AUTOPHAGY 2016, VOL. 12, NO. 8, 1355–1371 http://dx.doi.org/10.1080/15548627.2015.1034405 BASIC RESEARCH PAPER PSMD10/gankyrin induces autophagy to promote tumor progression through cytoplasmic interaction with ATG7 and nuclear transactivation of ATG7 expression Tao Luoa,b,*, Jing Fua,b,*,AnXua,b,*,BoSua, Yibing Rena,b, Ning Lia, Junjie Zhua, Xiaofang Zhaoa, Rongyang Daia, Jie Caoa, Bibo Wanga, Wenhao Qina, Jinhua Jianga, Juan Lid, Mengchao Wua, Gensheng Fenga, Yao Chena,b, and Hongyang Wanga,b,c aInternational Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China; bNational Center for Liver Cancer, Shanghai, China; cState Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; dDepartment of Nutrition and Endocrinology, Changhai Hospital, Shanghai, China ABSTRACT ARTICLE HISTORY Although autophagy is most critical for survival of cancer cells, especially in fast-growing tumors, the Received 27 May 2014 mechanism remains to be fully characterized. Herein we report that PSMD10/gankyrin promotes Revised 12 February 2015 autophagy in hepatocellular carcinoma (HCC) in response to starvation or stress through 2 complementary Accepted 25 February 2015 routes. PSMD10 was physically associated with ATG7 in the cytoplasm, and this association was enhanced KEYWORDS by initial nutrient deprivation. Subsequently, PSMD10 translocated into the nucleus and bound ATG7; autophagy; drug cooperatively with nuclear HSF1 (heat shock transcription factor 1) onto the ATG7 promoter, upregulated resistance; hepatocellular ATG7 expression in the advanced stage of starvation. Intriguingly, the type of PSMD10-mediated carcinoma; HSF1; PSMD10; autophagy was independent of the proteasome system, although PSMD10 has been believed to be an sorafenib indispensable chaperone for assembly of the 26S proteasome. A significant correlation between PSMD10 expression and ATG7 levels was detected in human HCC biopsies, and the combination of these 2 parameters is a powerful predictor of poor prognosis. The median survival of sorafenib-treated HCC patients with high expression of PSMD10 was much shorter than those with low expression of PSMD10. Furthermore, PSMD10 augmented autophagic flux to resist sorafenib or conventional chemotherapy, and inhibition of autophagy suppressed PSMD10-mediated resistance. We conclude that these results present a novel mechanism involving modulation of ATG7 by PSMD10 in sustaining autophagy, promoting HCC cell survival against starvation or chemotherapy. Targeting of PSMD10 might therefore be an attractive strategy in HCC treatment by suppressing autophagy and inducing HCC cell sensitivity to drugs. Introduction apoptotic pathway,8-10 indicating its emergence as a therapeutic Autophagy is a process in which intracellular membrane struc- target for cancer prevention. tures sequester proteins and intracellular components to PSMD10/gankyrin/p28GANK/p28 is one of the nonATPase degrade and recycle these materials in response to nutrient star- subunits of the regulatory complex of the human 26S proteasome. vation or environmental stress.1 Autophagy often takes place Interestingly, PSMD10 has been identified as an oncoprotein fre- under stress conditions, resulting in either adaptation and sur- quently overexpressed in HCC,11 and plays complicated roles in vival, or death. Increasing evidence implies the significance of HCC, which are yet to be fully elucidated. For example, PSMD10 autophagy in cancer.2 However, the role of autophagy in cancer controls the balance between cell cycle and apoptosis through deg- is controversial, especially due to its opposing functions in radation of RB1 and TP53.11,12 Overexpression of PSMD10 accel- tumor initiation and development.3 For example, deletion of erates HCC invasiveness and metastasis, serving as a valuable autophagy-related genes, such as Becn1, Atg7,andAtg5, enhan- predicting factor for recurrence and survival.13 Increasing evi- ces liver carcinogenesis,4,5 suggesting that autophagy acts as a dence indicates that the contribution of PSMD10 to HCC progres- tumor suppressor at the early stage of hepatocarcinogenesis. sion is closely associated with its resistance to chemotherapeutic Conversely, at advanced stages of tumor development, induc- agents.12,14 However, it is not clear whether autophagy contributes tion of autophagy promotes tumor progression under low-oxy- to the oncogenic role of PSMD10, especially in nutrient depriva- gen, nutrient-deprivation, and stressed conditions.6,7 Moreover, tion and drug resistance. Here we show that PSMD10 promotes accumulating data argue that autophagy could protect some autophagy through 2 complementary mechanisms in response to cancer cells against anticancer treatment by blocking the environmental stressors to promote HCC progression. CONTACT Hongyang Wang [email protected]; Yao Chen [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kaup. *These authors equally contributed to this work. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis 1356 T. LUO ET AL. Results regulation. The autophagy interaction network reveals the sig- nificance of interacting proteins during the autophagy pro- PSMD10 promotes autophagy in response to starvation or cess.19 We assessed the possibility that PSMD10 associated stress conditions with a series of ATG family members in the autophagic vacuole To assess the effect of PSMD10 on autophagy in HCC cells,15 process. In the SMMC-7721-PSMD10 stable cell lines, the we used Earle’s Balanced Salt Solution (EBSS) treatment to MYC-tagged PSMD10 was used to perform coimmunoprecipi- mimic starvation conditions. Overexpression of PSMD10 tation with the typical markers of autophagy after EBSS treat- caused increased autophagic flux as evidenced by conversion ment, including BECN1, the ATG12–ATG5 complex and from MAP1LC3B-I/LC3B-I to LC3B-II and degradation of ATG7 (Fig. S2A). We found that PSMD10 and ATG7, an E1- SQSTM1 in SMMC-7721 cells, while knockdown of PSMD10 like enzyme for LC3-conjugation systems,20,21 are present in a in HCC-LM3 cells reversed it (Fig. 1A). After blockage of the single complex (Fig. S2B). Furthermore, their interaction autophagosomal-lysosomal fusion process with chloroquine increased with the starvation stimuli (Fig. 2A). Their endoge- (CQ), overexpression of PSMD10 resulted in more accumula- nous interaction in SMMC-7721 cells was also detected in the tion of autophagosomes (Fig. 1B). To confirm the effect of precipitates (Fig. 2B). Confocal microscopy analysis revealed PSMD10 on autophagic flux, we examined GFP-LC3B and colocalization of PSMD10 with ATG7 mainly in the cytoplasm mRFP-GFP-LC3B puncta formation with a fluorescence (Fig. 2C). These exogenous and endogenous studies and results microscope. Consistently, PSMD10 promoted the autophago- therefore clearly showed the interaction of PSMD10 with somal-lysosomal fusion process (Fig. 1C, D). Furthermore, ATG7 under physiological conditions. To determine which autophagosomes increased by PSMD10 were directly visualized region of PSMD10 is required for its interaction with ATG7, a using transmission electron microscope (Fig. 1E). To confirm series of PSMD10 deletion mutants containing different regulation of autophagy by PSMD10 in vivo, we used trans- ankyrin repeats were used (Fig. S2C).18 As shown in Fig. 2D, genic overexpression of Psmd10 in mouse hepatocytes, which removing the last 3 ankyrin repeats or C-terminal tail domain was driven by the albumin (Alb) promoter (Fig. S1A). As impaired PSMD10 interaction with ATG7, implicating these shown in Fig. 1F, no effect of PSMD10 on the basal level of motifs essential for both associations. autophagy was observed in the liver tissues of littermates. How- The E1-like ubiquitin-conjugating activity of ATG7 is cru- ever, PSMD10 significantly enhanced autophagic flux upon cial for ATG7-mediated autophagy, which could be blocked by fasting (Fig. 1G). Given a possible protective role of autophagy ATG7 mutants with a Cys-to-Ser replacement at the active site D against stress conditions during tumor progression, we further (ATG7C571S), a deletion of the C terminus (ATG7C ), or com- D examined the effect of oncogenic PSMD10 on autophagy in bination of the 2 (ATG7C571S, C ) (Fig. S2D).22 However, the liver tumors from diethylnitrosamine (DEN) plus TCPOBOP- full-length ATG7, as well as the enzymatically deficient ATG7 induced HCC models. At 20 wk after DEN plus TCPOBOP- constructs, all appeared to bind to PSMD10 (Fig. 2E). The E1- treatment, 100% of littermates or Psmd10-transgenic mice like activity of ATG7 is correlated with LC3-II formation or its developed hepatocarcinomas. Interestingly, PSMD10 overex- recognition of LC3 and ATG12.22,23 In our present study, pression increased LC3B-II amounts and decreased SQSTM1 depletion of the PSMD10 motifs, indispensible for the interac- in tumors compared to the control (Fig. 1H). Thus, PSMD10 tion with ATG7, would never facilitate starvation-induced promoted autophagy in response to starvation. LC3B-II formation unlike full-length PSMD10 (Fig. 2F). More- Some evidence has shown the linkage of autophagy to over, coimmunoprecipitation experiments revealed that these cancer-associated dysregulation of TP53.16 Several stimuli of motifs are also essential for PSMD10 to regulate the interaction proteasome-mediated