Actin-related Protein 2/3 Complex Subunit 2-enriched Extracellular Vesicles Drive Liver Cancer Metastasis

Background and aimsHepatocellular carcinoma(HCC),the most frequent neoplasm among all primary liver cancer,is currently the third leading cause of cancer related deaths in China.Patients detected at an advanced stage are only eligible for palliative treatments and the overall life expectancy is less than one year.EVs are a heterogeneous population of membrane vesicles of various origins,which are present in biological fluids and involved in multiple oncogenesis and other pathological processes.EVs and their cargoes,including mRNAs,non-coding RNAs,and proteins,play pivotal roles in HCC tumor growth and metastasis.ARPC2(Actin-related protein 2/3 complex subunit 2,ARPC2),a key regulator of actin cytoskeletal structure,has been previously reported to mediate cell motility.However,the role of ARPC2 in HCC has not been elucidated.Herein,this article aims to identify proteins that contribute to the functionality of EVs derived from metastatic HCC cells and investigate the role of APRC2 delivered by EVs in HCC cell motility and cancer metastasis.MethodsSection 1EVs were collected from metastatic HCC cell lines MHCC97L and MHCCLM3,and human normal liver cell line MIHA using ultracentrifugation and subjected to mass spectrometry analysis.ARPC2 was chosen for further investigation.The expression of ARPC2 in EVs and TCL of different cell lines and HCC tissues was examined using immunoblotting and TCGA database,respectively and clinical relevance of ARPC2 in HCC was also revealed.Section 2EVs from metastatic MHCC97L and MHCCLM3 cells have been shown to enhance tumor development and augment metastasis in HCC.To study whether APRC2 contributes to the promoting capacity of MHCC97L-and MHCCLM3-EVs,Pimozide,an inhibitor of APRC2,was examined for its effect in EVs in functional assays and was further demonstrated in an experimental metastasis assay.Section 3To affirm the role of ARPC2 in EVs of HCC cells,stable ARPC2 knockout cells(ARPC2-KO1 and ARPC2-KO2)and nontarget knockout control cells(Control-KO)were generated in metastatic HCC cell line MHCC97L.The functional roles of EV-ARPC2 was then investigated using ARPC2 knockout stable clones both in vitro and in vivo.ResultsSection 1Proteomic profiling identified ARPC2 to be highly expressed in EVs of metastatic HCC cells.Results of immunoblotting showed that ARPC2 was highly expressed in EVs of metastatic cells but barely detected in non-metastatic HCC cells and human normal liver cells.Analysis of TCGA database of HCC revealed ARPC2 overexpression was correlated with poor prognosis of patients.Section 2Pimozide,an ARPC2 inhibitor,suppresses the promoting effects of EVs derived from metastatic HCC cells in cell growth,migration and invasiveness.Moreover,Pimozide also inhibited the promoting effect of EVs of metastatic cells in lung colonization of tumor cells in mice.Section 3ARPC2 was successfully knockout in metastatic HCC cell line MHCC97L.EVs derived from knockout cells displayed compromised activity in enhancing cell growth,motility and metastasis compared to EVs of control cells.ConclusionThis study reveals previously unreported expression and function of ARPC2 in EVs.The results demonstrated that EVs with highly expressed ARPC2 enhances cancer cell growth,motility and cancer metastasis.Overall,EV-APRC2 plays an imperative role in HCC metastasis and it is suggested that targeting EV-ARPC2 may play an anti-metastatic effect in HCC.In the long term,ARPC2 may provide a prospective target for the novel treatment of HCC patients.