Tumor-secreted Dickkopf2 Accelerates Aerobic Glycolysis And Promotes Angiogenesis In Colorectal Cancer

Background and Objective:Colorectal cancer(CRC)is the third most common malignant tumor in the world,and tumor angiogenesis is an important factor affecting tumorigenesis and metastasis.Angiogenesis is a fundamental process that involves in tumor progression and metastasis.Vascular endothelial growth factor(VEGF)family and their receptors are identified as the most prominent regulators of angiogenesis.However,the clinical efficacy of anti-VEGF/VEGFR therapy is not ideal,prompting the needs to further understand mechanisms behind tumor angiogenesis.Here,we found that Dickkopf associated protein2(DKK2),a secretory protein highly expressed in metastatic colorectal cancer tissues,could stimulate angiogenesis via a classic VEGF/VEGFR independent pathway.Method:DKK2 was screened out from microarray data analyzing gene expression profiles of eight pairs of non-metastatic and metastatic human colorectal cancer(CRC)tissues.and the expression level of DKK2 in fresh colorectal cancer samples was detected by fluorescence quantitative PCR(qRT-PCR)and western blot.Immunofluorescence histochemical staining of(IHC)was used to further detect the expression of DKK2 in CRC tissues,and the relationship between the expression and clinicopathological parameters was analyzed.By up-regulating or interfering with the expression of DKK2 in colorectal cancer cells,CCK8,scratch healing,Transwell migration test and subcutaneous tumor model in nude mice were used to observe the effect of DKK2 on the growth and metastasis of colorectal cancer cells in vivo and in vitro.Immunofluorescence histochemical staining(IHC)was used to detect the expression of DKK2 and angiogenesis in CRC tissues.Chicken chorioallantoic membrane(CAM)assay and Human umbilical vein endothelial cells(HUVEC)tubule formation assay was used for in vitro and in vivo angiogenesis study,respectively.Lactate and glucose concentration in the culture medium was measured by enzyme-linked immunosorbent assay(ELISA).The interaction between DKK2 and LRP6 was verified by co-immunoprecipitation experiment and immunofluorescence confocal experiment.Bioinformatics,double luciferase reporting test,western blot and immunohistochemical(IHC)were used to verify the 3'UTR binding of miR-493-5p and DKK2 to regulate tumor-related signaling pathways.Results:The expression of DKK2 in colorectal cancer tissues and cell lines was significantly higher than that in adjacent tissues and normal colonic mucosal epithelial cell lines NCM460,and its expression level was actively correlated with tumor size,lymph node metastasis,distant metastasis and TNM stage of colorectal cancer.DKK2 could stimulate angiogenesis via accelerating the aerobic glycolysis of CRC cells,through which lactate is produced from glucose and accumulated in tumor microenvironment.Lactate functions as the final executor of DKK2 to stimulate tube formation of endothelial cells,and blockage of lactate secretion by lactate transporter(MCT)inhibitors dramatically neutralize the progression and metastasis of CRC both in vitro and in vivo.DKK2 could cooperate with lipoprotein receptor-related protein6,which is a protein associated with WNT signal receptor,and activated the downstream mTOR signal pathway to accelerate lactate secretion.In addition,the expression of DKK2 is switched on via the methylation of MiR-493-5p,initiates its stimulatory role on CRC progression in an autocrine or paracrine manner.Conclusion:DKK2 promotes tumor metastasis and angiogenesis through a novel VEGF-independent,but energy metabolism related pathway.DKK2 might be a potential anti-angiogenic target in clinical treatment for the advanced CRC patients.