Roles of PI3K/Akt and c-Jun Signaling Pathways in Human Papillomavirus Type 16 Oncoprotein-Induced HIF-1α, VEGF, and IL-8 Expression and In Vitro Angiogenesis in Non-Small Cell Lung Cancer Cells

Background and Objectives: Human papillomavirus (HPV)-16 infection may be related to non-smoking associated lung cancer. Our previous studies have found that HPV-16 oncoproteins promoted angiogenesis via enhancing hypoxia-inducible factor-1 alpha (HIF-1 alpha), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) expression in non-small cell lung cancer (NSCLC) cells. In this study, we further investigated the roles of PI3K/Akt and c-Jun signaling pathways in it. Methods: Human NSCLC cell lines, A549 and NCI-H460, were stably transfected with pEGFP-16 E6 or E7 plasmids. Western blotting was performed to analyze the expression of HIF-1 alpha, p-Akt, p-P70S6K, p-P85S6K, p-mTOR, p-JNK, and p-c-Jun proteins. VEGF and IL-8 protein secretion and mRNA levels were determined by ELISA and Real-time PCR, respectively. The in vitro angiogenesis was observed by human umbilical vein endothelial cells (HUVECs) tube formation assay. Co-immunoprecipitation was performed to analyze the interaction between c-Jun and HIF-1 alpha. Results: HPV-16 E6 and E7 oncoproteins promoted the activation of Akt, P70S6K, P85S6K, mTOR, JNK, and c-Jun. LY294002, a PI3K inhibitor, inhibited HPV-16 oncoprotein-induced activation of Akt, P70S6K, and P85S6K, expression of HIF-1 alpha, VEGF, and IL-8, and in vitro angiogenesis. c-Jun knockdown by specific siRNA abolished HPV-16 oncoprotein-induced HIF-1 alpha, VEGF, and IL-8 expression and in vitro angiogenesis. Additionally, HPV-16 oncoproteins promoted HIF-1 alpha protein stability via blocking proteasome degradation pathway, but c-Jun knockdown abrogated this effect. Furthermore, HPV-16 oncoproteins increased the quantity of c-Jun binding to HIF-1 alpha. Conclusions: PI3K/Akt signaling pathway and c-Jun are involved in HPV-16 oncoprotein-induced HIF-1 alpha, VEGF, and IL-8 expression and in vitro angiogenesis. Moreover, HPV-16 oncoproteins promoted HIF-1 alpha protein stability possibly through enhancing the interaction between c-Jun and HIF-1 alpha, thus making a contribution to angiogenesis in NSCLC cells.