Abstract 1989: ASPP2 enhances DRAM induced autophagy to promote apoptosis in HepG2 cells.

Abstract

Abstract Background and Aims: ASPP2 (Apoptosis Stimulating Protein 2 of p53) binds to p53 and enhances p53 pro-apoptotic function. Autophagy may play a role in this process although the mechanisms remain to be clarified. Our aims were to explore the mechanisms by which the ASPP2-p53 pathway could promote apoptosis and autophagy in hepatocellular carcinoma cells in order to identify novel therapeutic targets in this disease. Methods: HepG2 cells were transfected with GFP-LC3 plasmid and then damaged with methyl methanesulfonate (MMS). Apoptosis was detected by cleavage of CK18 and PARP by immunocytochemical staining and Western blot. Real-time PCR and immunoblot were used to analyze changes in DRAM and LC3I/II expression. DRAM siRNA was used to inhibit DRAM function. Results: After MMS exposure for 24 hours, we identified more M30 positive cells and p85 formation in HepG2 cells compared to control cells. When HepG2 cells were transfected with a GFP-LC3 expression plasmid and then treated by MMS, we found that MMS treatment induced LC3 staining punctate structures, a marker for autophagy. Moreover, enforced expression of ASPP2 using adenovirus infection led to more autophagy and apoptosis in HepG2 after MMS treatment, under conditions where ASPP2 expressing adenovirus infection alone could not induce apoptosis and autophagy. Using real time PCR, we identified that MMS treatment could induce DRAM expression and that ASPP2 overexpression prior to MMS could further enhance DRAM expression compared to MMS alone. Immunoblot assay demonstrated that ASPP2 expression increased MMS-induced DRAM expression, LC3 II and p85 formation. Importantly, a DRAM specific siRNA blocked autophagy and apoptosis in HepG2 cells under these conditions when compared to a control siRNA. Conclusions: Our results suggest that ASPP2 can enhance MMS induced apoptosis via p53-dependent DRAM-mediated autophagy. Since DRAM is an important component of p53-mediated autophagy, our results demonstrate a new role for ASPP2 in this pathway. These findings open new avenues for investigation into the mechanisms of autophagy-based therapy in hepatocellular cancer (Supported by National Natural Science Foundation of China (30870853,30770742 and 30910103915), Beijing Municipal Natural Science Foundation (7092045 and 7101005) and Basic-Clinical Collaborative Research Fund, CCMU (12JL-L05)). Citation Format: Kai Liu, Tao Wen, Ming Ji Yin, Xin Lu Qiao, Ying Shi, Li Li, Jie Dao Liu, Chao Zhang, Ning Li, Charles Lopez, Xi De Chen. ASPP2 enhances DRAM induced autophagy to promote apoptosis in HepG2 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1989. doi:10.1158/1538-7445.AM2013-1989