The Role Of SLIT2 In Colorectal Cancer Metastasis And The Underlying Molecular Mechanisms

Colorectal cancer is a common malignancy mainly arising from adenomas. Over 1,000,000 pepole are diagnosed with colorectal cancer every year around the world. Surgery is the most effective therapeutic strategie of colorectal cancer, while 30% patients were diagnosed in advanced stage and which were unsuitable for operation. For unresectable colorectal cancer, the main therapeutic strategies are chemotherapy, radiotherapy, sometimes combined with interventional therapy. Despite therapeutic approaches have been improved in recent years, colorectal cancer remains one of the biggest causes of cancer-related death worldwide, which is mainly due to the high recurrence and metastasis rate.As dysregulation of a series of well known oncogenes and tumor suppressors were involved in development and progression of colorectal cancer, improved understanding of how these genes affect cancer biology may lead to an improved ability to predict clinical outcomes and the discovery of novel therapeutic strategies. The SLIT2 gene is located on chromosome 4p15.2. As a secreted glycoprotein, SLIT2 exerts its activity through binding to the receptor ROBO1 that launches the cellular signaling. Normally, the physiological function of SLIT2/ROBO1 signaling is to maintain the exact modulation of neuron migration through regulating the activity of Rho-GTPases such as Rac1 and cdc42. Several studies also suggested SLIT2 was responsible for vascular formation and angiogenesis. Recently, dysregulated downstream signaling of SLIT2 has been found to be implicated in various types of human cancers. Low level of SLIT2 expression was always detected in hepatocellular carcinoma, cervical cancer, lung cancer and breast cancer, which is due to the promoter methylation and associated with the malignancy conversion. The epigenetic inactivation of SLIT2 frequently happened in gliomas, and lost control of RhoGTPase signaling by SLIT2 inactivation promoted glioma cell migration and invasion. SLIT2 was also found to repress growth and metastasis in fibrosarcoma and squamous cell carcinoma, by regulating levels of Bcl-2, Cdk6 and other molecules that are related to apoptosis and proliferation.The epigenetic inactivation of SLIT2 is involved in colorectal cancer development, research showed that SLIT2 promoter region methylation was found in 72% of primary colorectal cancers. SLIT2 methylation was reversed and expression restored by treating colorectal tumor cell lines with the demethylating agent. Ectopic expression of SLIT2 diminished the ability to form colonies in colorectal tumor cell lines. However, other studies revealed that ectopic expression of Slit2 and Robol or recombinant Slit2 treatment of Robol-expressing colorectal epithelial carcinoma cells recruited an ubiquitin ligase Hakai for E-cadherin (E-cad) ubiquitination and lysosomal degradation, epithelial-mesenchymal transition (EMT), and tumor growth and liver metastasis.The results above indicated that the role of SLIT2 in colorectal cancer progression is complicated. While, in colorectal cancer, the functional role of SLIT2 in tumor metastasis has been little reported and the relevant molecular mechanisms remain elusive. To clarify the related issues, we examined 120 tumor samples from patients with colorectal cancer at different stages, and analyzed the relationship between the expression of SLIT2 and pathological features of colorectal cancer. The results showed that there is a negative correlation between the expression of SLIT2 and the progression of colorectal cancer. The addition of SLIT2 protein attenuated the migration capability of SW480 cells, while knockdown of SLIT2 induced increased motility and epithelial-to-mesenchymal transition (EMT) in NCM460 cells. Our further results revealed that the repressive effects of SLIT2 on tumor migration were modulated by AKT-GSK3β signaling pathway. Analysis of relationship between SLIT2 expression levels and clinicopathologic features of colorectal cancer patients further illustrated the suppressive role of SLIT2 in tumor metastasis.Part One SLIT2 expression level is down-regulated in metastasis-positive colorectal cancer patientsObjective:To investigate the relationship between SLIT2 and colorectal tumor metastasis.Methods:SLIT2 and β-catenin expression levels in metastasis colorectal tumors or non-metastasis colorectal tumors were examined. q-PCR was used to detect the expression of SLIT2 mRNA, immunohistochemistry was used to detect the expression of SLIT2, ROBO1 and β-catenin protein.Results:SLIT2 mRNA expression levels in Ⅲ and Ⅳ phase of colorectal tumors were depressed obviously; Accordingly, SLIT2 and its receptor protein ROBO1 erxpression levels were also downregulated; Meanwhile, β-catenin was upregulated in metastasis colorectal tumors.Conclusion:Colotectal tumor metastasis is relate with the deregulation of slit2, and β-catenin might be involved.Part Two SLIT2 inhibits migration and invasion of colorectal cancer cellObjective:To investigate the effects of SLIT2 on colorectal cancer cell migration and invasion.Methods:Colorectal cancer cell line SW480 and normal colorectal epithelial cell line NCM460 were introduced, by adding recombinant SLIT2 into cell culture or transfecting SLIT2 shRNA to overexpress or knockdown SLIT2, combined cell wound healing assay and Transwell assay to examine the effect of slit2 on colorectal cancer cell migration.Results:The results of cell wound healing showed that addition of purified proteins of SLIT2 lowered the healing speed, which means that SLIT2 inhibited cell migration in SW480 cells. And overexpressing SLIT2 by transfecting SLIT2 expression plasmid into SW480 with lipofectamin also resuled lower healing speed, which means SLIT2 inhibited SW480 cell migration in a cell-autonomous way. We also found that knockdown of SLIT2 with shRNA could speed wound healing. Accordingly, Transwell assay results revealed that the number of migrated cells was significantly increased in SLIT2-knockdown NCM460 cells and decreased in SLIT2-overexpression SW480 cells, demonstrating SLIT2 negatively regulated colorectal cancer cell migration.Conclusion:Increased SLIT2 expression levels led to the repression of cells migration, while decreased SLIT2 expression levels led to the promotion of cell migration, demonstrating SLIT2 play important roles in colorectal cancer metastasis.Part Three The role of SLIT2 in EMT of colonic epithelial cellObjective:To explore the role of SLIT2 in EMT of colonic epithelial cell.Methods:Colorectal cancer cell line SW480 and normal colorectal epithelial cell line NCM460 were introduced, by adding recombinant SLIT2 into cell culture or transfecting SLIT2 shRNA to overexpress or knockdown SLIT2, combined western blot and cell immunofluorescence to investigate the effects of SLIT2 on key proteins in EMT.Results:Western blot results showed that, overexpression of SLIT2 in SW480 cells led to increased Snail, and resulted in upregulation of E-cadherin. Meanwhile, β-catenin in NCM460 cell nucleus increased; Cell immunofluorescence results showed that knockdown SLIT2 in NCM460 resulted in β-catenin translocated from cell plasma membrane to cytoplasm and nucleus; In addition, upregualtion of N-cadherin and Vimentin were also detected in NCM460 cells with SLIT2 knockdown.Conclusion:Loss of SLIT2 leads to EMT in colonic epithelial cell.Part Four SLIT2 inhibits colonic cell migration through AKT-GSK3p signaling pathwayObjective:To investigate the related mechanism underlying the inhibition role of SLIT2 on colorectal cancer cells migrationMethods:Knockdown SLIT2 in NCM460 by using RNAi, then treated cells with AKT inhibitor triciribine, using Transwell assay to investigate the inactivity of AKT on colorectal cancer cell migration induced by SLIT2 dificiency, or collected the cells then using western blot to examine the inactivity of AKT on p-AKT, p-GSK3β, Snail, E-cadherin induced by SLIT2 dificiency.Results:After SLIT2 knockdown, levels of E-cadherin were decreased and levels of Snail-1 were increased, elevated phosphorylation level of AKT was induced. Accordingly, phosphorylation level of GSK-3 was also elevated with AKT activation. Treatment of AKT inhibitor abrogated changes of E-cadherin and Snail-1 resulted by SLIT2 knockdown. Meanwhile, results of Transwell migration analysis indicated block of AKT activation by AKT inhibitor repressed SLIT2 knockdown-induced enhanced motility of colonic cells.Conclusion:Collectively, these results demonstrated AKT signaling pathway may play a critical role on SLIT2-mediated migration in colonic cancer cells.