The Function And Molecular Mechanism Of ASAP3 In Colorectal Cancer

Objective:Colorectal cancer?CRC?is one of the most common malignancy with the highest morbidity and mortality in the world,which pose a great threat to human health.The initiation and development of colorectal cancer is a multi-step,multi-phase,multi-pathway pathological process,involving many genetic alterations.This process involves the proliferation,invasion and metastasis of tumor cells and a series of other molecular events.Further study on the mechanism of colorectal cancer may provide potential targets for the treatment of colorectal cancer.Our previous study confirmed that ArfGAP SH3 Domain Repeat,Ankyrin And PH Domain 3?ASAP3?can promote the secretion of gastric acid.It has been reported that ASAP3 can promote the metastasis of cancer,and its upregulation is associated with poor prognosis.However,the exact function of ASAP3 in CRC is not fully understood.The aim of this study was to investigate the biological function of ASAP3 and its possible molecular mechanism in CRC.Methods:?1?High throughput RNA-sequencing data of the CRC cohort from TCGA were analyzed to confirm the expression of ASAP3 in tumor tissues and in normal adjacent tissues.Real time PCR and Immunohistochemistry were used to detect the expression of ASAP3 in 90 cases of tumor tissues and corresponding adjacent tissues from RenJi hospital.The relationship between ASAP3 expression and various clinicopathological parameters was analyzed.Log-rank test was used to analyze the relationship between ASAP3 expression and prognosis,and Kaplan-Meier method was used to draw survival curve.COX proportional regression model was used to analyze the effect of clinicopathological parameters on prognosis of patients.To evaluate the potential capability of ASAP3 as a diagnostic biomarker for the prediction of patient survival,receiver operating characteristic?ROC?curves were conducted using AJCC stage,ASAP3 level,or a combination of both.?2?The expression levels of ASAP3 in different cancer cell lines were tested by Real time PCR and Western blotting.Human ASAP3 vector was used to induce the ASAP3low expression cells,human ASAP3 siRNA were transfected in the ASAP3 high expression cells.CCK-8 assay was used to evaluate the effect of ASAP3 on the proliferation of CRC cells.Flow cytometry analysis were applied to evaluate the effects of ASAP3 knockdown on the cell cycle and cell apoptosis.The effects of ASAP3 on cell migration and invasion were assessed by Transwell assays.?3?High throughput RNA-sequencing data of the CRC cohort were used to perform GSEA to probe the ASAP3-associated pathways in an unbiased manner.Western blotting and Real time PCR were used to detect the expression of genesin the related signaling pathways.COIP was used to investigate the possible molecular mechanism of ASAP3 in CRC.?4?Azoxymethan?AOM?and dextran sulfate sodium?DSS?were used in Asap3wild-type and knockout mice to verify the effect of ASAP3 on CRC growth.The number of tumors,tumor size,colon length and body weight change of the two groups were observed.Results:?1?ASAP3 expression level was significantly higher in tumor tissues than in normal adjacent tissues,and higher ASAP3 expression predicted poor prognosis.ASAP3 expression was correlated with tumor size,AJCC stage and lymph node metastasis.The combination of ASAP3 and AJCC stage was superior to ASAP3 or AJCC stage alone for the prediction of patient survival.?2?ASAP3 significantly enhanced the proliferation,invasion and migration ability in CRC cells.Suppression of ASAP3 inhibited cell proliferation by inducing G1-phase arrest without influencing apoptosis.The flow cytometry assays showed that ASAP3could promote cell proliferation by regulating the G1/S checkpoint,However,phycoerythrin-conjugated Annexin V staining revealed no alteration in cell apoptosis with the change of ASAP3.?3?GSEA showed that high expression of ASAP3 was related to NF-?B,cell cycle and Metastasis signaling pathway,which were associated with proliferation and metastasis.Upregulation of ASAP3 increased the phosphorylation and nuclear translocation of p65NF-?B subunit.ASAP3 could increase NF-?B downstream genes related to cell cycle and metastasis.ASAP3 interact with NEMO and could reduce the polyubiquitinylation of NEMO.Overall,ASAP3 might regulate NF-?B via binding to NEMO.?4?Tumors induced by AOM plus DSS were located mainly in the middle to distal colon.Asap3 knockout mice showed markedly decreased tumor size and number compared with the Asap3 wild type mice.The Asap3^+/+mice showed more body weight loss and colon shorten.Asap3 knockout mice had a longer overall survival time than wild-type mice.Conclusion:ASAP3 overexpression promotes the proliferation,invasion and metastasis of CRC,which influences the prognosis of patients.ASAP3 may involve in the activation of NF-?B canonical pathway by regulating the expression of NEMO.The activated NF-?B can regulate the transcription of downstream genes related to proliferation and metastasis,then promote the development of colorectal cancer.These findings enrich the molecular-biology contents of genesis and development of colorectal cancer,ASAP3 may serve as a potential prognostic factor and a therapy target for CRC.