BackgroundA substantial number of microRNAs (miRNAs) is subject to epigenetic silencing in cancer. Although epigenetic silencing of tumour suppressor genes is an important feature of cervical cancer, little is known about epigenetic silencing of miRNAs. Since DNA methylation-based silencing of hsa-miR-124 occurs in various human cancers, we studied the frequency and functional effects of hsa-miR-124 methylation in cervical carcinogenesis.ResultsQuantitative MSP analysis of all 3 loci encoding the mature hsa-miR-124 (hsa-miR-124-1/-2/-3) showed methylation in cervical cancer cell lines SiHa, CaSki and HeLa as well as in late passages of human papillomavirus (HPV) type 16 or 18 immortalised keratinocytes. Treatment of SiHa cells with a demethylating agent reduced hsa-miR-124 methylation levels and induced hsa-miR-124 expression. In HPV-immortalised keratinocytes increased methylation levels were related to reduced hsa-miR-124 expression and higher mRNA expression of IGFBP7, a potential hsa-miR-124 target gene. Ectopic hsa-miR-124 expression in SiHa and CaSki cells decreased proliferation rates and migratory capacity. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 139 cervical tissue specimens showed an increasing methylation frequency from 0% in normal tissues up to 93% in cervical carcinomas. Increased methylation levels of hsa-miR-124-1 and hsa-miR-124-2 were significantly correlated with reduced hsa-miR-124 expression in cervical tissue specimens. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 43 cervical scrapes of high-risk HPV positive women was predictive of underlying high-grade lesions.ConclusionsDNA methylation-based silencing of hsa-miR-124 is functionally involved in cervical carcinogenesis and may provide a valuable marker for improved detection of cervical cancer and its high-grade precursor lesions.

We recently identified MAL (T-lymphocyte maturation associated protein) as the most down-regulated gene in cervical oncogenesis. Here, we examined the mechanism underlying MAL silencing, its functional role in cervical carcinogenesis, and the relevance of detecting MAL alterations for risk assessment of hrHPV-positive women. MAL mRNA expression and promoter methylation were analysed in primary keratinocytes, hrHPV-immortalized keratinocytes, cervical cancer cell lines, biopsies, and scrapings by quantitative (methylation-specific) PCR. SiHa cells were transfected with MAL cDNA and assayed for proliferation, migration, and anchorage-independent growth. MAL mRNA was (nearly) undetectable in all HPV-immortalized and cervical cancer cells, but could be up-regulated upon methylation inhibition. MAL promoter methylation at two promoter regions (M1 and M2) was detected in all HPV-immortalized cells and cancer cells. Ectopic expression of MAL in SiHa cells suppressed proliferation, migration, and anchorage-independent growth. None (0/22) of normal cervical biopsies, 9% (6/66) of CIN1 lesions, 53% (34/64) of CIN3 lesions, 90% (85/94) of cervical squamous cell carcinomas (SCCs), and 93% (26/28) of cervical adenocarcinomas (AdCAs) demonstrated MAL promoter methylation at both promoter regions. Moreover, detection of MAL promoter methylation in cervical scrapings was predictive for underlying high-grade lesions. Both in biopsies and in scrapings, MAL promoter methylation was significantly correlated with reduced mRNA expression. MAL gene silencing by promoter methylation is a frequent and biologically essential event in HPV-induced cervical carcinogenesis. Hence, MAL promoter methylation and/or mRNA expression analysis on cervical scrapings may provide a valuable diagnostic tool to improve the detection of CIN3, SCC, and AdCA.

We previously showed that silencing of TSLC1, recently renamed CADM1, is functionally involved in high-risk HPV-mediated cervical carcinogenesis. CADM1 silencing often results from promoter methylation. Here, we determined the extent of CADM1 promoter methylation in cervical (pre)malignant lesions and its relation to anchorage-independent growth and gene silencing to select a CADM1-based methylation marker for identification of women at risk of cervical cancer. Methylation-specific PCRs targeting three regions within the CADM1 promoter were performed on high-risk HPV-containing cell lines, PBMCs, normal cervical smears, and (pre)malignant lesions. CADM1 protein expression in cervical tissues was analysed by immunohistochemistry. All statistical tests were two-sided. Density of methylation was associated with the degree of anchorage-independent growth and CADM1 gene silencing in vitro. In cervical squamous lesions, methylation frequency and density increased with severity of disease. Dense methylation (defined as >or= 2 methylated regions) increased from 5% in normal cervical samples to 30% in CIN3 lesions and 83% in squamous cell carcinomas (SCCs) and was significantly associated with decreased CADM1 protein expression (p < 0.00005). The frequency of dense methylation was significantly higher in >or= CIN3 compared with or= CIN3.

We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n ¼ 11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARb and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas.

BackgroundHigh-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation.MethodsCervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting.ResultsCervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation in vitro, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10.ConclusionThe present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.