HEPATOLOGY, VOL. 68, NO. 6, 2018 HEPATOBILIARY MALIGNANCIES Vacuolar Protein Sorting 33B Is a Tumor Suppressor in Hepatocarcinogenesis 1 2 2 2 1 3 1 4 Conghui Wang, * Yuqiang Cheng, * Xiuping Zhang, * Nan Li, Lin Zhang, Shengdian Wang, Xuemei Tong, Ying Xu, 4 2 1 1,5 Guo-qiang Chen, Shuqun Cheng, Xuemei Fan, * and Junling Liu * Polarity defects are frequently involved in liver diseases, such as chronic hepatitis and hepatocellular carcinoma (HCC). It was reported that vacuolar protein sorting 33B (Vps33b) plays critical roles in the maintenance of hepatocyte polarity; however, the functional roles and mechanisms of Vps33b in HCC occurrence and progression remain unknown. First of all, we showed that Vps33b is down-regulated in human and mouse liver cancer samples, and the low expression levels of Vps33b correlate with the poor prognosis of many HCC patients. Liver-specific Vps33b deficiency induces liver damage, progressive hepatitis, fibrosis, and HCC in male mice, indicating that Vps33b is a crucial contributory factor to hepatocarcinogenesis. Vps33b deficiency–caused liver damage was primarily due to the disorders of structural and functional hepatocyte polarity, which were reflected by the decreased protein levels of E-cadherin because of inaccurate location to lysosomes and polarity defects at both apical and lateral plasma membrane proteins. The results of a mechanism study revealed that Vps33b inter- acts with VPS33B-interacting protein, which is involved in polarity and apical protein restriction; vesicle-trafficking protein Sec22b; and Flotillin-1 in hepatocytes and is in charge of the normal distribution of polarity-determined proteins. Expression levels of Vps33b negatively correlated with the degree of inflammatory cell infiltration in livers from diethylnitrosamine- induced or transgenic HCC mouse models, and the inflammatory stimuli suppressed the expression of Vps33b in vitro. Conclusion: Down-regulation of Vps33b expression is a critical step for inflammation-driven HCC, and Vps33b serves as an important tumor suppressor in hepatocarcinogenesis. (Hepatology 2018;68:2239-2253). (2) epatocytes are highly polarized polygonal intracellular trafficking proteins. Hepatic polar- cells. The hepatic multipolar membrane ity is critical for supporting liver lobule structure system is a unique structure, with basal and normal physiological functions such as direc- membrane contacting liver sinusoidal endothelial tional secretion of bile acid into the bile canaliculi H (3) cells and multiple apical poles forming bile cana- and lipid transfer from sinusoidal blood. Defects (1) liculi. Establishment and maintenance of hepato- in hepatocyte polarization result in chronic hepa- cyte polarity require cell adhesion molecules, cell titis, cirrhosis, and even hepatocellular carcinoma (4,5) junctions, cytoskeleton, extracellular matrix, and (HCC). Abbreviations: ABCG, adenosine triphosphate–binding cassette subfamily G member; AFP, alpha-fetoprotein; ALP, alkaline phosphatase; ALT, alanine aminotransferase; ARC, arthrogryposis renal dysfunction cholestasis; CD, cluster of differentiation; CHEVI, class C homologs in endosome- vesicle interaction; DEN, diethylnitrosamine; DFS, disease-free survival; EMT, epithelial–mesenchymal transition; Flot1, Flotillin-1; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; H&E, hematoxylin and eosin; HEK293T, human embryonic kidney 293T; IHC, immunohistochemical; IL, interleukin; LDL, low-density lipoprotein; LDLR, LDL receptor; OS, overall survival; PBS, phosphate- buffered saline; SD, standard deviation; SRB1, scavenger receptor class B member 1; Stat3, signal transducer and activator of transcription 3; TBA, total bile acid; TC, total cholesterol; TFR, transferrin receptor; TNFα, tumor necrosis factor alpha; VIPAR, VPS33B-interacting protein involved in polarity and apical protein restriction; Vps33b, vacuolar protein sorting 33B. Received November 3, 2017; accepted April 30, 2018. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.30077/suppinfo. *These authors contributed equally to this work. Supported in part by the Program of National Natural Science Foundation of China (81525001, 81721004, and 91739302 to J.L.; 81600104, to X.F.), the National Key Basic Research Program “973 Project” (2015CB554000, to S.C.), and the Science Fund for Creative Research Groups (81521091, to S.C.). © 2018 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.30077 Potential conflict of interest: Nothing to report. 2239 WANG, CHENG, ET AL. HEPATOLOGY, December 2018 (10) HCC is a common cause of cancer-related deaths polarity. Vps33b is a member of the Sec1/Munc18 worldwide. HCC mostly develops from chronic hep- family of class C vacuolar protein sorting proteins. atitis virus infection (hepatitis B virus [HBV] or hep- Mutations of Vps33b cause arthrogryposis renal dys- atitis C virus [HCV]) or exposure to toxins (alcohol function cholestasis (ARC) syndrome, a severe autoso- or aflatoxin). HCC lesions always emerge with abnor- mal recessive multisystem disorder including neonatal mal tissue architecture, featuring polar modality with cholestatic jaundice, renal tubular acidosis, arthrogry- (11,12) deficiency or abnormal location of surface molecules. posis, and failure to thrive. Hepatocyte pigment Hepatocyte polarization and canalicular network accumulation, giant-cell transformation, hepatitis, and formation require coordinated expression of several significantly increased alkaline phosphatase (ALP) key evolutionarily conserved elements. E-cadherin is activity always present in ARC patient liver biop- an intercellular adhesion molecule that plays a piv- sies. Liver-specific Vps33b deficiency in mice caused otal role in apical junctional complex formation and abnormal location of apical membrane proteins and (6) the maintenance of hepatocyte polarity. Many loss of functional tight junction integrity, confirm- studies have shown that HBV and HCV infections ing that Vps33b is a crucial regulator of hepatocyte (10) affect hepatic polarity by suppression of E-cadherin polarity. expression through multiple mechanisms includ- Previously, Vps33b and its partner VPS33B- ing epigenetic modifications, down-regulation of interacting protein involved in polarity and apical pro- microRNA-373, and up-regulation of SNAIL expres- tein restriction (VIPAR) were proposed to comprise (7,8) sion. The down-regulation of E-cadherin and sub- class C core vacuole/endosome tethering (CORVET) sequent perturbation of polarity have a direct effect on and homotypic fusion and vacuole protein sorting promoting epithelial–mesenchymal transition (EMT) (HOPS) multiprotein complexes and to participate in and invasion in HCC. Moreover, HCV productive vesicle-mediated protein trafficking to the late endo- infection and HCV virion release occur mainly from somes/multivesicular bodies and lysosomal compart- (13,14) the basolateral domain of hepatocytes, and tight junc- ments. Recently, the Vps33b/VIPAR complex tion proteins claudin 1 and occludin were found to be was proposed as the class C homologs in endo- (9) important for HCV entry. Therefore, the polarity of some-vesicle interaction (CHEVI) complex, which hepatocytes is a key regulation factor for HCC malig- acts as a tethering complex in endosomal trafficking nant progression. in mammalian cells. A recent study suggests that the Recently, the intracellular trafficking protein vacu- Vps33b/VIPAR complex interacts with RAB11A olar protein sorting 33 homologue B (Vps33b) was to regulate membrane trafficking of recycling endo- (10) shown to play roles in the maintenance of hepatic somes, thereby maintaining hepatocyte polarity. ARTICLE INFORMATION: 1 From the Department of Biochemistry and Molecular Cell Biology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese 2 Ministry of Education, Shanghai Jiao Tong University School of Medicine, and Department of Hepatic Surgery VI, Eastern Hepatobiliary 3 Surgery Hospital, Second Military Medical University, Shanghai, China; Key Laboratory of Infection and Immunity, Institute of 4 Biophysics, University of Chinese Academy of Sciences, Beijing, China; Department of Pathophysiology, Key Laboratory of Cell 5 Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine; Collaborative Innovation Center of Hematology, Shanghai Jiao Tong University School of Medicine, Shanghai, China. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Junling Liu, Ph.D., or Xuemei Fan, Ph.D. Shuqun Cheng, M.D., Ph.D. Department of Biochemistry and Molecular Cell Biology Department of Hepatic Surgery VI, Eastern Hepatobiliary Surgery Shanghai Jiao Tong University School of Medicine Hospital 280 South Chongqing Road Second Military Medical University Huangpu District, Shanghai, 200025, China 225 Changhai Road E-mail: [email protected] or [email protected] Yangpu District, Shanghai, 200438, China or E-mail: [email protected] 2240 HEPATOLOGY, Vol. 68, No. 6, 2018 WANG, CHENG, ET AL. The Vps33b/VIPAR/RAB11A complex plays a key MICE role in sustaining liver physiological structure and f/f (15) function. Our previous studies showed that Vps33b Vps33b mice with loxP sites flanking Vps33b could also associate with VIPAR, Sec22b, GDI2, exons 2 and 3 in a C57BL/6 genetic background Flotillin-1 (Flot1), and RAB11A to regulate hemato- were bred with albumin-Cre mice