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STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

ABSTRACT: Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle.

SUBMITTER: Morgan EL

PROVIDER: S-EPMC5908086 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

Morgan Ethan L EL Wasson Christopher W CW Hanson Lucy L Kealy David D Pentland Ieisha I McGuire Victoria V Scarpini Cinzia C Coleman Nicholas N Arthur J Simon C JSC Parish Joanna L JL Roberts Sally S Macdonald Andrew A

PLoS pathogens 20180409 4

Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undiffe ...[more]

PMID: 29630659

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Project description:BackgroundHuman Papillomavirus type 18 (HPV18) is a high-risk HPV that is commonly associated with cervical cancer. HPV18 oncogenes E6 and E7 are associated with the malignant transformation of cells, thus the identification of human leukocyte antigen (HLA)-restricted E6/E7 peptide-specific CD8 + T cell epitopes and the creation of a HPV18 E6/E7 expressing cervicovaginal tumor in HLA-A2 transgenic mice will be significant for vaccine development.MethodsIn the below study, we characterized various human HLA class I-restricted HPV18 E6 and E7-specific CD8 + T cells mediated immune responses in HLA class I transgenic mice using DNA vaccines encoding HPV18E6 and HPV18E7. We then confirmed HLA-restricted E6/E7 specific CD8 + T cell epitopes using splenocytes from vaccinated mice stimulated with HPV18E6/E7 peptides. Furthermore, we used oncogenic DNA plasmids encoding HPV18E7E6(delD70), luciferase, cMyc, and AKT to create a spontaneous cervicovaginal carcinoma model in HLA-A2 transgenic mice.ResultsTherapeutic HPV18 E7 DNA vaccination did not elicit any significant CD8 + T cell response in HLA-A1, HLA-24, HLA-B7, HLA-B44 transgenic or wild type C57BL/6 mice, but it did generate a strong HLA-A2 and HLA-A11 restricted HPV18E7-specific CD8 + T cell immune response. We found that a single deletion of aspartic acid (D) at location 70 in HPV18E6 DNA abolishes the presentation of HPV18 E6 peptide (aa67-75) by murine MHC class I. We found that the DNA vaccine with this mutant HPV18 E6 generated E6-specific CD8 + T cells in HLA-A2. HLA-A11, HLA-A24 and HLA-b40 transgenic mice. Of note, HLA-A2 restricted, HPV18 E7 peptide (aa7-15)- and HPV18 E6 peptide (aa97-105)-specific epitopes are endogenously processed by HPV18 positive Hela-AAD (HLA-A*0201/Dd) cells. Finally, we found that injection of DNA plasmids encoding HPV18E7E6(delD70), AKT, cMyc, and SB100 can result in the development of adenosquamous carcinoma in the cervicovaginal tract of HLA-A2 transgenic mice.ConclusionsWe characterized various human HLA class I-restricted HPV18 E6/E7 peptide specific CD8 + T cell epitopes in human HLA class I transgenic mice. We demonstrated that HPV18 positive Hela cells expressing chimeric HLA-A2 (AAD) do present both HLA-A2-restricted HPV18 E7 (aa7-15)- and HPV18 E6 (aa97-105)-specific CD8 + T cell epitopes. A mutant HPV18E6 that had a single deletion at location 70 obliterates the E6 presentation by murine MHC class I and remains oncogenic. The identification of these human MHC restricted HPV antigen specific epitopes as well as the HPV18E6/E7 expressing adenosquamous cell carcinoma model may have significant future translational potential.

Project description:Keratinocyte infection with high-risk human papillomavirus genotypes has been linked to cancer development. In cervix, the alpha HPV16 and HPV18 have been reported as the mayor causative agents of cervical cancer. Oncogenic progression and chronic inflammation are closely related processes, with IL-6 as one of the main pro-inflammatory cytokines involved. However, there are limited studies about the regulation of IL-6 by low and high risk HPVs and the HPV proteins implicated in this modulation. In this work, we report the overexpression of IL-6 in HPV infected cervical cancer derived cell lines (HeLa and SiHa) compared to non-tumorigenic keratinocytes (HaCaT), and in Cervical Intraepithelial Neoplasia grade 1 HPV16 and HPV18 positive cervical samples compared to HPV negative samples without lesions. Moreover, we generated HaCaT keratinocytes that express E5, E6, and E7 from high risk (16 or 18) or low risk (62 and 84) HPVs. E5 proteins do not modify IL-6 expression, while E7 modestly increase it. Interestingly, E6 proteins in HaCaT cells upregulate IL-6 mRNA expression and protein secretion. Indeed, in HaCaT cells that express high risk HPV16E6 or HPV18E6 proteins, only the truncated E6* isoforms were expressed, showing the stronger IL-6 overexpression, while in HaCaT cells that express low risk HPV62 and HPV84 full length E6 proteins, IL-6 was also upregulated but not so drastically. Since HaCaT cells have a mutated p53 form that is not degraded by the introduction of E6 or E6/E7, it seems that E6/E7 regulate IL-6 by an additional mechanism independent of p53. In addition, basal keratinocytes showed a strong expression of IL-6R using immunohistochemistry, suggesting an autocrine mechanism over proliferative cells. Altogether, IL-6 cytokine expression in keratinocytes is upregulated by E6 and E7 proteins from HPVs 16, 18, 62, and 84, especially by high risk HPV16 and HPV18 E6*, which may contribute to promote a pro-inflammatory and highly proliferative microenvironment that can persist over time and lead to cervical tumorigenesis.

Dataset Information

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

Publications

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

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