CDK5RAP3 Inhibits Angiogenesis In Gastric Neuroendocrine Carcinoma By Modulating AKT/HIF-1?/VEGFA Signaling

Objective: Gastric neuroendocrine carcinoma(GNEC)is a uncommon gastric malignancy that is characterized of rich blood vessels,high malignancy,high invasiveness and easy metastasis,therefore,the clinical treatment effect is not good.Angiogenesis is closely related to the progression and metastasis of malignant tumors and is an important process in the development of malignant tumors.Tumor vascular targeted therapy can significantly inhibit the progression of malignant tumors and is a new strategy for tumor therapy.Research on the mechanism of tumor angiogenesis helps to discover new therapeutic targets.Cyclin-dependent kinase 5 activation of binding protein(CDK5RAP3)has a large difference in the expression of malignant tumor tissues,and both cancer-promoting and tumor-suppressing effects have been reported.Our previous study found that CDK5RAP3 was low expressed in gastric cancer tissues and could inhibit the progression of gastric cancer through the Wnt/-catenin signal pathway.However,the role of CDK5RAP3 in GNEC has not been reported.This study is to investigate the effects and mechanism of CDK5RAP3 in angiogenesis of gastric neuroendocrine carcinoma,providing new candidate targets and interventions for the treatment of gastric neuroendocrine cancer.Methods: Immunohistochemistry(IHC)was used to assess the expression of CDK5RAP3 in GNEC tissues and adjacent non-tumor tissues,and its correlation with the clinical and pathological parameters and long-term prognosis of patients was analyzed.IHC was used to detect CD31(vascular endothelial marker)and vascular endothelial growth factor A(VEGFA)in gastric neuroendocrine carcinoma to evaluate the expression of microvascular density and VEGFA.Cell lines with stable overexpression or knockdown of CDK5RAP3 were constructed using lentiviral transfection.Human umbilical vein endothelial cells(HUVECs)wound-healing assays,invasion and metastasis assays,tube formation assays were performed to investigate the effect of overexpression or knockdown of CDK5RAP3 in GNEC cells on vascular endothelial cell movement and tube formation.Tumor xenograft transplantation assays and subcutaneous tumor tissues IHC were performed to investigate the effect of CDK5RAP3 on tumor growth and angiogenesis in vivo.Real-time PCR(RT-PCR),ELISA,Western Blot analysis,and confocal-immunofluorescence staining were used to explore the molecular mechanism of CDK5RAP3 affect the angiogenesis.Results: 1.Compared with their respective adjacent non-tumor tissues,protein levels of CDK5RAP3 were significantly decreased in GNEC tissues(p<0.05).Furthermore,low expression of CDK5RAP3 was correlated with more advanced TNM stage,increased tumor microvessel density(MVD),and poor prognosis.2.In vitro assays showed that CDK5RAP3 overexpression in GNEC cells indirectly inhibited HUVECs migration and tube formation.3.CDK5RAP3 down-regulated AKT/ HIF-1?/VEGFA signaling pathway,and decreased the protein synthesis and secretion of VEGFA.4.Tumor xenograft transplantation assays found that CDK5RAP3 inhibited the growth of subcutaneous tumors and tumor angiogenesis in vivo.5.In GNEC tissues,CDK5RAP3 expression was negatively correlated with VEGFA expression.Patients with low expression of CDK5RAP3 and high expression of VEGFA had the worst prognosis.Patients with high expression of CDK5RAP3 and low expression of VEGFA had the best prognosis.Conclusions: This study found that CDK5RAP3 inhibited angiogenesis by downregulating AKT/HIF-1?/VEGFA signaling in GNEC,reducing synthesis and secretion of VEGFA.Combined analysis of CDK5RAP3 and VEGFA could more accurately assess the prognosis of patients,and targeting CDK5RAP3 and VEGFA signaling was expected to be a new therapeutic strategy for GNEC.