Methylation of p16 ink4a promoter is independent of human papillomavirus DNA physical state: a comparison between cervical pre-neoplastic and neoplastic samples

Abstract

BACKGROUND Epigenetic modifications in host cells, like p16 ink4a methylation, have been considered as putative complementary mechanisms for cancer development. Because only a small proportion of infected women develop cervical cancer, other factors might be involved in carcinogenesis, either independently or in association with high-risk human papillomavirus (HR-HPV) infections, including epigenetic factors. OBJECTIVES We hypothesised that p16 ink4a methylation might have a role in cancer development driven by HPV16, mainly in the presence of intact E1/E2 genes. Thus, our objectives were to assess the status of p16 ink4a methylation and the HPV16 E1/E2 integrity in samples in different stages of cervical diseases. METHODS Presence of HPV16 was determined by E6 type-specific polymerase chain reaction (PCR). Methylation status of the p16 ink4a promoter was assessed by methylation-specific PCR in 87 cervical specimens comprising 29 low-grade (LSIL), 41 high-grade (HSIL) lesions, and 17 cervical cancers (CC). Characterisation of E1 and E2 disruption (as an indirect indicator of the presence of episomal viral DNA) was performed by PCR amplifications. FINDINGS We observed a significantly increased trend (nptrend = 0.0320) in the proportion of methylated p16 ink4a in cervical samples during cancer development. Concomitant E1 and E2 disruptions were the most frequent pattern found in all groups: CC (76%), HSIL (54%), and LSIL (73%). No statistically significant differences between p16 ink4a methylation and E1/E2 integrity, in histological groups, was observed. MAIN CONCLUSIONS There was an increase in methylation of the p16 ink4a promoter from pre-neoplastic lesions to cancer. Additionally, a high frequency of E1/E2 disruptions in LSIL/HSIL suggested that viral DNA integration was an early event in cervical disease. Moreover, the methylation status was apparently independent of HPV16 integrity.

Association between oncogenic human papillomavirus type 16 and Killian polyp

“…Mutations at the p16 coding gene may explain its down-expression [74,75]. Alternatively, methylation at p16 promoter may silence the gene leading to decrease expression, as previously shown in HPV-positive samples carrying HPV in episomal form [76].…”

MiRNA in cervical cancer: Diagnosis to therapy: Systematic review

“…In contrast, E7, which is in charge of cell proliferation, binds to pRB protein to release E2F transcription factor and control the transition of the cell cycle from the G1 to the S phase, which is followed by DNA replication and ultimately leads to cell division. Additionally, E7 ″LR-HPV” is less effective at binding to pRB than E7 ″HR-HPVs,” and it is ineffective in assays for cell transformation and ras oncogene [ [29] , [30] , [31] ].

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New Trends in the Detection of Gynecological Precancerous Lesions and Early-Stage Cancers

“…Methylation rates usually correlated with the disease stage (being highest in invasive carcinomas). In precancerous lesions, frequently observed differences in the rate of methylation between LSILs and HSILs were reported, e.g., for the promoters of the SFRP gene family [ 64 ], namely CDKN2A [ 65 ], HS3ST2 [ 66 ], CADM1 , MAL [ 67 , 68 ], DAPK [ 69 ], and SOX1 [ 70 ]. Besides human genes, the methylation of HPV DNA has also been proposed as a novel biomarker for the triage of HPV-positive women.…”