nutlin-3a and Ovarian-Neoplasms

Ovarian cancer is the fifth leading cause of cancer-related female deaths. Due to serious side effects, relapse and resistance to standard chemotherapy, better and more targeted approaches are required. Mutation of the TP53 gene accounts for 50% of all human cancers. In the remaining malignancies, non-genotoxic activation of wild-type p53 by small molecule inhibition of the MDM2-p53 binding interaction is a promising therapeutic strategy. Proof of concept was established with the cis-imidazoline Nutlin-3, leading to the development of RG7388 and other compounds currently in early phase clinical trials. This preclinical study evaluated the effect of Nutlin-3 and RG7388 as single agents and in combination with cisplatin in a panel of ovarian cancer cell lines. Median-drug-effect analysis showed Nutlin-3 or RG7388 combination with cisplatin was additive to, or synergistic in a p53-dependent manner, resulting in increased p53 activation, cell cycle arrest and apoptosis, associated with increased p21WAF1 protein and/or caspase-3/7 activity compared to cisplatin alone. Although MDM2 inhibition activated the expression of p53-dependent DNA repair genes, the growth inhibitory and pro-apoptotic effects of p53 dominated the response. These data indicate that combination treatment with MDM2 inhibitors and cisplatin has synergistic potential for the treatment of ovarian cancer, dependent on cell genotype.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Survival; Cisplatin; DNA Repair; Female; Gene Expression Regulation, Neoplastic; Genotype; Humans; Imidazoles; Ovarian Neoplasms; para-Aminobenzoates; Piperazines; Proto-Oncogene Proteins c-mdm2; Pyrrolidines; Tumor Suppressor Protein p53

The Mdm2 oncogene is a negative regulator of the p53 tumor suppressor and recently identified inhibitor of DNA break repair. Nutlin-3 is a small-molecule inhibitor of Mdm2-p53 interaction that can induce apoptosis in cancer cells through activation of p53. Although this is a promising therapy for those cancers with wild-type p53, half of all human cancers have inactivated p53. Here, we reveal that a previously unappreciated effect of Nutlin is inhibition of DNA break repair, stemming from its ability to increase Mdm2 protein levels. The Nutlin-induced increase in Mdm2 inhibited DNA double-strand break repair and prolonged DNA damage response signaling independent of p53. Mechanistically, this effect of Nutlin required Mdm2 and acted through Nbs1 of the Mre11-Rad50-Nbs1 DNA repair complex. In ovarian cancer cells, where >90% have inactivated p53, Nutlin combined with the genotoxic agents, cisplatin or etoposide, had a cooperative lethal effect resulting in increased DNA damage and apoptosis. Therefore, these data demonstrate an unexpected consequence of pharmacologically increasing Mdm2 levels that when used in combination with genotoxic agents induces synthetic lethality in ovarian cancer cells, and likely other malignant cell types, that have inactivated p53.. Data reveal a therapeutically beneficial effect of pharmacologically increasing Mdm2 levels combined with chemotherapeutic agents for malignancies that have lost functional p53.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cisplatin; Comet Assay; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; Etoposide; Female; Fibroblasts; HEK293 Cells; Humans; Imidazoles; Mice; Mice, Transgenic; Mutagens; Ovarian Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Evolutionary Action analyses of The Cancer Gene Atlas data sets show that many specific p53 missense and gain-of-function mutations are selectively overrepresented and functional in high-grade serous ovarian cancer (HGSC). As homozygous alleles, p53 mutants are differentially associated with specific loss of heterozygosity (R273; chromosome 17); copy number variation (R175H; chromosome 9); and up-stream, cancer-related regulatory pathways. The expression of immune-related cytokines was selectively related to p53 status, showing for the first time that specific p53 mutants impact, and are related to, the immune subtype of ovarian cancer. Although the majority (31%) of HGSCs exhibit loss of heterozygosity, a significant number (24%) maintain a wild-type (WT) allele and represent another HGSC subtype that is not well defined. Using human and mouse cell lines, we show that specific p53 mutants differentially alter endogenous WT p53 activity; target gene expression; and responses to nutlin-3a, a small molecular that activates WT p53 leading to apoptosis, providing "proof of principle" that ovarian cancer cells expressing WT and mutant alleles represent a distinct ovarian cancer subtype. We also show that siRNA knock down of endogenous p53 in cells expressing homozygous mutant alleles causes apoptosis, whereas cells expressing WT p53 (or are heterozygous for WT and mutant p53 alleles) are highly resistant. Therefore, despite different gene regulatory pathways associated with specific p53 mutants, silencing mutant p53 might be a suitable, powerful, global strategy for blocking ovarian cancer growth in those tumors that rely on mutant p53 functions for survival. Knowing p53 mutational status in HGSC should permit new strategies tailored to control this disease.

Topics: Animals; Apoptosis; Cell Survival; DNA Copy Number Variations; Female; Humans; Imidazoles; Mice; Mutation; Ovarian Neoplasms; Piperazines; RNA, Small Interfering; Tumor Cells, Cultured; Tumor Suppressor Protein p53

FoxM1 is an oncogenic Forkhead transcription factor that is overexpressed in ovarian cancer. However, the mechanisms by which FoxM1 is deregulated in ovarian cancer and the extent to which FoxM1 can be targeted in ovarian cancer have not been reported previously. In this study, we showed that MDM2 inhibitor Nutlin-3 upregulated p53 protein and downregulated FoxM1 expression in several cancer cell lines with wild type TP53 but not in cell lines with mutant TP53. FoxM1 downregulation was partially blocked by cycloheximide or actinomycin D, and pulse-chase studies indicate Nutlin-3 enhances FoxM1 mRNA decay. Knockdown of p53 using shRNAs abrogated the FoxM1 downregulation by Nutlin-3, indicating a p53-dependent mechanism. FoxM1 inhibitor, thiostrepton, induces apoptosis in cancer cell lines and enhances sensitivity to cisplatin in these cells. Thiostrepton downregulates FoxM1 expression in several cancer cell lines and enhances sensitivity to carboplatin in vivo. Finally, FoxM1 expression is elevated in nearly all (48/49) ovarian tumors, indicating that thiostrepton target gene is highly expressed in ovarian cancer. In summary, the present study provides novel evidence that both amorphic and neomorphic mutations in TP53 contribute to FoxM1 overexpression and that FoxM1 may be targeted for therapeutic benefits in cancers.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carboplatin; Cell Line, Tumor; Drug Synergism; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; HEK293 Cells; Humans; Imidazoles; Mice; Mice, Nude; Molecular Targeted Therapy; Mutation; Ovarian Neoplasms; Piperazines; Thiostrepton; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Ovarian cancer (OVCa) is the leading cause of death from gynecological malignancies. Although treatment for advanced OVCa has improved with the introduction of taxane-platinum chemotherapy, the majority of patients will develop resistance to the treatment, leading to poor prognosis. One of the causes of chemoresistance is the reduced ability to undergo apoptosis. Cisplatin is a genotoxic drug that leads cells to apoptosis through the activation of the p53 pathway. Defective signaling in this pathway compromises p53 function, and thus cisplatin does not induce apoptosis. A new group of nongenotoxic small molecules called Nutlins have been developed to inhibit p53-Mdm2 binding, inducing apoptosis in chemoresistant tumors through the activation of the p53 pathway. The wild-type p53 cisplatin-resistant ovarian cancer cell-line A2780cis was used to test the effect of Nutlin-3a (Nut3a) on apoptosis response. The results showed that Nut3a synergized with cisplatin, inducing cell-cycle arrest in G2/M and potentiating apoptotic cell death. Increased apoptosis was also induced in wild-type TP53 primary OVCa cultures by double cisplatin-Nut3a treatment. In conclusion, Nut3a appears to sensitize chemoresistant OVCa cells to cisplatin, inducing apoptosis. As increased response was generalized in primary tumors, this cisplatin-Nut3a combination could be useful for the treatment of patients harboring wild-type TP53 who do not respond to standard chemotherapy.

Topics: Adenocarcinoma, Mucinous; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle; Cell Proliferation; Cisplatin; Cystadenocarcinoma, Serous; Drug Resistance, Neoplasm; Drug Synergism; Endometrial Neoplasms; Female; Flow Cytometry; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Neoplasm Grading; Ovarian Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Survivin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Loss of Pten in the Kras(G12D);Amhr2-Cre mutant mice leads to the transformation of ovarian surface epithelial (OSE) cells and rapid development of low-grade, invasive serous adenocarcinomas. Tumors occur with 100% penetrance and express elevated levels of wild-type tumor repressor protein 53 (TRP53). To test the functions of TRP53 in the Pten;Kras (Trp53+) mice, we disrupted the Trp53 gene yielding Pten;Kras(Trp53-) mice. By comparing morphology and gene expression profiles in the Trp53+ and Trp53- OSE cells from these mice, we document that wild-type TRP53 acts as a major promoter of OSE cell survival and differentiation: cells lacking Trp53 are transformed yet are less adherent, migratory, and invasive and exhibit a gene expression profile more like normal OSE cells. These results provide a new paradigm: wild-type TRP53 does not preferentially induce apoptotic or senescent related genes in the Pten;Kras(Trp53+) cancer cells but rather increases genes regulating DNA repair, cell cycle progression, and proliferation and decreases putative tumor suppressor genes. However, if TRP53 activity is forced higher by exposure to nutlin-3a (a mouse double minute-2 antagonist), TRP53 suppresses DNA repair genes and induces the expression of genes that control cell cycle arrest and apoptosis. Thus, in the Pten;Kras(Trp53+) mutant mouse OSE cells and likely in human TP53+ low-grade ovarian cancer cells, wild-type TRP53 controls global molecular changes that are dependent on its activation status. These results suggest that activation of TP53 may provide a promising new therapy for managing low-grade ovarian cancer and other cancers in humans in which wild-type TP53 is expressed.

Topics: Adenocarcinoma; Animals; Apoptosis; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mice; Mice, Knockout; Ovarian Neoplasms; Piperazines; Tumor Suppressor Protein p53