CHOP mediates ASPP2-induced autophagic apoptosis in hepatoma cells by releasing Beclin-1 from Bcl-2 and inducing nuclear translocation of Bcl-2

OPEN Citation: Cell Death and Disease (2014) 5, e1323; doi:10.1038/cddis.2014.276 & 2014 Macmillan Publishers Limited All rights reserved 2041-4889/14 www.nature.com/cddis CHOP mediates ASPP2-induced autophagic apoptosis in hepatoma cells by releasing Beclin-1 from Bcl-2 and inducing nuclear translocation of Bcl-2 K Liu1,2,3, Y Shi1,2,3, X Guo1,2, S Wang1,2, Y Ouyang1,2, M Hao1, D Liu1,2, L Qiao1,2, N Li1, J Zheng*,1 and D Chen*,1,2 Apoptosis-stimulating protein of p53-2 (ASPP2) induces apoptosis by promoting the expression of pro-apoptotic genes via binding to p53 or p73; however, the exact mechanisms by which ASPP2 induces apoptotic death in hepatoma cells are still unclear. Here, we show that the transient overexpression of ASPP2 induces autophagic apoptosis in hepatoma cells by promoting p53- or p73-independent C/EBP homologous protein (CHOP) expression. CHOP expression decreases the expression of Bcl-2; this change releases Beclin-1 from cytoplasmic Bcl-2-Beclin-1 complexes and allows it to initiate autophagy. However, transient overexpression of Beclin-1 can induce autophagy but not apoptosis. Our results show that ASPP2 induces the expression of damage-regulated autophagy modulator (DRAM), another critical factor that cooperates with free Beclin-1 to induce autophagic apoptosis. The effect of CHOP on the translocation and sequestration of Bcl-2 in the nucleus, which requires the binding of Bcl-2 to ASPP2, is also critical for ASPP2-induced autophagic apoptosis. Although the role of nuclear ASPP2–Bcl-2 complexes is still unclear, our results suggest that nuclear ASPP2 can prevent the translocation of the remaining Bcl-2 to the cytoplasm by binding to Bcl-2 in a CHOP-dependent manner, and this effect also contributes to Beclin-1-initiated autophagy. Thus, CHOP is critical for mediating ASPP2-induced autophagic apoptosis by decreasing Bcl-2 expression and maintaining nuclear ASPP2–Bcl-2 complexes. Our results, which define a mechanism whereby ASPP2 overexpression induces autophagic apoptosis, open a new avenue for promoting autophagy in treatments to cure hepatocellular carcinoma. Cell Death and Disease (2014) 5, e1323; doi:10.1038/cddis.2014.276; published online 17 July 2014 Apoptosis-stimulating protein of p53-2 (ASPP2) belongs to the evolutionarily conserved ASPP family. Members of this family are characterized by the presence of ankyrin repeats, an SH3 domain, and a protein-rich region.1,2 ASPP2 was previously identified as an interacting partner for several proteins, for example, Bcl-2 and p53.3 ASPP2 binds to p53 through its C terminus to stimulate the transactivation function of p53 on the promoters of pro-apoptotic genes, such as the genes encoding Bax, PIG3 and PUMA. ASPP2 is a haploinsufficient tumor suppressor; ASPP2 þ /  mice are prone to developing cancer.4 Recently, ASPP2 has been reported to bind RAS and to regulate autophagy by binding Atg5 via its N terminus.5,6 Autophagy is an evolutionarily conserved pathway for maintaining cellular homeostasis through the targeting of proteins and organelles to lysosomes for degradation.7 Upon induction, a small vesicular sac elongates and subsequently encloses a portion of the cytoplasm, forming a doublemembrane structure known as an autophagosome. The autophagosome then fuses with a lysosome to form an autolysosome in which the enclosed materials are degraded.8 The release of Beclin-1 from Bcl-2–Beclin-1 complexes is critical for inducing autophagy as free Beclin-1 is needed to interact with VPS34 and initiate autophagosome formation.9 1 Therapeutic approaches for hepatocellular carcinoma (HCC) that aim to increase the amount of autophagy have been successfully tested in vitro and in vivo.10 Furthermore, mice with heterozygous disruption of Beclin-1 have a high frequency of spontaneous HCC.11 These data suggest that maintaining and increasing autophagy is beneficial for preventing and curing HCC. The transcription factor C/EBP homologous protein (CHOP) regulates certain aspects of the cellular response to stress.12,13 CHOP has been implicated in the regulation of energy metabolism, cellular proliferation and differentiation and the expression of cell type-specific genes.14 CHOP expression is low in non-stressed conditions but is markedly increased in response to ER stress, amino-acid starvation and adipocytic differentiation.15–17 The overexpression of CHOP promotes apoptosis in several cell lines.18 CHOP reduces the expression of Bcl-2, an antiapoptotic factor that prevents the translocation of Bax to the mitochondria, which results in the induction of apoptosis.16 Bcl-2 is known to interact with Beclin-1, an initiator of autophagy.9 Autophagy is also regarded as an inducer of apoptosis, and a previous study reported that damage-regulated autophagy modulator (DRAM) expression is critical for inducing autophagic apoptosis.8 Beijing You’an Hospital, Capital Medical University, Beijing 100069, China and 2Beijing Institute of Hepatology, Beijing 100069, China *Corresponding authors: D Chen or J Zheng, Beijing Institute of Hepatology, Beijing You’an Hospital, Capital Medical University, No. 8 Xi Tou Tiao, You An Men Wai, Feng Tai, Beijing 100069, China. Tel: +86 10 83997392; Fax: +86 10 63057109; E-mail: (DC) or Tel: +86 10 83997328; Fax: +86 10 83997628; E-mail: (JZ) 3 These authors contributed equally to this work. Abbreviations: ASPP2, Apoptosis-stimulating protein of p53-2; HCC, hepatocellular carcinoma; DRAM, damage-regulated autophagy modulator; LC3, microtubuleassociated protein light chain 3; CHOP, C/EBP homologous protein; Bcl-2, B-cell lymphoma 2; Beclin-1, Bcl-2-interacting protein 1 Received 02.10.13; revised 25.5.14; accepted 29.5.14; Edited by GM Fimia ASPP2 induces autophagic apoptosis in HCC K Liu et al 2 The relationship between autophagy and apoptosis is still unclear, and the detailed mechanisms of ASPP2 function in autophagy and apoptosis have not been fully elucidated. In this study, we observed that transient overexpression of ASPP2 induces CHOP-mediated autophagic apoptosis in hepatoma cells. Results ASPP2 overexpression induces CHOP in a p53- or p73-independent manner; however, CHOP is still involved in ASPP2 overexpression-induced apoptosis in hepatoma cells. HCC cell lines (Hep1-6, HepG2, Hep3B and Huh7) were transfected with a plasmid expressing ASPP2 for 24 h. We used immunofluorescence to observe that ASPP2 overexpression induced the expression of CHOP (Figures 1a and b, Supplementary Figure 1a). ASPP2 overexpression-induced CHOP was located mostly in the nucleus (Figure 1a). The vector did not induce CHOP expression in every hepatoma cell line (data not shown). The results of immunoblotting, M30 immunofluorescence and flow cytometry showed that ASPP2 overexpression promoted apoptosis in all cell lines (Figures 1c–e and Supplementary Figures 1b and f). An MTT assay further showed that ASPP2 overexpression promoted cell death in all cell lines (Supplementary Figure 1c). (...truncated)