Evidence for Alteration of EZH2, BMI1, and KDM6A and Epigenetic Reprogramming in Human Papillomavirus Type 16 E6/E7-Expressing Keratinocytes

Abstract

A number of epigenetic alterations occur in both the virus and host cellular genomes during human papillomavirus (HPV)-associated carcinogenesis, and investigations of such alterations, including changes in chromatin proteins and histone modifications, have the potential to lead to therapeutic epigenetic reversion. We report here that transformed HPV16 E6/E7-expressing primary human foreskin keratinocytes (HFKs) (E6/E7 cells) demonstrate increased expression of the PRC2 methyltransferase EZH2 at both the mRNA and protein levels but do not exhibit the expected increase in trimethylated H3K27 (H3K27me3) compared to normal keratinocytes. In contrast, these cells show a reduction in global H3K27me3 levels in vitro, as well as upregulation of the KDM6A demethylase. We further show for the first time that transformation with the HPV16 E6 and E7 oncogenes also results in an increase in phosphorylated EZH2 serine 21 (P-EZH2-Ser21), mediated by active Akt, and in a downregulation of the PRC1 protein BMI1 in these cells. High-grade squamous cervical intraepithelial lesions also showed a loss of H3K27me3 in the presence of increased expression of EZH2. Correlating with the loss of H3K27me3, E6/E7 cells exhibited derepression of specific EZH2-, KMD6A-, and BMI1-targeted HOX genes. These results suggest that the observed reduction in H3K27me3 may be due to a combination of reduced activities/levels of specific polycomb proteins and increases in demethylases. The dysregulation of multiple chromatin proteins resulting in the loss of global H3K27me3 and the transcriptional reprogramming in HPV16 E6/E7-infected cells could provide an epigenetic signature associated with risk and/or progression of HPV16-associated cancers, as well as the potential for epigenetic reversion in the future.Human papillomavirus (HPV) infections account for approximately 5% of all cancers worldwide (33). Although vaccination against HPV types 16 (HPV16) and 18 (HPV18) prevents the acquisition of cervical dysplastic lesions among eligible women, cervical cancer is still the third most common cancer in women worldwide, with an estimated 529,000 new cases and 275,000 deaths occurring in 2008, predominantly in developing countries (13). It is now known that a number of epigenetic alterations occur during all stages of cervical carcinogenesis in both the HPV and host cellular genomes, and therefore investigations of such alterations could hold the promise for development of therapeutic interventions against the development and progression of this malignancy.The transformed phenotype of HPV16-positive human foreskin keratinocytes (HFKs) depends on the concerted action and constitutive expression of the viral E6 and E7 oncogenes. Recently, the methyltransferase enhancer of zeste homolog 2 (EZH2) (37) was identified as a novel downstream target of E7 and was shown to be elevated in HPV-positive dysplastic and tumorigenic cervical lesions in vivo (18). Increased expression of EZH2 may also be mediated through the loss of p53 (Holland) (and thus indirectly throu...

Epigenetics Markers of Metastasis and HPV-Induced Tumorigenesis in Penile Cancer

“…Aberrant methylation of genes regulated by the PRC2 complex has been observed in many cancer types, including head and neck, cervical, and prostate cancer but not previously in penile cancer. However, changes in the epigenetic regulation of PRC2 target genes has been noted during the HPV16 transformation of normal foreskin keratinocytes, with HPV16 infection resulting in the increased EZH2 expression and decreased global H3K27me3 (30). Furthermore, we also see overexpression of the members of the PRC2 complex (EZH2 and SUZ12) in penile cancers.…”

HOXA9 is Underexpressed in Cervical Cancer Cells and its Restoration Decreases Proliferation, Migration and Expression of Epithelial-to-Mesenchymal Transition Genes

“…Therefore, viral infection per se is not considered sufficient to generate a neoplastic process; it is, rather, a first alteration that predisposes cells to subsequent changes; if those do occur, the neoplasm is generated (Perez-Plasencia et al, 2008). It is evident that additional factors, such as genomic instability caused probably by viral oncogenes (E6 and E7), environmental factors, or the genetic background itself, are necessary for cervical tumorigenesis (Hyland et al, 2011). Alterations in developmental signaling pathways or transcription factors that regulate the ontogeny, such HOX genes, have recently been proposed as alternative secondary modifications that could lead to malignant transformation (Karamboulas and Ailles, 2013).…”

Tumor suppressor p16 INK4A is necessary for survival of cervical carcinoma cell lines

“…As evidenced by aberrant homeobox gene expression, HPV16 E7-expressing cells undergo profound epigenetic alterations as a consequence of enhanced KDM6A and KDM6B expression (17,27). Indeed, several homeobox genes (HOX) have been identified as bona fide oncogenes.…”