Vacuolar Protein Sorting-associated Protein 35 Promots Hepatocellular Carcinoma Progression By Activation Of PI3K/AKT Signaling Pathway

Objective:Hepatocellular carcinoma(HCC)is one of the most prevalent malignant solid tumors worldwide,with its incidence rates ranking the sixth of global cancer incidence,and its mortality rates ranking the fourth of global cancer deaths.China accounts for about 50%of the global cancer incidence and death cases.While the overall cancer incidence and mortality rates are declining,the incidence of liver cancer is still increasing at an annual growth rate of 2%-3%.Chronic Hepatitis B virus(HBV)infection is the main cause of liver cancer in China,accounting for more than 90%of the HCC cases.The treatment of hepatocellular carcinoma is still remains to be solved,especially those without significant vascular invasion,local lymph nodes or distant metastasis.The current 5-year survival rate for liver cancer ranges from 5%to 30%.Therefore,in-depth study of specific molecular events in HCC progression is expected to provide a novel target in HCC diagnosis and treatment.The development of HCC is a multistep process including initial hepatocyte injury,inflammation,and aberrant hepatocyte regeneration,which leads to the accumulation of genetic and epigenetic events during the formation of displastic nodules.Additional molecular alterations provide dysplastic cells with proliferative,invasive,and survival advantages,which ultimately leads to HCC occurrence.Genetic mutations and changes in their expression regulation are closely related to the occurrence and development of HCC.High throughput next-generation sequencing has delineated the molecular landscape of HCC.The driver genes closely related to the occurrence of human liver cancer include TERT,TP53,CTNNB1,NRAS,ARID1A,and AXIN1,which promoted the identification of cancer driver genes in HCC.Telomerase activation via TERT promoter mutations,HBV genome insertions,chromosomal translocation or gene amplification are the most prevalent driver gene alterations,which were observed in?60%of the HCC cases.Activation of the Wnt-?-catenin signalling pathway via mutations in CTNNB1 and AXIN1 or APC inactivation were observed in 30-50%of the HCC cases.Other frequent mutations or genetic alterations such as TP53,RB1,CCNA2,CCNE1 were involved in abrrent cell cycle regulation.Mutations in ARID1A,ARID2,KMT2 family genes altered epigenetic regulation.Amplification in FGF3,FGF4,FGF19 and inactivating mutations in TSC1,TSC2,and PTEN were involved in the activation of AKT-m TOR-MAPK signaling pathway.Mutation in NFE2L2 or KEAP1 were found to activate the oxidative stress pathway.Furthermore,amplifications in CCND1,FGF19,VEGFA,MYC or MET resulted in the activation of various oncogenic signaling(including receptor tyrosine kinases).Overall,each HCC has over 50 genomic aberrations,only 20-25%of patients with HCC have at least one driver mutation.Since HCC is a highly malignant tumor characterized by molecular heterogeneity,it is still necessary for us to explore the related driving events in the pathogenesis of HCC,and to further clarify its role and specific molecular mechanisms in the occurrence and development of HCC.The vesicle protein sorting-associated protein 35(VPS35)is a key component of the retromer complex,with its C ternimal associates with VPS29 and N terminal associtates with VPS26.The retromer complex was composed of a VPS29-VPS35-VPS26 heterotrimer and a Sorting nexin(SNX)heterodimer.The retromer complex was originally found to mediate the retrograde transport of the carboxypeptidase receptor from the endosome to the Golgi apparatus in yeast.Retromer complex mediates protein trafficking from the endosomes to the trans-Golgi network(TGN)or through direct recycling to the plasma membrane and has been implicated in neurodegenerative diseases.By mediating the sorting and trafficking of multiple cargo proteins on the cell membrane,the function of the retromer complex is closely related to important physiological processes such as autophagy,signal receptor regulation,and cell proliferation.In addition,the retromer participates in the regulation of intracellular signal pathways through its involvement in the sorting of signal receptors such as?₂-adrenergic receptor(?₂-AR),parathyroid hormone receptor(PTHR)and interferon receptor 2(IFNAR2).VPS35 has been reported to interact with N-Ras to promote the melanoma cell proliferation.Silencing of VPS35 down-regulated the mitogen-activated protein kinase(MAPK)signal and inhibited the proliferation of melanoma cells.Nevertheless,the role of VPS35 in HCC progression remains poorly understood.Here,we performed whole-exome and RNA sequencing in a small cohort of HBV-associated HCC.A graph clustering of the transcribed tumor-mutated alleles characterized overlapped functional clusters,and thus prioritized VPS35 as potentially novel oncogenes in HCC.We first clarified the expression level of VPS35 in clinical liver cancer samples,and demonstrated that overexpression of VPS35 promotes HCC cell proliferation.RNA sequencing was performed to explore the precise molecular mechanisms underlying VPS35 promoted HCC growth.Our study have identified potential novel oncogenes in HCC in a small cohort of HBV-associated HCC,provided new insights into the roles of retromer complex in tumorigenesis and is expected to provide important implications for personalized therapeutic strategies for HCC.Methods:1.Prioritization of potentially novel oncogenes.DNA and RNA sequencing were performed in 9 paired tumor and adjacent non-tumor tissues.Further functional verification of the hub genes in the network was performed and prioritized VPS35 as novel oncogene in HCC.We explored the clinical significance of VPS35 in the Cancer Genome Atlas(TCGA)database and the potential roles in signaling pathway.The expression level of VPS35 in HCC and non-tumor liver tissues were analyzed in TCGA HCC cohort.Then the correlation between VPS35 expression and the survival rate was analyzed.Real-time PCR,Western blot analysis,and immunohistochemical staining was performed to evaluate the expression of VPS35 between tumor tissues and adjacent non-tumor tissues.Then we further explored the potential mechanisms underlying the upregulation of VPS35 in tumors.2.The effect of VPS35 on HCC cell proliferation and cell cycle progression.VPS35 overexpression and VPS35 knock out(VPS35-KO)cell models were established through recombinant adenoviruses and CRISPR/Cas9 system,respectively.MTS,colony formation,and Ed U incorporation assay were performed to evaluate the effect of VPS35 on cell proliferation.Flow cytometry and Western blotting were performed to evaluate the role of VPS35 on hepatoma cell cycle.Then,the subcutaneous transplanted tumor model of VPS35-KO hepatoma cells was established in nude mice.The effect of VPS35-promoted tumor growth was determined by tumor growth,tumor weight and Ki67 staining.3.The effect of VPS35 on PI3K/AKT pathway was detected.Transcriptome sequencing were performed in VPS35-KO hepatoma cells.Pathway enrichment analysis revealed that target genes were enriched in lysosome and PI3K/AKT pathway.Real-time PCR was performed to verify the expression of enriched target genes and Western blot analysis was performed to demonstrate the activation of AKT signal.To further validate the participation of VPS35 in the PI3K/AKT signaling,a potent oral allosteric pan AKT inhibitor MK2206,was introduced.The role of MK2206 on VPS35-promoted HCC cell proliferation was validated via MTS and colony formation assays.The role of MK2206 on VPS35-promoted HCC cell cycle progression was validated via flow cytometric analysis and and Western blotting.Then,the orthotopic xenograft tumor model were established in nude mice with VPS35-overexpressing hepatoma cells.MK2206 were injected intraperitoneally,and the effect of MK2206 on VPS35-promoted tumor growth was determined by tumor size,Ki67 staining,immunoblot analysis and IHC staining.4.The specific mechanism by which VPS35 activates PI3K/AKT signals was studied.The TCGA HCC cohort indicated a pairwise correlation of VPS35 with the members of RTKs family(e.g.,FGFR3,FGFR2,and NTRK1).Flow cytometric analysis,Western blot and immuno-fluorescence(IF)staining was performed to identify the localization of FGFR3 in hepatoma cells.To futher study the role of VPS35 in the sorting and trafficking of FGFR3,VPS35 was re-expressioned in VPS35-KO hepatoma cells.Western blot and IF staining was performed to identificate the role of VPS35 on FGFR3 expression level and intracellular localization.Results:1.A total of 52 modules of the weighted protein complexes was identified in the constructed PPI network using graphclustering algorithms.An analysis for 52 hub genes based on GO analysis showed that they were significantly enriched in six GO terms,including retrograde transport,endosome to Golgi and retromer complex.Further functional verification of the hub genes in the network was performed and prioritized VPS35 as novel oncogene in HCC.We further investigated their clinical significance using TCGA provisional HCC cohort(n=375)and noted that somatic aberrations in VPS35 were significantly associated with poor survival.The data from TCGA database shows that VPS35 was upregulated in HCC tissues.Accordingly,real-time PCR,Western blot and IHC staining also showed that the expression of VPS35 in tumor tissues was significantly up-regulated.The potential mechanism underlying the up-regulation of VPS35 in HCC tissues:VPS35 expression were not significantly altered upon HBV infection;Two transcription factors:TBP and TCF3,were positively correlated with the expression of VPS35 in our sequenced cohort and TCGA HCC provisional dataset.Real-time PCR revealed that VPS35and TBP were significantly upregulated in tumor tissues than noncancerous tissues.Finally,using Meth HC,we noted that the methylation levels in the VPS35 promoter in TCGA HCC cohort were significantly lower than noncancerous samples(P=0.037).Taken together,our analyses suggested that the genetic or epigenetic regulation may drive the high expression of VPS35.In the TCGA HCC cohort,somatic mutations in VPS35 were occurred synergistically with genes in the PI3K/AKT signaling,including FGFR3(p=0.005).Besides,somatic mutations in VPS35 and an increased expression of VPS35 were significantly associated with poor prognosis in the TCGA HCC patients(n=360,P=0.002).Therefore,we prioritized VPS35 as a potentially novel oncogene and further validated its roles in HCC development both in vitro and in vivo.2.Overexpression of VPS35 significantly promoted the proliferation and G1/S phase transition of hepatoma cells,while knock out of VPS35significantly inhibits the proliferation of hepatoma cells and cell cycle was blocked in G1 phase.Nude mice bearing subcutaneous transplanted tumors showed that VPS35 knockout significantly inhibited tumor growth.3.RNA-seq in VPS35-KO and parental hepatoma cells indicated that differentially expressed genes were significantly enriched in the lysosome and the PI3K/AKT signaling.Real-time PCR and Western blotting demonstrated that overexpression of VPS35 activated RAS and PI3K/AKT signals,while knock out of VPS35 inhibited PI3K/AKT signal activation.The AKT inhibitor MK2206 can reverse the effects of VPS35 in promoting hepatoma cell growth and cell cycle progression both in vivo and in vitro.4.The TCGA LIHC cohort indicated a pairwise correlation of VPS35 with the members of RTKs family(e.g.,FGFR3,FGFR2,and NTRK1).Flow cytometry,Western blot and immunofluorescence staining revealed that localization of FGFR3 on the plasma membrane were declined in VPS35-KO HCC cells.It is worth noting that Western blot and immunofluorescence(IF)staining reveal that re-expression of VPS35 in VPS35 knockout hepatoma cells had rescued the down-regulation of FGFR3 on the cell membrane.These results provided evidences that VPS35 is essential for the sorting and trafficking of FGFR3 to the plasma membrane,thereby activating the downstream PI3K/AKT signal and promoting HCC cell proliferation.Conclusion:Here,we performed whole-exome and RNA sequencing in a small cohort of HBV-associated HCC.A graph clustering of the transcribed tumor-mutated alleles characterized overlapped functional clusters.We validated the function of the potentially novel oncogenes in vitro and in vivo.We showed that a component of the retromer complex-VPS35-promoted the proliferation of hepatoma cell through promoting cell cycle progression.Further study revealed that as a key component of the retromer complex,VPS35 promoted the sorting and trafficking of FGFR3 to the plasma membrane,therefore activating downstream PI3K/AKT signaling and promoting HCC cell cycle progression and cell proliferation.