Epigenetic Alterations Induced by Human Papillomavirus E6 and E7 Oncoproteins

Abstract

High-risk human papillomaviruses (HPVs) account for over 99 percent of cervical carcinomas as well as other anogenital tract and oropharyngeal carcinomas. The E6 and E7 oncoproteins are consistently expressed in cervical cancer and are small proteins with no enzymatic or DNA binding activity. Oncogenic transformation results from the modulation of host proteins and pathways by these proteins, most notably the degradation of tumor suppressors p53 and pRB by E6 and E7, respectively. To overcome the oncogenic activities of E7, the expression of the p16INK4A tumor suppressor is induced; thereby inhibiting CDK 4/6 activity leading to growth arrest and cellular senescence. However, E7 subverts this response through the degradation of the downstream effector pRB, resulting in aberrant proliferation. The mechanisms for E7 driven p16INK4A expression are not fully defined, but include epigenetic derepression through removal of repressive H3K27 trimethylation by the demethylase KDM6B. Here we show that epigenetic modulation of p16INK4A by E7 also involves removal of repressive H2AK119 monoubiquitination and deposition of activating H3K4 trimethyl and H3K27 acetyl marks, likely facilitated through the recruitment of the H2AK119 ubiquitin reader ZRF1 and H3K4 methyltransferase MLL1, respectively. Moreover, we report that p16INK4A expression persists despite the presence of repressive PRC2 components EZH2 and SUZ12 and H3K27 trimethylation, suggesting that presence of PRC2 complexes and H3K27 trimethylation are not sufficient to repress p16INK4A expression. We investigated the necessity of epigenetic factors on the survival of E6/E7 expressing cells. The expression of epigenetic factor KDM6B, and consequently p16INK4A, is necessary for the survival of E7 expressing cells; therefore, performing the first epigenetic RNAi screen in primary human foreskin keratinocytes, we identified and validated four factors, MLL1, ASXL1, BRD4, and ZRF1 that are necessary for the survival of E6/E7 expressing cells. Finally, we evaluated the necessity of p16INK4A and KDM6B expression in carcinomas that express high levels of p16INK4A. Depletion of p16INK4A in breast carcinoma cells selectively affected survival of p16INK4A-high expressing cells over p16INK4A-deleted cells. Furthermore, treatment of ovarian, lung, Merkel cell, and breast carcinoma cells with a KDM6B inhibitor resulted in decreased survival with the greatest effects in the high p16INK4A expressing cells.