WO2011150400A1 - Protéine de support nucléaire stip/tfip11 au titre de cible pour la thérapie anticancéreuse - Google Patents

Protéine de support nucléaire stip/tfip11 au titre de cible pour la thérapie anticancéreuse Download PDF

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WO2011150400A1
WO2011150400A1 PCT/US2011/038447 US2011038447W WO2011150400A1 WO 2011150400 A1 WO2011150400 A1 WO 2011150400A1 US 2011038447 W US2011038447 W US 2011038447W WO 2011150400 A1 WO2011150400 A1 WO 2011150400A1
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stip
usp7
protein
mdm2
cells
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PCT/US2011/038447
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English (en)
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Mao Ye
Ying Chen
Cheng-Han Huang
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New York Blood Center, Inc.
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Publication of WO2011150400A1 publication Critical patent/WO2011150400A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This present disclosure relates to methods of modulating the interaction between USP7 and STIP in cancer cells.
  • p53 is a multi-talented tumor suppressor and the most frequently mutated gene in human cancers. Extensive research has identified a complex network of p53 functions and revealed a central tenet at adapting its dynamic induction and activation. In normal cells, p53 is kept at low levels with a short half-life via ubiquitin-dependent proteosomal degradation. In response to stresses such as DNA damage, p53 is stabilized via decreased protein degradation and increased gene expression resulting in cell-cycle arrest, apoptosis or cellular senescence. Mdm2, an E3 ubiquitin ligase, is a key negative regulator of p53 to catalyze its ubiquitination.
  • Mdm2 and p53 act through reciprocal modulation. While under a positive feedback of p53, Mdm2 is itself subject to autoubiquitination. Recent results from Mdm2-RING knock-in mutant mice indicate a more intricate scenario regarding the maintenance of the endogenous level of Mdm2, implicating the participation of an unknown E3 ubiquitin ligase in its degradation. [0005] Recent studies also showed that p53 and Mdm2 ubiquitination is reversible prior to commitment to final proteosomal degradation.
  • USP7 a deubiquitinase mainly resident in the nucleus, binds to Mdm2 and p53 via their A erminal TRAF-domain in a mutually exclusive manner to remove ubiquitin and stabilize the two proteins.
  • the binding affinity of USP7 is several-fold higher for Mdm2 than for p53 in vitro.
  • Mdm2 is a physiologic substrate of USP7 in unstressed cells, but upon DNA damage, the affinity of USP7 for Mdm2 is reduced leading to Mdm2 degradation.
  • a pool of the p53-Mdm2-USP7 complex exists in vivo, and USP7 may deubiquitinate p53 in trans through an Mdm2-mediated indirect interaction.
  • STIP sepl/tuftelin interacting p/oteins
  • STIP are a unique class of multidomain proteins that share a G-patch, a coiled-coil and several short tryptophan-tryptophan repeats from the N to C-terminal region.
  • Proteomic studies identified STIP as a nuclear phosphoprotein in HeLa cells and a factor associated with the spliceosome.
  • RNAi RNA interference
  • STIP novel molecular function of STIP has been identified which links USP7 to the Mdm2-p53 pathway.
  • STIP and USP7 interact and co-localize in the native nuclear compartment and STIP overexpression stabilizes both Mdm2 and p53, while its depletion reduces the level of the two proteins.
  • STIP-mediated stabilization of p53 and Mdm2 depends on USP7 and there exists in vivo two ternary complexes, STIP-p53-USP7 and STIP-Mdm2-USP7. Given that STIP is not known to possess any enzymatic activity, STIP functions as a nuclear interaction platform that enables protein complex assembly and promotes p53-Mdm2 pathway integration.
  • STIP plays a critical role in the p53-Mdm2 axis by acting in concert with USP7-mediated regulation.
  • a method for screening for anti-cancer compounds comprising obtaining a potential anti-cancer compound; and determining the effects of said compound on the expression of STIP in a cell or tissue.
  • the determining step comprises determining the levels of at least one nucleic acid sequence encoding for STIP, p53 or Mdm2 in the cell or tissue. In another embodiment, the determining step comprises determining the levels of at least one STIP, p52 or Mdm2 protein in the cell or tissue. In another embodiment, the cell or tissue is a cancer cell or tissue.
  • a method for modulating the activity of USP7 comprising modulating the levels of STIP in a cell or tissue.
  • modulating the activity of USP7 comprises increasing the activity of USP7.
  • modulating the activity of USP7 comprises decreasing the activity of USP7.
  • modulating the levels of STIP comprises increasing the levels of STIP.
  • modulating the levels of STIP comprises decreasing the levels of STIP.
  • a method for modulating ubiquitination of a protein, the method comprising modulating the levels of STIP in a cell or tissue.
  • modulating ubiquitination of a protein comprises increasing the ubiquitination of said protein.
  • modulating ubiquitination of a protein comprises decreasing the ubiquitination of said protein.
  • modulating the levels of STIP comprises increasing the levels of STIP.
  • modulating the levels of STIP comprises decreasing the levels of STIP.
  • a method for inhibiting tumor cell proliferation by exposing a subject, a cell population or a tissue to an effective amount of an antagonist or inhibitor of STIP expression.
  • FIG. 1 depicts the expression and nuclear localization of endogenous STIP protein in human cells.
  • FIG. 1A Endogenous STIP expressed in ten human cell types by Western Blot (WB) analysis. In each lane 50 ⁇ g of total proteins were loaded and probed with anti-STIP antibody. Actin denotes protein loading. A protein band of STIP was seen in blots of SK-Mel-2, H1229 and HepG2 cells when exposed longer.
  • FIG. 1 B Nuclear location of endogenous STIP protein. A typical confocal image of U20S cells stained with anti-STIP and then Texas Red-conjugated anti-lgG (left) is shown along with its differential interference contrast microscopy image (DIC; middle) and merged (right) images.
  • DIC differential interference contrast microscopy image
  • FIG. 2 depicts co-localization and interaction of STIP with USP7.
  • FIG. 2A - STIP and USP7 colocalize to the nuclear stiposomes under in vivo conditions.
  • U20S cells were fixed and incubated with USP7 and STIP antibodies followed by reaction with FITC or Texas Red-conjugated IgG. Shown are USP7 and STIP (upper) and their DIC or merged confocal images (lower).
  • FIG. 2B - Overexpressed STIP interacts with USP7.
  • U20S (p53 +/+ ) and H1299 (p53 "/_ ) cells were transfected with a Flag-STIP plasmid.
  • FIG. 2C Endogenous STIP interacts with USP7. Lysates of U20S cells were precipitated with anti-USP7 (033A to 1-50aa; 034A to 1050-1 102aa) or control IgG (rabbit normal IgG and isotype-matching IgG * ). The immunoprecipitates were probed with USP7, STIP or p53 antibody. FIG. 2D - Overexpressed USP7 does not affect STIP levels. U20S cells were transfected with Flag-USP7 or control plasmid. Lysates were analyzed for expression of Flag-USP7, p53, Mdm2 and STIP. Actin denotes protein loading.
  • FIG. 3 depicts that STIP is associated with Mdm2 or p53.
  • FIG. 3A Overexpressed STIP interacts with Mdm2 or p53. Lysates from U20S cells transfected with Flag-STIP or control vector were precipitated with anti-Flag and IgG antibodies. Western blots of immunoprecipitates and total cell lysates probed with Mdm2 and p53 antibodies are shown.
  • FIG. 3B - Endogenous STIP interacts with Mdm2 or p53. Lysates from U20S cells were precipitated with the anti-Mdm2 (left), anti-p53 (right) or control IgG antibody. Immunoprecipitates and lysates were analyzed by Western blot with the indicated antibodies.
  • FIG. 4 depicts the identification of ternary complexes of STIP formed with USP7, Mdm2 and/or p53.
  • U20S cells were co-transfected with Flag-STIP plus Mdm2 or Flag-STIP plus p53 overnight followed by MG132 treatment.
  • Cell lysates were precipitated with anti-Flag M2 agarose and eluted with Flag peptides. Eluates were subject to a second precipitation with anti-Mdm2, anti-p53 or control IgG antibody.
  • Protein samples obtained from each step were analyzed by WB with the indicated antibody: FIG. 4A - ternary complex formation as STIP-USP7-Mdm2; FIG. 4B - ternary complex formation as STIP-USP7-p53.
  • FIG. 5 depicts that STIP controls the steady-state level of Mdm2 and p53.
  • FIG. 5A Overexpressed STIP increased the steady-state level of Mdm2 and p53.
  • U20S (p53 +/+ ), H1299 (p53 " ' “ ), HCT1 16 (p53 +/+ ) and HCT1 16 (p53 " ' “ ) cells were transfected with Flag-STIP (+) and control plasmid (-). The p53 genotypes of the cells are shown (top). The proteins in lysates were analyzed with the indicated antibody.
  • FIG. 5B siRNA-mediated downregulation of STIP decreases the steady-state level of Mdm2 and p53.
  • U20S cells were treated with control siRNA and increasing amount of STIP siRNA. The expression of the indicated proteins was examined by WB with the indicated antibody.
  • FIG. 6 depicts that STIP controls the stability of Mdm2 and p53.
  • FIG. 6A - STIP prolongs the half-life of Mdm2 and p53.
  • U20S cells transfected with Flag-STIP or control plasmid were treated with CHX for indicated time points and analyzed for the level of Mdm2, p53, USP7 and Flag-STIP proteins by WB.
  • FIG. 6B - STIP decreases the ubiquitination of endogenous Mdm2 and p53.
  • U20S cells transfected with Flag-STIP or control plasmid were incubated with 20 ⁇ MG132 for 4 hr. The ubiquitination of endogenous Mdm2 and p53 was analyzed.
  • FIG. 7 depicts that STIP-mediated stabilization of Mdm2 and p53 depends on USP7.
  • U20S cells treated with USP7 siRNA or control siRNA were transfected with Flag-STIP (+) and control plasmid (-), respectively. The expression of the indicated proteins was examined by WB.
  • FIG. 8 depicts that STIP interacts directly with USP7 and their binding is mediated through the A erminal sequence (aa1 -209) of STIP and the C-terminal sequence (aa601 -1 102) of USP7, respectively.
  • FIG. 8A Yeast colonies grown on the plate lacking four nutrient selection markers in the presence of STIP(1-209) bait and USP7 prey in various combinations. 1. STIP + USP7(1 -1 102); 2. STIP + USP7(601 -1 102); and 3. STIP + USP7(1-210). 4 and 5 are blank bait and prey vectors used as controls.
  • FIG. 8B Schematic representation of the interaction of STIP with USP7.
  • FIG. 9 depicts that overexpressed p53 or Mdm2 does not change the level of endogenous STIP.
  • FIG. 10 depicts that overexpressed STIP does not change the transcriptional level of p53 and Mdm2 genes.
  • STIP sipl/tuftelin interacting proteins
  • STIP is a unique class of multidomain nuclear proteins highly conserved in metazoans from C. elegans to humans. Although the function of this class is elusive, its disruption in C. elegans causes 100% lethality to 16-cell stage embryos, which can be rescued by a Drosophila or human stip gene.
  • STIP is a novel macromolecular scaffold that links USP7, a multifunctional deubiquitinase, to the Mdm2-p53 pathway. STIP and a fraction of USP7 interact and constitutively colocalize to the STIP-assembled polymers or stiposomes in the endogenous nuclear environment.
  • STIP overexpression promotes Mdm2 and p53 stability, whereas its downregulation reduces the level of the two proteins.
  • STIP stabilization of Mdm2 and p53 results from its decreasing ubiquitination and extending the half-life of the two proteins.
  • STIP functions as a macromolecular interaction platform enabling protein complex assembly and acting in concert with USP7 to coordinate p53-Mdm2 pathway regulation.
  • STIP plays a critical role in the p53-Mdm2 pathway and functions as a new regulatory mechanism operative at the level of scaffolding organization.
  • p53 At the center of the tumor suppressor network, p53 receives numerous signals from its pathway modules and integrates them to elicit tight control over genotoxic stresses and cell proliferation.
  • the multitude of p53 functions is achieved in different cellular compartments or organelles and is fine-tuned at various levels of gene expression and protein posttranslational modification.
  • the majority of p53 pathway-linked components identified thus far possess either catalytic activity or transcriptional activity, and they are themselves subject to stringent regulation.
  • STIP represents a new and critical scaffold protein class that unites the USP7 deubiquitinase and its targets, p53 and Mdm2, to coordinate their interaction and complex formation.
  • STIP The mode of action and structural features of STIP satisfy three main biochemical criteria set for scaffolds.
  • STIP does not intrinsically possess any catalytic activity, in view of the modular arrangement of its conserved domains that are known to engage protein-protein and/or protein-RNA interactions.
  • STIP associates with at least two different proteins, USP7 and Mdm2 or p53 which possess catalytic or transcriptional activity, but STIP is itself unaffected by such biological activity.
  • the STIP interaction partners, USP7 and Mdm2 or p53 have been shown to bind to each other directly, although their binding in vitro shows variable affinity.
  • the STIP-USP7 interaction and ternary complex formation are functionally significant because they reveal a new mechanism whereby USP7 executes its dual stabilization effect on Mdm2 and p53 via STIP scaffolding.
  • USP7 is a conserved member of the deubiquitinase family involved in cellular stress, epigenetic silencing, cell survival and viral infection. It resides mainly in the nucleus, albeit also occurs in the cytoplasm and mitochondria. Prior studies indicated that USP7 can be enriched as granules in association with the PML-nuclear body. In the nucleus, a fraction of USP7 is constitutively associated with STIP and accumulates in the stiposome.
  • STIP can naturally self-assemble into large stiposome bodies in the endogenous cellular environment.
  • the stiposome polymers may function as a macromolecular interaction platform to allow deposition and release of active components, thereby regulating their local intensity and positional proximity.
  • the scaffold organization enables STIP to actively recruit or tether USP7 and its targets Mdm2 and/or p53 from the nucleoplasm.
  • USP7 stabilizes p53 and Mdm2 through its deubiquitinase activity and thus plays a key role in regulating the levels of p53 and Mdm2 under normal and stressed conditions.
  • Daxx a death-domain adaptor
  • STIP stabilizes both p53 and Mdm2 by interacting with USP7 and promoting separate complex assembly.
  • STIP overexpression led to an increase of the levels of both p53 and Mdm2, while STIP depletion showed the opposite effect.
  • STIP stabilizes p53 and Mdm2 proteins by decreasing their ubiquitination and increasing their half-life.
  • STIP-USP7-Mdm2 and STIP-USP7-p53 were identified which are likely to exist in vivo.
  • a subpopulation of the p53-Mdm2-USP7 ternary complex also occurs in vivo.
  • STIP is likely to function in concert with, and depend on, USP7 to mediate complex assembly with Mdm2 or p53, promoting the deubiquitinase activity of USP7 and enabling USP7 to stabilize the two proteins.
  • STIP The STIP nuclear scaffold proteins (hereinafter referred to as STIP) and associated complexes described herein can be used: (1 ) to identify agents which modulate the level of or at least one activity of a protein, (2) to identify binding partners for a protein, (3) as an antigen to raise polyclonal or monoclonal antibodies, (4) as a therapeutic agent or target for cancer and (5) as a diagnostic agent or marker of cancer and other diseases associated with STIP.
  • the present disclosure further provides host cells transformed with a nucleic acid molecule that encodes the STIP or related proteins and associated complexes.
  • the host cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of a protein are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product.
  • Exemplary eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line.
  • Exemplary eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61 , NIH Swiss mouse embryo cells (NIH/3T3) available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines.
  • CHO Chinese hamster ovary
  • NIH/3T3 NIH Swiss mouse embryo cells
  • BHK baby hamster kidney cells
  • Any prokaryotic host can be used to express an rDNA molecule encoding a protein.
  • An exemplary prokaryotic host is Escherichia coli.
  • Transformation of appropriate cell hosts with an rDNA molecule of the present disclosure is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed. With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically employed.
  • Successfully transformed cells i.e., cells that contain a rDNA molecule
  • Another embodiment provides methods for identifying agents that modulate the expression of a nucleic acid encoding a STIP or related proteins and associated complexes. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids. As used herein, an agent is said to modulate the expression of a nucleic acid if it is capable of up- or down-regulating expression of the nucleic acid in a cell.
  • cell lines that contain reporter gene fusions between nucleotides from within the open reading frame defined by a STIP or related proteins and associated complexes and/or the 5' and/or 3' regulatory elements and any assayable fusion partner may be prepared.
  • Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase.
  • Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid encoding a STIP or related proteins or associated complexes.
  • Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a STIP or related proteins or associated complexes. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids. Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures.
  • the cells are derived from human tissue, for instance, biopsy tissue or cultured cells from patients with cancer.
  • Cell lines from tissue such as, but not limited to, breast, colon, lung, ovary, prostate, stomach, intestine, liver, skin, muscle, kidney, bladder, brain, bone, etc. may be used. Alternatively, other available cells or cell lines may be used.
  • Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids related to a STIP or related proteins or associated complexes. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency.
  • the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and probe:non-target hybrids.
  • Probes may be designed from the nucleic acids through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available.
  • Hybridization conditions are modified using known methods as required for each probe.
  • Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format.
  • total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences under conditions in which the probe will specifically hybridize.
  • nucleic acid fragments comprising at least one, or part of one of the sequences can be affixed to a solid support, such as a silicon chip, porous glass wafer or membrane.
  • the solid support can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize.
  • Such solid supports and hybridization methods are widely available.
  • agents which up- or down-regulate the expression of a nucleic acid encoding the STIP or related proteins or associated complexes are identified.
  • Hybridization for qualitative and quantitative analysis of mRNAs may also be carried out by using, for example, a RNase Protection Assay. Briefly, an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription.
  • a phage specific DNA dependent RNA polymerase promoter e.g., T7, T3 or SP6 RNA polymerase
  • the labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45°C overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCI and 1 mM EDTA.
  • the resulting hybrids are then digested in a buffer comprising 40 ⁇ g/ml ribonuclease A and 2 ⁇ g/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis.
  • cells or cell lines are first identified which express the gene products physiologically. Cells and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and/or the cytosolic cascades.
  • such cells or cell lines would be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) construct comprising an operable non-translated 5' promoter-containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag or other detectable marker.
  • an expression vehicle e.g., a plasmid or viral vector
  • Cells or cell lines transduced or transfected as outlined above are then contacted with agents under appropriate conditions.
  • the agent in a pharmaceutically acceptable excipient is contacted with cells in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and/or serum incubated at 37°C.
  • PBS phosphate buffered saline
  • BSS Eagles balanced salt solution
  • Said conditions may be modulated as deemed necessary by one of skill in the art.
  • the cells will be disrupted and the polypeptides of the lysate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot).
  • immunological assay e.g., ELISA, immunoprecipitation or Western blot.
  • the pool of proteins isolated from the "agent-contacted” sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent-contacted” sample compared to the control will be used to distinguish the effectiveness of the agent.
  • Another embodiment provides methods for identifying agents that modulate the level or at least one activity of a protein, such as the protein having the amino acid sequence of a STIP or related proteins or associated complexes. Such methods or assays may utilize any means of monitoring or detecting the desired activity and are particularly useful for identifying agents that treat cancer.
  • the relative amounts of a protein between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed.
  • probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations.
  • Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time.
  • Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.
  • Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co. (Rockford, IL), may be desirable to provide accessibility to the hapten.
  • the hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
  • Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art.
  • titers of antibodies are taken to determine adequacy of antibody formation.
  • Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard methods or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal antibodies or the polyclonal antisera which contain the immunologically significant (antigen-binding) portion can be used as antagonists, as well as the intact antibodies.
  • Use of immunologically reactive (antigen-binding) antibody fragments, such as the Fab, Fab', or F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or antigen-binding fragments may also be produced, using current technology, by recombinant means.
  • Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin, such as humanized antibodies.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of a protein alone or with its associated substrates, binding partners, etc.
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site.
  • the agents of the present disclosure can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. Dominant negative proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. "Mimic” as used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present disclosure.
  • the peptide agents disclosed herein can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.
  • Another class of agents disclosed herein are antibodies immunoreactive with critical positions of a STIP or related proteins or associated complexes.
  • Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.
  • the proteins and nucleic acids related to a STIP or related proteins or associated complexes may be differentially expressed in cancerous tissue.
  • Agents that up- or down-regulate or modulate the expression of the protein or at least one activity of the protein such as agonists or antagonists, may be used to modulate biological and pathologic processes associated with the protein's function and activity. This includes agents identified employing homologues and analogues.
  • a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein.
  • mammal is defined as an individual belonging to the class Mammalia.
  • Pathological processes refer to a category of biological processes which produce a deleterious effect.
  • expression of a protein may be associated with cell growth or hyperplasia.
  • an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process.
  • cancer may be prevented or disease progression modulated by the administration of agents which up- or down-regulate or modulate in some way the expression or at least one activity of a protein or complex disclosed herein.
  • the agents can be provided alone, or in combination with other agents that modulate a particular pathological process.
  • an agent can be administered in combination with other known drugs.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • the agents can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • compositions containing one or more agents which modulate expression or at least one activity of a protein or gene While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt, 0.1 to 10 ⁇ g/kg body wt. or 0.1 to 1 ⁇ g/kg body wt.
  • compositions disclosed herein may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • the compounds may be used alone or in combination, or in combination with other therapeutic or diagnostic agents.
  • the compounds disclosed herein may be co-administered along with other compounds typically prescribed for these conditions according to generally accepted medical practice.
  • the compounds can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
  • Another embodiment provides methods for isolating and identifying binding partners of a STIP or related proteins or associated complexes.
  • a disclosed protein or complex
  • a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein.
  • peptides, polypeptides, proteins or other molecules that have become associated with a protein are separated from the mixture.
  • the binding partner that bound to the protein can then be removed and further analyzed.
  • the entire protein for instance a protein comprising the entire amino acid sequence of the protein can be used.
  • a fragment of the protein can be used.
  • a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell.
  • the preferred source of cellular extracts will be cells derived from human tumors or transformed cells, for instance, biopsy tissue or tissue culture cells from carcinomas.
  • cellular extracts may be prepared from normal tissue or available cell lines.
  • a variety of methods can be used to obtain an extract of a cell.
  • Cells can be disrupted using either physical or chemical disruption methods.
  • physical disruption methods include, but are not limited to, sonication and mechanical shearing.
  • chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis.
  • a skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.
  • the extract is mixed with the disclosed protein under conditions in which association of the protein with the binding partner can occur.
  • conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell.
  • Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.
  • the bound complex is separated from the mixture.
  • a variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used.
  • the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.
  • the protein can be immobilized on a solid support.
  • the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found in the extract.
  • the identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using a Far-Western assay or identified through the use of epitope tagged proteins or GST fusion proteins.
  • nucleic acid molecules disclosed herein can be used in a yeast two-hybrid system or other in vivo protein-protein detection system.
  • the yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.
  • binding partners of the disclosed proteins, and homologues and analogues thereof, obtained using the above described methods can be used for a variety of purposes.
  • the binding partners can be used to generate antibodies that bind to the binding partner using techniques known in the art.
  • Antibodies that bind the binding partner can be used to assay the activity of the protein, as a therapeutic agent to modulate a biological or pathological process mediated by the protein, or to purify the binding partner. These uses are described in detail below.
  • Another embodiment provides methods for identifying agents that reduce or block the association of a protein or complex with a binding partner. Specifically, a protein is mixed with a binding partner in the presence and absence of an agent to be tested. After mixing under conditions that allow association of the proteins, the two mixtures are analyzed and compared to determine if the agent reduced or blocked the association of the protein with the binding partner. Agents that block or reduce the association of the protein with the binding partner will be identified as decreasing the amount of association present in the sample containing the tested agent.
  • an agent is said to reduce or block the association between a protein and a binding partner when the presence of the agent decreases the extent to which or prevents the binding partner from becoming associated with the protein.
  • One class of agents will reduce or block the association by binding to the binding partner while another class of agents will reduce or block the association by binding to the protein.
  • the binding partner used in the above assay can either be an isolated and fully characterized protein or can be a partially characterized protein that binds to the protein or a binding partner that has been identified as being present in a cellular extract. It will be apparent to one of ordinary skill in the art that so long as the binding partner has been characterized by an identifiable property, e.g., molecular weight, the present assay can be used.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • Another class of agents are antibodies immunoreactive with critical positions of the protein or the binding partner.
  • antibodies are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein or the binding partner, intended to be targeted by the antibodies.
  • Critical regions include the contact sites involved in the association of the protein with the binding partner.
  • the important minimal sequence of residues involved in activity of the disclosed proteins define a functional linear domain that can be effectively used as a bait for two hybrid screening and identification of potential associated molecules. Use of such fragments will significantly increase the specificity of the screening as opposed to using the full-length molecule and is therefore preferred.
  • this linear sequence can be also used as an affinity matrix also to isolate binding proteins using a biochemical affinity purification strategy.
  • the proteins and nucleic acids related to a STIP or related proteins or associated complexes disclosed herein may be differentially expressed in cancer tissue.
  • Agents that reduce or block the interactions of a protein, including those identified employing homologues and analogues of the protein, with a binding partner may be used to modulate biological and pathologic processes associated with the protein's function and activity.
  • compositions containing one or more agents that block association of a STIP or related proteins or associated complexes with a binding partner While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt, 0.1 to 10 ⁇ g/kg body wt or 0.1 to 1 ⁇ g/kg body wt.
  • the present disclosure further encompasses rational drug design and combinatorial chemistry.
  • Rational drug design involving polypeptides requires identifying and defining a first peptide with which the designed drug is to interact, and using the first target peptide to define the requirements for a second peptide. With such requirements defined, one can find or prepare an appropriate peptide or non-peptide that meets all or substantially all of the defined requirements.
  • one goal of rational drug design is to produce structural or functional analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, null compounds) in order to fashion drugs that are, for example, more or less potent forms of the ligand.
  • Combinatorial chemistry is the science of synthesizing and testing compounds for bioactivity en masse, instead of one by one, the aim being to discover drugs and materials more quickly and inexpensively than was formerly possible.
  • Rational drug design and combinatorial chemistry have become more intimately related in recent years due to the development of approaches in computer-aided protein modeling and drug discovery.
  • genetic therapy can be used as a means for modulating biological and pathologic processes associated with the protein's function and activity.
  • This comprises inserting into a cancerous cell a gene construct encoding a protein comprising all or at least a portion of the sequences of a STIP or related proteins or associated complexes, operably linked to a promoter or enhancer element such that expression of said protein causes suppression of said cancer and wherein said promoter or enhancer element is a promoter or enhancer element modulating said gene construct.
  • expression of the protein can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element.
  • CMV human cytomegalovirus
  • SV40 simian virus 40
  • metallothionein promoters e.g., metallothionein promoters
  • enhancers known to preferentially direct gene expression in neural cells, T cells, or B cells may be used to direct the expression.
  • the enhancers used could include, without limitation, those that are characterized as tissue or cell specific in their expression.
  • regulation may be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
  • Insertion of the construct into a cancerous cell is accomplished in vivo, for example using a viral or plasmid vector.
  • a viral or plasmid vector Such methods can also be applied to in vitro uses.
  • the methods disclosed herein are readily applicable to different forms of gene therapy, either where cells are genetically modified ex vivo and then administered to a host or where the gene modification is conducted in vivo using any of a number of suitable methods involving vectors especially suitable to such therapies.
  • Retroviral vectors may be used as a gene transfer delivery system for a therapeutic gene construct. Numerous vectors useful for this purpose are generally known. Retroviral vectors are particularly well developed and have been used in clinical settings. Non-viral approaches may also be employed for the introduction of therapeutic DNA into cells otherwise predicted to undergo cancer. For example, a gene may be introduced into a neuron or a T cell by lipofection, asialorosonucoid polylysine conjugation, or, less preferably, microinjection under surgical conditions.
  • the therapeutic nucleic acid construct is preferably applied to the site of the cancer event (for example, by injection). However, it may also be applied to tissue in the vicinity of the cancer event or to a blood vessel supplying the cells predicted to undergo cancer.
  • Transgenic animals containing mutant, knock-out or modified genes corresponding to the cDNA sequence of a STIP or related proteins or associated complexes, or fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues, are also included.
  • Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene.”
  • the nucleic acid sequence of the transgene may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at, the normal locus for the transgene.
  • the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
  • transgenic animals in which all or a portion of a gene is deleted may be constructed.
  • the gene contains one or more introns
  • the entire gene-all exons, introns and the regulatory sequences- may be deleted.
  • less than the entire gene may be deleted.
  • a single exon and/or intron may be deleted, so as to create an animal expressing a modified version of a protein.
  • germ cell line transgenic animal refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.
  • the alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection.
  • the method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art.
  • genes and proteins disclosed herein may be differentially expressed in cancer tissues compared to non-cancerous tissues, the genes and proteins may be used to diagnose or monitor cancer, to track disease progression, or to differentiate cancerous tissue from non-cancerous tissue samples.
  • One means of diagnosing cancer using the nucleic acid molecules or proteins disclosed herein involves obtaining tissue from living subjects.
  • Assays to detect nucleic acid or protein molecules disclosed herein may be in any available format.
  • Typical assays for nucleic acid molecules include hybridization or PCR-based formats.
  • Typical assays for the detection of proteins, polypeptides or peptides include the use of antibody probes in any available format such as in situ binding assays, etc.
  • the diagnostics can be classified according to whether the embodiment is a nucleic acid or protein-based assay. Some diagnostic assays detect mutations or polymorphisms in the nucleic acids or proteins, which contribute to cancerous aberrations. Other diagnostic assays identify and distinguish defects in protein activity by detecting a level of RNA or protein in a tested organism that resembles the level of RNA or protein in a organism suffering from a disease, such as cancer, or by detecting a level of RNA or protein in a tested organism that is different than an organism not suffering from a disease.
  • kits that incorporate the reagents and methods described in the following embodiments so as to allow for the rapid detection and identification of aberrations in protein activity or level are contemplated.
  • the diagnostic kits can include a nucleic acid probe or an antibody or combinations thereof, which specifically detect a mutant form of the protein or a nucleic acid probe or an antibody or combinations thereof, which can be used to determine the level of RNA or protein expression of one or more protein.
  • the detection component of these kits will typically be supplied in combination with one or more of the following reagents.
  • a support capable of absorbing or otherwise binding DNA, RNA, or protein will often be supplied. Available supports include membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents.
  • One or more restriction enzymes, control reagents, buffers, amplification enzymes, and non-human polynucleotides like calf-thymus or salmon-sperm DNA can be supplied in these kits.
  • nucleic acid-based diagnostic techniques include, but are not limited to, direct DNA sequencing, gradient gel electrophoresis, Southern Blot analysis, single-stranded confirmation analysis (SSCA), RNAse protection assay, dot blot analysis, nucleic acid amplification, allele-specific PCR and combinations of these approaches.
  • the starting point for these analyses is isolated or purified nucleic acid from a biological sample. It is contemplated that tissue biopsies would provide a good sample source.
  • the nucleic acid is extracted from the sample and can be amplified by a DNA amplification technique such as the polymerase chain reaction (PCR) using primers.
  • PCR polymerase chain reaction
  • antibodies are attached to a support in an ordered array wherein a plurality of antibodies are attached to distinct regions of the support that do not overlap with each other.
  • proteins are obtained from biological samples and are labeled by conventional approaches (e.g., radioactivity, calorimetrically, or fluorescently).
  • an investigator can accurately determine the concentration of the protein in a sample and from this information can assess the expression level of the particular form of the protein.
  • Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of such protein.
  • any addressable array technology known in the art can be employed with this aspect and display the protein arrays on the chips in an attempt to maximize antibody binding patterns and diagnostic information.
  • the presence or detection of a polymorphism in an gene or protein can provide a diagnosis of a cancer or similar malady in an organism.
  • Additional embodiments include the preparation of diagnostic kits comprising detection components, such as antibodies, specific for a particular polymorphic variant of the gene or protein.
  • the detection component will typically be supplied in combination with one or more of the following reagents.
  • a support capable of absorbing or otherwise binding RNA or protein will often be supplied.
  • One or more enzymes such as Reverse Transcriptase and/or Taq polymerase, can be furnished in the kit, as can dNTPs, buffers, or non-human polynucleotides like calf-thymus or salmon-sperm DNA. Results from the kit assays can be interpreted by a healthcare provider or a diagnostic laboratory. Alternatively, diagnostic kits are manufactured and sold to private individuals for self-diagnosis.
  • some diseases involving cancer result from skewed levels of protein or gene in particular tissues or aberrant patterns of protein expression.
  • a diagnosis can be made or a disease state can be identified.
  • a prognosis of health or disease can be made.
  • the levels of protein expression in various tissues from healthy individuals, as well as, individuals suffering from cancers are determined. These values can be recorded in a database and can be compared to values obtained from tested individuals.
  • disease state profiles are referred to as "disease state profiles" and by comparing one disease state profile (e.g. from a healthy or diseased individual) to a disease state profile from a tested individual, a clinician can rapidly diagnose the presence or absence of disease.
  • nucleic acid and protein-based diagnostic techniques described above can be used to detect the level or amount or ratio of expression of genes or proteins in a tissue.
  • quantitative Northern hybridizations, in situ analysis, immunohistochemistry, ELISA, genechip array technology, PCR, and Western blots for example, the amount or level of expression of RNA or protein for a particular protein (wild-type or mutant) can be rapidly determined and from this information ratios of expression can be ascertained.
  • the proteins to be analyzed can be family members that are currently unknown but which are identified based on their possession of one or more of the homology regions described above.
  • mRNA messenger RNA
  • Ribosomes read the sequence of the mRNA and create the protein for which it encodes. This process of new protein synthesis is known as translation.
  • a variety of factors affect the rate and efficiency of protein translation. Among the most significant of these factors is the intrinsic stability of the mRNA itself. If the mRNA is degraded quickly within the cell (such as before it reaches a ribosome), it is unable to serve as a template for new protein translation, thus reducing the cell's ability to create the protein for which it encoded.
  • the agent comprises nucleic acids involving in RNA interference (RNAi).
  • RNAi is a naturally-occurring mechanism for suppressing gene expression and subsequent protein translation. RNAi suppresses protein translation by either degrading the mRNA before it can be translated or by binding the mRNA and directly preventing its translation. This technology provides an avenue to suppress the expression and actions of molecules and their receptors within a given population of cells.
  • RNAi is mediated by double stranded RNA (“dsRNA”), short hairpin RNA (“shRNA”) or other nucleic acid molecules with similar characteristics. These nucleic acid molecules are processed or cut into smaller pieces by cellular enzymes including dicer and drosha. The smaller fragments of the nucleic acid molecules can then be taken up by a protein complex (the RISC complex) that mediates degradation of mRNAs. The RISC complex will degrade mRNA that complementarily base pairs with the nucleic acid molecules it has taken up. In this manner, the mRNA is specifically destroyed, thus preventing the encoded-for protein from being made.
  • dsRNA double stranded RNA
  • shRNA short hairpin RNA
  • nucleic acid molecules can include any type of nucleic acid molecule capable of mediating RNA interference, such as, without limitation, short interfering nucleic acid (siNA), short hairpin nucleic acid (shNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), and double-stranded RNA (dsRNA).
  • the nucleic acid molecules also include similar DNA sequences.
  • nucleic acid and nucleic acid molecules can contain unmodified or modified nucleotides. Modified nucleotides refer to nucleotides which contain a modification in the chemical structure of a nucleotide base, sugar and/or phosphate. Such modifications can be made to improve the stability and/or efficacy of nucleic acid molecules
  • RNAi The understanding of the mechanism of RNAi now allows geneticists to create nucleic acid molecules with sequences that are homologous to known gene sequences in order to suppress the expression or formation of certain proteins within a cell.
  • nucleic acid molecules that are homologous to a STIP or related protein or associated complexed mRNA sequences are introduced into cells locally to suppress expression of these proteins.
  • These nucleic acid molecules specifically suppress the expression of amino acid sequences that encode for a STIP or related proteins or associated complexes. Suppressing expression of these proteins locally treats cancer in an area without globally inhibiting other systems.
  • nucleic acid molecules include the sequences of the gene encoding for a STIP or related protein or associated complex, the reverse complement of those sequences and RNA-based sequences including uracils in the place of the listed thymidines.
  • any listing of RNAi sequences may be considered target sequences as well as sequences included in the nucleic acid molecules of gene encoding for a STIP or related protein or associated complex.
  • STIP is a critical nuclear scaffold linking USP7 to p53-Mdm2 pathway regulation
  • NHEM human epidermal melanocytes
  • A375, SK-Mel-2, Hs294T, HeLa, SK-N-As, LN-18, H 1299 and U20S cells were cultured in DMEM containing 10% FBS. All cells were maintained at 37°C in a humidified 5% C0 2 incubator. HCT1 16 cells were cultured. Transfection with various expression plasmids was done using HD FuGENE reagents (Roche).
  • anti-USP7 A300-033A and A300-034A, Bethyl
  • anti-p53 D01 , Santa Cruz; A300-247A, Bethyl
  • anti-Mdm2 SMP14, Santa Cruz; Ab-4, Calbiochem
  • anti-STI P N-12, Santa Cruz
  • anti-Flag M2, Sigma
  • anti-3-actin AC-15, Sigma
  • anti-ubiquitin 6C1 .17, BD Pharmingen
  • Immunoprecipitation was performed with a magnetic Co-IP kit (Active Motif). Briefly, 1 mg of crude extract was incubated with 3 ⁇ g of antibody or control IgG (4°C, overnight). 30 ⁇ of protein G magnetic beads were added, incubated for one hr and washed four times with Co-IP/wash buffer. The precipitated proteins were dissolved in 2xSDS sample buffer, boiled and subject to WB analysis.
  • the STIP protein complex was eluted with 300 ⁇ of IP buffer containing 250 mM NaCI and Flag peptide (300 ⁇ g/mL) (4°C, two hr).
  • IP buffer containing 250 mM NaCI and Flag peptide (300 ⁇ g/mL) (4°C, two hr).
  • anti-Mdm2, anti-p53 or control IgG was added to eluates from the first immunoprecipitation. The mix was kept overnight at 4°C and incubated with protein G-Sepharose for two hr at 4°C. Protein samples were subject to WB analysis with the indicated antibody.
  • RNA Interference Pre-designed siRNA duplexes of STIP and negative control were from Ambion (STIP siRNA sequence: CCUGUUAAGCAGGACGACUtt, SEQ ID NO:3). Cells were reverse-transfected with STIP siRNA or control siRNA as specified by the manufacturer. The efficiency of STIP knockdown was analyzed 48 hr later.
  • SMARTpool siRNA directed toward USP7 contained four pooled siRNA duplexes (Dharmacon) whose sense sequences are: CUAAGGACCCUGCAAAUUA (SEQ ID NO:4), GUGGUUACGUUAUCAAAUA (SEQ ID NO:5), UGACGUGUCUCUUGAUAAA (SEQ ID NO:6), and GAAGGUACUUUAAGAGAUC (SEQ ID NO:7).
  • Dharmacon pooled siRNA duplexes whose sense sequences are: CUAAGGACCCUGCAAAUUA (SEQ ID NO:4), GUGGUUACGUUAUCAAAUA (SEQ ID NO:5), UGACGUGUCUCUUGAUAAA (SEQ ID NO:6), and GAAGGUACUUUAAGAGAUC (SEQ ID NO:7).
  • Cells were incubated separately with USP7 siRNA and a control siRNA using the DharmaFECT transfection reagent. After 24 hr, cells were plated for
  • U20S cells were transfected with pCMV-Flag-STIP and pCMV-Tag2B. After 48 hr cells were treated with 20 ⁇ MG132 for four hr and then lysed in pre-boiled SDS buffer (50 mM Tris-HCI pH 7.5, 0.5 mM EDTA, 1 % SDS, 1 mM DTT). Lysate was boiled and cleared by centrifugation.
  • SDS buffer 50 mM Tris-HCI pH 7.5, 0.5 mM EDTA, 1 % SDS, 1 mM DTT
  • NP-40 buffer (0.5% NP-40, 150 mM NaCI, 50 mM NaF, 50 mM Tris pH7.5, 1 mM DTT, 1 mM PMSF, 1 mM Na 3 V0 4 and protease inhibitors). After incubation with anti-Mdm2 or anti-p53 antibody, the immunoprecipitate was subject to WB analysis with anti-ubiquitin antibody.
  • Yeast two-hybrid (Y2H) tests The coding sequence of STIP bait (amino acids or aa1-209) was PCR-amplified from a full-length human cDNA using high-fidelity pfu DNA polymerase and two primers with pre-designed restriction enzyme sites (Table 1 ). The PCR product was digested and subcloned into the compatible sites of BD bait vector pGBKT7. The ligated pGBKT7-STIP (1 -209) bait vector was transformed into E. coli DH5a for propagation and plasmid mini-preparation.
  • the coding sequence of USP7 was PCR-amplified from pENTR-USP7 in separate reactions to produce DNAs encoding the full-length (aa1 -1 102), the /V-terminal TRAF-domain (aa1-210) and the C-terminal (aa601 -1 102) region, respectively.
  • the four primers used for USP7 amplification are shown in Table 1.
  • the three DNA products were digested with proper restriction enzymes and subcloned into the compatible sites of AD prey vector pGADT7 as pGADT7-USP7(1 -1 102), pGADT7-USP7(1 -210), and pGADT7 (601 -1 102), respectively.
  • the vectors were propagated and isolated similarly as above. All the Y2H vectors used were sequenced to verify their correct open reading frame and proper orientation.
  • yeast cells were transformed sequentially with pGBKT7-STIP (1-209) bait and one of the three USP7 preys and were allowed to grow on agar plates with stepwise nutrient mark selection. The growth of yeast cells in form of colonies were observed and scored positive if they survived in the absence of four nutrients from the medium.
  • SABiosciences's RT 2 qPCR-Grade RNA Isolation Kit One microgram of total RNA from each of the two samples was reverse-transcribed using SABiosciences's RT 2 First Strand Kit followed by PCR amplification using pre-dispensed gene-specific primers and quantitative PCR master mix (SABosicences) containing SYBR Green and reference dyes.
  • PCR was done on MX3000P thermal cycler (Stratagene) according to the manufacturer's protocol with the following conditions: 95°C for 10 min, 40 cycles of 95°C for 15 sec, and 60°C for 1 min.
  • the fold change in gene expression was calculated by the 2 ⁇ (-AACt) method.
  • STIP was expressed in different human cells and forms large polymers in the nucleus. STIP is expressed in all developmental stages in C. elegans as shown by PCR and all adult tissues in mouse by Northern blot. Because its protein expression was less clear at cellular levels we analyzed ten human cell types by Western blot (WB). As shown (FIG. 1A), STIP was found in all cells, albeit with a low level in melanoma SK-Mel-2 cells, lung cancer H1299 cells, and liver carcinoma HepG2 cells. STIP localized to a rod-shaped nuclear polymer called stiposome through imaging overexpressed GFP-STIP proteins.
  • STIP expressed in U20S cells was labeled with the Texas Red-conjugated antibody against its /V-terminal epitopes and observed under confocal microscope. Consistently, endogenous STIP was found to form large polymers as similarly dispersed in the nuclear compartment (FIG. 1 B), confirming the results obtained from STIP overexpression.
  • STIP interacts with USP7 and the two proteins colocalize to the stiposomes in vivo.
  • yeast two-hybrid (Y2H) screen of human HeLa cell cDNA library (Clontech) multiple interaction partners of STIP were obtained after eliminating its middle domain (aa210-460), a self-activation sequence.
  • Y2H tests it was determined that the /V-terminal domain (aa1-209) of STIP interacted strongly with the full-length (aa1 -1 102) and C-terminal sequence (aa601 -1 102), not the /v-terminal TRAF-domain (aa1 -210) of USP7 (FIG. 8).
  • USP7 belongs to the family of deubiquitinating enzymes, which catalyze the removal of ubiquitins from their target proteins. The finding that STIP interacts with USP7 prompted the determination whether USP7 could promote STIP stability through its deubiquitinase activity. USP7 overexpression led to the stabilization of both Mdm2 and p53 proteins (FIG. 2D, panel 2 and 3). In contrast, no apparent change was observed with regard to the steady-state level of STIP when USP7 was overexpressed (FIG. 2D, panel 4). In addition, depletion of USP7 via RNAi knockdown did not appear to significantly affect the level or stability of either endogenous or overexpressed STIP.
  • STIP is associated with Mdm2 and p53 in vivo.
  • USP7 is known to bind to Mdm2 and p53 via its /V-terminal TRAF-domain in a mutually exclusive manner. Because STIP co-localizes and interacts with USP7 in vivo, it was determined whether STIP also associates with Mdm2 and/or p53. To test this, STIP was overexpressed in U20S cells and its association with Mdm2 or p53 was assayed by co-immunoprecipitation. As shown, both Mdm2 and p53 were readily co-immunoprecipitated with overexpressed STIP (FIG. 3A).
  • STIP mediates ternary complex assembly as STIP-USP7-Mdm2 or STIP-USP7-p53.
  • STIP mediates ternary complex assembly as STIP-USP7-Mdm2 or STIP-USP7-p53.
  • Mdm2 and/or p53 suggest that STIP possesses an intimate relationship with the USP7-Mdm2 and/or USP7-p53 complexes.
  • Transient co-expression of STIP with either Mdm2 or p53 in U20S cells was carried out.
  • a two-step assay of immunoprecipitation was employed to investigate whether Flag-STIP could assemble into different complexes with the other three proteins and, if so, to solve the molecular composition.
  • STIP influences the steady-state level of both Mdm2 and p53 but not that of USP7.
  • USP7 is a deubiquitinase toward p53 and Mdm2, and its overexpression causes p53 and Mdm2 stabilization.
  • STIP overexpression affects the steady-state level of p53, Mdm2, or USP7.
  • STIP overexpression was performed and its effect on the level of three endogenous proteins was assayed in U20S (p53 +/+ ) and H1299 (p53 "/_ ), and two HCT1 16 cell lines with p53 wild-type and null genotypes, respectively.
  • STIP overexpression elicited a positive effect on Mdm2 stability, because these four cell lines all manifested a significant increase in the steady-state level of Mdm2 regardless of the status of p53 expression (FIG. 5A).
  • STIP overexpression also mediated an elevation of the p53 steady-state level, although this effect was more apparent in U20S than HCT1 16 cells (FIG. 5A), possibly due to cell-type specificities. Nonetheless, STIP overexpression did not appear to affect USP7 levels under the same conditions.
  • STIP-specific siRNA was used in different concentrations to block its expression and assessed the knockdown effect. As expected, downregulation of STIP significantly diminished the endogenous level of Mdm2 and p53 in a dose-dependent manner in U20S cells (FIG. 5B), while USP7 was unaffected.
  • the cells overexpressing Flag-STIP were treated with CHX to inhibit protein biosynthesis and protein extracts from different time points were analyzed. As shown, STIP overexpression resulted in the lengthening of the half-life of p53 and Mdm2 but not that of USP7 (FIG. 6A).
  • STIP regulates the level of Mdm2 and p53 in a manner dependent on USP7 action. It was then determined how STIP simultaneously maintains Mdm2 and p53 stability, given its ability to form protein complexes and lack of catalytic activity. Since STIP and USP7 interact in vivo and display similar effects towards Mdm2 or p53, STIP regulation of Mdm2 and p53 stability can be mediated through USP7 action. Endogenous USP7 was depleted with gene-specific siRNA and its knockdown effect was compared to siRNA control in U20S cells where STIP was either overexpressed or in native state.

Abstract

La présente invention concerne des procédés d'inhibition de la prolifération des cellules tumorales par exposition d'un sujet, d'une population cellulaire ou d'un tissu à une quantité active d'un antagoniste ou d'un inhibiteur de l'expression de STIP, ainsi que des méthodes d'identification de composés présentant une activité anticancéreuse et/ou modulant USP7 ou STIP au sein d'une cellule ou d'un tissu.
PCT/US2011/038447 2010-05-28 2011-05-27 Protéine de support nucléaire stip/tfip11 au titre de cible pour la thérapie anticancéreuse WO2011150400A1 (fr)

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