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BackgroundCell death induced by poly(ADP-ribose) (PAR) and mediated by apoptosis-inducing factor (AIF) is well-characterized in models of ischemic tissue injury, but their roles in cancer cell death after chemotherapy are less understood.MethodsHere we investigated the roles of PAR and AIF by RNA interference (RNAi) in MDA-MB-231 and MCF-7 breast adenocarcinoma cells after chemotherapy. Differences in effects were statistically tested by analysis-of-variance and unpaired student’s t-test.ResultsSilencing of AIF by RNAi led to decreased MDA-MB-231 and MCF-7 breast cancer cell death after chemotherapy, which demonstrates a critical role for AIF. RNAi silencing of PAR glycohydrolase (PARG), the primary enzyme that catalyzes the hydrolysis of PAR, led to increased PAR levels but decreased cell death. Further investigation into the possible role of PAR in apoptosis revealed decreased caspase-3/7/8/9 activity in PARG-null cells. Interestingly, the pharmacologic inhibition of caspase activity in PARG-silenced breast cancer cells led to increased cell death after chemotherapy, which indicates that an alternative cell death pathway is activated due to elevated PAR levels and caspase inhibition. AIF silencing in these cells led to profound protection from chemotherapy, which demonstrates that the increased cell death after PARG silencing and caspase inhibition was mediated by AIF.ConclusionsThe results show a role for AIF in breast cancer cell death after chemotherapy, the ability of PAR to regulate caspase activity, and the ability of AIF to substitute as a primary mediator of breast cancer cell death in the absence of caspases. Thus, the induction of cell death by PAR/AIF may represent a novel strategy to optimize the eradication of breast tumors by activating an alternative cell death pathway.

Apoptosis-Inducing Factor (AIF) was previously characterized as a poly(ADP-ribose) (PAR) dependent, caspase independent cell death effector in a wide spectrum of diseases or conditions that involve cell death. The objective of this study was to determine the role of PAR and AIF in cancer cell death after chemotherapy. We have previously demonstrated the presence of AIF mediated caspase-independent cell death in MDA-MB-231 breast cancer cells following treatment with the DNA alkylating agent N-methyl-N’-nitrosoguanidine (MNNG). To gain further insight into the role of AIF in cell death induced by chemotherapy, we utilized RNA interference (RNAi) to knockdown expression of AIF and PAR glycohydrolase (PARG) in MDA-MB-231 and MCF-7 human breast cancer cells. UV-C irradiated AIF-silenced cells displayed increased cell viability. Moreover FACS analysis showed that AIF knockdown successfully protected cells against apoptosis induced by MNNG and the chemotherapeutic agents epirubicin and cyclophosphamide. Interestingly, the knockdown of PARG led to decreased apoptotic cell death after treatment with MNNG. However, inhibition of caspases using Q-VD-OPh in PARG RNAi treated cells led to an increase in apoptotic cell death, which suggests that caspase dependent cell death after chemotherapy is mediated in part by PARG. However, the knockdown of AIF in these cells led to significant protection, which indicates that cell death in the absence of caspase activity in PARG knockdown cells is mediated by AIF. Taken together, the results provide a paradigm where AIF can substitute as caspase executioner in the absence of PARG and caspases in breast cancer cells. Thus, the activation of AIF mediated cell death is a promising strategy to improve the treatment of breast cancer by providing an alternative pathway for tumor eradiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2019. doi:1538-7445.AM2012-2019