Hepatology, VOL. 68, NO. 6, 2018 HEPATOBILIARY MALIGNANCIES Vacuolar 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 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 . 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 (B6.Cg-Tg[Alb- poietically derived exosome maturation and secretion cre]21Mgn/J; Jackson Laboratory) to obtain liv- (16) and megakaryocytic α-granule formation. These er-specific, Vps33b-deficient mice. Genotyping was performed by PCR using genomic DNA isolated from results suggested a distinct mechanism of Vps33b- f/f + –/– the tails of Vps33b Alb-Cre (Vps33b ) mice and lit- mediated CHEVI function in diverse cell types and f/f – f/f organelles under different physiological and patholog- termate control mice, Vps33b Alb-Cre (Vps33b ). ical conditions. Vps33b deficiency was confirmed by immunoblotting in purified hepatocytes. Animals were housed in tem- Although the functions of Vps33b in maintaining perature-controlled rooms with alternating 12-hour hepatocyte polarity have been studied, the potential periods of light and dark and had free access to water roles and mechanisms of Vps33b in HCC progression and a normal chow diet. The Shanghai Jiao Tong are not yet clear. In this study, using HCC tissue sam- University School of Medicine Animal Care and Use ples as well as a liver-specific, Vps33b-deficient mouse Committee approved the animal research. model and biochemical technology, we elucidate the roles of Vps33b in HCC progression and underlying HISTOLOGICAL AND IHC potential mechanisms. ANALYSES Liver tissues from anti–cluster of differentiation Patients and Methods 137 (CD137) monoclonal antibody-treated HBV- (20) transgenic mice and diethylnitrosamine (DEN)– PATIENTS AND DESIGN (21) treated C57BL/6 wild-type mice were kindly A total of 171 HCC patients who underwent par- provided by S.W. and X.T., respectively. tial hepatectomy in Eastern Hepatobiliary Surgery Mouse liver tissues were fixed in 4% paraformalde- Hospital (Shanghai, China) were included from June hyde for 48 hours, embedded in paraffin wax, and sec- (17) 2009 to December 2010 according to the guidelines. tioned by a semiautomated rotary microtome (Leica; Each patient was diagnosed for the first time and had RM2235). Hematoxylin and eosin (H&E) staining received no antitumor treatment before surgery. All of was used to detect the morphologic changes of livers them were followed until September 2012, with the in Vps33b knockout mice. Distributions of Vps33b, longest observation time being 39 months. Overall sur- low-density lipoprotein (LDL) receptor (LDLR), vival (OS) and disease-free survival (DFS) were defined scavenger receptor class B member 1 (SRB1), as the interval between the dates of surgery and death E-cadherin, adenosine triphosphate–binding cassette or recurrence, respectively; if recurrence was not diag- subfamily G member 5 (ABCG5) and ABCG8 in nosed, patients were censored on the date of death or livers were detected by IHC following standard pro- (18) the last follow-up. For immunohistochemical (IHC) cedures. Sections were stained with ImmPACT DAB analysis, matched pairs of peritumor tissues and tumor Peroxidase Substrate (Vector Labs, Burlingame, CA), tissues of HCC were used in the construction of a tissue counterstained with hematoxylin, and mounted. microarray. The IHC staining of Vps33b was assessed as (19) described. Then, the median of the ratio of the IHC HEPATOCYTE ISOLATION AND score between peritumor tissues and tumor tissues was used as a cutoff point to divide patient into two groups: CULTURE lower group and normal group (ratio is equal to or greater Primary mouse hepatocytes were isolated using the or lower than median, respectively). All tissue samples of collagenase IV perfusion method, based on the mod- HCC, diagnosed by a pathologist, were obtained from ified two-step collagenase perfusion for isolation of (22) Eastern Hepatobiliary Surgery Hospital according to an intact hepatocytes. Briefly, mice were anesthetized, established protocol approved by the ethics committee spiled through the portal vein, and irrigated with 2+ of Eastern Hepatobiliary Surgery Hospital. Ca -free D-hanks (GIBCO), followed by 20 mL 0.5

2241 Wang, Cheng, et al. Hepatology, December 2018 g/L collagenase IV (GIBCO) digestion for 8 minutes. after hepatectomy. Kaplan-Meier survival analysis Then, the cell suspension was mixed with precooled showed the differences in OS and DFS between the 1 × phosphate-buffered saline (PBS) for termination two groups. Patients in the lower Vps33b expression of digestion. After filtering through a 100-μm pore group revealed shorter DFS (median DFS time, 14.3 size mesh nylon filter, the suspension was centrifuged and 18.4 months, respectively; difference, 4.1 months; at 500 rpm at 4°C for 5 minutes and washed with P = 0.031) and OS (median OS time, 23.9 and 29.4 1×PBS 3 times. Finally, the pellet was resuspended months, respectively; difference, 5.5 months; P = and cultured in Hepatocyte Medium (Sciencell). 0.008) than those in the normal expression group (Fig. After 24 hours, hepatocytes were washed with pre- 1C,D). Then, we examined the prognostic value of warmed PBS and replaced with Dulbecco’s modified Vps33b expression by multivariate analysis using the Eagle’s medium containing 10% fetal bovine serum, Cox proportional hazards model. The results revealed 4.5 g/L glucose, 4 mM L-glutamine, and 100 μg/mL that Vps33b expression was an independent risk fac- penicillin/streptomycin (Hyclone), for additional time tor of poor outcome of HCC patients (Supporting to culture. Table S2). Moreover, the data in Supporting Table S1 showed STATISTICAL ANALYSIS the percentage of cases with low Vps33b expression to be 25% (4/16) and 53% (82/155) in female and male Data are shown as the mean ± standard deviation HCC groups, respectively (P = 0.034). In the low- (SD). Statistical differences between control groups Vps33b expression group, 95.3% (82/86) were male and knockout groups were analyzed by two-tailed but only 4.7% (4/86) were female. This indicated that Student t tests. Statistical analyses were performed Vps33b is more correlated with hepatocarcinogenesis using GraphPad Prism (San Diego, CA). in male patients.

Results LIVER-SPECIFIC Vps33b DEFICIENCY CAUSES HCC DOWN-REGULATION OF VPS33B EXPRESSION CORRELATES WITH Patients with ARC syndrome died in infancy, mostly in the first 6 months of life due to failure of THE PROGNOSIS OF HCC (23,24) thrive ; therefore, it is impractical to observe the To investigate Vps33b expression in HCC tis- function of Vps33b in chronic hepatic pathology. Then, sues, tissue microarray analysis from 171 patients liver-specific, Vps33b-defient mice were generated by who underwent hepatectomy was performed. The crossing mice carrying Vps33b exons 2-3 flanked by results showed that the average expression level of loxP sites with albumin promoter Cre recombinase f/f – f/f Vps33b was significantly lower in tumor tissues than transgenic mice. Vps33b Alb-Cre (Vps33b ) and f/f + –/– that in peritumoral tissues (Fig. 1A,B). According to Vps33b Alb-Cre (Vps33b ) mice were genotyped the IHC results, all 171 HCC patients were divided by PCR. Primary hepatocytes were separated from –/– f/f into two groups: normal Vps33b expression (n = 85) 4-month-old Vps33b and Vps33b livers, and west- and low Vps33b expression (n = 86). Clinical char- ern blotting results showed that Vps33b was fully –/– acteristics of the patients are shown in Supporting deleted in Vps33b mouse hepatocytes (Fig. 2A). No –/– Table S1. We found that patients with lower Vps33b significant differences were found between Vps33b f/f had significantly poor pathologic differentiation (P = and Vps33b mice born at our animal facility. 0.011), more satellite node (P = 0.004), portal vein To study the spontaneous development of potential –/– f/f tumor thrombus (P = 0.039), and higher alpha-feto- liver pathologies, Vps33b and Vps33b mice were protein (AFP) levels (P = 0.006) than those in the monitored for up to 18 months under conventional –/– f/f normal group. housing conditions. Vps33b and Vps33b mice were In view of the above-mentioned Vps33b involve- matched for age and gender. At the age of 1 month, ment in the occurrence and development of liver can- the weight, liver morphology, and relative liver weight –/– cer, we further examined whether Vps33b expression of Vps33b mice showed no difference from those f/f levels correlated with the outcome of HCC patients of Vps33b littermate control mice (Fig. 2B-D).

2242 Hepatology, Vol. 68, No. 6, 2018 Wang, Cheng, et al.

FIG. 1. Down-regulated expression of Vps33b is associated with poor prognosis of HCC. (A) IHC analysis of Vps33b in HCC samples and corresponding peritumoral tissues. (B) Vps33b staining scores in the tissue microarray. All data represent means ± SD (n = 171). *P < 0.05, **P < 0.01, and ***P < 0.001. (C,D) Kaplan-Meier analysis of correlations between DFS (C) or overall survival (D) of 171 HCC patients and Vps33b expression level. Based on Vps33b staining analysis of the liver samples, HCC patients were divided into normal-Vps33b expression (n = 85) and low-Vps33b expression (n = 86) groups. Survival of patients in the normal-Vps33b group was significantly higher than that of patients in the low-Vps33b group (P < 0.001, log-rank test).

–/– However, the weight of Vps33b mice became slightly hyperchromasia, loss of cytoplasmic staining (Fig. 2F), f/f heavier than their Vps33b counterparts as they aged and necrosis (data not shown). And most of the nod- (Fig. 2B). Vps33b-deficient mice had a significant ules were AFP-positive, which confirmed HCC for- –/– –/– hepatauxe appearance, and some Vps33b mouse liv- mation in Vps33b mice (Supporting Fig. S1A). ers presented a rough surface and massive large nod- To further examine the tumor-suppressive role of f/f –/– ules (Fig. 2C). The incidence of male mice harboring Vps33b, Vps33b and Vps33b mice were injected liver nodules was approximately 32% at the age of 12 with DEN. At the age of 5 months, AFP staining was –/– months, and over the age of 18 months the incidence already positive in Vps33b livers, while AFP stain- f/f reached 69% (Fig. 2E; Supporting Table S3). ing was negative in Vps33b mice (Supporting Fig. –/– –/– Histopathologic examination of Vps33b mouse S4C), indicating that Vps33b mice were also more liver nodules revealed many characteristics of susceptible to a chemically induced HCC develop- –/– HCC. Cytologically, Vps33b nodules showed an ment. Taken together, these results reveal a direct link increase in cell size and nuclear to cytoplasmic ratio, between Vps33b and hepatic carcinogenesis.

2243 Wang, Cheng, et al. Hepatology, December 2018

FIG. 2. Liver-specific Vps33b deficiency causes HCC. (A) Breeding strategy based on a combination of the Alb-Cre/LoxP recombination system. Mice harboring two loxP sites flanking Vps33b exons 2-3 were crossed with Alb-Cre transgenic mice to generate +/f + f/f – f/f f/f + –/– liver-specific Vps33b knockout mice. Genotyping of Vps33b Alb-Cre , Vps33b Alb-Cre (Vps33b ), and Vps33b Alb-Cre (Vps33b ) –/– f/f mice used PCR. Primary hepatocytes were separated from 4-month-old Vps33b and Vps33b livers, and western blotting results –/– –/– showed that Vps33b was fully deleted in Vps33b mouse hepatocytes. (B) Body weight curve of Vps33b mice compared with control littermates at the age of 1 month (n = 5), 4 months (n = 5), 8 months (n = 5), and 12 months (n = 5). (C) Representative liver photographs f/f –/– of Vps33b and Vps33b mice at the age of 1 month and 12 months. (D) Relative liver weight (liver weight/body weight) curve of –/– Vps33b mice compared with control littermates at the age of 1 month (n = 5), 4 months (n = 5), 8 months (n = 5), and 12 months (n f/f –/– –/– = 5). (E) HCC incidence in 12-month-old Vps33b and Vps33b mice. Eight of 25 Vps33b male mice developed HCC, whereas –/– –/– Vps33b female mice lacked HCC. (F) Liver H&E staining of 12-month-old Vps33b mice. Tumor area is framed with dashed line. Abbreviations: FRT, flippase recognition target; WT, wild type.

To evaluate the roles of Vps33b in tumor cell Vps33b DEFICIENCY CAUSES LIVER proliferation and tumor growth, Vps33b was over- DAMAGE AND PROGRESSIVE expressed in the SMMC-7721 hepatocarcinoma cell HEPATITIS AND FIBROSIS line. Cell proliferation in vitro was measured by the Cell Counting Kit-8 assay, and tumor growth was Hanley et al. recently reported that Alfp-­ observed by subcutaneously injecting the cells into Cre-driven, hepatocyte-specific Vps33b deficiency nude mice. Interestingly, no significant differences caused slight enhancement of alanine aminotrans- were found between Vps33b overexpression and ferase (ALT) activity and significant increases of control cells (Supporting Fig. S3C), suggesting that the levels of ALP activity, total bile acid (TBA), and Vps33b may serve as an important tumor suppressor total cholesterol (TC) in plasma from 14-week-old (10) in carcinogenesis of HCC instead of tumor growth. ­deficient mice due to the hepatobiliary injury.

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Alfp-Cre mice delete expression under Cre- progressively increased from the age of 4 months, and recombinase completely in the fetal liver, while Alb- an approximate 10-fold increase was observed at the Cre mice completely delete gene expression at 2 weeks age of 12 months (Fig. 3A). ALP, a test index indicat- (26) after birth. To observe the effects of Alb-Cre-driven ing possible problems in the liver, was also signifi- –/– Vps33b deficiency on postembryonic hepatocyte injury, cantly elevated in Vps33b mice compared with the the plasma levels of ALT, ALP, TBA, and TC at the littermate control group at the indicated times over 1 ages of 1 month, 4 months, 8 months, and 12 months year (Fig. 3A). Cholestasis is one of the main features –/– (27) were measured in Vps33b mice. Serum level of ALT is of Vps33b mutation, so plasma TBA levels were (25) the most sensitive index of liver injury. Though the also measured. The data presented in Fig. 3A demon- –/– levels of ALT showed no difference between Vps33b- strate that TBA increased in the Vps33b mice in an deficient and littermate control mice at a young age age-dependent manner. These results confirmed that –/– (1 month), ALT levels in Vps33b mice plasma Vps33b deficiency causes liver injury.

FIG. 3. Vps33b deficiency causes liver damage and progressive hepatitis and fibrosis. (A) Serum levels of liver function and blood f/f –/– fat (including ALP, ALT, TBA, and TC) in Vps33b and Vps33b mice from 1 month to 12 months old were monitored. Data are the means ± SD (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Representative H&E staining of healthy controls (1-month-old –/– and 12-month-old mice) and Vps33b mice (1-month-old, 4-month-old, 8-month-old, and 12-month-old mice). (C) Liver leukocyte f/f –/– counts in Vps33b and Vps33b mouse liver tissues from 1 month to 12 months (n = 6). (D) Representative Masson staining of –/– healthy controls (1-month-old and 12-month-old mice) and Vps33b mice (1-month-old, 4-month-old, 8-month-old, and 12-month- f/f –/– old mice). (E) Fibrosis area in Vps33b and Vps33b mouse liver tissues from 1 month to 12 months. Abbreviation: HPF, high-power field.

2245 Wang, Cheng, et al. Hepatology, December 2018

–/– Pathological changes were observed by H&E stain- (TFR) in the Vps33b liver. Therefore, Vps33b defi- –/– f/f ing of Vps33b and Vps33b mouse liver sections. ciency caused polarity defects at both apical and lat- The H&E staining results in Fig. 3B demonstrate eral plasma membrane. that there were no significant differences in the tis- We postulated that abnormal serum cholesterol sue structure and inflammatory cell infiltration in liv- might be caused by disruption of apical and lateral –/– f/f ers between Vps33b and Vps33b mice at the age plasma membrane transporters. LDLR and SRB1 –/– of 1 month. However, Vps33b mice showed sig- are the hepatocyte sinusoid side membrane proteins. nificant changes of liver structure and inflammatory LDLR is responsible for the endocytosis of choles- cell infiltration starting from 4 months of age (Fig. terol-rich LDL and regulation of cholesterol metab- –/– 3B,C). And livers from 12-month-old Vps33b mice olism. SRB1 can facilitate the uptake of cholesteryl showed severe inflammatory cell infiltration and dis- esters from high-density lipoproteins in the liver and tinct necrotic areas (Fig. 3B). The inflammatory cells the movement of cholesterol from peripheral tissues consisted of macrophages and neutrophils, as indi- toward the liver. ABCG8 and ABCG5 are members cated by IHC staining of CD68 and Gr1, respectively of the superfamily of adenosine triphosphate-binding (Supporting Fig. S1B,C). The positive trichrome cassette (ABC) transporters. ABCG8 and ABCG5 –/– f/f staining of liver sections from Vps33b and Vps33b are strongly expressed in the canalicular hepatocyte mice revealed remarkable fibrosis in Vps33b-deficient membrane and play an important role in hepatobi- livers at age >4 months (Fig. 3D,E). This fibrotic liary cholesterol transport and sterol efflux into the change may indicate a response to inflammation bile. First of all, the expression levels of LDLR, SRB1, caused by Vps33b defects. ABCG8, and ABCG5 were tested in 4-month-old f/f –/– Hepatitis and fibrosis were found in all of the Vps33b and Vps33b mouse hepatocytes; the results –/– 12-month-old Vps33b mice, while only 32% of in Fig. 4C and Supporting Fig. S2A,B demonstrate mice developed HCC at this age, and the incidence that Vps33b deficiency has no effects on the expres- increased as aging (Supporting Table S3). These sion levels of LDLR, SRB1, ABCG8, and ABCG5. results suggest that Vps33b deficiency in hepatocytes Then, the expression profiles of these membrane sequentially leads to liver injury, inflammation, fibro- proteins in liver biopsy specimens were measured. sis, and eventual HCC. IHC results revealed that ABCG8, normally localized f/f in the apical domain of hepatocytes in Vps33b mice, was abnormally localized in the hepatocyte cytoplasm Vps33b DEFICIENCY CAUSES –/– of 12-month-old Vps33b mice, while no obvious POLARITY DEFECTS AT BOTH –/– APICAL AND LATERAL PLASMA IHC abnormalities of ABCG5 in Vps33b mice were found (Fig. 4D). Interestingly, the immunostaining MEMBRANE PROTEINS results showed that the distribution pattern of LDLR –/– Vps33b has been shown to regulate the traffick- and SRB1 was significantly altered in Vps33b mice f/f ing of apical (canalicular) membrane proteins and, compared with Vps33b mice (Fig. 4E). (28) therefore, bile canaliculi structure and function. To These results suggested that abnormal polarity dis- confirm the role of Vps33b in bile canaliculi structure tribution of ABCG8, LDLR, and SRB1 might con- –/– and function, transmission electron micrographs of tribute to the hypercholesterolemia in Vps33b mice. f/f –/– bile canaliculi in Vps33b and Vps33b mouse livers were captured. The images in Fig. 4A show that Alb- Vps33b DEFICIENCY CAUSES Cre-driven Vps33b deficiency causes an apparent lack INACCURATE LOCATION OF of canalicular microvilli, and surprisingly there is a E-CADHERIN TO LYSOSOMES significant increase of lysosomes in Vps33b-deficient hepatocytes. Double-immunofluorescence staining Adherens junction protein E-cadherin is important was further performed to characterize the fault loca- for polarization formation of hepatocytes, and inacti- tion of specific apical and lateral membrane proteins; vation of human E-cadherin is strongly associated with (29,30) the results in Fig. 4B demonstrate the disorganized tumorigenesis and increased cancer invasiveness. localization of both canalicula-specific protein CD26 It was reported that E-cadherin decreased immuno- (15) and lateral transferring receptor histochemically in patients with ARC syndrome.

2246 Hepatology, Vol. 68, No. 6, 2018 Wang, Cheng, et al.

FIG. 4. Vps33b deficiency causes polarity defects at apical and lateral plasma membrane domains. (A) Transmission electron f/f –/– micrographs of bile canaliculi in Vps33b and Vps33b mouse liver. Solid line with an arrowhead indicates bile canaliculi, and dotted f/f –/– line with an arrowhead indicates lysosomes. (B) Immunofluorescence analysis of CD26 and TFR in Vps33b and Vps33b mouse f/f liver tissues. (C) Immunoblotting analysis of ABCG8, ABCG5, LDLR, and SRB1 in primary hepatocytes from 3 Vps33b and 3 –/– Vps33b mice, respectively. Glyceraldehyde 3-phosphate dehydrogenase served as a loading control. (D,E) IHC analysis of ABCG5, –/– ABCG8, LDLR, and SRB1 in liver samples of Vps33b mice compared with control mice. Abbreviations: DAPI, 4′,6-diamidino-2- phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

–/– Abnormal expression of E-cadherin and defective epi- 4-month-old Vps33b mouse liver. And the mRNA –/– thelial polarization in ARC syndromes with Vps33b levels of E-cadherin were not affected in Vps33b defects may link the human Vps33b complex to com- mouse liver (Fig. 5B), suggesting that Vps33b defi- (15) mon diseases such as inflammation and cancer. ciency–driven low expression of E-cadherin was not Therefore, the expression and cellular distribution of through a transcription-dependent pathway. f/f –/– E-cadherin in Vps33b and Vps33b mice liver were To further study the cause of decreased expres- –/– studied. The results presented in Fig. 5A showed that sion of E-cadherin in Vps33b mouse liver, the expression of E-cadherin dramatically decreased in cellular location of E-cadherin was analyzed by

2247 Wang, Cheng, et al. Hepatology, December 2018

FIG. 5. Vps33b deficiency causes inaccurate location of E-cadherin to lysosomes. (A) Immunoblotting analysis of E-cadherin in f/f –/– f/f –/– Vps33b and Vps33b mouse livers. Samples were from six Vps33b and six Vps33b mice. (B) Quantitative mRNA and protein –/– expression of E-cadherin in Vps33b and control littermate liver samples (n = 6). Data are presented as mean ± SD. P > 0.05. (C) –/– Images of E-cadherin (green) and 4′,6-diamidino-2-phenylindole (blue) immunofluorescence in Vps33b and control littermate liver –/– samples. (D) E-cadherin (green) and LAMP1 (red) staining in Vps33b and control littermate liver samples. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. immunofluorescence. The results in Fig. 5C show Vps33b INTERACTS WITH that E-cadherin mostly located on the lateral plasma f/f Vps16b, Sec22b, AND FLOT1 IN membrane in Vps33b mouse liver; however, most HEPATOCYTES of the E-cadherin loss was at the lateral location, with detectable E-cadherin located in the cyto- Our previous studies showed that Vps33b can asso- –/– plasm in Vps33b mouse liver. Further costaining of ciate with Vps16b, Flot1, Sec22b, GDI2, RAB11a, E-cadherin and lysosome-associated membrane pro- and RAB27a in human embryonic kidney 293T tein 1 (Lamp1), a marker of late endosome or lyso- (HEK293T) cells. Vps33b complex can regulate mega- some, showed that most of the detectable E-cadherin karyocyte α-granule formation and exosomal autocrine (31) colocalized with augmented lysosome in the cyto- signaling for hematopoiesis and leukemogenesis. –/– plasm in Vps33b mouse liver (Fig. 5D; Supporting Here, the association of Vps33b with Vps16b, Flot1, Fig. S2C). Therefore, Vps33b is a critical regulator of and Sec22b in hemagglutinin-Vps33b-transfected the posttranslational location of E-cadherin. HepG2 or primary mouse hepatocytes was confirmed Loss of E-cadherin leads to EMT induction and using coimmunoprecipitation experiments (Fig. 6A- (30) up-regulation of stem cell markers. IHC with C). Western blotting after pull-down confirmed inter- the mesenchymal marker vimentin and the stem actions of exogenous Vps33b with Vps16b, Sec22b, f/f cell marker CD44 was performed in Vps33b and and Flot1; and mouse immunoglobulin G was used –/– Vps33b livers. The results in Supporting Fig. S3A,B as a negative control (Fig. 6B). To confirm the endog- show that vimentin and CD44-positive cells appeared enous binding of Vps33b with Vps16b, Sec22b, and –/– in 8-month-old Vps33b livers, indicating that Flot1, coimmunoprecipitation of the Vps33b com- EMT-like transformation occurred and hepatocytes plexes was carried out; and the results showed that regained stem cell features. Vps33b could associate endogenously with Vps16b,

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FIG. 6. Vps33b interacts with Vps16b, Sec22b, and Flot1 in hepatocytes. (A,B) HepG2 cells were transfected with hemagglutinin- empty vector or hemagglutinin-Vps33b construct. Immunoprecipitation experiments were followed by western blot to assess the interacting proteins with exogenous Vps33b. The immunoprecipitation experiment was performed using the indicated antibodies. (C) The immunoprecipitation experiment was performed on primary mouse hepatocyte lysates to confirm the interacting proteins with endogenous Vps33b. (D) Immunoprecipitations were carried out using HEK293T cells transfected with different truncated forms of the Vps33b gene, amino acids 146-615 (59 kDa), amino acids 1-146 (16 kDa), amino acids 1-304 (33 kDa), amino acids 1-566 (62 kDa), and hemagglutinin-tagged full-length Vps33b (amino acids 1-615, 72 kDa). Western blot experiments were used to detect the associations of different domains of Vps33b with Vps16b, Sec22b, and Flot1. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, hemagglutinin; IgG, immunoglobulin G; IP, immunoprecipitate; WB, western blot.

Sec22b, and Flot1 in primary mouse hepatocytes facilitated hepatocarcinogenesis, the mechanism (Fig. 6C). Our previous study already showed that underlying down-regulation of Vps33b expression Sec22b bound to full-length Vps33b and Vps33b1 in HCC remains unclear. Persistent inflammation is (L146-A617) but not to the other truncated forms of known to promote and exacerbate malignancy. HCC Vps33b. Vps16b bound firmly to full-length Vps33b is a clear example of inflammation-related cancer as and slightly to Vps33b1 and Vps33b4. Here, we fur- >90% of HCCs arise in the context of hepatic injury ther tested the binding ability of Flot1 to bind to full- and inflammation. First of all, the expression lev- length and various truncated forms of Vps33b. We els of Vps33b and the leukocyte counts were mea- found that Flot1 only binds to full-length Vps33b but sured by IHC. The results presented in Fig. 7A-D not to the truncated forms of Vps33b (Fig. 6D). These and Supporting Fig. S4A,B show that expression of results suggested that the binding domains of Vps33b Vps33b was significantly suppressed in CD137 anti- for Vps16b, Sec22b, and Flot1 involve more than the body–treated HBV transgenic male mice or DEN/ SEC1-like domain, which was reported to be import- high fat–treated wild-type male mice. The expression ant in the regulation of soluble N-ethylmaleimide- level of Vps33b showed a negative correlation with sensitive factor attachment protein receptor (SNARE) leukocyte infiltration, indicating that liver inflamma- (32,33) complex formation. tion probably suppresses the expression of Vps33b in hepatocytes. EXPRESSION OF Vps33b IS The inflammatory cytokines interleukin-6 (IL-6) DOWN-REGULATED IN HCC and tumor necrosis factor alpha (TNFα) have been (34) MOUSE MODELS PROBABLY shown to be important for HCC development.  BY INFLAMMATORY FACTOR IL-6 and TNFα levels were much higher in tumor INTERLEUKIN-6 AND TUMOR than peritumoral tissues in HCC patients (Supporting Fig. S5A,B). To examine the mechanism of Vps33b NECROSIS FACTOR ALPHA down-regulation, primary hepatocytes were purified and Although we have identified that down-regula- treated with IL-6 and TNFα, respectively, or in combi- tion of Vps33b expression correlated with the prog- nation. The results in Fig. 7E and Supporting Fig. S5C nosis of HCC patients and that Vps33b deficiency demonstrate that IL-6 and TNFα can significantly

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FIG. 7. Proinflammatory factor IL-6 and TNFα down-regulate the expression of Vps33b. (A,C) Vps33b expression in livers of two HCC mouse models. Liver samples from (A) anti-CD137 monoclonal antibody–treated, HBV-transgenic mice and (C) DEN-induced liver cancer mice were used to analyze Vps33b expression levels. Representative photomicrographs of IHC analysis of Vps33b expression in liver cancer tissues and their paired normal tissues are shown. (B) Liver leukocyte counts and relative Vps33b expression levels in (A) (n = 6). (D) Liver leukocyte counts and relative Vps33b expression levels in (C) (n = 6). (E) Relative Vps33b mRNA expression of 50 ng/mL IL-6, 50 ng/mL TNFα, and IL-6+TNFα-treated hepatocytes (n = 4). **P < 0.01 and ***P < 0.001. (F) Schematic model of mechanisms of Vps33b in inflammation and HCC pathogenesis. Abbreviations: HPF, high-power field; tg, transgenic; WT, wild type. suppress the expression of Vps33b in mouse hepato- cytes and that the combination of IL-6 and TNFαhas Discussion synergistic inhibitory effects on Vps33b expression. HCC is one of the most deadly human malignan- The phosphorylation of signal transducer and activa- cies diagnosed worldwide. Many risk factors, includ- tor of transcription 3 (Stat3) and p65 were significantly ing hepatitis viral infection, aflatoxin B1, and alcohol enhanced in primary hepatocytes treated with IL-6 and consumption, have been identified as causes of HCC TNFα, respectivelyS31-21 can block the DNA bind- pathogenesis. Although an increasing number of ing activity of Stat3. JSH-23 is a specific inhibitor of therapeutic strategies for patients and molecules for nuclear factor kappa B’s (NFκB’s) transcription activ- predicting HCC prognosis have been developed, the ity. The data in Supporting Fig. S5D show that S31- 5-year postoperative survival rate remains poor. This 21 and JSH-23 blocked the inhibitory effect of IL-6 is mainly due to the key and molecular mecha- and TNFα on Vps33b mRNA expression, respectively. nisms involved in HCC development and progression Thus, IL-6 and TNFα may suppress Vps33b expression remaining unclear. through the transcription activity of Stat3 and NFκB. Vps33b is a member of the Sec-1 domain family. Therefore, the down-regulation of Vps33b Our previous work showed that Vps33b regulates expression in HCC was probably due to the liver von Willebrand factor-positive vesicular trafficking in inflammation. megakaryocytes and exosomal autocrine signaling to

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(31) mediate hematopoiesis and leukemogenesis. In the liver. Abnormal distribution of LDLR and SRB1 the current study, we found that the expression lev- may disturb the absorption of cholesterol from plasma els of Vps33b significantly decreased in tumor from to liver, which may also be partially responsible for (37) liver cancer mouse models and HCC patients. And abnormal serum cholesterol levels. Therefore, lower Vps33b expression was related to a poor prog- Vps33b plays very important roles in the maintenance nosis and advanced stages of HCC, which suggests of hepatic polarity and cholesterol homeostasis. that abnormality of Vps33b expression is associated Among hepatocellular carcinogenesis mechanisms, with a poor prognosis of hepatocellular carcinogen- alterations of cell adhesion junctions are important (38) esis. In our study, Alb-Cre-driven, Vps33b-deficient events. Transmembrane protein E-cadherin, the mice developed hepatitis, which progressed into major constituent of adherens junctions, is essential hepatic fibrosis and finally HCC, further indicating for the generation and maintenance of hepatocyte that Vps33b plays critical roles in the entire process of polarity. Recent studies have established links between hepatocarcinogenesis. The much higher incidence of abnormal expression of E-cadherin and hepatocellu- liver cancer in male subjects is well known. Male mice lar carcinogenesis and invasiveness. In our study, we produce far more IL-6 in response to liver injury than showed decreased expression of E-cadherin in livers –/– female mice, which has been proposed as the cause of of Vps33b mice due to mis-sorting toward lyso- their high incidence of liver cancer. In our study, the somes. This improper translocation of E-cadherin proinflammatory factor IL-6 and TNFα significantly to lysosomes is a probable partial casualty of abnor- suppressed Vps33b expression, and HCC occurred mal distribution of canalicula-specific protein CD26, only in male Vps33b-deficient mice, which reflects ABCG8, and lateral membrane protein TFR, LDLR, not only an insight into Vps33b’s involvement in and SRB1 and, therefore, abnormal hepatocyte polar- inflammation-associated HCC but also that Vps33b ity and hepatocarcinogenesis. is another factor in the gender bias of HCC. Further Vesicle-mediated protein sorting plays an import- investigation of the mechanism of the gender bias of ant role in the segregation of intracellular molecules Vps33b in HCC incidence is required. to distinct organelles. Vps33b, sharing high homol- Apical plasma membranes with their microvilli ogy with yeast class C vacuolar protein sorting (vps) and bile canaliculi network formation play import- protein, belongs to the Sec1-Munc18 family. Class (2) ant roles in hepatocyte structural polarity. A recent C vps protein usually interacts with multiple pro- study showed that Vps33b plays a role in the main- teins to form two complexes, CORVET and HOPS. tenance of the apical-basolateral polarity of hepato- Recently, it was reported that the Vps33b/Vps16b (10) (39) cytes. Consistently, we showed mislocalization of complex was proposed as the CHEVI complex. ABCG8 in hepatocyte cytoplasm instead of canaliculi In our previous study, we identified that Vps33b asso- –/– membrane in livers of Vps33b mice, confirming that ciates with Vps16b, Flot1, Sec22b, GDI2, RAB11a, (16) Vps33b plays critical roles in maintaining the normal and RAB27a in HEK293T cells. Here, we found location of apical membrane proteins and bile canalic- that Vps33b, Vps16b, Flot1, and Sec22b form a com- uli structure of hepatocytes. ABCG8 is a member of plex in HepG2 cells. Flot1 was previously reported as a the ABC transporters and forms a heterodimer with -associated integral membrane protein essen- ABCG5 to excrete cholesterol and plant sterols into tial for vesicular endocytosis and cellular cholesterol (35,36) (40) bile. Mislocalization of ABCG8 may be partially uptake. So we speculate that the Vps33b complex (37) responsible for abnormal serum cholesterol levels. interacts with Flot1 to regulate ABCG8, LDLR, and Although it has not been reported that there are polar- SRB1 trafficking and therefore cholesterol transport ity defects at the basal membrane domains in ARC in hepatocytes. Sec22b is a vesicle trafficking protein patients’ liver samples, interestingly we found that the thought to complex with SNARE and plays a role in location of hepatic lateral proteins LDLR and SRB1 the mutual targeting and fusion of Golgi and endo- is also abnormal in Vps33b-deficient mouse liver. plasmic reticulum–derived transport vesicles as well LDLR mediates the endocytosis of cholesterol-rich as in phagosomal maturation and plasma membrane (41,42) LDL by recognizing the apoprotein B100. SRB1 is expansion. Association of Vps33b with Sec22b an integral membrane protein, facilitating the uptake may play a critical role in E-cadherin trafficking in of cholesteryl esters from high-density lipoproteins in hepatocytes. These results reflect the complexity of

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