referred to as sensitive cells in the study, display high levels of tumor suppressor DAB2 expression compared to the generated resistant cells, and DAB2 attenuates WNT signaling by regulating the endocytic fate of the LRP6-CD44v6 signalosome (See Example 1).
- CD44v6 requires nuclear localization sites (NLS) to associate with LRP6 in lipid, and CD44v6 regulates WNT/LRP6/p-catenin promoter TOPFlash activation (see Example 1).
- NLS nuclear localization sites
- PN and IL17A recruit WNT3A by inhibiting the tumor-suppressor protein DKK1.
- DKK1 and DAB2 inhibit WNT3 A-dependent signaling by promoting LRP6 internalization through the clathrin pathway.
- DAB2 interacts with the intracellular domain of LRP6 (Jiang, Y., et al., 2012, EMBO J, 31:2336-2349), while DKK1 interacts with the extracellular domain of LRP6 (Yamamoto, H., et al., 2008, Dev Cell, 15:37-48). It was also observed that DKK1 does not suppress PN- or IL17A-induced accumulation of b- catenin when clathrin-microdomain-mediated endocytosis is disrupted using MDC (Figure 29C-G).
- methylation and loss of expression of GATA5 are associated with promoter methylation of its target genes including DAB2 (Akiyama, Y., et al., 2003, Mol Cell Biol, 23:8429-8439).
- DAB2 Akiyama, Y., et al., 2003, Mol Cell Biol, 23:8429-8439.
- Similar regulations may occur in chronic FOLFOX exposure to CAFs.
- PN, or IL-17A or WNT3A neutralizing antibodies moderately stimulated the efficacy of FOLFOX chemotherapy on the growth of tumor established by injecting CICs in combination with CAFs subcutaneously in immunocompromised mice (Figure 31).
- CD44v6 is a molecule that could be clinically exploited both as a biomarker and an effective therapeutic modulation of CRC.
- circulating CICs can enter secondary sites of tumor tissue and, together with CAFs (cancer activated fibroblasts), can reprogram these niches to become secondary niches that result in the cancer returning.
- Periostin (PN) and IL17A secreted by CAFs contribute to the tumorigenic reprogramming of secondary niches by upregulating the expression of CD44v6 through a reciprocal inductive interaction between CICs and CAFs through a WNT3 A signaling mechanism.
- the elevated expression of CD44v6 is shown to directly regulate CIC maintenance. Not all potential secondary tissue sites can be expected to fully facilitate generation of a tumor niche, which would be consistent with a non-random distribution of metastases in organs.
- the returning tumor can then be expected to demonstrate drug resistance due to over production of CD44v6 via its downstream WNT3 A/p-catenin/TCF4 mediated MDR1 and CD44v6 gene expressions.
- Therapeutic intervention can be done in two ways. Rather than attacking all the tumor cells or the elements secreted by CAFs at once (as is the case for traditional cancer therapy), a strategy is propose in which tissue specific delivery of therapeutic drugs that are targeted to CD44v6+CICs would slowly shrink the tumor, since the CICs would no longer be able to replenish (or reprogram) cells that naturally die within the cancer niche.
- Example 3 Selection of efficient CD44v6 shRNA among old and new sets of CD44v6 shRNAs comparing their CD44v6-silencing efficiency and subsequent inhibition of CD44v6 signaling for reduction of xenograft growth.
- CD44v6 shRNAl New and CD44v6 shRNA2 New are better silencing agents for tumor growth suppression by inhibiting MDR1 activation as compared to CD44v6shRNA old.
- Transferrin-nanoparticle delivery of pSicoCD44v6 shRNA plus Fabpl-Cre was used herein and it was determined whether silencing CD44v6- variants can inhibit the tumor cell survival and growth by reducing WNT-CD44v6-MDRl signaling.
- pSicoCD44v6 shRNAs plus Fabpl-Cre nanoparticles were used in vivo to examine the therapeutic potential of targeting CD44v6 in tumors implanted from CICs plus CAFs isolated from FOLFOX resistant xenograft tumors (SW480-FR/SQ tumor; schematic schedule in Figure 40A).
- Cre-mediated CD44v6-shRNA nanoparticles prevented tumor growth (Figure 40B) and inhibited CD44v6 and MDR1 expression (Figure 40C, 100 pg/ml plasmid concentration) having a predominant effect in reducing tumor growth and inhibiting MDR1 expression.
- CD44v6 shRNAl New and CD44v6 shRNA2 New are better silencing agents for reduction of Pcatenine-STAT3-MDR1 activation as compared to CD44v6shRNA old.
- CRC colorectal cancer
- a high frequency of RAS, BRAF, PIK3CA, HER2, FGFR1, PDGFRa, and MAP2K1 mutation often confers metastatic tumors after Cetuximab (anti -EGFR) therapy.
- HGF-dependent MET activation contributes to cetuximab resistance in CRC cells, as HGF can activate RTK signaling in parallel with EGF.
- the intracellular portion of CD44v6 assists in linking the MET cytoplasmic domain to actin microfilaments and intermediating ezrin, radixin, and moesin proteins, thus facilitating the activation of RAS.
- Chemotherapy activates CAFs to secrete cytokines including HGF and EGF in the microenvironment to stimulate CIC growth and subsequently these activated CAFs stimulate tumor growth (see Example 2 above). It has also been shown that WNT- CD44v6-P-catenin pathway regulates MDRl activation and FOLFOX resistance (see Example 1 above). Further, additional evidence suggests that CD44v6-Pcatenin-STAT3 signaling is linked to MDRl activation to induce FOLFOX resistance and CRC tumorigenesis (data not shown).
- EGF EGF, or HGF affects b-catenin and STAT3 activation
- 20 ng/ml EGF or HGF was added to sensitive CRC cells and it was found that both EGF and HGF stimulate b-catenin and STAT3 activation (Figure 41) and these activations are associate with PI3K/AKT and GSK ⁇ activation.
- Figure 42 the effect of Old and New CD44v6 shRNAs on the expression of active-betacatenin, p-STAT3, MDRl expression was tested ( Figure 42).
- CD44v6 shRNAl New and CD44v6 shRNA2 New appear to be better silencing agents compared to that of CD44v6 shRNA Old with respect to reduction of active ⁇ catenin, pSTAT3, and MDRl expression in FOLFOX resistant SW480-FR cells ( Figure 42).
- Figure 43 results of Figure 43 show that, CD44v6 shRNAl New and CD44v6 shRNA2 New efficiently reduce the expression of active ⁇ catenin, pSTAT3, and MDRl expression in FOLFOX resistant SW480-FR cells.
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Abstract
La présente invention concerne de manière générale des compositions comprenant un ou plusieurs modulateurs d'une ou plusieurs molécules favorisant la chimiorésistance, une ou plusieurs molécules favorisant la chimiosensibilité, ou une combinaison de celles-ci, et leurs méthodes d'utilisation. La présente invention concerne également des compositions d'inhibiteur de CD44v6 et des méthodes d'utilisation pour traiter ou prévenir une ou plusieurs maladies ou troubles, tels que le cancer.
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