Sphingosine Kinase 1 Promoting Colorectal Cancer Metastasis Through Connexin 43 Upregulating Vasculogenic Mimicry Formation

Objective To investigate the expression of Sphingosine kinase 1(SphK1),and Connexin 43(Cx43)in colorectal cancer tissues and the associations between each of the proteins and pathological parameters and prognosis,as well as their inter-relationships,and to explore its role in the development of colonrectal cancer and possible molecular mechanism.Methods(1)Immunohistochemistry assay was conducted to detect the expression of SphK1 and Cx43 in 92 cases of colorectal cancer and pericarcinoma tissues.Immunohistochemistry and CD34-periodic acid-Schiff(PAS)dual staining was used to detect the VM formation.The correlation of SphK1,Cx43 and VM formation with clinicpathological parameters and overall survival of colorectal cancer patients was evaluated.Western Blotting was used to confirm the expression of SphK1 and Cx43 in 40 cases of colorectal cancer and pericarcinoma tissues.(2)Human colon cancer HT-29,HCT116 and SW620cell lines were routinely cultured in vitro.Western Blotting was used to detect the protein expression of SphK1 among them,the lowest expression of SphK1cell line was selected to transfect with lentivirus overexpressed SphK1.Stable transfectants were screened by puromycin,and the transfection efficiency was confirmed by Western Blotting.To verify the effect of overexpression of SphK1on cell function,MTT assay and colony formation assay were used to detect the proliferation of cells.Wound healing assay was used to detect the migration capability.Transwell assay was used to detect the ability of cell migration and invasion.Western blotting was used to detect the protein expression of SphK1,Cx43,ZEB-1,AKT and p-AKT(Ser473).(3)The PI3K/AKT inhibitor LY294002 was used to inhibit the AKT activity,the changes of cell proliferative and movement and the related proteins were examined using the methods above.Results(1)Immunohistochemistry results show:SphK1 was mainly localized to the cytoplasm,and was highly expressed in colorectal cancer tissues.The positive expression rate of colorectal cancer samples was 85.87%(79/92),more than that of the pericarcinoma tissues 33.70%(31/92),showing a significant statistical difference(?~2=52.081,P=0.000).Overexpression of SphK1in colorectal cancer were all related with differentiation(P=0.041),TNM staging(P=0.009)and lymph node metastasis(P=0.033).It is worth noting that there's no statistically significant difference of SphK1 expression in distant metastasis(?~2=13.525,P=0.142),but in all 17 colorectal cancer tissues with distant metastasis the expression of SphK1 was all positive.However,in previous studies of our research group,it was found that SphK1 expression was significantly associated with distant metastasis.The difference owed to the difference of selected samples.Cx43 localized to the plasma membrane and cytoplasm,and some of them strongly expression forming particles.Cx43showed low expression in colorectal cancer tissues,and the positive expression rate was 58.70%(54/92),less then that of pericarcinoma tissues(92.39%(85/92)).There was only differentiation has statistically significant of Cx43 in colorectal cancer samples(?~2=13.525,P=0.001).While the age,gender and tumor volume,TNM stage,lymph node metastasis,invasion depth,and distant metastasis were irrelevant(all P>0.05).Patients with SphK1 positive tumors tended to have decreased overall survival,while there's no significant correlation of Cx43.There was a significantly correlation between SphK1 and Cx43 expression levels(r=0.595,P=0.000).Western Blotting results show that SphK1 was overexpression in colorectal cancer while Cx43 expression is lower than pericarcarcinoma tissues.Vasculogenic mimicry(VM)is an alternative microvascular system which tumor cells orchestrate,independent of endothelial cell-mediated angiogenesis.There were 29 patients with VM,the positive rate is31.52%,and VM is correlated with TNM stage,lymph node metastasis and short survival time(all P<0.05),no statistically relation of age,gender,differentiation,tumor size,invasive depth,and distant metastasis(all P>0.05);VM was significantly associated with SphK1 and Cx43 protein expression(P=0.020 and 0.023,respectively).(2)Vitro tests'results show that the expression levels of SphK1 were gradually increased in three colon cancer cell lines HT-29,HCT116 and SW620,HT-29 expressed the lowest level of SphK1;then HT-29 cells was selected to transfect with the lentivirus overexpressed of SphK1,stably transfected strains was obtained by staining with 0.5?g/L puromycin for 14 days.The expression of SphK1 protein after transfected with SphK1 was significantly increased by Western Blotting,indicating successful construction of a stable overexpressing SphK1 HT-29 cell line,named SphK1strain;cell lines transfected with negative control were named Vector strain;the untransfected cell strain was named HT-29 strain.(3)MTT results showed that the proliferative capacity of SphK1 cells was significantly enhanced compared with HT-29 cells.SphK1 cells showed stronger colony formation than HT-29cells.The migration and invasion capabilities of SphK1 cells were stronger than that of HT-29 cells.HT-29 cells did not express Cx43 while overexpression of SphK1 promoted the expression of Cx43 and formed a large number of gap junction plaques.The protein expression of AKT in both cells did not change significantly.However,the protein expression levels of p-AKT(Ser473),ZEB-1,and Cx43 were significantly higher in SphK1 cells than in HT-29 cells.(4)After inhibition of AKT activity in both SphK1 and HT-29 cells using the PI3K inhibitor LY294002(50?mol/L),the proliferative capacity of both cells was found to be significantly weaken than before intervention.It was difficult to form clones,and the clones were smaller in size and had looser structure;there was no significant difference in the proliferation and colony forming ability of the two cells.The same results was also found in the migration and invasion assay.The formation of gap junction plaques was also reduced.There was no significant difference in the protein expression levels of AKT between the two cells,but the protein expression levels of p-AKT(Ser473),ZEB-1 and Cx43were significantly lower than those of the non-intervention group.Conclusions 1.SphK1 and Cx43 may be closely related to the occurrence and development of colon cancer,and may participate in the invasion and metastasis of colon cancer by promoting the formation of vasculogenic mimicry.2.SphK1 can increase the malignant phenotype of HT-29 cells and promote the proliferation,migration and invasion of HT-29 cells.It may due to promote the formation of vasculogenic mimicr by increasing the gap junction splaques.3.SphK1 may regulate the invasion and metastasis of human colorectal cancer in vitro through the AKT/ZEB-1/Cx43 signaling pathway.