Activation Of Slit2/Robo1 Signaling Promotes Tumor Metastasis In Colorectal Carcinoma Through Activating TGF-β/Smads Pathway

Colorectal carcinoma（CRC）is a common malignant tumor.CRC is currently one of the three most common cancers in the world,with mortality rates ranking second.In recent years,the incident rate has increased rapidly and the patients are getting younger.It is also one of the leading causes of cancer-related deaths worldwide.Approximately 20%of patients have demonstrated metastases at the time of diagnosis and have a poor prognosis for highly metastatic colorectal cancer.Therefore,in-depth research on tumor metastasis mechanisms and targeted drug development will contribute to the future clinical treatment of patients.Slit2 is a secreted protein that binds to the Roundabout1（Robo1）receptor to mediate downstream signaling and plays an important role in neuronal axon and neuronal cell migration.However,there are also differences in the expression and function of the Slit2/Robo1 signal in different tumors.In CRC tumor tissues,Slit2 expression is inconsistent.Previous studies in our laboratory have shown that Slit2/Robo1 signaling is elevated in tumor tissues of CRC patients,promotes tumor growth and metastasis through Hakai-mediated E-cadherin degradation.It also promotes tumorigenesis by activating Src which then activates Wnt/β-catenin signaling.However,the literature has pointed out that in the CRC tumor,when the Slit2 promoter is abnormally methylated,its expression can be reduced.Therefore,the study of Slit2/Robo1 signal expression and function in CRC tumors will contribute to the development of targeted precise treatment of CRC patients.Transforming growth factor-beta（TGF-β）regulates various biological processes such as cell proliferation,migration and apoptosis by binding to its receptor binding protein Smads protein family.A large number of studies have shown that TGF-β/Smads signaling can regulate the occurrence of epithelial-mesenchymal transition（EMT）and EMT is closely related to tumor metastasis.Transforming growth factor-beta（TGF-β）not only plays a role in maintaining the physiological function of normal colon cells,but also plays a regulatory role in the progression of CRC and its metastasis.TGF-β/Smads signaling is activated in CRC,inhibits tumor cell proliferation in early tumor stage,promotes tumor cell growth and EMT in the late stage of tumor development,and promotes tumor metastasis.Previous studies in our laboratory found that Slit2/Robo1 can activate TGF-β/Smads signaling pathway to promote liver fibrosis.However,it is unclear whether Slit2/Robo1 regulates the TGF-β/Smads signaling pathway in CRC.In order to explore the problem,this paper uses serum from clinical CRC patients,animal models and tumor cells to elucidate the expression of Slit2/Robo1 and its relationship with TGF-β/Smads signaling pathway,and to elucidate its role in CRC tumor metastasis.In this paper,we analyzed the bioinformatics database（MERAV）to determine the expression of Slit2 and TGF-β1 in CRC tumor tissues,and it was positively correlated with tumor stage.The expression of Slit2 was increased in CRC patients as shown by ELISA detection of serum Slit2 levels in patients with clinical CRC.The serum Slit2 level in different pathological processes of Apc^Min/+spontaneous intestinal adenoma mice was detected by ELISA.The results showed that the serum Slit2 content of the tumor mice was higher than that of the control mice,and the serum Slit2 content gradually increased with the pathological processes.We further used the Apc^Min/+mouse and LoVo cell mouse lung metastasis model to explore the role of Slit2/Robo1 signaling in CRC development and metastasis,by using R5 antibody to specifically block Slit2/Robo1 signal,we found that the number of microadenomas in Apc^Min/+mouse is reduced and the pathological processes is slowed down;the number of metastases in the lung metastasis model of LoVo cells was also reduced.In vitro results showed that R5 antibody specifically blocked the Slit2/Robo1signal in the LoVo（highly invasive）and SW480（medium invasive）cells resulted in significantly reduced proliferation,migration and infiltration,and the expression of TGF-β1/Smad2/3 signaling pathway protein was significantly reduced.At the same time,we also found that the inhibition of TGF-β1/Smad2/3 signaling by P144 significantly reduced the migration and infiltration ability of LoVo and SW480 cells,but tumor cell proliferation was not affected.Inhibition of LoVo cell migration and invasion by R5 can be restored by supplementing the cell culture with TGF-β1.Throughout this study,it was confirmed that the expression of Slit2 was elevated in CRC clinical patients,and activation of Slit2/Robo1 in CRC could promote tumor growth and metastasis;more importantly,tumor metastasis is promoted by activating TGF-β1/Smad2/3 signaling.In addition,the Slit2/Robo1 signal specific blocking antibody R5,was shown to be able to significantly inhibit tumor growth and metastasis,and is expected to be developed into a targeted clinical treatment for CRC.