CD147 Enhances HCC Progression By Vesicle Recycling And Sequential Proteolysis

Known as “King of Cancer”,hepatocellular carcinoma(HCC)a is very dangerous disease characterised as malignant,difficult to cure,and of high mortality.HCC threatenes the health of human with its mortality ranking 3rd worldwide and 2nd in China.Previously,CD147 was identified as a tumor associated antigen and potential target for tumor treatment in our lab.CD147 is expressed in various tumors including HCC,and its high expression is correlated with poor prognosis clinically.Indeed,CD147 functions both as an inducer and as an effector in promoting tumor malignant development by participating tumor cell growth,invasion,metastasis,angiogenesis,chemo-resistance,and resistant to cell death.Under physiological and pathological conditions,membrane CD147 could be internalized,sorted,and recycled back to the membrane.Besides,CD147 is detected in exosomes,and membrane CD147 also releases its extracellular domain into ECM(extracellular matrix)by a process termed shedding.Recently,deregulated vesicle recycle is gradually recegnized as a hallmark of tumor progression,but it remains elusive whether CD147 endocytosis and recycle contributes to HCC progression.Moreover,uncovering the destiny and roles of the residual C-terminal portion of CD147 after shedding is essential to fully understand the role of CD147 in promoting tumor progression.To further confirm the role of CD147 in being a potential target for tumor treatment,the following study was performed:In the first part of this study,based on that CD98 was upregulated and correlated with cancer progression and malignancy in HCC,the role of CD98 was first confirmed in liver cancer cells by cell spreading in vitro and tumorigenicity by nude mice xenograft tumor assay in vivo;then we found that CD147 could influence the membrane expression of CD98 in SMMC-7721 by FCAS;thirdly,we found the co-localization of CD98 and CD147 on the human HCC cell membrane,and there exists a direct association between CD147 and CD98 in vitro with pull down and SPR analysis;Moreover,we found Ds Red1 tagged CD98 was blocked in the cytoplasm in K7721(whose CD147 was knockn out)and had a well colocalization with ER and Rab5 a positive recycling endosomes,mainwhile CD147 transfection could rescue blocked CD98;finally,we observed the internalization of CD147 and CD98 was flotillin-1-regulated,and their recycle at early steps was Arf6-mediated.In this part,we demonstrated that CD147 can directly bind to CD98,mediating CD98 redistribution on the HCC cell membrane and activating the downstream integrin signaling pathway.These findings indicate that CD147,as a redistribution chaperone of CD98 through vesicle recycle,plays a critical role in promoting cell spreading and HCC progression.In the second part of this study,we investigated the the converging point between internalized CD147 and its soluble form.Treatment with the cholesterol chelator M?CD significantly inhibited CD147 endocytosis,but FACS results showed that the level of membrane CD147 was decreased in cholesterol-depleted cells.Further study uncovered that ADAM10 is responsible for the CD147 shedding induced by cholesterol depletion,and HAb18 targeting CD147 inhibited CD147 shedding mediated by ADAM10.Moreover,we found residual CD147(CD147-?ECD)was transported to lysosomes for further processing after shedding.Together with the former shedding of CD147 mediated by ADAM10,the processing of CD147-?ECD in the lysosomes discussed here might constitute a novel degradation pathway of CD147.Therefore,the level of cholesterol influences not only the endocytosis but also sequential proteolysis of CD147,while its sequential proteolysis may also produce a functional CD147-ICD(intracellular domain of CD147).In the third part of this study,we found a highly conserved sequence,consistent with NLS(nuclear leading sequence),in CD147-ICD proximal to the membrane.And ectopic CD147-ICD was localized in the nucleus.After construction of a CD147-expressing vector with its N-terminal fused with GFP and its C-terminal fused with m Cherry and its transfection into 293 T cells,nuclear accumulation of the m Cherry signal revealed regulated intramembrane proteolysis of CD147.By blocking autophagy with 3-MA or chloroquine,we found the production and accumulation of CD147-ICD is related to cell autophagy process.Of note,ectopic CD147-ICD could also enhance autophagy of HCC cells through NF-?B-TRAIL-caspase8-ATG3 axis,while CD147-ICD transfected cells gained resistance to chemotherapy with enhanced autophagy.Finally,we confirmed HAb18 significantly improved the chemosensitivity of SMMC-7721 cells to cisplatin in vivo,which is partly attributed to HAb18 inhibiting CD147 sequential proteolysis.Taken together,we confirmed that CD147 promoted the membrane localization of CD98 and its downstreaming ?1 integrin signalling through vesicle recycle.The sequential proteolysis of CD147 is mediated by ADAM10 and lysosome,and this is a novel mechanism revealing proteostasis not limited to CD147.Lastly,the critical role of CD147-ICD in autophagy by NF-?B-TRAIL-caspase8-ATG3 axis through its nuclear localization was uncovered,and it is a potential mechanism of HAb18 improving chemosensitivity of cancer cells.