Re-expression of HPV16 E2 in SiHa (human cervical cancer) cells potentiates NF-κB activation induced by TNF-α concurrently increasing senescence and survival.

Re-expression of E2 in human papillomavirus (HPV) transformed tumour cells can induce apoptosis; however, some evidences also attribute an important role to E2 in sustaining tumorigenesis. In the present paper, we studied the effects of tumour necrosis factor (TNF)-α-mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) activation on E2-induced senescence in HPV16-integrated SiHa cells. The results show that E2 inhibits endogenous E6 gene expression and sensitizes SiHa cells to TNF-α-induced NF-κB activation. Under this condition there was an increase in the expression of senescent proteins p53, p21, p27 and p16 and senescence-associated (SA)-ß-galactosidase activity indicating that TNF-α augments E2-mediated senescence. Re-expression of E2 expression with TNF-α treatment resulted in an increase in the expression of anti-apoptotic Bcl2 (B-cell lymphoma 2) protein and other pro-survival genes like cyclin D1 (cyc D1), survivin and hTERT (human telomerase reverse transcriptase). Concomitantly, E2 + TNF-α combination increased the survival of SiHa cells by positive changes in viability, proliferation and colony formation. E2-induced apoptotic tendency shifted towards senescence in presence of TNF-α by arresting the cells at both G0/G1 and G2/M phases, thus enhancing cell survival. Another observation in the present study is the significant up-regulation of key senescence messaging factors regulated by NF-κB namely interleukin (IL)-6, IL-8, high-mobility group protein A (HMGA)1 and B (HMGB)1 in E2-transfected cells treated with TNF-α. Our data provide a mechanistic basis and a new insight for the role of TNF-α and E2 in linking cellular senescence, tumorigenesis and HPV re-infection.

HPV16 E2 enhances the expression of NF-κB and STAT3 target genes and potentiates NF-κB activation by inflammatory mediators.

HPV-transformed cells exhibit activation of NF-κB and STAT3 (mediators of inflammation), but very little is known about their regulation under inflammatory conditions before HPV integration. This study reports that cervical tissues with stromal inflammation and intact HPV16 E2 gene show increased expression of target genes of NF-κB and/or STAT3 which can regulate cell survival (cyclin D1, c-Myc, survivin and Bcl2) and inflammatory responses (TNF-α, IL-1ß, IL-6, IL-8 and CCR2). Increased expression of RelA, p-IκBα, STAT3, p-STAT3 (Ser727), Pin1 (peptidyl-prolyl cis/trans isomerase) and MCM2 in the squamous epithelia of cervices with stromal inflammation supports early activation of NF-κB-STAT3. Furthermore, HPV16 E2 potentiated NF-κB activation induced by inflammatory mediators, IL-1ß and SDF-1α, in HEK293 cells. These results reveal a novel role for E2 in regulating the activities of NF-κB and STAT3 that may have implications in carcinogenic progression of HPV16-infected cells under conditions of stromal inflammation.

HPV16 E2-mediated potentiation of NF-κB activation induced by TNF-α involves parallel activation of STAT3 with a reduction in E2-induced apoptosis.

Human papilloma virus is associated with cervical and other tumors, and several cellular conditions also play an important role in carcinogenesis. Human papilloma virus (HPV)-infected cells exhibit activation of NF-κB and STAT3 (mediators of inflammation), but little is known about their regulation by HPV. This study attempts to understand the role of HPV16 E2, an important early protein of HPV16, in the regulation of NF-κB and STAT3 by reporter assays, quantitative reverse transcriptase-polymerase chain reaction, and immunoblotting. We demonstrate that E2 enhances NF-κB activation induced by TNF-α, a proinflammatory cytokine, in both non-tumor- and tumor-derived epithelial cell lines besides potentiating STAT3 transcriptional activity induced by TNF-α in HEK293 cells. E2 increases the expression of RelA and its transcriptional activation, and retention of E2 was observed in the nucleus with significant interaction with RelA (immunoprecipitation) upon TNF-α treatment. Transfection with shRNA-RelA or pretreatment with a STAT3 inhibitor had a negative effect on the ability of E2 to enhance TNF-α-induced NF-κB activation. Experiments with co-expression of a mutant of STAT3 with E2 also suggested that the activation of STAT3 is indispensible for TNF-α-induced NF-κB activation. Inhibition of STAT3 activation enhanced E2-induced apoptosis, whereas parallel activation of NF-κB and STAT3 by the combined action of E2 and TNF-α increased the expression of their common targets, cyclin-D1, c-Myc, survivin, and Bcl-2, leading to a decrease in E2-induced apoptosis (viability and cell cycle). Our results reveal novel mechanisms by which E2 may regulate NF-κB and STAT3 activation in the presence of TNF-α with implications on the survival of HPV-infected cells.