Some Novel Insights on HPV16 Related Cervical Cancer Pathogenesis Based on Analyses of LCR Methylation, Viral Load, E7 and E2/E4 Expressions

Damayanti Das Ghosh,Bornali Bhattacharjee,Sharmila Sengupta,Indranil Mukhopadhyay,Sudipta Roy,Shrinka Sen,Laikangbam Premi,Rahul Roy Chowdhury,Giuseppe Viglietto

doi:10.1371/journal.pone.0044678

Abstract

This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1∧E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1∧E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 CT/E4 CT. Relative quantification based on comparative CT (theshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 CT (p = 0.007) and E2 CT (p<0.0001), respectively, each normalized with ACTB CT, among episomal cases only. The k-means clustering analysis considering E7 CT from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

Highlights

human papillomavirus 16 (HPV16) appears to be the most common high risk HPV type identified in CaCx cases, precancerous cervical lesions, and in cytologically normal cervical samples [1,2]
Based on a comparison between fifteen HPV16 positive cytologically normal and fifty-seven HPV16 positive CaCx cases with intact E2 gene, we proposed that loss of E2 repressor activity in such cases, predominantly of the E-lineage, could be attributable to CpG methylation at nucleotide 58 within E2 binding site I (E2BS-I) next to the p97 promoter, attenuating the binding of E2 to this site [7,9]
Our study provided novel insights into alternative mechanisms of loss of E2 repressor activity, which could be related to E2BS-I/II methylation, presence of the transcript (E7-E1‘E4) that encodes E2 repressor protein, high viral load and E7 expression among HPV16 positive CaCx cases with episomal viral genomes and not among the cases with purely integrated viral genomes

Summary

Introduction

HPV16 appears to be the most common high risk HPV type identified in CaCx cases, precancerous cervical lesions, and in cytologically normal cervical samples [1,2]. It is established that the HPV genome exists in the episomal form in low-grade lesions, the viral genome gets integrated, with increasing grades of lesion [5] Despite this observation, analysis of the association of HPV infections with CaCx development reveals the presence of both integrated as well as episomal forms of HPV, HPV16, in CaCx cases [6]. Over 60% of the CaCx cases harbor intact E2, despite the fact that HPV16 E2 protein negatively regulates transcription of the E6 and E7 genes [7,8] This prompted us to explore new paradigms of cervical carcinogenesis, which would offer insights into mechanisms involved in sustained E6/E7 mRNA expression even with the E2 gene intact, i.e. presence of concomitant viral genomes (both episomal and integrated) or purely episomal viral genomes [7,9,10,11]

Methods

Results

Conclusion