STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.
STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.
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DOI：
10.1371/journal.ppat.1006975
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发表时间：
2018-04
期刊：
影响因子：
6.7
通讯作者：
Macdonald A
中科院分区：
文献类型：
--
作者：
Morgan EL;Wasson CW;Hanson L;Kealy D;Pentland I;McGuire V;Scarpini C;Coleman N;Arthur JSC;Parish JL;Roberts S;Macdonald A
人乳头瘤病毒（HPV）激活许多宿主因子来控制其分化依赖的生命周期。转录因子信号转导和转录激活因子(STAT)-3在细胞周期的进展和细胞对细胞因子和生长因子的反应中起重要作用。STAT3除了需要Tyr705磷酸化外，还需要Ser727磷酸化才能具有转录活性。在这项研究中，我们发现STAT3对于未分化和分化角质形成细胞的HPV生命周期至关重要。与正常角质形成细胞相比，含有高风险HPV18基因组的原代人角质形成细胞显示出增强的STAT3磷酸化。E6癌蛋白的表达足以诱导STAT3在Ser727和Tyr705位点的双重磷酸化，其机制需要Janus激酶和MAPK家族成员。e6介导的STAT3激活诱导STAT3应答基因的转录，包括cyclin D1和Bcl-xL。通过siRNA沉默STAT3蛋白表达或通过小分子抑制剂抑制STAT3激活，或通过表达显性STAT3阴性磷酸化位点突变体，导致细胞周期进程受阻。缺乏活性STAT3会损害未分化角质形成细胞中HPV基因的表达并阻止片段的维持，在分化后，缺乏活性STAT3会消除病毒基因组扩增和晚期基因表达。含有表达磷酸化位点STAT3突变的角质形成细胞的HPV18的器官型筏培养显示出基底上增生的显著减少，这与细胞周期蛋白B1表达的缺失和分化的增加有关。最后，与对照样本相比，在HPV阳性宫颈疾病活检中观察到STAT3表达和磷酸化增加，突出了STAT3激活在宫颈癌发生中的作用。总之，我们的数据提供了STAT3在HPV18生命周期中起关键作用的证据。人类乳头瘤病毒（HPV）是全球病毒诱导癌症的主要原因。人乳头瘤病毒是导致宫颈癌和越来越多的头颈癌的病原体。HPV感染依赖于宿主细胞的复制操作，这可能导致癌症等疾病。STAT3转录因子是一种已知的癌症进展驱动因子，在hpv相关癌症中经常过度活跃；然而，它在HPV生命周期中的作用尚未被研究。利用原代细胞培养模型，我们提供了第一个证据，证明HPV增加了STAT3的磷酸化和活性，这是病毒基因表达和复制所必需的。重要的是，小分子抑制剂抑制STAT3或表达不能磷酸化的STAT3突变体会损害HPV的生命周期。最后，我们证明了STAT3磷酸化在宫颈疾病进展过程中增加，突出了STAT3作为hpv相关癌症的新治疗靶点的潜力。
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. Human papillomaviruses (HPV) are the leading cause of viral induced cancers worldwide. HPV are the causative agents of cervical cancers and an increasing number of head and neck cancers. HPV infections are dependant on the manipulation of the host cell for their replication and this may result in diseases such as cancer. The STAT3 transcription factor, a known driver of cancer progression, is often over active in HPV-associated cancers; however, its role in the life cycle of HPV has not been studied. Using primary cell culture models we provide the first evidence demonstrating that HPV increases both the phosphorylation and activity of STAT3 and that this is required for viral gene expression and replication. Importantly, inhibition of STAT3 by small molecule inhibitors or expression of STAT3 mutants that cannot be phosphorylated impairs the HPV life cycle. Finally, we demonstrate that STAT3 phosphorylation is increased during cervical disease progression, highlighting the potential of STAT3 as a novel therapeutic target in HPV-associated cancers.