DNA Hypermethylation Profiles in Squamous Cell Carcinoma of the Vulva

Abstract

Gene silencing through promoter hypermethylation is a growing concept in the development of human cancers. In this study, we examined the contribution of aberrant methylation of promoter regions in methylation-prone tumor suppressors to the pathogenesis of vulvar cancer. Thirteen cell lines from 12 patients with squamous cell carcinoma of the vulva (SCV) were evaluated for aberrant methylation status and gene copy number alterations, concomitantly, using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Of the 22 tumor suppressor genes examined, aberrant methylation was observed for 9 genes: TP73, FHIT, VHL, APC, ESR1, CDKN2B, DAPK1, GSTP1 andI GSF4. The most frequently methylated genes included TP73 in 9 of 13 cell lines, and IGSF4, DAPK1 and FHIT in 3 of 13 cell lines. Methylation specific polymerase chain reaction (MSP) was performed for TP73 and FHIT to confirm aberrant methylation by MS-MLPA. In the context of gene copy number and methylation status, both copies of the TP73 gene were hypermethylated. Loss or decreased mRNA expression of TP73 and IGSF4 by RT-PCR confirmed aberrant methylation. Frequent genetic alterations of loss and gain ofgene copy number included gain of GSTP1 and MEN1, and loss of MFHAS1 and IGSF4 in over 50% of the SCV cell lines. These findings underscore the contribution of both genetic and epigenetic events to the underlying pathogenesis of squamous cell carcinoma of the vulva.

Genome-wide methylation and expression differences in HPV(+) and HPV(-) squamous cell carcinoma cell lines are consistent with divergent mechanisms of carcinogenesis

“…These differences were marked by overall greater DNA methylation across the genic and other non-repetitive genome in HPV(+) cells compared to HPV(-) cells, also observed in a sample of primary tumors. LINE-1 methylation, a measure of global methylation, was also higher in HPV(+) cell lines and in a larger population of tumor specimens, consistent with the differences of two genes which had both higher methylation and higher expression in HPV(+): ESR1 (due to its significance and differential promoter methylation in other cancers), [28][29][30] and DCC (identified as biologically important in head and neck cancer). 31 Illumina Infinium platform data showed ESR1 and DCC average methylation of 4% and 10% in HPV(-) tumors compared to 82% and 87% respectively in HPV(+) tumors.…”

Patterns of cellular and HPV 16 methylation as biomarkers for cervical neoplasia

“…ESR1 was chosen due to its significance and differential promoter methylation in other cancers (Archer et al, 2010; Heldring et al, 2007; Stephen et al, 2009). Additionally, Wisman et al identified more ESR1 promoter hypermethylation in cervical scrapings from cancer patients compared to normal subjects (64% versus 5.3% hypermethylated; p<0.0005) (Wisman et al, 2006).…”

“…Analyses focused on three tumor suppressor genes, ESR1 (estrogen receptor 1), p16 (cyclin-dependent kinase inhibitor 2A) and DCC (deleted in colorectal carcinoma), based on reported associations of hypermethylation of these genes with either cervical cancer or cancers at other sites (Archer et al, 2010; Carvalho et al, 2006; Furtado et al, 2010; Heldring et al, 2007; Kim et al, 2010a; Stephen et al, 2009; Wisman et al, 2006). Specifically, the following candidate gene sites were examined in all participants for whom adequate DNA was available (n=151): 3 CpG sites in the ESR1 promoter region, 5 in an intronic region of DCC , and 4 in the promoter region of p16 .…”

Increased expression of HIF-1A and its implication in the hypoxia pathway in primary advanced uterine cervical carcinoma

“…The levels of the VHL transcript are increased in cervical carcinoma, which is the opposite of the demonstrated lower expression of VHL in renal cell carcinoma (49), multiple myeloma (50), pancreatic endocrine tumors (51), chronic lymphocytic leukemia (52) and squamous cell carcinoma of the vulva (53). The observed abnormal expression of VHL in cervical cancer could be associated with increased levels of HIF-1A, because we found a statistically significant, positive Spearman's rank correlation between HIF-1A and VHL expression.…”