1. Institute of Biochemistry and Molecular Biology, Guangdong Medical University, Zhanjiang 524023, P.R. China
2. Collaborative innovation center for antitumor active substance research and development, Guangdong Medical University, Zhanjiang 524023, P.R. China
3. Dongguan Key Laboratory of Medical Bioactive Molecular Developmental and Translational Research, Guangdong Medical University, Dongguan 523808, P.R. China
4. Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University, Dongguan 523808, P.R. China.
* These authors contribute equally to this work.
Background: Our previous studies have demonstrated that human papillomaviruse (HPV)-16 oncoproteins promoted epithelial-mesenchymal transition (EMT), leading to non-small cell lung cancer (NSCLC) progression, but the underlying molecular mechanisms still remain unclear. PI3K/Akt/HIF-1α signaling pathway has been reported to mediate hypoxia-induced EMT. In this study, we further explored the role of PI3K/Akt/HIF-1α signaling pathway in HPV-16 oncoprotein-induced EMT in NSCLC cells.
Methods: A549 and NCI-H460 NSCLC cells were transiently transfected with pEGFP-HPV-16 E6 or E7 constructs. Western blotting and RT-qPCR were respectively performed to determine the protein and mRNA expression of EMT-related transcription factors. HPV-16 E6 or E7-transfected NSCLC cells were co-transfected with specific HIF-1α-siRNA or pretreated with different concentrations of LY294002, a specific PI3K inhibitor, followed by the analysis of expression of EMT-related transcription factors. The correlation between HIF-1α and EMT-related transcription factors in NSCLC tissues was analyzed by immunohistochemical staining and Spearman rank correlation coefficient.
Results: HPV-16 E6 and E7 oncoproteins upregulated the expression of Slug and Twist1, the EMT-related transcription factors, at both protein and mRNA levels in A549 and NCI-H460 cells. The co-transfection with specific HIF-1α-siRNA, but not the non-specific (NS)-siRNA, significantly abrogated HPV-16 oncoprotein-induced upregulation of ZEB1, Snail1, Slug, and Twist1 at both protein and mRNA levels. Additionally, pretreatment with LY294002 obviously blocked HPV-16 E6- and E7-induced Snail1, Slug, and Twist1 protein expression in A549 and NCI-H460 cells. Further analysis of clinical specimens showed that HIF-1α protein was strongly expressed in NSCLC tissues, which was positively correlated with ZEB1, Snail1, Slug, and Twist1 protein expression.
Conclusions: PI3K/Akt/HIF-1α may contribute to the progression of HPV-associated NSCLC via mediating the expression of EMT-related transcription factors in NSCLC cells.
Keywords: PI3K/Akt/HIF-1α, human papillomaviruse (HPV)-16, non-small cell lung cancer (NSCLC), epithelial-mesenchymal transition (EMT), transcription factor