1. Cancer Research Center, School of Medicine, Xiamen University, Xiamen, Fujian, China.
2. Department of Pathology, The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China.
3. Department of Gastrointestinal Surgery, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian, China.
4. Department of Pathology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian, China.
5. Organ Transplantation Institute of Xiamen University, Fujian Provincial Key Laboratory of Organ and Tissue Regeneration, School of Medicine, Xiamen University, Xiamen, Fujian, China.
*These authors contributed equally to this work
Background: Metastasis is the leading cause of death in colorectal cancer (CRC) patients. It is regulated mainly by tumor cell angiogenesis, and angiogenesis is caused by the binding of vascular endothelial growth factor (VEGF) to vascular endothelial growth factor receptor 2 (VEGFR2). Tumor necrosis factor-α-induced protein 8 (TNFAIP8, hereto after TIPE) plays an important role in tumorigenesis, development, and prognosis. However, the relationship between TIPE and VEGFR2 in CRC angiogenesis and the mechanism of action remain unknown.
Method: In this study, we used quantitative real-time PCR, Western blotting and immunohistochemistry to detect TIPE and VEGFR2 expression in 55 specimens from CRC patients. We also used HCT116 CRC cells and human umbilical vein endothelial cells (HUVECs) for in vitro experiments by stably transducing shTIPE and shRNA control lentivirus into HCT116 cells, detecting VEGFR2 expression after TIPE knockdown and repurposing the culture supernatant as conditioned medium to stimulate angiogenesis of HUVECs. In vivo experiments with chicken chorioallantoic membranes (CAMs) and a nude mouse matrix subcutaneous tumor model were performed to validate the effects of TIPE on angiogenesis. Additionally, we analyzed the expression and phosphorylation levels of PDK1 and blocked PDK1 expression using inhibitors to determine whether TIPE-induced changes in VEGFR2-mediated angiogenesis acted via the PI3K-Akt pathway.
Results: We found that TIPE and VEGFR2 are highly expressed in CRC and act as oncogenes. TIPE knockdown also downregulated VEGFR2 expression, which resulted in simultaneous inhibition of cell proliferation, cell migration and angiogenesis. Then, in vivo experiments further demonstrated that TIPE promotes angiogenesis in CRC. Finally, we found that TIPE promotes VEGFR2-mediated angiogenesis by upregulating PDK1 expression and phosphorylation and that blocking PDK1 expression can inhibit this process.
Conclusion: TIPE promotes angiogenesis in CRC by regulating the expression of VEGFR2, which may be a target for antiangiogenic cancer therapy.
Keywords: CRC, TIPE, angiogenesis, VEGFR2, PDK1, PI3K-Akt