ERK Signaling Pathway Is Involved in HPV-16 E6 but not E7 Oncoprotein-Induced HIF-1α Protein Accumulation in NSCLC Cells

Abstract

Extracellular signal-regulated kinase (ERK)1/2 signaling pathway plays a critical role in regulating tumor angiogenesis. Our previous studies have demonstrated that HPV-16 oncoproteins enhanced hypoxia-inducible factor-1α (HIF-1α) protein accumulation and vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) expression in non-small cell lung cancer (NSCLC) cells, thus contributing to angiogenesis. In this study, we further investigated the role of ERK1/2 signaling pathway in HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis in NSCLC cells. Our results showed that HPV-16 E6 and HPV-16 E7 oncoproteins promoted the activation of ERK1/2 signaling pathway in A549 and NCI-H460 cells. Moreover, PD98059, a specific inhibitor of ERK1/2, blocked in vitro angiogenesis stimulated by HPV-16 E6 but not E7 oncoprotein. Additionally, HIF-1α protein accumulation and VEGF and IL-8 expression in NSCLC cells induced by HPV-16 E6 but not E7 oncoprotein were significantly inhibited by PD98059. Taken together, our results suggest that ERK1/2 signaling pathway is involved in HPV-16 E6 but not E7 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression in NSCLC cells, leading to the enhanced angiogenesis in vitro.

Figure 1

Overexpression of HPV-16 oncoproteins promoted the activation of ERK1/2 signaling pathway in NSCLC cells. (A) The green fluorescence signals derived from stable-transfected A549 and NCI-H460 cells were observed under a fluorescence microscope (10×). (B) p-ERK protein levels were determined by Western blot analysis in stable-transfected A549 and NCI-H460 cells.

Figure 2

PD98059 blocked HPV-16 E6 but not E7 oncoprotein-induced angiogenesis in vitro. NSCLC cells overexpressing HPV-16 oncoproteins were treated with different concentrations of PD98059 (5 µg/ml, 50 µg/ml) for 24 h. Left: the formation of capillary tube was observed under a phase-contrast microscope (20×). Right: the quantification of the total tube length pixel value in three random view fields per well. All data are presented as mean ± SD of three separate experiments. *p < 0.05.

Figure 3

PD98059 inhibited HPV-16 E6 but not E7 oncoprotein-induced HIF-1α protein accumulation in NSCLC cells. Stable-transfected A549 and NCI-H460 cells overexpressing HPV-16 E6 or HPV-16 E7 oncoprotein were pretreated with different concentrations of PD98059 for 24 h. Western blot analysis was performed to detect the expression of HIF-1α protein in NSCLC cells. (A and B) HIF-1α protein levels in NSCLC cells overexpressing HPV-16 E6 oncoprotein. (C and D) HIF-1α protein levels in NSCLC cells overexpressing HPV-16 E7 oncoprotein.

Figure 4

PD98059 inhibited HPV-16 E6 but not E7 oncoprotein-induced VEGF and IL-8 protein secretion. A549 cells, transfected with HPV-16 E6 or HPV-16 E7, were exposed to various concentrations of PD98059 for 24 h. (A and B) VEGF (A) and IL-8 (B) protein concentration in the conditioned media derived from A549 cells overexpressing HPV-16 E6 oncoprotein was determined by ELISA. (C and D) VEGF (C) and IL-8 (D) protein concentration in the conditioned media derived from A549 cells overexpressing HPV-16 E7 oncoprotein was determined by ELISA. *p < 0.05.

Figure 5

PD98059 downregulated the mRNA levels of VEGF and IL-8 induced by HPV-16 E6 but not E7 oncoprotein. Real-time PCR was performed to determine VEGF and IL-8 mRNA levels in transfected A549 cells treated with different concentrations of PD98059. (A and B) The mRNA levels of VEGF (A) and IL-8 (B) in A549 cells overexpressing HPV-16 E6 oncoprotein. (C and D) The mRNA levels of VEGF (C) and IL-8 (D) in A549 cells overexpressing HPV-16 E7 oncoprotein. *p < 0.05.