SEMA3F Down-regulates ASCL2-CXCR4 Axis To Inhibit Metastasis Of Colorectal Cancer

Colorectal cancer(CRC) is the third most common cancer and the fourth most frequent cause of cancer-related deaths around the world, with the majority of death due to distant metastases. The 5-year survival rate for patients without metastasis is 80–90%, whereas that of those with distant metastasis is around 10-20%. Therefore, discovery of biomarkers and therapeutic targets for CRC metastasis is of paramount importance.Class 3 Semaphorin family is a group of secreted axon guidance molecules that plays critical roles in neuronal development. Accumulating evidence has indicated that these Semaphorins including SEMA3 F are widely expressed outside the central nervous system, and they play important roles in the tumor context such as angiogenesis, tumor growth, and metastasis. We previously reported that SEMA3 F expression was significantly reduced in CRC tissues and cell lines, and that SEMA3 F inhibited the growth and metastasis of CRC. However, the prognostic value of SEMA3 F and the molecular mechanisms underlying its anti-metastasis effects in human CRC are poorly known.Chemokine receptor CXCR4 is the first identified chemokine receptor playing a crucial role in determining the metastatic destination of breast cancer to the bone and lungs where its ligand CXCL12 is abundant. Following binding with CXCL12, CXCR4 induces intracellular signaling to initiate signals related to chemotaxis, migration and invasion, proliferation and stemness. Blocking CXCR4 function reduced metastasis of mouse colon cancer cell line CT-26 to the liver and lung. Clinical studies showed that CXCR4 expression in CRC was associated with increased recurrence, poor survival and liver metastasis. Further study demonstrated that CXCL12 secreted by myofibroblasts derived from hepatic satellite cells promoted liver metastasis of CRC through the CXCL12/CXCR4 axis, and AMD3100, a potent CXCR4 antagonist, significantly suppressed tumor formation in the liver.Achaete scute-like 2(ASCL2), a basic helix-loop-helix(bHLH) transcription factor, is a downstream target of WNT signaling pathway in intestinal stem cells. ASCL proteins bind to the canonical E-box(‘CANNTG’) element through their basic domains following dimerization through their helix–loop–helix domains. ASCL2 gene is located at chromosome 11p15.5 and loss of imprinting at this locus is frequent during CRC tumorigenesis. It has also been reported that ASCL2 is overexpressed in CRC and associated with CRC stem cells. Knockdown of ASCL2 inhibits the proliferation, colony formation and invasion of CRC cells.In the present study, specimens from 303 patients with follow-up information were used to analyze the prognostic value of SEMA3 F expression level in colorectal cancer by Kaplan-Meier analysis. The effects of SEMA3 F expression on CXCR4 expression were examined in CRC cells by quantitative reverse transcriptase-PCR(qRT-PCR), western blot, flow cytometry and immunofluorescent assays. The underlying mechanisms were investigated by gene over-expression and knock-down studies. The main results and conclusions are as follows:1. Low expression of SEMA3 F is correlated with metastasis and poor prognosis in CRC51.2%(155/303) of the CRC specimens showed low expression of SEMA3 F. We found that low expression of SEMA3 F is correlated with distant metastasis(P=0.006), recurrence(P=0.008), TNM stage(P=0.024), and lymph node metastasis(P=0.044), but not with gender, location, tumor size, differentiation degree or T-stage. Results from IHC studies demonstrated that SEMA3 F expression level was significantly reduced in:(i) primary CRC samples as compared to normal human intestine mucosa(P< 0.01);(ii) in high-grade as compared to low-grade CRC;(iii) in lymph node metastases as compared to primary CRC tissues(P< 0.01). Kaplan-Meier analyses showed that patients with reduced SEMA3 F expression had significantly shorter overall survival(P<0.01).2. SEMA3 F down-regulates functional CXCR4 expressionWe investigated the mechanisms by which SEMA3 F regulates metastasis using lentivirus expressing either short hairpin RNA targeting SEMA3 F or SEMA3 F for gene knockdown or overexpression. Knockdown of SEMA3 F could increase, and overexpression of SEMA3 F could decrease CXCR4 mRNA and protein levels in CRC cells. In contrast, knockdown or overexpression of CXCR4 had no effect on SEMA3 F expression and secretion, as determined by Western blot of the cell lysate and ELISA assay of the culture medium. Similarly, treatment with CXCR4 antagonist AMD3100 did not affect expression and secretion of SEMA3 F.We then examined whether CXCR4/CXCL12 contributes to SEMA3F-mediated regulation of invasion. Our results demonstrated that knockdown of SEMA3 F increased and overexpression of SEMA3 F decreased invasion of CRC cells. Treatment with CXCL12(50 or 100 ng/ml) enhanced both invasion and migration of SEMA3F-knockdown cells but not SEMA3F-overexpression cells. We then either suppressed CXCR4 in SEMA3F-knockdown cells or overexpressed CXCR4 in SEMA3F-overexpressing cells. Knockdown or overexpression of CXCR4 was confirmed by western blot. Our results demonstrated that suppression of CXCR4 abrogated SEMA3F-knockdown mediated increase in cell invasion and migration. Consistently, overexpression of CXCR4 reversed SEMA3F-overexpression mediated decrease of invasion and migration in CRC cells. These results demonstrated that SEMA3 F down-regulates functional CXCR4 in CRC cells.3. SEMA3 F down-regulates CXCR4 by inhibiting the expression of the transcription factor ASCL2 via PI3K/AKT pathwayKnockdown of SEMA3 F up-regulated while overexpression of SEMA3 F downregulated ASCL2 in CRC cells. Knockdown of ASCL2 decreased CXCR4 expression and overexpression of ASCL2 increased CXCR4 expression in CRC cells. To determine whether ASCL2 affects the transcription of CXCR4, we performed luciferase assay and found that overexpression of ASCL2 significantly enhanced the transcription of CXCR4. Since ASCL2 binds to the E-box of the regulatory regions of a gene, we tested whether ASCL2 may bind the promoter region of CXCR4 gene. We divided the promoter region of CXCR4 into eight segments and found that ASCL2 bound to the eighth segment of the CXCR4 promoter region shown by ChIP. Thus, SEMA3 F down-regulates CXCR4 in CRC cells via depressing the expression of transcription factor ASCL2.SEMA3F is known to reduce the phosphorylation of AKT, JNK and ERK1/2 in some malignancies. We thus examined the effect of SEMA3 F on CRC cells, and found that knockdown of SEMA3 F significantly increased the phosphorylation of AKT but not ERK1/2 and JNK. Conversely, overexpression of SEMA3 F in CRC cells significantly decreased the phosphorylation of AKT. Furthermore, the PI3 K inhibitor LY294002 significantly reduced the protein abundance of both ASCL2 and CXCR4, but had no effect on SEMA3 F expression in CRC cells. The m RNA level of ASCL2 and CXCR4 were also significantly reduced after treatment with LY294002 in CRC cells. These results demonstrate that SEMA3 F down-regulates ASCL2/CXCR4 axis in CRC cells through PI3K/AKT pathway.4. ASCL2 predicts prognosis of CRC patients and enhances metastasis of CRC cellsTo evaluate the function of ASCL2 in CRC, we examined ASCL2 expression in the specimens from 119 CRC patients.47.1%(56/119) of these samples had high ASCL2 expression. High expression of ASCL2 in CRC was correlated with distant metastasis(p=0.004), TNM stage(p=0.024), tumor size(p=0.022), and tumor location(p=0.038), but not with gender, differentiation degree, lymph node metastasis or T-stage. Kaplan-Meier analyses showed that CRC patients with reduced SEMA3 F expression had significantly shorter overall survivals. Knockdown of ASCL2 not only significantly decreased invasion of CRC cells, but also decreased the chemotactic effect of CXCL12 towards CRC cells. On the contrary, overexpression of ASCL2 increased invasion of CRC cells and the chemotactic effect of CXCL12 towards CRC cells. In vivo, knockdown of ASCL2 significantly attenuated liver metastasis of HCT116 cells, and overexpression of ASCL2 significantly enhanced liver metastasis of HCT116 cells. These data revealed that ASCL2 enhanced invasion and metastasis of CRC cells and predicts metastasis and poor prognosis of CRC patients.5. Blocking CXCR4 activation inhibits SEMA3 F knockdown-induced invasion and metastasisThe CXCR4 antagonist AMD3100 dose-dependently inhibited the invasiveness of CRC cells with or without SEMA3F-knockdown. The invasion of SEMA3F-knockdown CRC cells were reduced to the level of control cells after treatment with AMD3100. In the CRC liver metastasis nude mouse model, treatment with AMD3100 decreased the percentage of liver metastases caused by injection in the spleen of control or SEMA3F-knockdown CRC cells. We also counted the number of metastatic nodules in each liver, and found that SEMA3F-knockdown CRC cells established increased number of metastatic nodules in the liver as compared with control cells. The increased liver metastasis of SEMA3F-knockdown CRC cells was inhibited by AMD3100 treatment. Moreover, overexpression of SEMA3 F in CRC cells significantly decreased their liver metastasis. These results confirm that SEMA3 F inhibits the invasion and metastasis of CRC cells through down-regulating CXCR4.6. SEMA3 F is negatively correlated with CXCR4 in human CRC samplesBioinformatic analysis of gene expression of 229 CRC samples from The Cancer Genome Atlas(TCGA) revealed a negative correlation between the expression of SEMA3 F and CXCR4. Negative correlation between SEMA3 F and CXCR4 expression was observed. To further validate the negative correlation, primary tumor tissues of 85 patients were examined for SEMA3 F and CXCR4 expressions by IHC. Kaplan-Meier survival curves showed that CRC patients with SEMA3Flow/CXCR4 high had the shortest survival time in the four subgroups. The ratio of both distant metastasis and lymph node metastasis in CRC patients with SEMA3Flow/CXCR4 high is highest in the four subgroups. These results suggest that CRC patients with SEMA3Fhigh/CXCR4 low appear to survive longer due to reduced metastasis.These results demonstrated that SEMA3 F functioned as a CRC suppressor through down-regulating ASCL2/CXCR4 axis via PI3K/AKT signaling. ASCL2 promotes the transcription of CXCR4 via binding to the promoter region of CXCR4 gene. Moreover,ASCL2 predicts prognosis of CRC patients and enhances metastasis of CRC cells.