CD147 Induces Hepatocyte Polarity Loss To Promote Hepatocellular Carcinoma Development

Hepatocytes have a unique polarization arrangement in which each of two adjacent cells form an apical membrane(i.e.bile canaliculus)and sinusoidal membrane representing basolateral plasma membrane.Establishment and maintenance of hepatocyte polarity is essential for normal cell physiology and liver tissue homeostasis which requires carefully orchestrated cooperation between cell adhesion molecules,polarity protein complexes,extracellular matrix,and intracellular protein trafficking machinery.Growing evidences suggest that loss of epithelial cell polarity leads to the reduction in cell adhesion and excessive proliferation,then induces epithelial-mesenchymal transition(EMT)and finally promotes tumor progression including hepatocellular carcinoma(HCC).However,the hepatocyte polarity-associated structural and functional components involved in HCC progress are rarely recognized.CD147 is a type I transmembrane glycoprotein and highly expressed in liver tumor cells.Whether CD147 is also expressed in liver nonparenchymal cells and associated with HCC development was unknown.Our previous reports have demonstrated that a positive autoregulatory loop of transforming growth factor-?1(TGF-?1)-CD147 signaling induces EMT and promotes the carcinogenesis and metastasis of HCC.A sorting signal,leucine-252 in the C-terminal domain of CD147 is identified in Madin-Darby canine kidney cells,which dictates its basolateral localization.CD147 carrying the sorting information contributes to polarized targeting of the monocarboxylate transporter1/CD147 hetero-complex in the basolateral membrane of kidney cells.Very little is known regarding whether CD147 has the polarized localization in human hepatocytes and the pathways that involved in the hepatocyte depolarization during HCC development.In view of the above scientific issues,this study aims to identify 1)the cell expression pattern of CD147 of liver tissue in the development of HCC;2)the localization of CD147 on the hepatocyte membrane and its clinical significance;3)the influence of CD147 expression on hepatocyte polarity and its molecular mechanism about promoting HCC.The study is divided into three parts.Part I:Cell expression patterns of CD147 in mouse liver tissues during HCC progress.We used the N-diethylnitrosamine/phenobarbital(DEN/PB)two-stage protocol to explore the time-dependent development of HCC in vivo.Tumor formation was induced at the fifth month of post-DEN/PB stimulation and developed in the following administration months.Liver samples collected at month 1-12 of post-DEN/PB administration were assessed the localization of CD147 in hepatocytes,endothelial cells,hepatic stellate cells,and macrophages.Immunohistochemistry analysis showed that CD147 was upregulated in liver tumors during month 1-8 of DEN/PB induction.Expression of CD147 was positively correlated with cytokeratin 18(CK18),a hepatocyte marker(r=0.7857,P=0.0279),CD31,an endothelial cell marker(r=0.9048,P=0.0046),and CD68,a macrophage marker(r=0.7619,P=0.0368).A significant correlation was also observed between CD147 and alpha-smooth muscle actin(?-SMA,r=0.8857,P=0.0333)at DEN/PB initiation and early stage of tumor formation.Immunofluorescence and fluorescence in situ hybridization showed that CD147coexpressed with CK18,CD31,?-SMA,and CD68.Moreover,there existed positive correlations between CD147 and microvessel density(r=0.7857,P=0.0279),CD147and Ki-67(r=0.9341,P=0.0022)in the development of DEN/PB-induced HCC.Part 2:CD147 induces hepatocyte polarity loss.In HepG2 3D spheroids and mouse HCC tissues,we found CD147 co-localized with the basolateral protein markers Na~+/taurocholate cotransporting polypeptide(NTCP),or integrin?1 but not co-localized with the apical protein markers multidrug resistance-associated protein 2(MRP2)or F-actin by immunofluorescence.CD147 was basolaterally polarized in hepatocyte membrane.In liver tissues of 192 human HCC,CD147 localization was classified into polarity and non-polarity by immunohistochemistry.CD147-polarized localization positively correlated with differentiation grade in HCC(r=0.2060,P=0.004).HCC patients with CD147-polarized localization had significantly better overall survival than patients with CD147 non-polarity(P=0.021).Therefore,the polarized distribution of CD147 has clinical significance.Then,in order to clarify the effect of CD147 expression on the hepatocyte polarity,we established HCC cell lines with CD147 overexpression and CD147 knockdown and knockout.Using these cell lines,we demonstrated that CD147promoted not only the TGF-?1-mediated hepatocyte polarity loss,but also the reduction of E-cadherin and Par3.Importantly,CD147 overexpression could directly induce hepatocyte polarity loss.Part 3:The molecular mechanism of CD147-induced hepatocyte polarity loss.CD147 overexpression induced endocytosis and downregulation of E-cadherin,while Par3was down-regulated,which contributed to hepatocyte depolarization.On the contrary,E-cadherin and Par3 expressions were up-regulated after knockdown or knockout of CD147.Overexpression of CD147 induced Src activation,subsequently recruited ubiquitin ligase Hakai for E-cadherin ubiquitination and lysosomal degradation,leading to decrease of Par3 expression and?-catenin nuclear translocation.This signal transduction was initiated by competitively binding of CD147 with integrin?1 that interrupted the interaction between Arg-Gly-Asp motif of fibronectin and integrin?1.The specific antibodies targeting integrin?5 and?1 reversed the decrease of E-cadherin and Par3 levels induced by CD147 overexpression.Moreover,mRNA data analysis of HCC tissues showed the significant negative correlations between CD147 and Par3(r=-0.3230,P=0.0221),CD147 and E-cadherin(r=-0.2824,P=0.0469),and the positive correlations between CD147 and integrin?5(r=0.2957,P=0.0371),CD147 and integrin?1(r=0.3043,P=0.0317).All the above results suggest an important role of the apical-basal cell polarity machinery in HCC progression,with a focus on the cancer-associated CD147.1)CD147is upregulated in the liver parenchymal and mesenchymal cells and involved in angiogenesis and tumor cell proliferation in the development of DEN/PB-induced HCC.2)CD147 localizes at basolateral membrane of human hepatocytes,which closely relates to the HCC progression.3)CD147 mediates hepatocyte polarity through the signaling pathway of“CD147–intergrin?5?1–E-cadherin ubiquitination–Par3 decrease&?-catenin translocation”.Therefore,this study replenishes the mechanism of HCC development and supplies new theories for the treatment of HCC based on targeting CD147.