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.