The Role of Activating Transcription Factor 3 (ATF3) in Chemotherapeutic Induced Cytotoxicity

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity