The Role And Molecular Mechanisms Of FBX8 In Regulating Hepatic Metastatic Dormancy Of Colorectal Cancer Cells

Background and objectiveColorectal carcinom(CRC)is one of the most common malignant tumors and liver metastasis is the main reason for the cause of deaths.CRC can metastasize to liver through clinically invisible micrometastases even after treating.This is the period calls dormancy and can be activated under certain conditions which will lead to drug resistance and metastasis inevitably.FBX8,belonging to the ubiquitin-proteasome system,can inhibit the proliferation,invasion and metastasis of CRC tumor cells[37].However,its role and mechanism in the regulation of hepatic metastases in colorectal cancer is not clear yet.Therefore,we aim to explore the role and molecular mechanism of FBX8 in regulating the dormancy of liver metastases from colorectal cancer which can provide new potential therapeutic targets for tumor metastasis and also provide a new treatment strategy for the prevention and treatment of clinical tumor metastasis.MethodsShort-term chemotherapy experiments are used to construct the dormant model of cancer cells in vitro.Mouse cecum planted in situ is used to construct a dormant model of liver metastasis from colorectal cells in vivo.Live animal imaging technology is used to observe the growth of tumor cells in mouse.The GST-pull down assay is used to detect the binding between FBX8 and substrate of ubiquitination.ResultsWe found that FBX8 expression was significantly lower in liver metastases of CRC than in primary cancer(P<0.001).Survival analysis showed that in CRC patients with lymph node or liver metastases,the prognosis of group expressing high FBX8 was significantly better than that group of low FBX8 expression(P<0.001).Bioinformatics analysis showed that high expression of FBX8 in patients with colorectal cancer,breast cancer or renal clear cell carcinoma have a high relapse free survival rate(P<0.05,P<0.01,P<0.05).We detected genes expression related to cancer cell dormancy in subcutaneous tumor tissue of nude mice,such as CK,E-cadherin,Sox-2 and caspase-3 and found that were higher in group with FBX8 overexpression,and the opposite result was found when it comes to genes expression related to activation of tumor dormant cells such as Vimentin,C-myc,CDK4,Cyclin D1,HIF-l?,and VEGF.In the dormant model in vivo and vitro,the overexpression of FBX8 group significantly prolonged the dormancy period of CRC.The dormancy period of CMT93 cells after MG 132 treatment was significantly shorter than the FBX8 overexpressing group in C57.Gene enrichment analysis showed that FBX8 was enriched for hypoxia,cell cycle,and MYC-related genes which are associated with tumor dormancy.Co-IP assay showed that FBX8 could combine with HIF-1?,CDK4,and C-myc respectively.Immunofluorescence showed that FBX8 was co localized with HIF-1,CDK4 and C-myc which were more significantly in MG 132 treatment group Comparing with the control group.GST-pull down assay showed that the Sec-7 domain of FBX8 directly binds to HA-HIF-l?-2,HA-C-myc-2 and HA-CDK4.The ubiquitination and half life test in vitro showed that FBX8 could increased the ubiquitination of HIF-1 a,CDK4 and C-myc in FBX8 overexpression group comparing with MG132 treatment group.Rescue test showed that the density of tumor microvasculature increased in FBX8 and HIF-1? co-expressing group and the number of lumen formation increased as well when comparing with FBX8 overexpression group.The proportion of cells in G0/G1 phase decreased in FBX8 and CDK4 co-expressing group comparing with control group.The FBX8 and C-myc co-expressing group showed that the cell proliferation index increased,the proportion of apoptosis decreased,the number of stem cell balling increased,and the expression of SOX-2,CD44,and CD133 increased comparing with the FBX8 overexpression group(P<0.01).Correlation analysis of endogenous expression of colorectal cancer cell lines showed that FBX8 was negatively correlated with HIF-1,CDK4 and C-myc.The correlation analysis of 20 pairs of matched fresh colorectal cancer tissues and liver metastases showed that the expression of FBX8 was negatively correlated with the expression of HIF-1?,CDK4 and C-myc(P<0.01).ConclusionFBX8 degraded HIF-1?,CDK4 and C-myc by Ubiquitination Pathway and the down-regulation of them respectively could promote angiogenesis,cell cycle progression and cell proliferation which could lead to the dormancy of liver metastasis from colorectal cancer.Our study has revealed a new mechanism of FBX8 regulate the dormancy of liver metastasis from colorectal cancer,which makes it possible for FBX8 to be a potential biomarker and target for tumor metastasis and provides new theories and treatment strategies for the prevention and treatment of tumor metastasis.Innovation points of this paper1.This study confirmed and proposed the key role and mechanism of FBX8 in regulating cell dormancy in colorectal cancer liver metastases;2.This project will provide potential effective markers for tumor metastasis and provide new strategies for the prevention and treatment of tumor metastasis.