Overexpression of Human Papillomavirus Type 16 Oncoproteins Enhances Hypoxia-Inducible Factor 1α Protein Accumulation and Vascular Endothelial Growth Factor Expression in Human Cervical Carcinoma Cells

Abstract

Purpose: Human papillomavirus (HPV)-16 oncoproteins, E6 and E7, are associated with enhanced tumor angiogenesis in human cervical cancers. The purpose of this study was (a) to investigate whether expression of HPV-16 E6 and E7 oncoproteins induces hypoxia-inducible factor 1a (HIF-1a) and vascular endothelial growth factor expression in cervical cancer cells; and (b) to assess the effect of resveratrol on 16 E6-and E7-induced HIF-1a and VEGF gene expression. Experimental Design: Human cervical cancer cell lines C-33A and HeLa were transiently cotransfected with pSG5-HPV-16 E6 or 16 E7 constructs along with HIF-1a small interfering RNA (siRNA) or nonspecific siRNA. The expression of HIF-1a/VEGF was measured using realtime PCR, Western blot analysis, or ELISA. The in vitro angiogenic activity induced by 16 E6-and E7-transfected cells was examined. The effect of resveratrol on oncoprotein-induced HIF-1a/VEGF expression and in vitro angiogenesis was investigated. Results: HPV-16 E6-and E7-transfected cervical cancer cells express increased HIF-1a protein andVEGF expression.These stimulatory effects were abrogated by cotransfection with either HIF1a siRNA or treatment with resveratrol. Blocking extracellular signal-regulated kinase 1/2 (ERK 1/ 2) and phosphoinositide-3-kinase by PD98059 and LY294002, respectively, abolished 16 E6-and E7-induced HIF-1aand VEGF expression. Functionally, we showed that HPV-16 E6-and E7-transfected cervical cancer cells stimulated in vitro capillary or tubule formation, and these angiogenic effects could be abolished either by cotransfection with HIF-1asiRNA or by treatment with resveratrol. Conclusion: HPV-16 oncoproteins contribute to enhanced angiogenesis in cervical cancer cells via HIF-1a^dependentVEGF expression. Resveratrol suppresses 16 E6-and E7-induced HIF-1am ediated angiogenic activity and, thus, is a promising chemotherapeutic agent for human cervical cancer.

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

“…A few studies have shown that HPV E6/E7 could regulate VEGF expression. [36][37][38] However, the current result indicates that VEGF may also modulate HPV E7 expression.…”

HPV-16 E6 upregulation of DNMT1 through repression of tumor suppressor p53

“…1). The tumor suppressor p53 is one of the most wellcharacterized target genes of HPV-16 E6 (8,30,31) besides the PDZ domain containing proteins (25), human telomerase reverse transcriptase (hTERT) (32), vascular endothelial growth factor (VEGF) (33,34), and hypoxia-inducible factor 1· (HIF-1· ) (34). p53 was recently reported to repress the DNMT1 level in human colon carcinoma HCT116 cells (19).…”

BC-box protein domain-related mechanism for VHL protein degradation

“…IFN-induced HIF 1α stabilization, as VHL loss, could cooperate in organizing host defenses and have cellular consequences that in some cases can inhibit viral replication (49,50). However, in other instances of persistent viral infection, HIF-1 activation can lead to the activation of the proangiogenic program and oncogenic transformation as a consequence (11,(51)(52)(53)(54). Therefore, considering the primary role of HIF 1α in immune defense, it is not surprising that cells have evolved auxillary mechanisms to rapidly sustain the stabilization of HIF 1α, for instance through the degradation of VHL protein.…”