Host proteome linked to HPV E7-mediated specific gene hypermethylation in cancer pathways

Abstract

Background: Human papillomavirus (HPV) infection causes around 90% of cervical cancer cases, and cervical cancer is a leading cause of female mortality worldwide. HPV-derived oncoprotein E7 participates in cervical carcinogenesis by inducing aberrant host DNA methylation. However, the targeting specificity of E7 methylation of host genes is not fully understood but is important in the down-regulation of crucial proteins of the hallmark cancer pathways. In this study, we aim to link E7-driven aberrations in the host proteome to corresponding gene promoter hypermethylation events in the hope of providing novel therapeutic targets and biomarkers to indicate the progression of cervical cancer. Methods: HEK293 cells were transfected with pcDNA3.1-E7 plasmid and empty vector and subjected to mass spectrometry-based proteomic analysis. Down-regulated proteins (where relative abundance was determined significant by paired T-test) relevant to cancer pathways were selected as gene candidates for mRNA transcript abundance measurement by qPCR and expression compared with that in SiHa cells (HPV type 16 positive). Methylation Specific PCR was used to determine promoter hypermethylation in genes downregulated in both SiHa and transfected HEK293 cell lines. The FunRich and STRING databases were used for identification of potential regulatory transcription factors and the proteins interacting with transcription factor gene candidates, respectively. Results: Approximately 400 proteins totally were identified in proteomics analysis. The transcripts of six genes involved in the host immune response and cell proliferation (PTMS, C1QBP, BCAP31, CDKN2A, ZMYM6 and HIST1H1D) were down-regulated, corresponding to proteomic results. Methylation assays showed four gene promoters (PTMS, C1QBP, BCAP31 and CDKN2A) were hypermethylated with 61, 55.5, 70 and 78% increased methylation, respectively. Those four genes can be regulated by the GA-binding protein alpha chain, specificity protein 1 and ETS-like protein-1 transcription factors, as identified from FunRich database predictions. Conclusions: HPV E7 altered the HEK293 proteome, particularly with respect to proteins involved in cell proliferation and host immunity. Down-regulation of these proteins appears to be partly mediated via host DNA methylation. E7 possibly complexes with the transcription factors of its targeting genes and DNMT1, allowing methylation of specific target gene promoters.

CIGB-300 peptide targets the CK2 phospho-acceptor domain on Human Papillomavirus E7 and disrupts the Retinoblastoma (RB) complex in cervical cancer cells

“…To further evaluate the in vivo CIGB-300-E7 interaction between both molecules, we performed in vivo pull-down. We employed HEK293 as an epithelial cell model with high transfection efficiency that has been previously used for studying E6 and E7 interaction partners [ 37 ]. Using HEK293 cells overexpressing HPV-16 E7, we observed physical interaction between CIGB-300 and E7 protein.…”

Association between promoter methylation and gene expression of CGB3 and NOP56 in HPV‑infected cervical cancer cells

“…Therefore, it was hypothesized that there would be genes other than CCNA1 and CADM1 whose promoters are methylated by HPV 16 E7, and that there would be specific transcription factors binding with HPV 16 E7 on the gene promoter. A study by Na Rangsee et al ,used STRING database protein network analysis, showed that E7 possibly forms a complex with the set of transcription factors containing a YY1 domain ( 26 ). In the present study, the binding site of HPV 16 E7 on the CCNA1 promoter was analyzed to identify the putative transcription factors.…”

“…Moreover, their study indicated that the E7 protein can bind to the promoter region of CADM1 , and chromatin immunoprecipitation indicated that it may bind to DNMT1 through the same mechanism as CCNA1 ( 25 ). Na Rangsee et al ( 26 ), using readings from mass spectrometry followed by STRING database protein network analysis, showed that E7 possibly formed a complex with a set of transcription factors, including SP1, which links with the Yin yang 1 (YY1) transcription factor contributing to the E7 mediated hypermethylation of the genes. This highlighted the possibility of E7 combining with DNMT1 through a transcription factor to the promoter region of the CCNA1 gene, resulting in promoter methylation.…”

HPV-16 E6 and E7 expression drives transcription of p14(ARF) and p16(INK4a) prior to Intragenic methylation of CDKN2A.

“…These changes in DNMT1 activity have effects on other downstream targets critical in tumorigenic pathways, including E-cadherin and cyclin A1 45,46 , and may be responsible for the intragenic CDKN2A methylation we observed in this study. It is also likely that additional oncoproteinmediated alterations in gene expression are linked to host DNA methylation 47 .…”