The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3) as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogen-activated protein kinase (MAPK) pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38) resulted in decreased ATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/- murine embryonic fibroblasts (MEFs) were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin's cytotoxic effects.

This study compares Breast Cancer 1 (BRCA1) and excision repair cross complementation group 1 (ERCC1) expression as predictive markers and evaluates the in vitro enhancement of platinum sensitivity using targeted agents in sporadic ovarian cancer (OC). A retrospective study was performed of advanced stage OC patients receiving platinum-based chemotherapy. BRCA1 and ERCC1 mRNA expression was determined from frozen tissue of 51 patients. Median overall survival (OS) was longer for patients with lower BRCA1 vs. higher BRCA1 (46 vs.33 months, p 5 0.03). High BRCA1 was predictive of poorer OS specifically in patients with residual disease (RD) <2 cm (p 5 0.03). There was a non-significant association for patients with lower ERCC1 and RD <2 cm in favor of improved OS and time to progression. Patients who expressed higher levels of both BRCA1 and ERCC1 mRNA had a shorter OS compared to patients with lower levels of either or both transcript (33 vs.46 months, p 5 0.04). When Cox proportional modeling was used by representing BRCA1 and ERCC1 mRNA expression as a continuous variable, both emerge as potential predictors of survival. OC cell lines were exposed to chemotherapy in combination with DNA repair pathway inhibitors and cell viability was assessed. In vitro histone deacetylase (HDAC) inhibition increased the sensitivity of A2780s/cp cells to cisplatin and carboplatin but not to taxol, coincident with a significant decrease in BRCA1 and ERCC1 expression, suggesting that this compound directly targets DNA repair. In summary, this study shows that low BRCA1 and ERCC1 expression correlate with improved survival in advanced OC and HDAC inhibition induces synergistic cytotoxicity with platinum in vitro. ' 2008 Wiley-Liss, Inc.Key words: BRCA1; ERCC1; sporadic ovarian cancer; DNA repair; predictive marker Epithelial ovarian cancer (OC) is generally diagnosed at advanced stage resulting in a 5-year survival of only 20-30%. The standard treatment of OC is surgical debulking followed by platinum and taxane chemotherapy. Although 70% of patients with advanced disease initially respond to the first-line regimen, early recurrences and platinum resistance are common obstacles in the management of this disease. Identifying reliable predictors of response and the use of novel therapeutic agents to enhance platinum efficacy are needed to improve the management of OC. Platinum resistance is multifactorial, 1 and an important mechanism of resistance is increased tolerance to platinum-DNA damage and enhanced repair of damaged DNA. In a variety of malignancies including OC, enhanced expression of the DNA repair proteins, Breast Cancer 1 (BRCA1) and excision repair cross complementation group 1 (ERCC1), have correlated with resistance to platinum. [2][3][4][5][6] In vitro models have shown that targeting the expression of both BRCA1 and ERCC1 sensitizes cells to platinum-based chemotherapeutic agents, suggesting a potential clinical application to help overcome platinum resistance in OC.Mutations in the BRCA1 tumor suppressor gene are ...

Background: The inhibition of Breast Cancer 1 (BRCA1) expression sensitizes breast and ovarian cancer cells to platinum chemotherapy. However, therapeutically relevant agents that target BRCA1 expression have not been identified. Our recent report suggested the potential of the histone deacetylase (HDAC) inhibitor, M344, to inhibit BRCA1 expression. In this study, we further evaluated the effect of M344 on BRCA1 mRNA and protein expression, as well as its effect on cisplatin-induced cytotoxicity in various breast (MCF7, T-47D and HCC1937) and ovarian (A2780s, A2780cp and OVCAR-4) cancer cell lines. Results: With the addition of M344, the platinum-sensitive breast and ovarian cancer cell lines that displayed relatively high BRCA1 protein levels demonstrated significant potentiation of cisplatin cytotoxicity in association with a reduction of BRCA1 protein. The cisplatin-resistant cell lines, T-47D and A2780s, elicited increased cytotoxicity of cisplatin with M344 and down regulation of BRCA1 protein levels. A2780s cells subjected to combination platinum and M344 treatment, demonstrated increased DNA damage as assessed by the presence of phosphorylated H2A.X foci in comparison to either treatment alone. Using Chromatin Immunoprecipitation, A2780s and MCF7 cells exposed to M344 alone and in combination with cisplatin, did not demonstrate enhanced acetylated Histone 4 at the BRCA1 promoter, suggesting an indirect effect on this promoter.

Introduction: Targeting BRCA1 expression has been shown to sensitize Breast-Cancer 1 (BRCA1)-expressing tumor cells to platinum-based chemotherapies. The improved outcome of (BRCA1)-deficient breast and ovarian cancer is believed to be linked to the impaired ability to repair chemotherapy-induced DNA damage. However, therapeutically relevant agents that target BRCA1 expression have yet to be identified. In this study, we explore the effect of histone deacetylase inhibition (HDACi) on BRCA1 expression and platinum sensitivity in a breast and ovarian cancer cell line model. Experimental procedures: BRCA1 mRNA and protein expression was determined by Q-PCR and Western blot, respectively. The efficacy of the HDACi, M344, to potentiate the cytotoxicity of platinum-based chemotherapeutics was evaluated in a range of breast and ovarian tumor cell lines using the MTT cell viability assay and confirmed by flow cytometry. The effect on DNA damage was measured by immunofluorescence staining and flow cytometry for H2A.X foci, a hallmark for the presence of DNA double strand breaks. Results: Baseline BRCA1 mRNA and protein expression was variable in three breast (MCF7, T47D and HCC1937) and three ovarian (A2780s, A2780cp a cisplatin-resistant variant and OVCAR4) cancer cell lines. Expression of BRCA1 protein decreased in response to the addition of the HDACi, M344, in the four cell lines that express detectable levels of BRCA1 by Western blot analysis. BRCA1 mRNA levels decreased with the addition of this HDACi in all the breast and ovarian cancer lines evaluated. Treatment with M344 also increased the sensitivity of these tumor cells to cisplatin and carboplatin treatment in the cell lines with high BRCA1 expression. Furthermore, expression of BRCA1 protein and mRNA decreased in cells treated with the combination of HDACi and cisplatin. A2780s cells subjected to combination platinum and HDACi treatment demonstrated increased levels of DNA damage as assessed by the presence of phosphorylated H2A.X foci in comparison to either treatment alone. Conclusion: This study supports a novel mechanism of BRCA1 targeting using an HDACi resulting in enhanced platinum sensitivity in breast and ovarian cancer cells. Further studies are needed to determine if a HDACi/platinum combination has potential clinical relevance in a subset of breast and ovarian cancer patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B174.

e22017 Background: The improved outcome of Breast-Cancer 1 (BRCA1)-deficient breast and ovarian cancer may be linked to the impaired ability to repair double strand breaks caused by DNA-damaging chemotherapy (CTX), such as platinum compounds. Therapeutically relevant agents that target BRCA1 expression to sensitize tumors to platinum have not been identified. In this study, we explore the effect of histone deacetylase inhibition (HDACi) on platinum sensitivity and BRCA1 expression in a breast and ovarian cancer cell line model. Methods: The efficacy of HDACi to potentiate the cytotoxicity of platinum-based chemotherapeutics was evaluated in a range of breast and ovarian tumor cell lines using the MTT cell viability assay and confirmed by flow cytometry. BRCA1 mRNA and protein expression was determined by Q-PCR and Western blot, respectively. The effect on DNA damage was measured by immunofluorescence staining and flow cytometry for γH2A.X foci, a hallmark for the presence of DNA double strand breaks. Results: Baseline BRCA1 expression was variable in two ovarian (A2780s, cisplatin-sensitive and A2780cp, cisplatin-resistant) and four breast cancer cell lines (MCF7, T47D, BT549 and HCC1937) with minimal and absent protein expression in BT549 and HCC1937, respectively. The addition of the HDACi, M344 increased the sensitivity of cells to cisplatin and carboplatin treatment in those cell lines with significant BRCA1 levels. Expression of BRCA1 protein decreased in response to the addition of HDACi to platinum in all cell lines. BRCA1 mRNA levels decreased with the addition of HDACi to platinum in all breast cancer lines and in A2780cp. A2780s and MCF7 cells subjected to combination platinum and HDACi treatment demonstrated increased levels of DNA damage, as assessed by the presence of phosphorylated γH2A.X foci. Conclusions: This study supports a novel mechanism of HDAC inhibition to sensitize breast and ovarian cancer cells to platinum via inhibition of the DNA repair protein BRCA1. BRCA1 expression changes may represent a novel biomarker to assess the activity of this combinational therapeutic approach in clinical evaluations of breast and ovarian cancer patients. No significant financial relationships to disclose.

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