ATF3 regulates lovastatin-induced apoptosis in squamous cell carcinoma cells

2267 Background: In this study, we examined the role of activating transcription factor 3
 (ATF3) as a regulator of lovastatin-induced apoptosis. We recently demonstrated the role of the integrated stress response (ISR) in mediating apoptotic effects of lovastatin in squamous cell carcinoma (SCC) cells. The major events of the ISR include phosphorylation of eIF2-α by several kinases, including GCN2, followed by inhibition of global protein translation and selective translational induction of ATF4 and its target genes, such as ATF3 and CHOP. ATF3, a member of the ATF/cyclic AMP response element-binding family of transcription factors has been identified as an oncogene or pro-apoptotic factor in a cell type- or stimulant-dependent manner.
 Methods: Mouse embryonic fibroblasts (MEFs) lacking ATF3, ATF4, and GCN2 or wild type MEFs were subjected to 0-50μM lovastatin treatment from 3-24hrs. As a positive control for the induction of the ISR, cells were also treated with thapsigargin and MG132. Cells were subjected to cell viability assays and protein lysates were extracted for immunoblotting assays. MCF-7 (breast carcinoma) and SCC25 (head and neck squamous cell carcinoma), a nonresponsive and responsive tumor derived cell line respectively, were subjected to lovastatin treatment for 24hrs followed by immunoblotting or real-time PCR analysis.
 Results: In ATF3-/- MEFs, lovastatin-induced apoptosis was attenuated indicating a role for ATF3 in this response. Time course experiments showed a strong correlation between the cleavage of PARP-a marker of apoptotic cell- and induction of ATF3 following lovastatin treatment. Moreover, lack of PARP cleavage in ATF3 nulls was in agreement with their attenuated response to lovastatin induced cell death. Using ATF4 -/- MEFs, we identified this gene as a prerequisite for induction of ATF3 by lovastatin. In agreement with our previous data, immunoblot assays in GCN2 -/- cells showed that induction of ATF3 by lovastatin similar to its upstream gene, ATF4 is highly dependent on the presence of the eIF2-α kinase, GCN2. Furthermore, using Western blotting and immunofluorescence microscopy, we demonstrate that induction of CHOP by lovastatin-a target of ATF3- and its nuclear localization is respectively disrupted by deletion of ATF3 in null cell lines. Interestingly, the level of ATF3 expression in MCF-7, unlike SCC25 cells, was not induced in response to lovastatin while thapsigargin treatment led to potent induction of ATF3 in MCF7 cells.
 Conclusions: In this report we demonstrate for the first time a strong pro-apoptotic role for ATF3 in lovastatin-induced apoptosis in a GNC2 dependent manner. These data suggest that targeting the ISR and particularly ATF3 expression represents a more refined therapeutic approach in lovastatin-sensitive tumor cells.