HPVE6-USP46 Mediated Cdt2 Stabilization Reduces Set8 Mediated H4K20-Methylation to Induce Gene Expression Changes. HPVE6-USP46 Mediated Cdt2 Stabilization Reduces Set8 Mediated H4K20-Methylation to Induce Gene Expression Changes

Cervical cancer is one of the most common cancers in women. More than 95% of the cervical cancers are caused by the human papilloma viruses. Earlier we had identified a protein target, USP46 that can be inhibited to block the cancer growth. We found that the viral E6 and human USP46 complex increases the levels of Cdt2 in cervical cancers. In this paper we compared real cervical tumors with matched normal tissue from same patients and found that the effects of E6 mediated USP46 activation are indeed reflected as increased levels of Cdt2 and decreased levels of Set8 protein in the real tumors. We also found that the HPV protein E6 alone can stimulate the enzymatic activity of human USP46. By doing this it activates several cellular pathways that are required for the growth of cancers. One such pathway is the EGFR pathway that is normally upregulated in cervical cancers. We find that E6-USP46 contributes to the activation of EGFR by inducing epigenetic changes on the DNA by degrading the Set8 protein. These findings illuminate the role of viral E6 protein in inducing cancers and substantiate the candidature of USP46 as a drug target for HPV induced cancers. Overall design: HeLa cells were transfected with control siRNAs or HPV-E6 siRNA or HPV-E6 and Set8 siRNAs. Cells were collected 48 h after siRNA transfection and RNA was extracted using the Qiagen RNA extraction kit. RNA samples were sent to Beijing Novogene Co., Ltd. (Beijing, China ) for library preparation and next generation RNA sequencing. More than 50 million paired end reads were obtained for each sample. Three experimental replicates were prepared for each sample and RNA integrity numbers (RIN) derived from a Bioanalyzer were used to ascertain the quality of RNA.

宫颈癌是女性最常见的恶性肿瘤之一，超过95%的宫颈癌由人乳头瘤病毒（human papillomavirus，HPV）引发。此前我们已鉴定出蛋白靶点USP46，抑制该靶点可阻断肿瘤生长。我们发现，病毒E6蛋白与人类USP46形成的复合物可升高宫颈癌组织中Cdt2的蛋白水平。本研究中，我们将真实宫颈肿瘤组织与同患者配对的正常组织进行比对，发现E6介导的USP46激活效应确实可在真实肿瘤中表现为Cdt2水平升高与Set8蛋白水平降低。我们还发现，仅HPV E6蛋白即可激活人类USP46的酶活性，借此激活肿瘤生长所必需的多条细胞信号通路。其中一条典型通路即为在宫颈癌中通常呈上调状态的表皮生长因子受体（epidermal growth factor receptor，EGFR）信号通路。我们发现，E6-USP46复合物可通过降解Set8蛋白诱导DNA发生表观遗传修饰，进而促进EGFR通路激活。上述研究阐明了病毒E6蛋白在肿瘤发生中的作用，并证实USP46可作为HPV相关宫颈癌的药物研发靶点。整体实验设计：将海拉（HeLa）细胞分别转染对照小干扰RNA（small interfering RNA，siRNA）、HPV-E6 siRNA或HPV-E6与Set8 siRNA的组合。于siRNA转染48小时后收集细胞，使用Qiagen RNA提取试剂盒提取总RNA。将RNA样品送至中国北京诺禾致源科技股份有限公司（Novogene Co., Ltd.，北京）进行文库构建与下一代RNA测序。每个样本均获得超过5000万条双端测序读段。每个样本设置三次生物学重复，并通过生物分析仪测得的RNA完整性数（RNA integrity number，RIN）评估RNA样品质量。