The Functional Role Of Cancer-associated Fibroblast Expressed RAB31 In Promoting Colorectal Cancer Metastasis

BackgroundColorectal cancer(CRC)is a common malignant tumor of the digestive system.Liver metastasis is the leading cause of death in CRC.Revealing the mechanism of metastasis may facilitate the development of cancer CRC treatment strategies.The progression and metastasis of cancer are not only caused by gene mutations in the tumor cells,but are also associated with the microenvironment of the primary or potential metastatic tumor site.Cancer Associated Fibroblast(CAF)is the main cellular component in the tumor microenvironment.Over the past decade,studies have revealed that CAFs play an important role in tumor development and metastasisIn an early attempt to identify key proteins implicated in the development of CRC,our group conducted an integrated analysis of differentially expressed proteins across stage ? to stage ? CRC using proteomics(ITRAQ mass spectrometry)and gene microarray.We found that RAB31 protein was significantly elevated in stage IV CRC tissue.Ras-related Protein 31(RAB31)is a highly conserved small GTPase that belongs to the RAB family as well as Ras superfamily.It is implicated in endocytosis,vesicular trafficking in cells and plays an important role in a variety of physiological processes.However,the specific functions of RAB31 in CRC is still unclear.This study aims to study the role of RAB31 in CRC development and metastasisMethodsTissue microarray of 98 CRC patients was analyzed to explore the relationship between RAB31 expression and patient's prognosis.The expression of RAB31 in tumor tissues was detected by immunohistochemistry and immunofluorescence.A RAB31-GFP fusion protein construct was transfected intoprimary CRC CAFs,and subcellular localization of RAB31 was determined by immunofluorescence staining.The effect of CAF-RAB31 on cancer cell proliferation,migration and liver metastasis was verified by CCK8 proliferation assay,Transwell cell migration assay,and a murine xenograft model.Transwell migration assay was used to screen for CAF secreted factors that promote RKO migration,and the secretion of hepatocyte growth factor(HGF)in CAF-RAB31 was detected by ELISA.Phosphorylase inhibitors or shRNA-MET were used to inhibit HGF-MET signaling in RKO to further confirm that RAB31 in CAFs regulate HGF secretion which promotes CRC progression through the activation of HGF/MET signaling pathway in tumor cells.Results1.RAB31 is expressed in tumor tissues,and the expression of RAB31 in cancer cells does not correlate withprognosis.Elevated expression of RAB31 in the tumor stroma is associated with poor prognosis(HR:1.85,p=0.02).In the tumor stroma,RAB31 is expressed in CAFs,and RAB31 is partially localized to Golgi in CAF and partially expressed in the cytoplasm in a cluster-like form.2.We successfully cultured primary colorectal tumor-associated fibroblasts in vitro,and constructed RAB31 overexpressing CAF cell lines.3.Conditioned medium from RAB31 overexpressiing CAFs(CAF-RAB31)had no effect on the proliferation of CRC cells,but significantly promoted the migration of colorectal cancer cells(p=0.0067).Physically separated co-culturing of CAF-RAB31 with RKO promoted the migration of RKO in a scratch healing assay(p=0.012),and co-injection of CAF-RAB31 and RKO cells promoted liver metastasis in a murine xenograft model.4.CAF-RAB31 secreted more HGF than wild-type CAF.By blocking HGF-MET signaling pathway in RKO cell,the pro-migration effect of CAF CM was significantly attenuated(p<0.05).ConclusionElevated expression of RAB31 in the tumor stroma is associated with poor prognosis in CRC patients.In the CRC stroma,RAB31 is expressed in CAF and localizes to the Golgi and specific vesicles of CAF.Overexpressing Rab31 in CRC CAF promotes the secretion of HGF which promotes RKO migration and distant metastasis through HGF/MET signaling.