人乳头瘤病毒抑制肝激酶B1的分子机制研究

HPV infection is necessary for the development of cervical cancer, as oncogenic HPV DNA sequences are detected in more than 99% of cervical cancers. The high-risk (HR) HPV oncoproteins E6 and E7 initiate the transformation of lesion progression to carcinomas by abrogating cell cycle control and apoptosis. However, current knowledge cannot completely explain the transforming potential of the viral oncogenes. Therefore, the long-term objective of this project is to identify additional cellular targets of E6/E7, the modulation of which contributes to the oncogenic processes. LKB1 (liver kinase B1) is a tumor suppressor mutationally inactivated in Peutz-Jeghers syndrome as well as in a variety of cancers. Our preliminary studies showed that HPV16 E6/E7 dramatically reduced LKB1 expression and disrupted LKB1-mediated AMPK-mTOR signaling. Ectopic expression of LKB1 suppressed the growth of HPV18-positive cervical cancer HeLa cells. We found that HPV16 E6/E7 increased the expression of the miRNA-106b/miR-93/miR-25 cluster, which is predicted to target LKB1. We hypothesize that LKB1 may be a target of HPVs E6/E7 and that disruption of LKB1 by HPVs via the induction of miRNA-106b/miR-93/miR-25 may play a central role in HPV oncogenic activities. We further hypothesize that the presence of HPV affects cellular sensitivity to LKB1-based treatments, creating an “Achilles’ heel” that can be exploited to develop improved, individualized therapies against cervical cancer. We design three specific aims to address our hypothesis: (1) To determine the functional interactions between LKB1 and HPV oncogenes; (2) To determine the role of miR-106b/miR-93/miR-25 cluster in HPV oncogene-mediated downregulation of LKB1 and elucidate the molecular mechanisms by which LKB1 dysregulation mediates the oncogenic activity of HPV. (3) To test the synthetic lethality by targeting of LKB1-deficient/HPV+ cells with PARP inhibitors in vitro and in vivo. Our previous studies showed that LKB1-deficient cells exhibited a delayed DNA damage response, reduced DNA repair capability, and enhanced sensitivity to DNA-damage agents. Hence, we expect that, by inactivating LKB1, HPV E6/E7 expression renders the cells more susceptible to standard platinum-based therapy and, in particular, to compounds that further impair DNA repair, such as poly(ADP-ribose) polymerase (PARP) inhibitors. We will test this hypothesis in HPV-positive and -negative cells in the presence or absence of LKB1 and in a mouse model of HPV-associated cancer. Our studies may eventually contribute to the improvement of chemotherapeutic strategies by selectively exposing HPV positive/LKB1 inactive patients to platinum and PARP inhibitors.

宫颈癌是造成女性死亡的第二大肿瘤。97%的宫颈癌为高危型人类乳头瘤病毒（HR HPV）感染引起。HPV癌蛋白E6和E7分别可引起p53和pRb失活，诱发癌变。然而，p53和pRb失活并不能完全诠释HPV E6和E7诱导的细胞恶性转化。我们的前期研究提示肿瘤抑制因子肝激酶B1（LKB1）为HPV的一个靶基因。LKB1受抑可能介导了HPV的致癌活性。在本项目中，我们将深入研究HPV对LKB1的抑制作用，明确LKB1下调对HPV致癌活性的影响，阐明HPV抑制LKB1及LKB1表达降低介导HPV致癌活性的分子机制，探讨LKB1缺乏增强PARP抑制剂对宫颈癌细胞毒活性的体内、外效应。成功完成本项目将为HPV的致癌特性提供新的机制解释，为抑制HPV的致癌作用提供新的手段，为治疗HPV感染所致肿瘤提供新的策略。

宫颈癌是造成女性死亡的第二大肿瘤。97%的宫颈癌为高危型（HR） HPV感染引起。有研究发现，20%的原发性宫颈癌存在LKB1突变。LKB1是一种肿瘤抑制因子。TCGA数据库分析显示，29%宫颈癌患者LKB1 mRNA水平下降。我们之前的研究发现，宫颈癌患者LKB1不但在mRNA水平降低，更多患者在蛋白水平亦出现LKB1表达降低或缺失。..为明确LKB1在HPV感染及宫颈癌中的作用，我们进行了三方面的研究。第一部分，我们明确了HPV对LKB1的抑制作用，确定 LKB1 下调介导了 HPV 癌基因活性。我们的研究发现HPV E6/E7表达下调LKB1，致LKB1-AMPK信号途径失活，该作用为HR HPV特有。LKB1下调介导了HPV引起的细胞转化及恶性表型的维持。..第二部分，阐明了HPV抑制LKB1及LKB1表达降低促进 HPV 活性的分子机制。我们的研究发现，HPV E6/E7癌基因表达上调miRNA-106b/-93/-25簇，后者靶向LKB1 3’-UTR，降低LKB1水平。LKB1表达抑制HPV癌基因引起的有氧糖酵解及磷酸戊糖途径激活，降低HPV的致癌活性。这些观察在细胞、动物及宫颈癌患者组织标本水平得到了验证。..第三部分，我们评价了LKB1缺乏对HPV感染细胞基于PARP 抑制剂的抗肿瘤治疗反应。我们的研究表明，LKB1缺失增加DNA损伤，致敏HPV感染细胞及小鼠宫颈癌肿瘤模型对PARP抑制剂和/或顺铂的处理效果。..总之，我们成功地完成了本项目研究计划的实验设计内容，验证了我们的科学假说，为最终阐明LKB1受抑在HPV致癌活性中的作用奠定了坚实基础。研究还为HPV的致癌特性研究提供新的机制解释，为抑制HPV的致癌作用提供新的手段，为治疗HPV感染所致的肿瘤提供新的策略。