Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Akt - NFκB - IL-6 signalling axis

Persistent human papillomavirus (HPV) infection is the leading cause of cervical cancer. Although the fundamental link between HPV infection and oncogenesis is established, the specific mechanisms of virus-mediated transformation remain poorly understood. We previously demonstrated that the HPV encoded E6 protein increases the activity of the proto-oncogenic transcription factor STAT3 in primary human keratinocytes; however, the molecular basis for STAT3 activation in cervical cancer remains unclear. Here, we show that STAT3 phosphorylation in HPV positive cervical cancer cells is mediated primarily via autocrine activation by the pro-inflammatory cytokine Interleukin 6 (IL-6). Antibody-mediated blockade of IL-6 signalling in HPV positive cells inhibits STAT3 phosphorylation, whereas both recombinant IL-6 and conditioned media from HPV positive cells leads to increased STAT3 phosphorylation within HPV negative cervical cancer cells. Interestingly, we demonstrate that non-conventional activation of the transcription factor NFκB, involving the protein kinase Akt, is required for IL-6 production and subsequent STAT3 activation. Our data provides new insights into the molecular re-wiring of cancer cells by HPV E6. We reveal that activation of an IL-6 signalling axis drives the autocrine and paracrine phosphorylation of STAT3 within HPV positive cervical cancers cells. Greater understanding of this pathway provides a potential opportunity for the use of existing clinically approved drugs for the treatment of HPV-mediated cervical cancer. Author Summary Persistent infection with HPV is the predominant cause of anogenital and oral cancers. Transformation requires the re-wiring of signalling pathways in infected cells by virus encoded oncoproteins. At this point a comprehensive understanding of the full range of host pathways necessary for HPV-mediated carcinogenesis is still lacking. In this study we describe a signalling circuit resulting in the aberrant production of the IL-6 cytokine. Mediated by the HPV E6 oncoprotein, it requires activation of the NFκB transcription factor. The autocrine and paracrine actions of IL-6 are essential for STAT3 activation in HPV-positive cervical cancers. This study provides molecular insights into the mechanisms by which a virus encoded oncoprotein activates an oncogenic pathway, and illuminates potential targets for therapeutic intervention.

126 or CaSKi-CM induced increased STAT3 nuclear accumulation within C33A cells ( Fig   127 2C and D). These data indicate that a secreted factor in the media from HPV+ cells 128 induces STAT3 phosphorylation in target cells. 136 higher than in C33A cells (Fig 3A), with IL-6 showing the greatest increase. Building 137 on this, we analysed IL-6 mRNA expression in all six cervical cancer cell lines. In both 138 HPV16+ and HPV18+ cervical cancer cells, a significantly higher level of IL-6 mRNA 139 expression was observed compared with HPV-cervical cancer cells (Fig 3B), which 140 correlated with intracellular IL-6 protein expression when analysed by western blot 141 ( Fig 3C). Finally, to confirm that the IL-6 protein detected was secreted, an IL-6 specific 142 ELISA was performed. IL-6 was undetectable in the culture medium of HPV-cells; in 143 contrast a significant quantity of IL-6 could be detected the culture medium of both 144 HPV16+ and HPV18+ cells (SiHa, p = 0.03; CaSKi, p = 0.0004; SW756, p = 0.03; 145 HeLa, p = 0.00004). These data suggest that IL-6 expression and secretion is much 146 higher in HPV+ cervical cancer cells compared with uninfected lines.

147
148 IL-6 binding to gp130-containing receptor complexes is required for STAT3 149 phosphorylation and nuclear translocation in cervical cancer cells. IL-6 signalling 150 is initiated by an interaction between IL-6 and the IL-6 receptor (IL-6R) -gp130 co-151 receptor complex [17]. In cervical cancer cells, blocking antibodies against IL-6 and 152 gp130 were utilised to confirm that they were required for STAT3 phosphorylation.
153 Firstly, we confirmed that IL-6 was the mediator of STAT3 phosphorylation secreted 154 by HPV+ cells by pre-incubating C33A cells with the gp130 blocking antibody before 8 155 treating them with HeLa or CaSKi-CM. Separately, we added the neutralising IL-6 156 antibody to the CM before addition to cells. CM from HPV+ cells induced STAT3 157 phosphorylation in HPV-cells; however, pre-incubation of C33A cells with gp130 158 blocking antibody, or the addition of CM containing the neutralising IL-6 antibody, 159 blocked the ability of HPV+ conditioned media to induce STAT3 phosphorylation ( Fig   160 4A) and nuclear translocation (Fig 4B). This suggests that IL-6 secretion from HPV+ 161 cervical cancer cells is able to induce the paracrine activation of STAT3 in HPV-162 cervical cancer cells via IL-6/gp130 signalling.
163 Finally, to confirm that IL-6/gp130 signalling was required for the autocrine activation 164 of STAT3 in HPV+ cervical cancer cells, HeLa cells were pre-incubated with IL-6 and 165 g130 neutralizing antibodies. Incubation with either neutralising antibody led to a 166 reduction in STAT3 phosphorylation on both phosphorylation sites, accompanied by a 167 block in STAT3 nuclear translocation, suggesting that IL-6 is required for the autocrine 168 activation of STAT3 (Fig 4C and D). Taken together, these data demonstrate that HPV 169 induces autocrine and paracrine IL-6/STAT3 signalling in cervical cancer. . Therefore, the ability of E6 to induce IL-6 expression in cervical cancer cells was 176 assessed. To this end, we first transfected C33A with an IL-6 promoter luciferase 177 reporter in combination with a GFP-E6 expression plasmid or the GFP vector.
178 Expression of HPV18 E6 significantly increased IL-6 promoter activity by 4-fold 179 compared with the GFP control ( Fig 5A; p= 0.002). This corresponded to a 4-fold 11 229 230 NFB is required for STAT3 phosphorylation in E6 expressing cells.
231 As NFB was required for the increase in IL-6 expression observed in HPV+ cancer 232 cell lines, it was necessary to next test whether NFB was also required for the 233 activation of STAT3. First, we tested the ability of an inducer of NFB activation, TNFα, 234 to induce STAT3 phosphorylation. Treatment of serum starved C33A cells with TNFα 235 increased p65 phosphorylation, which peaked at 0.5 hours after treatment (Fig 7A; 236 lane 3). TNFα treatment also induced IL-6 expression; starting at 0.5 hours post 237 treatment and remained high up to 24 hours post treatment. TNFα treatment also led 238 to an increase in STAT3 phosphorylation at both Y705 and S727 residues; however, 239 this peaked later, at 2 hours ( Fig 7A; lane 5). TNFα-dependent nuclear translocation 240 of STAT could also be observed 2 hours post treatment ( Fig 7B).
241 To assess the importance of NFB activation for STAT3 phosphorylation, HPV E6 was 242 first overexpressed in C33A cells, with or without treatment with the NFB inhibitor 243 IKKi, or co-expression of IBm. E6 overexpression noticeably increased p65 and 244 STAT3 phosphorylation and inhibition of NFB using either approach led to a loss of 245 STAT3 phosphorylation (Fig 7C), Additionally, blockade of NFB also led to a 246 reduction in STAT3 phosphorylation in HeLa cells (Fig 7D and E), suggesting that E6 247 mediated STAT3 phosphorylation is depended on NFB activity. Finally, to ascertain 248 if NFB was essential for the paracrine activation of STAT3 in C33A cells, we took 249 conditioned media from HeLa cells in which NFB was inhibited and added this to 250 C33A cells. This conditioned media failed to induce STAT3 phosphorylation ( Fig 7F) 251 and nuclear translocation ( Fig 7G). Importantly, inhibition of NFB activity had no 252 effect on STAT3 phosphorylation or nuclear translocation mediated by treatment with 12 253 exogenous IL-6 (Supp Fig 2A and 2B), demonstrating that NFB is upstream of IL-6 254 secretion. Together, these data suggest that NFB is required for the autocrine and 255 paracrine activation of STAT3. 275 To confirm that the Akt-mediated increase in IL-6 was transduced via NFB, we 276 measured p65 phosphorylation levels in C33A cells expression HPV E6 treated with 277 either Akti or co-transfected with Akt-DN. We observed a loss of Akt phosphorylation, 13 278 indicating that the inhibition strategy was successful, and this was coupled with a 279 reduction in IL-6 protein expression ( Fig 8D) and a partial reduction in p65 280 phosphorylation, suggesting that Akt may act upstream of NFB in the regulation of 281 IL-6.