(19)
(11)EP 2 475 378 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
24.07.2019 Bulletin 2019/30

(21)Application number: 10816095.3

(22)Date of filing:  09.09.2010
(51)Int. Cl.: 
A61K 38/16  (2006.01)
A61K 31/7105  (2006.01)
A61K 38/17  (2006.01)
A61P 35/00  (2006.01)
(86)International application number:
PCT/US2010/048302
(87)International publication number:
WO 2011/031884 (17.03.2011 Gazette  2011/11)

(54)

USP10 modulation for use in the treatment of cancer

Modulation von USP10 zur Verwendung bei der Behandlung von Krebs

Modulation de USP10 pour son utilisation dans le traitement du cancer


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

(30)Priority: 10.09.2009 US 241152 P
12.11.2009 US 260637 P

(43)Date of publication of application:
18.07.2012 Bulletin 2012/29

(73)Proprietor: MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH
Rochester, MN 55905 (US)

(72)Inventors:
  • LOU, Zhenkun
    Rochester, Minnesota 55902 (US)
  • YUAN, Jian
    Rochester, Minnesota 55902 (US)

(74)Representative: Conroy, John et al
Fish & Richardson P.C. Highlight Business Towers Mies-van-der-Rohe-Straße 8
80807 München
80807 München (DE)


(56)References cited: : 
WO-A2-2007/149484
US-A1- 2008 038 768
US-A1- 2009 017 006
US-A1- 2009 093 430
US-A1- 2008 038 768
US-A1- 2008 167 229
US-A1- 2009 093 430
  
  • Anonymous: "Principles of cnacer therapy" In: Anonymous: "The Merck Manual of Diagnosis and Therapy, 18th edition", 2006, Merck Research Laboratories, NJ, XP002688087, ISBN: 0911910182 pages 1157-1171, * the whole document *
  • SHISHAN DENG ET AL: "Over-expression of genes and proteins of ubiquitin specific peptidases (USPs) and proteasome subunits (PSs) in breast cancer tissue observed by the methods of RFDD-PCR and proteomics", BREAST CANCER RESEARCH AND TREATMENT, vol. 104, no. 1, 27 September 2006 (2006-09-27), pages 21-30, XP019498588, KLUWER ACADEMIC PUBLISHERS, BO ISSN: 1573-7217
  • JESSICA M GRUNDA ET AL: "Increased Expression of Thymidylate Synthetase (TS), Ubiquitin Specific Protease 10 (USP10) and Survivin is Associated with Poor Survival in Glioblastoma Multiforme (GBM)", JOURNAL OF NEURO-ONCOLOGY, KLUWER ACADEMIC PUBLISHERS, BO, vol. 80, no. 3, 14 June 2006 (2006-06-14), pages 261-274, XP019453042, ISSN: 1573-7373, DOI: 10.1007/S11060-006-9191-4
  • KELIE M. REECE ET AL: "A novel regulator (USP10) of p53: Implications for tumor suppression and therapeutic targeting", CANCER BIOLOGY & THERAPY, vol. 9, no. 8, 15 April 2010 (2010-04-15), pages 583-584, XP055045739, ISSN: 1538-4047, DOI: 10.4161/cbt.9.8.11690
  • JIAN YUAN ET AL: "USP10 Regulates p53 Localization and Stability by Deubiquitinating p53", CELL, vol. 140, no. 3, 1 February 2010 (2010-02-01), pages 384-396, XP055045731, ISSN: 0092-8674, DOI: 10.1016/j.cell.2009.12.032
  • Nomura ET AL: "Antiteratogenic effects of tumor inhibitors, caffeine, antipain, and retinoic acid in mice.", Cancer research, vol. 43, no. 11, 1 November 1983 (1983-11-01), pages 5156-5162, XP55127631, ISSN: 0008-5472
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

BACKGROUND


1. Technical Field



[0001] This document relates to methods and materials involved in modulating deubiquitinases and ubiquitinated polypeptides (e.g., tumor suppressors such as wild-type p53 polypeptides). For example, this document relates to methods and materials for increasing or decreasing deubiquitinase expression or activity, methods and materials for stabilizing or de-stabilizing ubiquitinated polypeptides, and compositions and materials for treating cancer.

2. Background Information



[0002] p53 is a tumor suppressor that is mutated in more than 50% of human cancers and whose major function is regulating cell fate following cellular stress and repressing the propagation of damaged cells (Lane, 1992; Riley et al., 2008; Vogelstein et al., 2000). p53 functions as a transcription factor, and through its target genes regulates a variety of cellular functions, from cellular senescence, to energy metabolism, DNA repair, cell differentiation, cell cycle progression and apoptosis. In addition to the activation of transcription, p53 can also act as a repressor of transcription, as it does in the suppression of CD44, a protein implicated in tumorigenesis (Godar et al., 2008). Finally, p53 also has transcription-independent functions, such as regulating apoptosis through protein-protein interactions (Moll et al., 2005).

SUMMARY



[0003] The present invention is defined by the independent claims. The dependent claims depict other embodiments of the invention.

[0004] This document relates to methods and materials involved in modulating deubiquitinases (e.g., USP10 polypeptides) and/or ubiquitinated polypeptides (e.g., tumor suppressor polypeptides or mutant versions of tumor suppressor polypeptides). For example, this document provides methods and materials for increasing deubiquitinase (e.g., a USP10 polypeptide) expression or activity, methods and materials for decreasing deubiquitinase (e.g., a USP10 polypeptide) expression or activity, methods and materials for stabilizing tumor suppressor polypeptides (e.g., wild-type p53 polypeptides), methods and materials for de-stabilizing mutant versions of tumor suppressor polypeptides (e.g., mutant p53 polypeptides), and compositions and materials for reducing cancer cell proliferation, increasing cancer cell apoptosis, and/or treating cancer (e.g., cancers having reduced levels of wild-type p53 polypeptides or cancers having increased levels of mutant p53 polypeptides). This document also provides methods and materials for identifying agonists or antagonists of USP10 mediated stabilization of p53 polypeptides.

[0005] Some cancer cells can express reduced levels of p53 polypeptides, while other cancer cells can express average or elevated levels of a mutant version of a p53 polypeptide. As described herein, USP10 polypeptides can interact with and deubiquinate wild-type or mutant p53 polypeptides, thereby increasing their stability. In the cases of cancer cells having reduced levels of wild-type p53 polypeptides, the methods and materials provided herein can be used to increase USP10 polypeptide expression or activity, thereby increasing the stability of the wild-type p53 polypeptides. This can result in an increased level of wild-type p53 polypeptides within the cancer cells, thereby resulting in reduced cancer cell proliferation and increased cancer cell apoptosis. In the cases of cancer cells that express mutant p53 polypeptides, the methods and materials provided herein can be used to decrease USP10 polypeptide expression or activity, thereby decreasing the stability of the mutant p53 polypeptides. This can result in a decreased level of mutant p53 polypeptides within the cancer cells, thereby resulting in reduced cancer cell proliferation and increased cancer cell apoptosis.

[0006] In general, one aspect of this document features a composition for use in reducing cancer cell proliferation in a mammal having cancer cells. The composition for use comprises, or consists essentially of, administering a composition to the mammal under conditions wherein the composition modulates USP10 polypeptide expression or activity within the cancer cells, thereby reducing cancer cell proliferation. The cancer cells can have a reduced level of wild-type p53 polypeptide expression, and the composition can
increase USP10 polypeptide expression or activity. The composition for use can comprise nucleic acid encoding a USP10 polypeptide. The composition for use can comprise nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The cancer cells can express a mutant version of a p53 polypeptide, and the composition can decrease USP10 polypeptide expression or activity. The composition can comprise an antagonist of USP10 polypeptide mediated stabilization of p53 polypeptides as defined in the claims. The antagonist can comprise nucleic acid having the ability to induce RNA interference against expression of the USP10 polypeptide. The USP10 polypeptide can be a human USP10 polypeptide.

[0007] In another aspect, this document features a composition for use in a method for treating cancer in a mammal. The method comprises, or consists essentially of, (a) identifying a mammal as having cancer cells that express a reduced level of wild-type p53 polypeptides or that express a mutant p53 polypeptide, (b) administering a USP10 polypeptide or a composition that increases USP10 polypeptide expression or activity within the cancer cells if the mammal is identified as having cancer cells that express the reduced level of wild-type p53 polypeptides, and (c) administering a composition that decreases USP10 polypeptide expression or activity within the cancer cells if the mammal is identified as having cancer cells that express the mutant p53 polypeptide.

[0008] In another aspect, this document features an in vitro method, an in vitro cell-based assay, an in vitro cell free assay, and/or an in vivo non-human animal model for identifying an antagonist of USP10 polypeptide mediated stabilization of p53 polypeptides. The in vitro method, the in vitro cell-based assay, the in vitro cell free assay, and/or the in vivo non-human animal model comprise, or consist essentially of, determining if the stabilization level of a ubiquinated p53 polypeptide contacted with a USP10 polypeptide in the presence of a test agent is less than the stabilization level of the ubiquinated p53 polypeptide contacted with the USP10 polypeptide in the absence of the test agent, wherein the presence of the stabilization level of the ubiquinated p53 polypeptide contacted with the USP10 polypeptide in the presence of the test agent that is less than the stabilization level of the ubiquinated p53 polypeptide contacted with the USP10 polypeptide in the absence of the test agent indicates that the test agent is the antagonist.

[0009] In another aspect, this document features an in vitro method, an in vitro cell-based assay, an in vitro cell free assay, and/or an in vivo non-human animal model for identifying an agonist of USP10 polypeptide mediated stabilization of p53 polypeptides. The in vitro method, the in vitro cell-based assay, the in vitro cell free assay, and/or the in vivo non-human animal model comprise, or consist essentially of, determining if the stabilization level of a ubiquinated p53 polypeptide contacted with a USP10 polypeptide in the presence of a test agent is greater than the stabilization level of the ubiquinated p53 polypeptide contacted with the USP10 polypeptide in the absence of the test agent, wherein the presence of the stabilization level of the ubiquinated p53 polypeptide contacted with the USP10 polypeptide in the presence of the test agent that is greater than the stabilization level of the ubiquinated p53 polypeptide contacted with the USP10 polypeptide in the absence of the test agent indicates that the test agent is the agonist.

[0010] Herein described is a method for assessing the p53 genotype of a cancer cell. The method comprises, or consists essentially of, determining the level of USP10 polypeptide expression in the cancer cell, diagnosing the cancer cell as having wild-type p53 if the cancer cell contains a reduced level of USP10 polypeptide expression, and diagnosing the cancer cell as having mutant p53 if the cancer cell contains an increased level of USP10 polypeptide expression.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

DESCRIPTION OF DRAWINGS



[0012] 

Figure 1. USP10 interacts with p53. (A and D) U2OS cell lysates were subjected to immunoprecipitation with control IgG or anti-USP10 antibodies. The immunoprecipitates were then blotted with anti-p53, anti-Mdm2, or anti-USPIO antibodies. (B and C) HCT116 p53+/+ and p53-/- cell lysates were subjected to immunoprecipitation with control IgG or anti-USP10 antibodies (B) or anti-p53 antibodies (C). The immunoprecipitates were then blotted with anti-p53 or anti-USPIO antibodies. (HC: Heavy Chain). (E). Purified FLAG-tagged USP10 was incubated with GST or GST-p53 coupled to GSH-Sepharose. Proteins retained on Sepharose were then blotted with indicated antibodies. (F) Constructs encoding FLAG-tagged full-length (FL)
or deletion mutations of USP10 were transfected into H1299 cells. Forty-eight hours after transfection, cells were lysed, and cell lysates were incubated with GST or GST-p53 coupled to GSH-Sepharose. Proteins retained on Sepharose were analyzed with the indicated antibody.

Figure 2. USP10 stabilizes and deubiquitinates p53. (A) HCT116 cells were transfected with vectors or constructs encoding FLAG-tagged USP10. Forty-eight hours later, cells were lysed, and cell lysates were blotted with indicated antibody. (B) HCT116 cells were infected with lentivirus encoding indicated shRNAs. 72 hours later, cells were lysed, and cell lysates were blotted with indicated antibody. (C) HCT116 cells were stably expressing control shRNA, USP10 shRNA, or USP10 shRNA together with shRNA-resistant USP10. Cells were treated with cycloheximide (0.1 mg/mL) and harvested at the indicated time. The upper panels show immunoblots of p53 and USP10. β-actin was included as a control. Lower panel: quantification of the p53 protein levels relative to β-actin. (D) HCT116 cells were transfected with indicated plasmids. Forty-eight hours later, cells were lysed, and cell lysates were blotted with indicated antibody. (E-G) Regulation of p53 ubiquitination levels in vivo by USP10. H1299 cells transfected with indicated constructs (E) or stably expressing control or USP10 shRNA (F) were transfected with FLAG-p53. Forty-eight hours later, cells were treated with MG132 for 4 hours before harvest. p53 was immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-p53 antibodies. (G) Deubiquitination of p53 in vitro by USP10. Ubiquitinated p53 was incubated with purified USP10 or USP10CA in vitro and then blotted with anti-p53 antibodies.

Figure 3. Regulation of the subcellular localization of p53 by USP10. (A) Subcellular localization of USP10 and a ubiquitin-specific protease, HAUSP. U2OS cells were transfected with constructs encoding FLAG-USP10 or FLAG-HAUSP. Forty-eight hours later, cells were fixed and stained with indicated antibodies and DAPI. (B) H1299 cells were cotransfected with indicated constructs. Forty-eight hours later, cells were treated with MG132, harvested, and fractionated as described herein. Cellular fractions were then blotted with indicated antibodies. (C, cytoplasmic; N, nuclear). A cytoplasmic marker protein (GAPDH) and a nuclear marker protein (Histone3) were used as controls to confirm the quality of fractionations. (C) H1299 cells were transfected with indicated constructs. Forty-eight hours later, the cells were treated with MG132, fixed, and stained with the indicated antibodies and DAPI. (D) U2OS cells were infected with lentivirus encoding control shRNA or USP10 shRNA. 72 hours later, cells were treated with MG132, fixed, and stained with the indicated antibodies or DAPI. (C-D) Right panels: Quantification of cells with different p53 subcellular localization. Nuc: Nucleus only; Cyto + Nuc: both cytoplasm and nucleus. The data represent the average of three experiments, and 150 cells were monitored in each experiment.

Figure 4. Effects of USP10 on p53-mediated transcriptional activity, cell growth repression, and apoptosis. (A) p53 reporter constructs for the p21 promoter were cotransfected with indicated constructs into HCT116 p53+/+ and HCT116 p53-/- cells. Reporter activity was then determined as described herein. (B) p53 reporter assay was performed in HCT116 p53+/+ and HCT116 p53-/- cells stably expressing control shRNA or USP10 shRNA. (C) H1299 cells were transfected with the indicated constructs. Forty-eight hours later, apoptotic cells were determined as described herein. (D) HCT116 p53+/+ and HCT116 p53-/- cells stably expressing control shRNA or USP10 shRNA were plated, and cell proliferation was then quantified at the indicated time. (E) Soft agar colony-formation assay was performed using HCT116 p53+/+ and HCT116 p53-/- cells stably expressing control shRNA, USP10 shRNA, or USP10 shRNA together with shRNA-resistant USP10. Right panel: quantification of colonies formed in soft agar. Bars, 400 µm. (A-E). Error bar represents the mean ±SEM of triplicate experiments. ** represents P < 0.01 two tailed student's t test.

Figure 5. USP10 translocates into the nucleus and regulates p53 activity following DNA damage. (A) HCT116 cells stably expressing control shRNA or USP10 shRNA were irradiated (10 Gy), and cells were harvested at the indicated time. Cell lysates were then blotted with the indicated antibodies. (B) HCT116 cells were left untreated or treated with 10 Gy radiation. Four hours later cells were stained with anti-USP10 antibody. (C) HCT116 cells were irradiated (10 Gy) or left untreated. After four hours, cells were harvested and fractionated as described herein. Cellular fractions were then blotted with the indicated antibodies. (D) HCT116 p53+/+ or p53-/- cells stably expressing control shRNA or USP10 shRNA were left untreated or treated with 10 Gy radiation. After 48 hours, apoptotic cells were determined as described herein. Error bar represents the mean ±SEM of triplicate experiments. ** represents P < 0.01 two tailed student's t test. (E) The same cells in (D) were treated with 10 Gy radiation, then harvested at the indicated time. Cell cycle progression was examined by FACS.

Figure 6. USP10 phosphorylation by ATM regulates USP10 stabilization, translocation, and p53 activation following DNA damage. (A) HCT116 cells were irradiated (10 Gy) and harvested at the indicated times. Cell lysates and mRNA were then extracted and analyzed by Western blot or RT-PCR, respectively. (B) HCT116 cells were left untreated or irradiated. Cells were then treated with cycloheximide (0.1 mg/mL) and harvested at the indicated times. Cell lysates were then blotted with the indicated antibodies. (C) HCT116 cells were transfected with FLAG-tagged USP10. Forty-eight hours later, the cells were left untreated or treated with 10 Gy radiation, 40 J/m2 UV, or 20 mM etoposide. After an additional 1 hour, the cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and immunobloted with phospho-SQ/TQ (pSQ/TQ) antibody. (D) HCT116 cells were transfected with FLAG-tagged USP10 and pretreated with DMSO, 25 mM Ku55933, or 3 mM caffeine. After 2 hours of incubation, cells were left untreated or treated with 10 Gy radiation. The phosphorylation of USP10 was examined as in (C). (E) ATM+/+ or ATM-/- cells were irradiated (10 Gy) or left untreated. After one hour, the cells were harvested, and cell lysates were subjected to immunoprecipitation with anti-USP10 antibody and blotted with pSQ/TQ antibody. (F). ATM+/+ or ATM-/- cells were left untreated or irradiated (10 Gy) and were harvested at the indicated time. Cell lysates were then blotted with the indicated antibodies. (G) HCT116 cells stably expressing USP10 shRNA were reconstituted with shRNA resistant FLAG-tagged USP10 WT (wild type), T42A, S337A or 2SA (T42A and S337A double mutation). Cells were left untreated or irradiated (10 Gy) and harvested at the indicated time. Cell lysates were then blotted with the indicated antibodies. (H) HCT116 cells stably expressing USP10 shRNA were reconstituted with shRNA resistant FLAG-tagged USP10 WT or 2SA. Cells were left untreated or treated with 10 Gy radiation. USP10 phosphorylation was examined by pSQ/TQ antibody. (I) Cells the same as (H) were irradiated (10 Gy) or left untreated. After four hours, cells were harvested and fractionated as described herein. (J) Cells the same as (H) were left untreated or irradiated (10 Gy). Cells were harvested at the indicated time, and cell lysates were blotted with the indicated antibodies. (K) Cells the same as (H) were left untreated or irradiated. Apoptotic cells were determined 48 hours later.

Figure 7. USP10 is downregulated in renal cell carcinoma. (A) Expression of USP10 and p53 in human renal tubular epithelial cell line (HK-2) and renal cell carcinoma (RCC) cell lines. (B) 11 pairs of fresh frozen RCC tissues and corresponding normal tissues were lysed, and cell lysates were blotted with the indicated antibodies. (N: normal tissue; T: tumor tissue) (C) Immunohistochemical staining of USP10 in normal renal tissues and renal cell carcinoma. Lower table: quantification of USP10-positive or USP10-negative renal cell carcinoma cases. (ccRCC: clear cell Renal Cell Carcinoma). (D-E) Soft agar colony-formation assay was performed using CAKI-1 and CAKI-2 cells stably expressing S/FLAG-USP10 (D) and 786-O cells stably expressing S/FLAG-USP10 or USP10 shRNA (E). Lower panels: quantification of colonies formed in soft agar. Error bar represents the mean ±SEM of triplicate experiments. ** represents P < 0.01 two tailed student's t test. Bars represent 400 µm. (F) A schematic of a model showing how USP10 regulates p53.

Figure 8. (A) H1299 cells stably transfected with control or USP10 shRNA were transfected with GFP-p53. Forty-eight hours later, the cells were treated with MG132, fixed, and stained with the indicated antibodies and DAPI. Right panels: Quanitification of cells with different p53 subcellular localization. Nuc: Nucleus only; Cyto+nuc: both cytoplasm and nucleus. The data represent the average of three experiments, and 150 cells were monitored in each experiment. (B) Cells the same as Figure 6H were left untreated or treated with 10 Gy radiation. After four hours, the cells were fixed and stained with anti-FLAG antibody or DAPI. Right panels: Quantification of cells with different USP10 subcellular localization. Cyto: cytoplasm only; Cyto+Nuc: cytoplasm and nucleus. The data represent the average of three experiments, and 150 cells were monitored in each experiment. (C) Cells the same as Figure 7D-E were lysed, and cell lysates were blotted with the indicated antibodies.

Figure 9 is a listing of a nucleic acid sequence (SEQ ID NO:1) that encodes a human USP10 polypeptide.

Figure 10 is a listing of an amino acid sequence of a human USP10 polypeptide (SEQ ID NO:2).

Figure 11 contains a list of shRNAs that can target nucleic acid encoding a human USP10 polypeptide.

Figure 12 is a listing of a nucleic acid sequence (SEQ ID NO:3) that encodes a human p53 polypeptide.

Figure 13 is a listing of an amino acid sequence of a human p53 polypeptide (SEQ ID NO:4).

Figure 14 is a listing of a nucleic acid sequence (SEQ ID NO:5) that encodes a mutant version of a human p53 polypeptide.

Figure 15 is a listing of an amino acid sequence of a mutant version of a human p53 polypeptide (SEQ ID NO:6).

Figure 16 contains photographs of Western Blots for β-actin and USP10 polypeptides in pancreatic cancer cell lines (A), breast cancer cell lines (B), and pancreatic tissues (C). The N represents normal tissue, while the T represents tumor tissue.

Figure 17A is a concentration curve graph plotting the level of deubiquitination of Ub-AMC observed following incubation with the indicated amounts of USP10 polypeptide (µg). Figure 17B is a graph plotting the level of deubiquitination of Ub-AMC in the presence (right) or absence (left) of 4 µg/mL of USP10 polypeptides and the indicated compound.

Figure 18 is a photograph of an immunoprecipitation of HCT116 cell lysates with an anti-USP10 polypeptide antibody (or control antibody, IgG) and immunoblotted with anti-G3BP1 polypeptide antibodies or anti-USP10 polypeptide antibodies.

Figure 19A is a schematic diagram of USP10 polypeptides (e.g., full length and fragments of full length USP10 polypeptides) with a table indicating that both G3BP1 polypeptides and p53 polypeptides interact with the N-terminal region (e.g., 1-100 amino acids) of USP10 polypeptides. Figure 19B is a photograph of an immunoprecipitation of HCT116 cell lysates with an anti-p53 polypeptide antibody (or control antibody, IgG) and immunoblotted with anti-USP10 polypeptide antibodies, anti-p53 polypeptide antibodies, or anti-G3BP1 polypeptide antibodies. The HCT116 cell lysates were obtained from cells treated with MG132 and either control shRNA (Ctrl) or shRNA designed to reduce G3BP1 polypeptide expression (G3BP1). Figure 19C is a photograph of an immunoprecipitation of HCT116 cell lysates with an anti-p53 polypeptide antibody (or control antibody, IgG) and immunoblotted with anti-USP10 polypeptide antibodies, anti-p53 polypeptide antibodies, or anti-FLAG antibodies. The HCT116 cell lysates were obtained from cells treated with MG132 and either a control vector (Vector) or a vector designed to overexpress G3BP1 polypeptides (FLAG-G3BP1).

Figure 20 is a photograph of HCT116 cell lysates immunoblotted with anti-FLAG antibodies, anti-USPIO polypeptide antibodies, anti-p53 polypeptide antibodies, or anti-β-actin antibodies. The HCT116 cell lysates were obtained from cells stably transfected with either a control construct (Ctrl) or an shRNA construct designed to reduce USP10 polypeptide expression (USP10) that were transfected with an empty vector (Vector), a vector designed to over-express G3BP1 polypeptides (FLAG-G3BP1), or a vector designed to over-express G3BP2 polypeptides (FLAG-G3BP2).

Figure 21 is a graph plotting cell growth (fold of growth, set cell number at day 1 as 1) versus time (days) for HCT116 cells stably expressing either control or an shRNA construct designed to reduce expression of USP10 polypeptides (USP10shRNA) and transfected with a control vector (Vector) or a FLAG-G3BP1 construct (G3BP1).

Figure 22 is a photograph of an immunoprecipitation of HCT116 cell lysates with an anti-USP10 polypeptide antibody (or control antibody, IgG) and immunoblotted with anti-G3BP1 polypeptide antibodies and anti-USP10 polypeptide antibodies. The HCT116 cell lysates were obtained from untreated cells or cells treated with 10 Gy irradiation.


DETAILED DESCRIPTION



[0013] This document relates to methods and materials involved in modulating deubiquitinases (e.g., USP10 polypeptides) and/or ubiquitinated polypeptides (e.g., tumor suppressor polypeptides or mutant versions of tumor suppressor polypeptides). For example, this document provides methods and materials for increasing deubiquitinase (e.g., a USP10 polypeptide) expression or activity, methods and materials for decreasing deubiquitinase (e.g., a USP10 polypeptide) expression or activity, methods and materials for stabilizing tumor suppressor polypeptides (e.g., wild-type p53 polypeptides), methods and materials for de-stabilizing mutant versions of tumor suppressor polypeptides (e.g., mutant p53 polypeptides), and methods and materials for use in reducing cancer cell proliferation, increasing cancer cell apoptosis, and/or treating cancer (e.g., cancers having reduced levels of wild-type p53 polypeptides or cancers having increased levels of mutant p53 polypeptides). This document also provides methods and materials for identifying agonists or antagonists of USP10 mediated stabilization of p53 polypeptides.

[0014] In one embodiment, this document provides compositions and materials related to treating mammals (e.g., humans) having cancer. Examples of mammals that can be treated as described herein include, without limitation, humans, monkeys, dogs, cats, cows, horses, pigs, rats, and mice. Examples of cancers that can be treated as described herein include, without limitation, renal cancers (e.g., renal cell carcinomas), pancreatic cancers, breast cancers, and glioma. A mammal can be identified as having cancer using any appropriate cancer diagnostic techniques. In some cases, a cancer can be assessed to determine if the cancer is a cancer with a reduced level of p53 polypeptides (e.g., wild-type p53 polypeptides). Any appropriate method can be used to assess the level of p53 polypeptides within cancer cells. For example, nucleic acid detection techniques such as RT-PCR or microarray assays can be used to assess the level of p53 mRNA within cancer cells or polypeptide detection techniques such as immunohistochemistry or ELISAs can be used to assess the level of p53 polypeptides within cancer cells.

[0015] As described herein, cancer having a reduced level of wild-type p53 polypeptides can be treated by increasing the level of USP10 polypeptide expression or activity. The increased level of USP10 polypeptide expression or activity can stabilize wild-type p53 polypeptides within the cancer cells, thereby resulting in reduced cancer cell proliferation and increased cancer cell apoptosis. In some cases, the level of USP10 polypeptide within cancer cells can be increased by administering a composition containing USP10 polypeptides. In some cases, the level of USP10 polypeptide expression or activity within cancer cells can be increased by administering a USP10 polypeptide or a nucleic acid encoding a USP10 polypeptide to the cancer cells. Such a nucleic acid can encode a full-length USP10 polypeptide such as a human USP10 polypeptide having the amino acid sequence set forth in SEQ ID NO:2, or a biologically active fragment of a USP10 polypeptide having amino acid residues 520 to 793 of the sequence set forth in SEQ ID NO:2. A nucleic acid encoding a USP10 polypeptide or fragment thereof can be administered to a mammal using any appropriate method. For example, a nucleic acid can be administered to a mammal using a vector such as a viral vector.

[0016] Vectors for administering nucleic acids (e.g., a nucleic acid encoding a USP10 polypeptide or fragment thereof) to a mammal are known in the art and can be prepared using standard materials (e.g., packaging cell lines, helper viruses, and vector constructs). See, for example, Gene Therapy Protocols (Methods in Molecular Medicine), edited by Jeffrey R. Morgan, Humana Press, Totowa, NJ (2002) and Viral Vectors for Gene Therapy: Methods and Protocols, edited by Curtis A. Machida, Humana Press, Totowa, NJ (2003). Virus-based nucleic acid delivery vectors are typically derived from animal viruses, such as adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, vaccinia viruses, herpes viruses, and papilloma viruses. Lentiviruses are a genus of retroviruses that can be used to infect cells (e.g., cancer cells). Adenoviruses contain a linear double-stranded DNA genome that can be engineered to inactivate the ability of the virus to replicate in the normal lytic life cycle. Adenoviruses and adeno-associated viruses can be used to infect cancer cells.

[0017] Vectors for nucleic acid delivery can be genetically modified such that the pathogenicity of the virus is altered or removed. The genome of a virus can be modified to increase infectivity and/or to accommodate packaging of a nucleic acid, such as a nucleic acid encoding a USP10 polypeptide. A viral vector can be replication-competent or replication-defective, and can contain fewer viral genes than a corresponding wild-type virus or no viral genes at all.

[0018] In addition to nucleic acid encoding a USP10 polypeptide, a viral vector can contain regulatory elements operably linked to a nucleic acid encoding a USP10 polypeptide. Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, or inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid. The choice of element(s) that may be included in a viral vector depends on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in a viral vector to facilitate transcription of a nucleic acid encoding a USP10 polypeptide. A promoter can be constitutive or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a USP10 polypeptide in a general or tissue-specific manner. Tissue-specific promoters include, without limitation, enolase promoter, prion protein (PrP) promoter, and tyrosine hydroxylase promoter.

[0019] As used herein, "operably linked" refers to positioning of a regulatory element in a vector relative to a nucleic acid in such a way as to permit or facilitate expression of the encoded polypeptide. For example, a viral vector can contain a neuronal-specific enolase promoter and a nucleic acid encoding a USP10 polypeptide. In this case, the enolase promoter is operably linked to a nucleic acid encoding a USP10 polypeptide such that it drives transcription in neuronal tumor cells.

[0020] A nucleic acid encoding a USP10 polypeptide also can be administered to cancer cells using non-viral vectors. Methods of using non-viral vectors for nucleic acid delivery are known to those of ordinary skill in the art. See, for example, Gene Therapy Protocols (Methods in Molecular Medicine), edited by Jeffrey R. Morgan, Humana Press, Totowa, NJ (2002). For example, a nucleic acid encoding a USP10 polypeptide can be administered to a mammal by direct injection (e.g., an intratumoral injection) of nucleic acid molecules (e.g., plasmids) comprising nucleic acid encoding a USP10 polypeptide, or by administering nucleic acid molecules complexed with lipids, polymers, or nanospheres.

[0021] A nucleic acid encoding a USP10 polypeptide can be produced by standard techniques, including, without limitation, common molecular cloning, polymerase chain reaction (PCR), chemical nucleic acid synthesis techniques, and combinations of such techniques. For example PCR or RT-PCR can be used with oligonucleotide primers designed to amplify nucleic acid (e.g., genomic DNA or RNA) encoding a USP10 polypeptide.

[0022] In some cases, a nucleic acid encoding a USP10 polypeptide can be isolated from a healthy mammal or a mammal having cancer. For example, a nucleic acid that encodes a wild type USP10 polypeptide having the amino acid sequence set forth in SEQ ID NO:2 can be isolated from a human containing that nucleic acid. The isolated nucleic acid can then be used to generate a viral vector, for example, which can be administered to a mammal so that the level of a USP10 polypeptide in cancer cells within the mammal is increased.

[0023] In some cases, a cancer can be assessed to determine if the cancer is a cancer that expresses a mutant version of a p53 polypeptide. Examples of mutant p53 polypeptide include, without limitation, those having the amino acid sequence as set forth elsewhere ("The UMD-p53 database: New mutations and analysis tools," Christophe Béroud and Thierry Soussi, Human Mutation, Volume 21:p. 176-181; and Berglind et al., Cancer Biol. Ther., 7(5):699-708 (2008)). Any appropriate method can be used to assess cancer cells for a mutant version of a p53 polypeptide. For example, nucleic acid detection techniques such as RT-PCR or microarray assays can be used to assess cancer cells for a mutant version of a p53 polypeptide or polypeptide detection techniques such as immunohistochemistry or ELISAs can be used to assess cancer cells for a mutant version of a p53 polypeptide.

[0024] As described herein, cancers that express a mutant version of a p53 polypeptide can be treated by decreasing the level of USP10 polypeptide expression or activity. The decreased level of USP10 polypeptide expression or activity can destabilize mutant p53 polypeptides within the cancer cells, thereby resulting in reduced cancer cell proliferation and increased cancer cell apoptosis. In some cases, the level of USP10 polypeptide expression or activity within cancer cells can be decreased by administering a USP10 polypeptide antagonist as claimed to the cancer cells. Examples of USP10 polypeptide antagonists that can have the ability to decrease or inhibit the level of USP10 polypeptide activity within a cell include, without limitation, N-ethylmaleimide, Z-phe-ala fluoromethyl ketone, chymostatin, E-64 (trans-Epoxysuccinyl-L-leucylamido (4-guanidino)butane, E-64d ((2S, 3S)-trans-Epoxysuccinyl-L-leuclamido-3-methylbutane ethyl ester), antipain dihydrochloride, cystatin, and cyano-indenopyrazine. In some cases, a USP10 polypeptide antagonist can be a nucleic acid molecule designed to induce RNA interference (e.g., an RNAi molecule or a shRNA molecule). Examples of such shRNA molecules include, without limitation, those set forth in Figure 11. Nucleic acid molecules designed to induce RNA interference against USP10 polypeptide expression can be administered to a mammal using any appropriate method including, without limitation, those methods described herein. For example, a nucleic acid designed to induce RNA interference against USP10 polypeptide expression can be administered to a mammal using a vector such as a viral vector.

[0025] In some cases, the USP10 polypeptide inhibitor G3BP1 polypeptide (also known as a RasGap Sh3 domain Binding Protein 1) can be used to decrease or inhibit the level of USP10 polypeptide activity within a cell. Examples of G3BP1 polypeptides include, without limitation, human G3BP1 polypeptides (e.g., a human G3BP1 polypeptide encoded by the nucleic acid sequence set forth in GenBank® Accession Nos. NM_005754.2 (GINo. 38327550) or NM_198395.1 (GI No. 38327551), rat G3BP1 polypeptides (e.g., a rat G3BP1 polypeptide encoded by the nucleic acid sequence set forth in GenBank® Accession Nos. NM_133565.1 (GI No. 281306780), and mouse G3BP1 polypeptides (e.g., a mouse G3BP1 polypeptide encoded by the nucleic acid sequence set forth in GenBank® Accession Nos. NM_013716.2 (GI No. 118130851).

[0026] In some cases, an USP10 polypeptide antagonist can be a nucleic acid-based molecule such as an shRNA or RNAi molecule. In some cases, an USP10 polypeptide antagonist can be a non-G3BP1 polypeptide molecule (e.g., a nucleic acid-based molecule such as an shRNA or RNAi molecule).

[0027] This document also provides methods and materials related to identifying agonists or antagonists of USP10 polypeptide mediated stabilization of p53 polypeptides. For example, this document provides methods and materials for using USP10 polypeptides and p53 polypeptides (e.g., ubiquinated p53 polypeptides) to identify agents that increase or decrease the ability of the USP10 polypeptides to stabilize the p53 polypeptides. In some cases, the stability of ubiquinated p53 polypeptides treated with USP10 polypeptides in the presence and absence of a test agent can be assessed to determine whether or not the test agent increases or decreases the stability of the ubiquinated p53 polypeptides. An agent that increases the stability of the ubiquinated p53 polypeptides in a manner dependent on the USP10 polypeptide can be an agonist of USP10 polypeptide mediated stabilization of p53 polypeptides, and an agent that decreases the stability of the ubiquinated p53 polypeptides in a manner dependent on the USP10 polypeptide can be an antagonist of USP10 polypeptide mediated stabilization of p53 polypeptides. The stability of ubiquinated p53 polypeptides can be assessed using polypeptide assays capable of detecting intact full-length polypeptide or degraded polypeptides. USP10 polypeptide agonists and antagonists can be identified by screening test agents (e.g., from synthetic compound libraries and/or natural product libraries). Test agents can be obtained from any commercial source and can be chemically synthesized using methods that are known to those of skill in the art. Test agents can be screened and characterized using in vitro cell-based assays, cell free assays, and/or in vivo non-human animal models.

[0028] USP10 agonists or antagonists can be identified using an in vitro screen that includes using purified His-tagged USP10 polypeptide together with ubiquitin-AMC (BIOMOL) as the substrate. Ubiquitin-AMC is a fluorogenic substrate for a wide range of deubiquitinylating enzymes (Dang et al., Biochemistry, 37:1868 (1998)). This fluorescence can allow high-throughput screen of USP10 agonists and antagonists in vitro.

[0029] In some cases, the expression level of USP10 polypeptides can be used to assess the p53 genotype of a cancer cell. For example, identification of cancer cells having an increased level of USP10 polypeptide expression can indicate that the cancer cells contain mutant p53, while identification of cancer cells having a decreased level of USP10 polypeptide expression can indicate that the cancer cells contain wild-type p53.

[0030] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES


Example 1 - USP10 Regulates p53 Localization and Stability by Deubiquitinating p53


Cell Culture, Plasmids, and Antibodies



[0031] H1299, HCT116 p53+/+, HCT116 p53-/-, U2OS, and HEK293 cells were cultured in RPMI supplemented with 10% FBS. Caki-1 and Caki-2 cells were cultured in McCoy's 5A supplemented with 10% FBS. A-498 cells was cultured in MEM supplemented with 10% FBS. 786-O and 769-P cells were cultured in DMEM supplemented with 10% FBS. ATM+/+ and ATM-/- MEFs were culture in DMEM supplemented with 15% FBS.

[0032] USP10 was cloned into the p3xFLAG-CMV vector (Sigma) and the pET-28a vector (Novagen). Mdm2 was cloned into the pCMV-HA vector (Clontech). p53 was cloned into the pCMV-Myc vector (Clontech). pBABE-S/FLAG/SBP (streptavidin binding peptide)-tagged USP10 was constructed using Invitrogen's Gateway System. pcDNA3-FLAG-p53 (Addgene plasmid 10838, provided by Dr. T. Roberts)(Gjoerup et al., J. Virol., 75:9142-9155 (2001)), GFP-p53 (Addgene plasmid 12091, provided by Dr. T. Jacks) (Boyd et al., Nat. Cell Biol., 2:563-568 (2000)), GST-p53 (Addgene plasmid 10852, provided by Dr. PM Howley) (Huibregtse et al., Embo J., 10:4129-4135 (1991)), p21 promoter A (Addgene plasmid 16462, provided by Dr. B. Vogelstein) (el-Deiry et al., Cancer Res., 55:2910-2919 (1995)) and pCI-neo Flag HAUSP (Addgene plasmid 16655, provided by Dr. B. Vogelstein)(Cummins and Vogelstein, Cell Cycle, 3:689-692 (2004)) were obtained from Addgene. Deletion mutants were generated by site-directed mutagenesis (Stratagene).

[0033] Rabbit anti-USP10 antibodies were raised by immunizing rabbits with GST-USP10 (amino acids 1-200). The antisera were affinity-purified with AminoLink Plus immobilization and purification kit (Pierce). Anti-FLAG (m2) and anti-HA antibodies were purchased from Sigma. Anti-p53 (DO-1) antibodies were purchased from SantaCruz. Anti-MDM2 monoclonal antibody was purchased from Calbiochem.

RNA Interference



[0034] USP10 shRNAs having the sequences set forth in SEQ ID NOs:7 and 8 were purchased from Openbiosystems (RHS4533-NM_005153). Lentivirus USP10 shRNAs were made using a commercially available protocol provided by OpenBiosystems as described elsewhere (Moffat et al., Cell, 124:1283-1298 (2006); Stewart et al., RNA, 9:493-501 (2003); Zufferey et al., Nat. Biotechnol., 15:871-85 (1997); Zufferey et al., J. Virol., 72:9873-80 (1998); and Yamamoto and Tsunetsugu-Yokota, Curr. Gene Ther., 8(1):1-8 (2008)). Briefly, 293T cells (80% confluency) were transfected with the pLKO.1 vector (3 µg) together with packaging plasmid (1.5 µg) and envelope plasmid (1.5 µg) using lipofectamine 2000. Media were changed after 20 hour (RPMI media with 30% FBS). Supernatants containing viruses were collected an additional 24 hours and 48 hours later and filleted (0.45 µm low-protein binding filter). Cells were infected with virus in the presence of 8 µg/mL polybrene.

Co-immunoprecipitation Assay



[0035] Cells were lysed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing 50 mM β-glycerophosphate, 10 mM NaF, and 1 mg/mL each of pepstatin A, and aprotinin. Whole cell lysates obtained by centrifugation were incubated with 2 µg of antibody and protein A or protein G Sepharose beads (Amersham Biosciences) for 2 hours at 4°C. The immunocomplexes were then washed with NETN buffer three times and separated by SDS-PAGE. Immunoblotting was performed following standard procedures.

GST Pull-downs



[0036] GST fusion proteins were prepared following a standard protocol as described elsewhere (Einarson and Orlinick, Identification of Protein-Protein Interactions with Glutathione S-Transferase Fusion Proteins. In Protein-Protein Interactions: A Molecular Cloning Manual, Cold Spring Harbor Laboratory Press, pp. 37-57 (2002); Einarson, Detection of Protein-Protein Interactions Using the GST Fusion Protein Pulldown Technique. In Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, pp.18.55-18.59 (2001); and Vikis and Guan, Glutathione-S-Transferase-Fusion Based Assays for Studying Protein-Protein Interactions. In Protein-Protein Interactions, Methods and Applications, Methods in Molecular Biology, 261, Fu, H. Ed. Humana Press, Totowa, N.J., pp. 175-186 (2004)). For in vitro binding assays, p53 GST fusion proteins bound to the GSH Sepharose were incubated with cell lysates. After washing, the bound proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies.

Protein Stability Assay



[0037] Cycloheximide was purchased from Sigma. For protein turnover analysis, cycloheximide was added to cell culture medium at the final concentration of 0.1 mg/mL, and cells were harvested at the indicated time points. Cells were then lysed, and cell lysates were resolved by SDS-PAGE and analyzed by Western blot.

Ubiquitination of p53 in vivo and in vitro



[0038] The ubiquitination levels of p53 were detected essentially as described elsewhere (Li et al., Nature, 416:648-653 (2002)). For the in vivo deubiquitination assay, H1299 cells were transfected with FLAG-p53 or in combination with different expression vectors as indicated. After 48 hours, cells were treated for 4 hour with a proteasome inhibitor MG132 (50 µM) before being harvested. The cell extracts were subjected to immunoprecipitation with anti-FLAG antibody and blotted with anti-p53 antibodies.

[0039] For the preparation of a large amount of ubiquitinated p53 as the substrate for the deubiquitination assay in vitro, HEK293 cells were transfected together with the FLAG-p53, pCMV-Mdm2, and HA-UB expression vectors. After treatment as described above, ubiquitinated p53 was purified from the cell extracts with anti-FLAG-affinity column in FLAG-lysis buffer (50 mM Tris-HCl pH 7.8, 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100, 0.2% Sarkosyl, 1 mM DTT, 10% glycerol and fresh proteinase inhibitors). After extensive washing with the FLAG-lysis buffer, the proteins were eluted with FLAG-peptides (Sigma). The recombinant His-USPIO and USP10CA were expressed in BL21 cells and purified on the His-tag purification column (Novagen). For the deubiquitination assay in vitro, ubiquitinated p53 protein was incubated with recombinant USP10 in a deubiquitination buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, 10 mM DTT, 5% glycerol) for 2 hours at 37°C.

Cell Fractionation



[0040] H1299 cells were transfected with the indicated constructs. Forty-eight hours later, cells were treated for 4 hours with a proteasome inhibitor, MG132 (50 µM) before being harvested. Cytoplasmic and unclear fractions were separated by using Paris Kit (Ambion).

Immunofluorescence



[0041] For the p53 translocation assay, H1299 cells were plated on glass coverslips and transfected with the indicated plasmid. Forty-eight hours after transfection, 50 µM of proteasome inhibitors (MG132) was added for 4 hours before fixation. Cells were then fixed in 4% paraformaldehyde for 10 minutes at room temperature and stained using standard protocols.

Luciferase Assay



[0042] HCT116 p53+/+ and HCT116 p53-/- cells were seeded at 8x104 cells/well on 24-well plates. The next day, cells were transfected with 200 ng of p21 reporter construct and other indicated plasmids. pRL-TK (50 ng) was included as an internal control. Luciferase assays were carried out according to the manufacturer's instructions (Dual-Luciferase Reporter Assay System; Promega). Results were normalized for expression of pRL-TK as measured by Renilla luciferase activity.

Cell Growth Assay



[0043] Cell growth was analyzed using MTS reagent (Promega) according to the manufacturer's directions. HCT116 p53+/+ and HCT116 p53-/- cells stably infected with lentivirus encoding control shRNA or USP10 shRNA (1,000 cells/well) were plated on 96-well plates and grown on 10% serum containing media. Cell proliferation was estimated after 1, 2, 3, 4, 8, and 10 days.

Colony and Soft Agar Colony-Formation Assays



[0044] The soft agar colony-formation assay was performed as described elsewhere (Shim et al., Proc. Natl. Acad. Sci. USA, 94:6658-6663 (1997)). Briefly, cells were infected with lentivirus encoding control, USP10shRNA, or USP10shRNA together with FLAG-tagged USP10. Cells were then plated in 0.3% top agarose in 35 mm dishes and cultured for two weeks. Colonies were counted at room temperature under a light microscope (ECLIPSE 80i; Nikon) using a 4x NA 0.10 objective lens (Nikon). Images were captured with a camera (SPOT 2 Megasample; Diagnostic Instruments) and processed using SPOT 4.6 software (Diagnostic Instruments). Adobe Photoshop and Illustrator were used to generate figures.

Apoptosis Assay



[0045] Cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 15 minutes. For DAPI staining, cells were stained with 50 µg/mL DAPI. The number of apoptotic cells with nuclear morphology typical of apoptosis was scored in at least 400 cells in each sample by using fluorescence microscopy. The reader was blinded to the actual groups in the fluorescence microscopy.

Tissue Microarray



[0046] The tissue array of kidney cancer samples was purchased from US Biomax (KD 2083, KD991t, KD804, KD241, KD208t). Immunohistochemical staining against USP10 (dilution 1:500) was carried out with a IHC Select® HRP/DAB kit (Cat. DAB50, Millipore). The degree of immunostainining was determined by board certified pathologists using a four-tier grading system (0 = negative, 1 = weak, 2 = moderate, and 3 = strong staining intensity) in a blinded manner.

Results


USP10 interacts with p53 and stabilizes p53



[0047] As shown in Figure 1A-B, USP10 coimmunoprecipitated with p53 in U2OS and HCT116 p53+/+ cells, but not HCT116 p53-/- cells. Reciprocal immunoprecipitation with anti-p53 also brought down USP10 in HCT116 p53+/+, but not in HCT116 p53-/- cells (Figure 1C). Unlike HAUSP, USP10 did not interact with Mdm2 (Figure 1D). These results suggest a specific interaction between USP10 and p53 in vivo. However, it is not clear whether the USP10-p53 interaction is direct. To test this, recombinant USP10 and p53 were generated and purified. Purified USP10 was able to interact with p53 under cell-free conditions, suggesting a direct interaction between USP10 and p53 (Figure 1E). Further mapping of the USP10-p53 interaction revealed that the N-terminal region (AA1-AA101), but not the enzymatic domain of USP10, is required for the interaction between USP10 and p53 (Figure 1F).

[0048] USP10 was overexpressed in cells to determine if USP 10 could function to stabilize p53. As shown in Figure 2A, overexpression of USP10 significantly increased the levels of endogenous p53 and the p53 target p21. To confirm these results, USP10 expression was knocked-down using USP10 specific shRNA. The downregulation of USP10 decreased p53 and p21 levels (Figure 2B). A second USP10 shRNA also exhibited similar effects (Figure 2B). These results indicate that USP10 can upregulate p53 levels, most likely by deubiquitinating and consequently stabilizing p53. To further confirm that USP10 affects p53 stability, control cells or cells stably expressing USP10 shRNA were treated with cycloheximide (CHX), and p53 stability was examined. p53 stability was decreased in cells stably expressing USP10 shRNA, while reconstitution with shRNA-resistant USP10 restored p53 stability (Figure 2C). These results demonstrate that USP10 stabilizes p53 in cells.

USP10 deubiquitinates p53



[0049] USP10 may function to deubiquitinate p53 to counteract the action of E3 ubiquitin ligases such as Mdm2. Indeed, as shown in Figure 2D, although overexpression of Mdm2 significantly induced the degradation of p53, coexpression of USP10 effectively rescued p53 from Mdm2-induced degradation. Whether USP10 regulates the levels of p53 ubiquitination in cells was also examined. As shown in Figure 2E, Mdm2 induced the ubiquitination of p53; however, p53 ubiquitination was significantly diminished by coexpression of USP10. On the other hand, coexpression of USP10-C488A (USP10CA), a catalytic-inactive USP10 mutant containing a mutation at the core enzymatic domain (Soncini et al., Oncogene, 20:3869-3879 (2001)), lost the ability to reverse p53 ubiquitination induced by Mdm2 (Figure 2E). Conversely, downregulation of USP10 increased p53 ubiquitination (Figure 2F). These results indicate that USP10 negatively regulates p53 ubiquitination induced by Mdm2 in cells. However, from this data alone it is not clear whether USP10's effect on p53 is direct, since it is possible that USP10 affects another protein, which in turn affects p53 ubiquitination. To directly examine the deubiquitination activity of USP10 toward p53, it was determined whether USP10 could deubiquitinate p53 in a cell free system. USP10 and USP10CA were purified from bacteria, and ubiquitinated p53 was purified from cells expressing FLAG-p53, pCMV-Mdm2, and HA-ub. USP10 and ubiquitinated p53 were then incubated in a cell-free system. As shown in Figure 2G, purified wild-type USP10, but not the catalytically inactive USP10CA, effectively deubiquitinated p53 in vitro. These results demonstrate that USP10 deubiquitinates p53 both in vitro and in vivo.

USP10 localizes in the cytoplasm and counteracts Mdm2 action



[0050] Previous studies suggest that ubiquitination of p53 by Mdm2 could induce p53 translocation from nucleus to cytoplasm (Boyd et al., Nat. Cell. Biol., 2:563-568 (2000); Geyer et al., Nat. Cell. Biol., 2:569-573 (2000); Li et al., Science, 302:1972-1975 (2003); and Stommel et al., Embo J., 18:1660-1672 (1999)). In addition, the cytoplasmic ubiquitin ligase Parc can ubiquitinate p53 and trap p53 in the cytoplasm (Nikolaev et al., Cell, 112:29-40 (2003). However, it is not clear whether the cytoplasmic p53 can be deubiquitinated and returned to the nucleus, since HAUSP is mainly localized in the nucleus and no cytoplasmic ubiquitin-specific protease against p53 has been identified. Unlike HAUSP, USP10 is predominantly localized to the cytoplasm (Figure 3A). This result suggests that USP10 is the cytoplasmic deubiquitinase for p53. Thus, it is possible that USP10 could reverse Mdm2-induced nuclear export of p53. To test this, cell fractionation experiments were performed. Expression of Mdm2 was found to induce ubiquitination and nuclear export of p53, which was reversed by USP10 coexpression (Figure 3B). To confirm this result, immunofluorescence assays were performed to detect the subcellular localization of p53. When H1299 cells were transfected with GFP-tagged p53, GFP-p53 was readily detected in the nucleus. As previously demonstrated, when cells were cotransfected with Mdm2, Mdm2 induced cytoplasmic translocation of p53 (Boyd et al., Nat. Cell. Biol., 2:563-568 (2000); Geyer et al., Nat. Cell. Biol., 2:569-573 (2000); Li et al., Science, 302:1972-1975 (2003); and Stommel et al., Embo J., 18:1660-1672 (1999)). However, coexpression of wild-type USP10, but not catalytically inactive USP10 (USP10CA), reversed Mdm2-induced cytoplasmic translocation of p53 (Figure 3C). These results demonstrate that USP10 counteracts Mdm2 by deubiquitinating p53 and inducing p53 translocation from the cytoplasm back to the nucleus. Therefore, a balance between USP10 and Mdm2 could determine p53 localization. If so, downregulation of USP10 could have a similar effect as Mdm2 overexpression. Consistent with this finding, downregulation of USP10 itself induced nuclear export of endogenous p53 (Figure 3D). Similar results were obtained using GFP-p53 (Figure 8A). These results support a role of USP10 in regulating homeostasis of p53 in cells.

USP10 regulates p53 function



[0051] USP10's effects on p53 stabilization and nuclear import raised the possibility that USP10 regulates p53-dependent transcriptional activity, cell transformation, and apoptosis. As shown in Figure 4A, overexpression of wild-type USP10, but not catalytically inactive USP10 (USP10CA), increased p21 promoter activity in HCT116 p53+/+ cells, but not in HCT116 p53-/- cells. Conversely, stable knockdown of USP10 by shRNA inhibited p21 promoter activity in HCT116 p53+/+ cells, but had little effect in HCT116 p53-/- cells (Figure 4B). These results demonstrate that USP10 regulates p53-dependent transcription activity. Furthermore, experiments were performed to test whether USP10 directly affects p53-dependent apoptosis. As shown in Figure 4C, overexpression of p53 induced apoptosis, while Mdm2 strongly reduced p53-dependent apoptosis. However, coexpression of USP10, but not catalytically inactive USP10, significantly reversed the inhibitory effect of Mdm2 on p53-mediated apoptosis. How USP10 affects cell proliferation was also investigated. As shown in Figure 4D, downregulation of USP10 increased cancer cell proliferation in p53+/+ cells, but not p53-/- cells. A similar effect was observed when cancer cells were culture in soft agar (Figure 4E). On the other hand, reconstitution of USP10 in cells with USP10 downregulation inhibited cancer cell proliferation (Figure 4E), suggesting the effect of USP10 knockdown is specific. Overall, these results demonstrate that USP10 potentiates p53 function in cells.

USP10 is upregulated and translocates to the nucleus following DNA damage and regulates p53-dependent DNA damage response



[0052] The results provided herein reveal that USP10 can regulate p53 homeostasis in unstressed cells. Since p53 plays a role in DNA damage response and becomes stabilized following DNA damage, it was examined whether USP10 is involved in p53 stabilization after DNA damage. Interestingly, downregulation of USP10 significantly decreased p53 stabilization and the expression of p53 target genes p21 and Bax after DNA damage (Figure 5A), suggesting that USP10 also regulates p53 stabilization after DNA damage. Furthermore, it was observed that the expression of USP10 itself was increased after DNA damage. These results can be rather surprising, since most DNA damage signaling is thought to occur in the nucleus. How does USP10, which is located in the cytoplasm, affect p53 stabilization during DNA damage response? It is possible that p53 is still actively exported out of the nucleus and gets degraded in the cytoplasm during DNA damage response, although there is lack of evidence to support this. Alternatively, USP10 could translocate into the nucleus to participate in DNA damage response. Indeed, USP10 also localized in the nucleus following DNA damage as determined by immunofluorescence (Figure 5B). To confirm the translocation of USP10, cell fractionation assays were performed. As shown in Figure 5C, increased amounts of USP10 were detected in the nucleus following DNA damage, confirming a DNA damage-induced translocation of USP10 into the nucleus.

[0053] Since USP10 regulates p53 stabilization following DNA damage, whether USP10 is required for p53-dependent function during DNA damage response was examined. As shown in Figure 5D, downregulation of USP10 inhibited IR-induced apoptosis in HCT116 p53+/+ cells. The IR-induced apoptosis in HCT116 p53-/- cells was blunted, however, downregulation of USP 10 did not have a further effect. Furthermore, knockdown of USP10 in HCT116 p53+/+ cells resulted in defective DNA damage-induced G1 arrest (Figure 5E). These results are consistent with decreased Bax and p21 expression in cells with USP10 downregulation (Figure 5A), and suggest that USP10 is required for p53 activation following DNA damage.

USP10 phosphorylation by ATM is required for its stabilization and translocation following DNA damage



[0054] The following experiments were performed to determine the molecular mechanisms that regulate USP10 upregulation and translocation. Initial experiments indicated that unlike p21, the upregulation of USP10 occurred without any change in USP10 mRNA (Figure 6A), suggesting it is not regulated at the transcriptional level, and might be regulated at the posttranslational levels. To examine whether USP10 polypeptides become stabilized, cells were irradiated, and cells were treated with cycloheximide. As shown in Figure 6B, USP10 became more stable in irradiated cells, suggesting USP10 accumulation after DNA damage is due to increased stability.

[0055] Phosphorylation is a major posttranslational modification of the DNA damage response pathway, and it has been shown to enhance protein stability and activity. For example, p53 is phosphorylated at Ser20 by the checkpoint kinase Chk2 after IR, which results in p53's dissociation from Mdm2 and its subsequent stabilization (Chehab et al., Genes Dev., 14:278-288 (2000); Hirao et al., Science, 287:1824-1827 (2000); and Shieh et al., Genes Dev., 14:289-300 (2000)). ATM can also directly phosphorylate p53 at Ser15, so regulating p53 transcriptional activity and localization (Canman et al., Science, 281:1677-1679 (1998); Siliciano et al., Genes Dev., 11:3471-3481 (1997); and Zhang and Xiong, Science, 292:1910-1915 (2001)). Therefore, it was examined whether USP10 is phosphorylated following DNA damage, which might be responsible for its stabilization and localization. As shown in Figure 6C, following IR, UV, or etoposide treatment, USP10 became phosphorylated at SQ/TQ motifs (USP10 polypeptide levels were equalized to specifically examine USP10 phosphorylation in experiments of Fig 6C-E). The SQ/TQ motifs are consensus phosphorylation sites for PI3-kinase like kinases (PIKKS), such as ATM, ATR, and DNA-PK (Abraham, Genes Dev., 15:2177-2196 (2001)), the major upstream kinases of the DNA damage response pathway. Experiments were performed to determine whether PIKKs are required for USP10 phosphorylation using the pan-PIKK inhibitor caffeine (Sarkaria et al., Cancer Res., 59:4375-4382 (1999)). As shown in Figure 6D, caffeine inhibited USP10 phosphorylation after DNA damage. In addition, a specific ATM inhibitor KU55933 (Hickson et al., Cancer Res., 64:9152-9159 (2004)) also inhibited USP10 phosphorylation after IR. These results suggest that PIKKS, likely ATM, regulate USP10 phosphorylation after DNA damage. The role of ATM in USP10 phosphorylation was further confirmed using ATM+/+ or ATM-/- cells. As shown in Figure 6E, USP10 failed to be phosphorylated at the SQ/TQ motifs in ATM-/- cells. Furthermore, USP10 levels did not increase following DNA damage in ATM-/- cells (Figure 6F). These results indicate that USP10 is phosphorylated by ATM following DNA damage, which might contribute to its stabilization.

[0056] Experiments were performed to determine the ATM phosphorylation sites of USP10. ATM specifically phosphorylates SQ/TQ motifs, of which there are two candidate sites in USP10: T42Q and S337Q. Mutation at either T42 or S337 partially affects USP10 stabilization, and mutating both T42 and S337 (USP10 2SA) abolished USP10 stabilization following DNA damage (Figure 6G). Mutation of both T42 and S337 (USP10 2SA) also abolished USP10 phosphorylation by ATM (Figure 6H). In addition, the USP10 2SA mutant failed to translocate into the nucleus following DNA damage (Figure 6I and Figure 8B). These results indicate that ATM-mediated phosphorylation of USP10 is required for USP10 translocation and stabilization.

[0057] The functional significance of USP10 phosphorylation by ATM was examined. HCT116 cells stably expressing USP10 shRNA were reconstituted with shRNA-resistant wild-type USP10 or USP10 2SA. As shown in Figure 6J, cells expressing the USP10 2SA mutant exhibited defective p53 stabilization and poor induction of Bax and p21 following DNA damage. In addition, reconstitution with wild-type USP10, but not the USP10 2SA mutant, restored DNA damage-induced apoptosis (Figure 6K). These results establish the role of USP10 phosphorylation in p53 activation following DNA damage.

USP10 is downregulated in renal cell carcinoma



[0058] Since p53 is a tumor suppressor that regulates cell proliferation and USP10 potentiates p53 function by deubiquitinating p53, it is possible that USP10 also acts as a tumor suppressor. The results shown in Figures 4D and E demonstrate USP10's ability to inhibit cancer cell proliferation and lend support to the hypothesis that USP10 functions as a tumor suppressor in vivo. To further test this hypothesis, the expression of USP10 in a panel of renal cell carcinoma (RCC) cell lines was examined. RCC was selected to study USP10 expression because a very low percentage of RCC cases has been found to have p53 mutations ((Soussi et al., Hum. Mutat., 15:105-113 (2000)). See, also, the p53 database at the International Agency for Research on Cancer. Given the function of p53 in tumor suppression, it is possible that the p53 pathway is compromised in RCC through other mechanisms, such as the downregulation of USP10. Indeed, USP10 expression was found to be significantly decreased in several RCC cell lines including A498, Caki-1, and Caki-2 cells, all of which contain wild-type p53 (Figure 7A). p53 expression was also lower in these cells than that of normal renal cells. However, in RCC cell lines with mutant p53, USP10 levels were increased. USP10 levels were also decreased in a majority of fresh frozen RCC tissues compared to corresponding normal tissues (Figure 7B). The RCC samples with USP10 downregulation all contained wild-type p53 gene (T1-T9), although p53 levels were decreased. These results suggest that downregulation of USP10 might be an alternative way to suppress p53 activity in RCC. Interestingly, similar as RCC cell lines, USP10 was overexpressed in some RCC tissues, and these tissues contained mutant p53 (T10, T11). These results suggest that increased USP10 levels in a mutant p53 background might be beneficial to tumor growth.

[0059] The expression of USP10 was further examined using RCC tissue microarray. The staining of USP10 was scored from 0-3, with a score of 0-1 being negative and a score of 2-3 being positive. Representative staining and scores were shown in Figure 7C. Strikingly, close to 90% of clear cell carcinoma exhibited negative staining of USP10. About 50% of chromophobe and 20% of papillary RCC exhibited negative USP10 staining. These results suggest that USP10 is downregulated in RCC cases, especially clear cell carcinoma.

[0060] To confirm the role of USP10 in tumor suppression, USP10 was reconstituted in RCC cells with USP10 downregulation, and tumor cell growth was examined using soft agar assay. Reconstitution of USP10 in CAKI-1 and CAKI-2 clear cell carcinoma cell lines, which contain wild-type p53, restored p53 expression and increased p21 expression (Figure 8C). Furthermore, cell proliferation was inhibited with USP10 reconstitution (Figure 7D). These results are consistent with the hypothesis that USP10 functions as a tumor suppressor by stabilizing p53.

[0061] USP10 is overexpressed in RCC cell lines and tissues with mutant p53, correlating with increased p53 levels. This is consistent with a phenomena that mutant p53 is often overexpressed in many cancers. Since mutant p53 is often dominant and displays gain of function, increased p53 levels could be advantageous to cancer. In contrast to cells with wild-type p53, increased expression of USP10 in mutant p53 background could be beneficial to cancer cell proliferation. Indeed, increased expression of USP10 in 786-O cells, which contain mutant p53, resulted in increased cell proliferation, while downregulation of USP10 inhibited cell proliferation (Figure 7E and Figure 8C). These results suggest that USP10 regulates p53 and cancer cell proliferation in a context-dependent manner.

[0062] The expression of USP10 in breast and pancreatic cancer cell lines was examined. As shown in Figure 16A-B, USP10 was downregulated in a subset of breast and pancreatic cancer cell lines. In addition, USP10 expression was lost in many pancreatic cancer tissues (Figure 16C). These results further confirm that USP10 might function as a tumor suppressor in multiple cancers.

[0063] In summary, the results provided herein indicate that in unstressed cells, USP10 localizes in the cytoplasm and regulates p53 homeostasis. Following DNA damage, a fraction of USP10 translocalizes to the nucleus and contributes to p53 activation (Figure 7F). USP10, through its regulation of p53, plays a role in tumor suppression.

Example 2 - Inhibiting USP10 polypeptide activity



[0064] Ubiquitin-AMC (Ub-AMC; BIOMOL), which is a fluorogenic substrate for a wide range of deubiquitinylating enzymes (Dang et al., Biochemistry, 37:1868 (1998)), was used as a substrate of USP10 polypeptides to demonstrate that the deubiquitination of Ub-AMC by USP10 polypeptides is dose dependent. Briefly, the amount of Ub-AMC deubiquitination in vitro increased as the concentration of USP10 polypeptides increased (Figure 17A).

[0065] N-ethylmaleimide (1 mM), Z-phe-ala fluoromethyl ketone (80 µM), antipain dihydrochloride (10 µg/mL), E-64 (10 µM), chymostatia (100 µM), phenylmethanesulfonyl fluoride (40 µM), E-64d (0.5 µM), and cystatin (36 µg/mL) were tested for the ability to inhibit USP10 polypeptide activity using USP10 polypeptide (4 µg/mL) and Ub-AMC (300 nmol/L). Briefly, both enzyme (USP10 polypeptide) and substrate (Ub-AMC) were freshly prepared in USP10 reaction buffer (50 mmol/L Tris-HCl (pH 7.6), 0.5 mmol/L EDTA, 5 mmol/L DTT, 0.01% Triton X-100, and 0.05 mg/mL serum albumin) for each run. Each well (except substrate control wells) in a typical assay contained 4 µg/mL of USP10, the compound, or 2% DMSO. The wells were incubated for 30 minutes to attain equilibrium, and the enzymatic reaction was then initiated by adding the substrate (300 nmol/L of Ub-AMC). The reaction mixture was incubated at room temperature for 2 hours, and the reaction was stopped by adding 250 mmol/L acetic acid.

[0066] The deubiquitinating activity of USP10 polypeptides was significantly inhibited (p<0.01) by N-ethylmaleimide (1 mM), Z-phe-ala fluoromethyl ketone (80 µM), and antipain dihydrochloride (10 µg/mL) as compared to incubation with DMSO (Figure 17B).

Example 3 - G3BP1 polypeptides inhibit USP10 polypeptide activity



[0067] Co-immunoprecipitation assays were performed as follows. Cells were lysed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing 50 mM β-glycerophosphate, 10 mM NaF, and 1 mg/mL each of pepstatin A and aprotinin. Whole cell lysates obtained by centrifugation were incubated with 2µg of antibody and protein A or protein G Sepharose beads (Amersham Biosciences) for 2 hours at 4°C. The immunocomplexes were then washed with NETN buffer three times and separated by SDS-PAGE. Immunoblotting was performed following standard procedures.

[0068] Cell growth assays were performed as follows. Cell growth was analyzed using MTS reagent (Promega) according to the manufacturer's directions. HCT116 cells stably infected with lentivirus encoding control shRNA or shRNA designed to reduce USP10 polypeptide expression (1,000 cells/well) were transfected with indicated constructs. After 24 hours, the cells were plated on 96-well plates and grown on 10% serum containing media. Cell proliferation was estimated after 1, 2, 3, 4 and 5 days.

[0069] HCT116 cells were harvested and lysed. The resulting cell lysates were subjected to immunoprecipitation with anti-USP10 polypeptide antibody and immunoblotted with anti-G3BP1 polypeptide antibodies or anti-USP10 polypeptide antibodies (Figure 18). These results demonstrate that G3BP1 polypeptides interact with USP10 polypeptide in vivo. In addition, experiments using full length and fragments of full length USP10 polypeptides indicated that both G3BP1 polypeptides and p53 polypeptides interact with the N-terminal region (e.g., 1-100 amino acids) of USP10 polypeptides (Figure 19A).

[0070] In one experiment, HCT116 cells were treated with MG132 for 4 hours and were depleted of G3BP1 polypeptide expression using shRNA having the following sequence: 5'-ATGTTTCATTCATTGGAAT-3' (SEQ ID NO:12). MG132 is a specific, potent, reversible, and cell-permeable proteasome inhibitor. In another experiment, HCT116 cells transfected with a vector designed to express a FLAG-G3BP1 polypeptide were treated with MG132 for 4 hours. In both cases, the cells were lysed, and cell lysates were subjected to immunoprecipitation with anti-p53 polypeptide antibodies and immunoblotted with anti-USP10 polypeptide antibodies, anti-p53 polypeptide antibodies, anti-G3BP1 antibodies, and/or anti-FLAG antibodies.

[0071] G3BP1 polypeptides competed with p53 polypeptides for USP10 polypeptide binding (Figures 19B and 19C). Depletion of G3BP1 polypeptide expression by shRNA significantly increased the binding between USP10 polypeptides and p53 polypeptides (Figure 19B). Over-expression of G3BP1 polypeptides reduced the binding between USP10 polypeptides and p53 polypeptides (Figure 19C). These results indicate that G3BP1 polypeptides compete with p53 polypeptides for USP10 polypeptide binding.

[0072] In another experiment, HCT116 cells stably transfected with either a control construct or an shRNA construct designed to reduce USP10 polypeptide expression were transfected with an empty vector, a vector designed to over-express FLAG-tagged G3BP1 polypeptides, or a vector designed to over-express FLAG-tagged G3BP2 polypeptides. The shRNA designed to reduce USP10 polypeptide expression had the following sequence: 5'-GCCTCTCTTTAGTGGCTCTTT-3' (SEQ ID NO:13). 48 hours later, the cells were lysed, and cell lysates were blotted with anti-FLAG antibodies, anti-USP10 polypeptide antibodies, anti-p53 polypeptide antibodies, or anti-β-actin antibodies. Overexpression of G3BP1 polypeptides, but not G3BP2 polypeptides, decreased the level of p53 polypeptides (Figure 20). Overexpression of G3BP1 polypeptides did not change the level of p53 polypeptides in cells depleted of USP10 polypeptides (Figure 20). These results indicate that G3BP1 polypeptides regulate p53 polypeptide through USP10 polypeptides.

[0073] In another experiment, HCT116 cells stably expressing either control construct or an shRNA construct designed to reduce expression of USP10 polypeptides (USP10shRNA) were transfected with a control vector or a FLAG-G3BP1 vector. 24 hours later, the cells were plated, and cell growth was measured by MTS assay at days 1, 2, 3, 4, and 5. Over-expression of G3BP1 polypeptides significantly enhanced cell growth in HCT116 cells, but not in HCT116 cells with depleted USP10 polypeptides (Figure 21). These results demonstrate that G3BP1 polypeptides regulate cancer cell growth through USP10 polypeptides.

[0074] In another experiment, HCT116 cells were left untreated or were treated with 10 Gy irradiation. Two hours later, the cells were lysed. The resulting cell lysates were subjected to immunoprecipitation with an anti-USP10 polypeptide antibody and immunoblotted with anti-G3BP1 polypeptide antibodies and anti-USP10 polypeptide antibodies. X-Ray Irradiation dramatically decreased the interaction between USP10 polypeptides and G3BP1 polypeptides (Figure 22). These results demonstrate that DNA damage relieves USP10 polypeptides from G3BP1 polypeptide inhibition.

SEQUENCE LISTING



[0075] 

<110> Mayo Foundation for Medical Education and Research

<120> METHODS AND MATERIALS FOR MODULATING
DEUBIQUITINASES AND UBIQUITINATED POLYPEPTIDES

<130> 07039/0955WO1

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Claims

1. A composition for use in reducing cancer cell proliferation in a mammal having cancer cells that have a reduced level of wild-type p53 polypeptide expression,
wherein said composition comprises nucleic acid encoding a USP10 polypeptide, and
wherein said composition increases USP10 polypeptide expression within said cancer cells, thereby reducing cancer cell proliferation.
 
2. The composition for use of claim 1,
wherein said composition comprises nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:2.
 
3. A composition for use in reducing cancer cell proliferation in a mammal having cancer cells,
wherein said cancer cells express a mutant version of a p53 polypeptide, and said composition comprises an antagonist of USP10,

(i) wherein said antagonist comprises a nucleic acid having the ability to induce RNA interference against expression of said USP10 polypeptide, or

(ii) wherein said antagonist is selected from the group consisiting of N-ethylmaleimide, Z-phe-ala fluoromethyl ketone, chymostatin, E-64 (trans-Epoxysuccinyl-L-leucylamido (4-guanidino)butane, E-64d ((2S, 3S)-trans-Epoxysuccinyl-L-leuclamido-3-methylbutane ethyl ester), cystatin, and cyano- indenopyrazine, or

(iii) wherein said antagonist is the G3BP1 polypeptide,

wherein said composition decreases said USP10 polypeptide expression or activity, thereby reducing cancer cell proliferation.
 
4. The composition for use of claim 3,
wherein said USP10 polypeptide is a human USP10 polypeptide.
 
5. The composition for use of claim 3,
wherein the nucleic acid has one of the sequences set forth in SEQ ID NO: 7, 8, 9, 10, or 11.
 
6. The composition for use of claim 3,
wherein the G3BP1 polypeptide is a human G3BP1 polypeptide, a rat G3BP1 polypeptides, or a mouse G3BP1 polypeptides.
 
7. An in vitro method, an in vitro cell-based assay, an in vitro cell free assay, and/or an in vivo non-human animal model for identifying an antagonist of USP10 polypeptide mediated stabilization of p53 polypeptides,
wherein said method comprises determining if the stabilization level of a ubiquinated p53 polypeptide contacted with a USP10 polypeptide in the presence of a test agent is less than the stabilization level of said ubiquinated p53 polypeptide contacted with said USP10 polypeptide in the absence of said test agent,
wherein the presence of said stabilization level of said ubiquinated p53 polypeptide contacted with said USP10 polypeptide in the presence of said test agent that is less than said stabilization level of said ubiquinated p53 polypeptide contacted with said USP10 polypeptide in the absence of said test agent indicates that said test agent is said antagonist.
 
8. An in vitro method, an in vitro cell-based assay, an in vitro cell free assay, and/or an in vivo non-human animal model for identifying an agonist of USP10 polypeptide mediated stabilization of p53 polypeptides,
wherein said method comprises determining if the stabilization level of a ubiquinated p53 polypeptide contacted with a USP10 polypeptide in the presence of a test agent is greater than the stabilization level of said ubiquinated p53 polypeptide contacted with said USP10 polypeptide in the absence of said test agent,
wherein the presence of said stabilization level of said ubiquinated p53 polypeptide contacted with said USP10 polypeptide in the presence of said test agent that is greater than said stabilization level of said ubiquinated p53 polypeptide contacted with said USP10 polypeptide in the absence of said test agent indicates that said test agent is said agonist.
 
9. The method of claim 7 or 8,
wherein the USP10 agonists or antagonists are identified using an in vitro screen that includes using purified His-tagged USP10 polypeptide together with ubiquitin-AMC as the substrate.
 


Ansprüche

1. Zusammensetzung zur Verwendung bei der Verringerung von Krebszellproliferation bei einem Säuger mit Krebszellen, die ein herabgesetztes Wildtyp-p53-Polypeptid-Expressionsniveau aufweisen,
wobei die Zusammensetzung ein USP10-Polypeptid codierende Nukleinsäure umfasst und
wobei durch die Zusammensetzung die Expression von USP10-Polypeptid in den Krebszellen gesteigert wird, wodurch Krebszellproliferation verringert wird.
 
2. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die Zusammensetzung ein Polypeptid mit der Aminosäuresequenz gemäß SEQ ID NO:2 codierende Nukleinsäure umfasst.
 
3. Zusammensetzung zur Verwendung bei der Verringerung von Krebszellproliferation bei einem Säuger mit Krebszellen,
wobei die Krebszellen eine mutierte Version eines p53-Polypeptids exprimieren und die Zusammensetzung einen Antagonisten von USP10 umfasst,

(i) wobei der Antagonist eine Nukleinsäure mit der Fähigkeit, RNA-Interferenz gegen die Expression des USP10-Polypeptids zu induzieren, umfasst oder

(ii) wobei der Antagonist ausgewählt ist aus der Gruppe bestehend aus N-Ethylmaleimid, Z-Phe-Ala-Fluormethylketon, Chymostatin, E-64 (trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butan, E-64d ((2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutanethyl-ester), Cystatin und Cyanoindenopyrazin oder

(iii) wobei es sich bei dem Antagonisten um das G3BP1-Polypeptid handelt.


 
4. Zusammensetzung zur Verwendung nach Anspruch 3, wobei es sich bei dem USP10-Polypeptid um ein menschliches USP10-Polypeptid handelt.
 
5. Zusammensetzung zur Verwendung nach Anspruch 3, wobei die Nukleinsäure eine der Sequenzen gemäß SEQ ID NO: 7, 8, 9, 10 oder 11 aufweist.
 
6. Zusammensetzung zur Verwendung nach Anspruch 3, wobei es sich bei dem G3BP1-Polypeptid um ein menschliches G3BP1-Polypeptid, ein Ratte-G3BP1-Polypeptid oder ein Maus-G3BP1-Polypeptid handelt.
 
7. In-vitro-Verfahren, In-vitro-Test auf Zellbasis, zellfreier In-vitro-Test und/oder nichtmenschliches In-vivo-Tiermodell zur Identifizierung eines Antagonisten von durch USP10-Polypeptid vermittelter Stabilisierung von p53-Polypeptiden,
wobei das Verfahren Bestimmen, ob das Stabilisierungsniveau eines ubiquinierten p53-Polypeptids, das mit einem USP10-Polypeptid in Gegenwart einer Testsubstanz in Kontakt gebracht wurde, niedriger ist als das Stabilisierungsniveau des ubiquinierten p53-Polypeptids, das mit dem USP10-Polypeptid in Abwesenheit der Testsubstanz in Kontakt gebracht wurde, umfasst,
wobei das Vorliegen des Stabilisierungsniveaus des ubiquinierten p53-Polypeptids, das mit dem USP10-Polypeptid in Gegenwart der Testsubstanz in Kontakt gebracht wurde, das niedriger ist als das Stabilisierungsniveau des ubiquinierten p53-Polypeptids, das mit dem USP10-Polypeptid in Gegenwart der Testsubstanz in Kontakt gebracht wurde, anzeigt, dass es sich bei der Testsubstanz um den Antagonisten handelt.
 
8. In-vitro-Verfahren, In-vitro-Test auf Zellbasis, zellfreier In-vitro-Test und/oder nichtmenschliches In-vivo-Tiermodell zur Identifizierung eines Agonisten von durch USP10-Polypeptid vermittelter Stabilisierung von p53-Polypeptiden,
wobei das Verfahren Bestimmen, ob das Stabilisierungsniveau eines ubiquinierten p53-Polypeptids, das mit einem USP10-Polypeptid in Gegenwart einer Testsubstanz in Kontakt gebracht wurde, höher ist als das Stabilisierungsniveau des ubiquinierten p53-Polypeptids, das mit dem USP10-Polypeptid in Abwesenheit der Testsubstanz in Kontakt gebracht wurde, umfasst,
wobei das Vorliegen des Stabilisierungsniveaus des ubiquinierten p53-Polypeptids, das mit dem USP10-Polypeptid in Gegenwart der Testsubstanz in Kontakt gebracht wurde, das höher ist als das Stabilisierungsniveau des ubiquinierten p53-Polypeptids, das mit dem USP10-Polypeptid in Gegenwart der Testsubstanz in Kontakt gebracht wurde, anzeigt, dass es sich bei der Testsubstanz um den Agonisten handelt.
 
9. Verfahren nach Anspruch 7 oder 8,
wobei die USP10-Agonisten bzw. -Antagonisten unter Verwendung eines In-vitro-Screen-Verfahrens, das die Verwendung von aufgereinigtem USP10-Polypeptid mit His-Tag zusammen mit Ubiquitin-AMC als Substrat beinhaltet, identifiziert werden.
 


Revendications

1. Composition pour utilisation dans la réduction de la prolifération de cellules cancéreuses chez un mammifère ayant des cellules cancéreuses qui ont un taux réduit d'expression de polypeptide p53 de type sauvage, ladite composition comprenant un acide nucléique codant pour un polypeptide USP10, et
ladite composition augmentant l'expression de polypeptide USP10 dans lesdites cellules cancéreuses, de façon à réduire la prolifération de cellules cancéreuses.
 
2. Composition pour utilisation selon la revendication 1,
ladite composition comprenant un acide nucléique codant pour un polypeptide ayant la séquence d'acides aminés décrite dans SEQ ID NO: 2.
 
3. Composition pour utilisation dans la réduction de la prolifération de cellules cancéreuses chez un mammifère ayant des cellules cancéreuses,
lesdites cellules cancéreuses exprimant une version mutante d'un polypeptide p53, et ladite composition comprenant un antagoniste d'USP10,

(i) dans laquelle ledit antagoniste comprend un acide nucléique ayant la capacité d'induire une interférence par ARN contre l'expression dudit polypeptide USP10, ou

(ii) dans laquelle ledit antagoniste est choisi dans le groupe constitué des N-éthylmaléimide, Z-phe-ala-fluorométhylcétone, chymostatine, E-64 (trans-époxysuccinyl-L-leucylamido(4-guanidino)butane, E-64d (ester éthylique d'acide (2S,3S)-trans-époxysuccinyl-L-leuclamido-3-méthylbutane), cystatine et cyano-indénopyrazine, ou

(iii) dans laquelle ledit antagoniste est le polypeptide G3BP1,

ladite composition diminuant l'expression ou l'activité dudit polypeptide USP10, de façon à réduire la prolifération de cellules cancéreuses.
 
4. Composition pour utilisation selon la revendication 3,
ledit polypeptide USP10 étant un polypeptide USP10 humain.
 
5. Composition pour utilisation selon la revendication 3,
dans laquelle l'acide nucléique a une des séquences décrites dans SEQ ID NO: 7, 8, 9, 10 ou 11.
 
6. Composition pour utilisation selon la revendication 3,
dans laquelle le polypeptide G3BP1 est un polypeptide G3BP1 humain, un polypeptide G3BP1 de rat, ou un polypeptide G3BP1 de souris.
 
7. Procédé in vitro, dosage à base de cellules in vitro, dosage sans cellules in vitro et/ou modèle animal non humain in vivo pour identifier une stabilisation de polypeptides p53 médiée par un antagoniste de polypeptide USP10,
ledit procédé comprenant la détermination du fait que le niveau de stabilisation d'un polypeptide p53 ubiquitiné mis en contact avec un polypeptide USP10 en présence d'un agent d'essai est ou non inférieur au niveau de stabilisation dudit polypeptide p53 ubiquitiné mis en contact avec ledit polypeptide USP10 en l'absence dudit agent d'essai,
dans lequel la présence dudit niveau de stabilisation dudit polypeptide p53 ubiquitiné mis en contact avec ledit polypeptide USP10 en présence dudit agent d'essai qui est inférieur audit niveau de stabilisation dudit polypeptide p53 ubiquitiné mis en contact avec ledit polypeptide USP10 en l'absence dudit agent d'essai indique que ledit agent d'essai est ledit antagoniste.
 
8. Procédé in vitro, dosage à base de cellules in vitro, dosage sans cellules in vitro et/ou modèle animal non humain in vivo pour identifier une stabilisation de polypeptides p53 médiée par un agoniste de polypeptide USP10,
ledit procédé comprenant la détermination du fait que le niveau de stabilisation d'un polypeptide p53 ubiquitiné mis en contact avec un polypeptide USP10 en présence d'un agent d'essai est ou non supérieur au niveau de stabilisation dudit polypeptide p53 ubiquitiné mis en contact avec ledit polypeptide USP10 en l'absence dudit agent d'essai,
dans lequel la présence dudit niveau de stabilisation dudit polypeptide p53 ubiquitiné mis en contact avec ledit polypeptide USP10 en présence dudit agent d'essai qui est supérieur audit niveau de stabilisation dudit polypeptide p53 ubiquitiné mis en contact avec ledit polypeptide USP10 en l'absence dudit agent d'essai indique que ledit agent d'essai est ledit agoniste.
 
9. Procédé selon la revendication 7 ou 8,
dans lequel les agonistes ou antagonistes d'USP10 sont identifiés en utilisant un criblage in vitro qui comprend l'utilisation d'un polypeptide USP10 étiqueté par His purifié conjointement avec ubiquitine-AMC en tant que substrat.
 




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REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description