Sec62 Promotes Gastric Cancer Metastasis Through Mediating UPR-induced Autophagy Activation

Background:Gastric cancer(GC)is one of the leading causes of cancer-related death worldwide.The number of new cases and deaths of GC in China both rank first all over the world in2020.Owing to the continuous updating of early diagnosis and treatment strategies,especially the development of endoscopic technologies,the prognosis of early GC has greatly improved.However,the 5-year survival rate in patients with advanced GC is still less than 30%.Tumor invasion and metastasis are the main cause of disease progression,treatment dilemma,poor quality of life and mortality.After decades of efforts,the mechanism of GC invasion and metastasis remains largely unknown and key breakthroughs are needed.During tumor onset and development,many stress stimuli,such as upregulated cell metabolism,microenvironment hypoxia,microenvironment acidification and increased demand for protein biosynthesis will impact the function of endoplasmic reticulum(ER)in protein processing and folding,collectively called ER stress.Thereby,cells will activate unfolded protein response(UPR)to counteract the ER stress and rebuild protein homeostasis.Notably,UPR is characterized by the expansion of ER and accumulation of unfolded or misfolded proteins.At present,UPR activation has been identified in a variety of tumors,including GC.Moreover,UPR is found to be associated with tumor malignant features such as proliferation and metastasis.There is an optimum size for every type of animal,similarly,the size is important for cell organelles.When the stress stimulation attenuates or cells gradually get used to the stress,expanded ER needs to decrease to its original size.Also,the accumulated misfolded proteins need to be removed.Recent studies have found that Sec62,an ER membrane protein can promote UPR resolving by mediating the recognition and binding between piecemeal ER,which contains the wrapped misfolded proteins,and autophagosomes.Then the degradation of expanded ER and misfolded proteins will be accomplished by autophagosomes.Interestingly,in our recent research,mass spectrometry for GC and adjacent tissues showed Sec62 was significantly upregulated in GC when compared with adjacent control.Although until 2006 the role of Sec62 in tumor biology was first reported,several studies have found that Sec62 is highly expressed in many kinds of tumors,and Sec62 upregulation can promote tumor cell migration and invasion.In this study,we investigated the functional role of Sec62 in GC metastasis and the underlying mechanism.Aims:1.To determine the expression of Sec62 in GC tissues and cell lines,and to explore the correlation between Sec62 expression and the clinicopathological features of GC as well as survival of GC patients.2.To study the effects of Sec62 expression on GC malignant phenotypes such as proliferation,invasion,migration and apoptosis.3.To investigate the association between UPR and Sec62.Also,the role of UPR in Sec62 modulating GC metastasis was assessed.4.To explore the mechanism that Sec62 employs to regulate GC metastasis.Especially,to clarify whether autophagy is involved in mediating GC cell migration and invasion as well as the key molecules involved.5.To explore the potential feasibility of Sec62 as a therapeutic target to inhibit the invasion and migration of GC cells.Methods:1.GEPIA2 database was consulted to analyze the expression of Sec62 in GC and adjacent normal tissues.KM-plotter database was used to determine the correlation between Sec62 level and the prognosis of GC patients.Protein and RNA were extracted from GC cell lines,immortalized normal gastric mucosal epithelial cell GES and GC tissue samples,then Western Blot(WB)and q RT-PCR assays were used to detect the protein and m RNA expression of Sec62.Also,immunohistochemical staining(IHC)was performed to assess Sec62 level in GC and adjacent normal tissues using tissue microarray and clinical tissue specimens.Moreover,the correlation between Sec62 level and GC clinicopathological features as well as the prognosis of GC patients was analyzed using tissue microarray data.2.Lentivirus transfection was used to construct GC cell line models with stable overexpression and knockdown of Sec62.Transfection efficiencies were verified by examining Sec62 protein expression using WB.Transwell assay,scratch healing assay and nude mice tumor metastasis model were performed to assess the effect of Sec62 levels on GC cell migration and invasion.CCK8 and colony formation assays were used to detect the effect of Sec62 level on GC cell proliferation.Flow cytometry was used to examine the effect of Sec62 on GC cell apoptosis.3.WB assay was used to determine the correlation between Sec62 and matrix metalloproteinases(MMPs)and epithelial-mesenchymal transition(EMT)markers including E-cadherin and Vimentin in lentivirus-transfection constructed GC cell lines.In addition,immunofluorescence assay was used to validate the relationship between Sec62 and EMT markers.Elisa assay was performed to examine the level of soluble TIMPs and MMP2/9,and gelatin zymography assay was adapted to evaluate the protease activity of MMP2/9 in the cell culture supernatants.4.WB assay was performed to detect the correlation between Sec62 and several key molecules in the UPR signalling pathway and autophagy marker LC3 II.Transmission electron microscope was used to observe the effect of Sec62 on autophagy activation measured by occurrence of autophagy-related membrane structures such as phagophore,autophagosomes and autophagolysosomes.The effect of Sec62 on autophagy flux was detected by m RFP-GFP-LC3 double-labeled adenovirus transfection.The intracellular binding of Sec62 and LC3 II was assessed by co-immunoprecipitation(CO-IP).Immunofluorescence co-localization assay was used to confirm the findings of CO-IP.Further,the correlation between Sec62 and several key proteins involved in autophagy signalling pathways was detected by WB assay.5.Previously constructed GC cell models were treated with rapamycin and hydroxychloroquine to activate or inhibit autophagy,respectively.Then the effect of autophagy activation and inhibition were verified by WB assay,transmission electron microscope and m RFP-GFP-LC3 double-labeled adenovirus transfection.After the effect of autophagy induction and inhibition was confirmed,Transwell migration and invasion assays and scratch healing assay were used to examine the effects of autophagy on cell migration and invasion.Additionally,the correlation between autophagy and MMP2/9were also investigated.Results:1.Among different human tumors,Sec62 expression level in GC is significantly higher than that in other tumors.Sec62 expression in GC tissues is significantly upregulated when compared to adjacent normal tissues.Also,Sec62 level in GC cell line is higher than that in normal epithelial cells.Cox regression indicates Sec62 upregulation is an independent risk factor for short survival in GC patients.2.Transwell,scratch healing assay and animal experiment showed that high expression of Sec62 could significantly promote GC cell migration and invasion in vitro and in vivo.CCK8 and colony formation experiments suggested that Sec62 had no effect on the GC cell proliferation.Similarly,flow cytometry assay demonstrated that Sec62 did not affect GC cell apoptosis.3.WB assay showed that Sec62 expression was positively correlated with that of MMP9 and MMP2,while not with EMT markers E-cadherin and Vimentin.No correlation between Sec62 and EMT markers was also verified with immunofluorescence assay.Elisa assay demonstrated Sec62 could affect TIMP-1 and MMP2/9 levels but showed no effect on TIMP-2 and TIMP-4 in the cell culture supernatant.Furthermore,gelatin zymography indicated Sec62 upregulated the activity of MMP2/9 in the supernatant of cell medium.4.WB assays showed Sec62 expression was found positively correlated with PERK、ATF4 and autophagy marker LC3 II.While no correlation was identified between Sec62 and other UPR molecules such as IRE1 and ATF6.Transmission electron microscope and m RFP-GFP-LC3 double labeled adenovirus transfection also showed that high level of Sec62 could enhance the autophagy activity.Further,CO-IP and immunofluorescence co-localization assays suggested that Sec62 could directly bind to LC3 II.Moreover,WB assay showed that Sec62 level was positively correlated with the expression of several key molecules involved in autophagy pathway including FIP200,Beclin-1 and Atg5.5.WB assay,transmission electron microscope and m RFP-GFP-LC3 double labeled adenovirus transfection showed that the level of autophagy significantly inhibited after hydroxychloroquine treatment,and positively correlated with MMP2 and MMP9.Furthermore,to explore the effects of autophagy on the GC cell migration and invasion,Transwell and scratch healing assays were performed and demonstrated that the migration and invasion ability of GC cells were also inhibited after autophagy blockage.Notably,autophagy blockage combined with Sec62 inhibition could exert synergetic anti-metastatic effects on GC metastasis,suggesting a promising therapeutic strategy.On the contrary,after rapamycin treatment,increased autophagy assessed by WB assay,transmission electron microscope and m RFP-GFP-LC3 double labeled adenovirus transfection was verified.Consistently,Transwell and scratch healing assays showed autophagy activation could promote GC cell migration and invasion.And the level of autophagy was still positively correlated with the level of MMP2 and MMP9.Conclusion:This study confirms the high expression of Sec62 in GC tissues and cells,and upregulation of Sec62 can promote GC cell migration and invasion.The mechanism that Sec62 employs to promote GC metastasis may be concluded as follow: Sec62 promotes GC metastasis by activating autophagy and subsequently regulating the TIMP-1 and MMP2/9 balance.The activation of autophagy by Sec62 may involve the UPR-related PERK/ATF4 pathway and binding of LC3 II during UPR recovery involving FIP200/Beclin-1/Atg5 upregulation,although the exact mechanism remains to be elucidated.In particular,the dual inhibition of Sec62 and autophagy may be a promising therapeutic strategy for GC metastasis.