Saskia M. Wilting

Methylation-mediated silencing and tumour suppressive function of hsa-miR-124 in cervical cancer

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.

Increased gene copy numbers at chromosome 20q are frequent in both squamous cell carcinomas and adenocarcinomas of the cervix

Genome‐wide microarray‐based comparative genomic hybridization (array CGH) was used to identify common chromosomal alterations involved in cervical carcinogenesis as a first step towards the discovery of novel biomarkers. The genomic profiles of nine squamous cell carcinomas (SCCs) and seven adenocarcinomas (AdCAs), as well as four human papillomavirus (HPV)‐immortalized keratinocyte cell lines, were assessed. On a genome‐wide scale, SCCs showed significantly more gains than AdCAs. More specifically, there was a striking and highly significant difference between the two histological types for gain at 3q12.1–28, which was predominantly observed in SCC. Other frequent alterations included gains of 1q21.1–31.1 and 20q11.21–13.33, and losses of 11q22.3–25 and 13q14.3–21.33. Subsequent FISH analysis for hTR, located at 3q26, confirmed the presence of 3q gain in SCCs and HPV‐immortalized cell lines. Fine mapping of chromosome 20q using multiplex ligation‐dependent probe amplification (MLPA) showed copy number increases for a number of genes located at 20q11–q12, including DNMT3B and TOP1. For DNMT3B, this correlated with elevated mRNA expression in 79% of cases. In conclusion, the assessment of frequent genomic alterations resulted in the identification of potential novel biomarkers, which may ultimately enable a better risk stratification of high‐risk (hr)‐HPV‐positive women. Copyright

Association between dense CADM1 promoter methylation and reduced protein expression in high-grade CIN and cervical SCC†

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 ≥ 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 ≥ CIN3 compared with ≤ CIN1 (p = 0.005), as well as in SCCs compared with adenocarcinomas (83% versus 23%; p = 0.002). Detection of dense CADM1 promoter methylation will contribute to the assembly of a valuable marker panel for the triage of high‐risk HPV‐positive women at risk of ≥ CIN3. Copyright

Combined CADM1 and MAL promoter methylation analysis to detect (pre‐)malignant cervical lesions in high‐risk HPV‐positive women

Given the lower specificity for high‐grade cervical lesions of high‐risk human papillomavirus (hrHPV) testing compared to cytology, additional triage testing for hrHPV test‐positive women is needed to detect high‐grade cervical lesions. Here, we tested whether combined methylation analysis for cell adhesion molecule 1 (CADM1) and T‐lymphocyte maturation associated protein (MAL), both functionally involved in cervical carcinogenesis, could serve as such a triage marker. Four quantitative methylation‐specific PCRs (qMSP), two for CADM1 (regions M12 and M18) and MAL (regions M1 and M2) each, were applied to 261 cervical tissue specimens ranging from no neoplasia to carcinoma. When qMSPs were combined and positivity for at least one of the qMSPs in the combination was taken into account, the highest positivity rates for cervical intraepithelial neoplasia grade 3 (CIN3) lesions (97%) and squamous cell‐ and adeno‐carcinomas (99%) were obtained by combining a single CADM1 marker with a single MAL marker. Subsequent qMSP analysis of 70 GP5+/6+‐PCR hrHPV‐positive scrapings revealed that a two‐marker panel consisting of CADM1‐M18 and MAL‐M1 was most discriminative, detecting 90% of women with CIN3 (n = 30), whereas it showed a positive result in only 13.5% of women without cervical disease (n = 40). Finally, we applied hrHPV GP5+/6+‐PCR testing followed by CADM1‐M18/MAL‐M1 methylation analysis to a cohort of 79 women visiting the outpatient colposcopy clinic. hrHPV testing revealed a sensitivity of 97% and a specificity of 33% for CIN3+. Additional CADM1‐M18/MAL‐M1 methylation analysis on the hrHPV‐positive women increased the specificity to 78% with a sensitivity of 70%. In conclusion, the methylation marker panel CADM1‐M18 and MAL‐M1 may serve as an alternative molecular triage tool for hrHPV‐positive women.

Repression of MAL tumour suppressor activity by promoter methylation during cervical carcinogenesis

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. Copyright

Integrated Genomic and Transcriptional Profiling Identifies Chromosomal Loci with Altered Gene Expression in Cervical Cancer

For a better understanding of the consequences of recurrent chromosomal alterations in cervical carcinomas, we integrated genome‐wide chromosomal and transcriptional profiles of 10 squamous cell carcinomas (SCCs), 5 adenocarcinomas (AdCAs) and 6 normal controls. Previous genomic profiling showed that gains at chromosome arms 1q, 3q, and 20q as well as losses at 8q, 10q, 11q, and 13q were common in cervical carcinomas. Altered regions spanned multiple megabases, and the extent to which expression of genes located there is affected remains unclear. Expression analysis of these previously chromosomally profiled carcinomas yielded 83 genes with significantly differential expression between carcinomas and normal epithelium. Application of differential gene locus mapping (DIGMAP) analysis and the array CGH expression integration tool (ACE‐it) identified hotspots within large chromosomal alterations in which gene expression was altered as well. Chromosomal gains of the long arms of chromosome 1, 3, and 20 resulted in increased expression of genes located at 1q32.1‐32.2, 3q13.32‐23, 3q26.32‐27.3, and 20q11.21‐13.33, whereas a chromosomal loss of 11q22.3‐25 was related to decreased expression of genes located in this region. Overexpression of DTX3L, PIK3R4, ATP2C1, and SLC25A36, all located at 3q21.1‐23 and identified by DIGMAP, ACE‐it or both, was confirmed in an independent validation sample set consisting of 12 SCCs and 13 normal ectocervical samples. In conclusion, integrated chromosomal and transcriptional profiling identified chromosomal hotspots at 1q, 3q, 11q, and 20q with altered gene expression within large commonly altered chromosomal regions in cervical cancer.

Genomic profiling identifies common HPV-associated chromosomal alterations in squamous cell carcinomas of cervix and head and neck

BackgroundIt is well known that a persistent infection with high-risk human papillomavirus (hrHPV) is causally involved in the development of squamous cell carcinomas of the uterine cervix (CxSCCs) and a subset of SCCs of the head and neck (HNSCCs). The latter differ from hrHPV-negative HNSCCs at the clinical and molecular level.MethodsTo determine whether hrHPV-associated SCCs arising from different organs have specific chromosomal alterations in common, we compared genome-wide chromosomal profiles of 10 CxSCCs (all hrHPV-positive) with 12 hrHPV-positive HNSCCs and 30 hrHPV-negative HNSCCs. Potential organ-specific alterations and alterations shared by SCCs in general were investigated as well.ResultsUnsupervised hierarchical clustering resulted in one mainly hrHPV-positive and one mainly hrHPV-negative cluster. Interestingly, loss at 13q and gain at 20q were frequent in HPV-positive carcinomas of both origins, but uncommon in hrHPV-negative HNSCCs, indicating that these alterations are associated with hrHPV-mediated carcinogenesis. Within the group of hrHPV-positive carcinomas, HNSCCs more frequently showed gains of multiple regions at 8q whereas CxSCCs more often showed loss at 17p. Finally, gains at 3q24-29 and losses at 11q22.3-25 were frequent (>50%) in all sample groups.ConclusionIn this study hrHPV-specific, organ-specific, and pan-SCC chromosomal alterations were identified. The existence of hrHPV-specific alterations in SCCs of different anatomical origin, suggests that these alterations are crucial for hrHPV-mediated carcinogenesis.

Focal aberrations indicate EYA2 and hsa-miR-375 as oncogene and tumor suppressor in cervical carcinogenesis.

Cervical cancer results from persistent infection with high‐risk human papillomavirus (hrHPV). Common genetic aberrations in cervical (pre)cancers encompass large genomic regions with numerous genes, hampering identification of driver genes. This study aimed to identify genes functionally involved in HPV‐mediated transformation by analysis of focal aberrations (<3 Mb) in high‐grade cervical intraepithelial neoplasia (hgCIN). Focal chromosomal aberrations were determined in high‐resolution array comparative genomic hybridization data of 60 hgCIN. Genes located within focal aberrations were validated using 2 external gene expression datasets or qRT‐PCR. Functional roles of candidate genes EYA2 (20q13) and hsa‐miR‐375 (2q35) were studied by siRNA‐mediated knock‐down and overexpression, respectively, in hrHPV‐containing cell lines. We identified 74 focal aberrations encoding 305 genes. Concurrent altered expression in hgCIN and/or cervical carcinomas compared with normal cervical samples was shown for ATP13A3, HES1, OPA1, HRASLS, EYA2, ZMYND8, APOBEC2, and NCR2. Gene silencing of EYA2 significantly reduced viability, migratory capacity, and anchorage‐independent growth of HPV16‐transformed keratinocytes. For hsa‐miR‐375, a direct correlation between a (focal) loss and significantly reduced expression was found. Downregulation of hsa‐miR‐375 expression was confirmed in an independent series of cervical tissues. Ectopic expression of hsa‐miR‐375 in 2 cervical carcinoma cell lines reduced cellular viability. Our data provide a proof of concept that chromosomal aberrations are actively contributing to HPV‐induced carcinogenesis and identify EYA2 and hsa‐miR‐375 as oncogene and tumor suppressor gene, respectively.

Chromosomal Signatures of a Subset of High-Grade Premalignant Cervical Lesions Closely Resemble Invasive Carcinomas

Cervical cancer develops from precancerous high-grade cervical intraepithelial neoplasia (CIN) harboring a transforming infection with high-risk human papillomavirus, which is characterized by p16(INK4a) overexpression. Once such a lesion has developed, progression toward an invasive squamous cell carcinoma (SCC) may take one or more decades, underlining the heterogeneity of these lesions in terms of duration of existence and progression risk. We performed array-based comparative genomic hybridization (array CGH) on 46 p16(INK4a) immunopositive CIN2/3 lesions to determine whether this heterogeneity is reflected in their chromosomal profiles. Chromosomal profiles of CIN2/3 lesions were related to those of invasive cervical SCC and promoter methylation of CADM1, a tumor suppressor gene known to be functionally involved in the tumorigenic phenotype of cervical cancer cells. Frequent alterations found in CIN2/3 lesions included gains located at chromosome 1, 3, 7, and 20 and losses located at 4, 11, 16, 17, and 19. Unsupervised hierarchical clustering identified two subsets of CIN2/3 lesions, chromosomal profiles of one of which closely resembled invasive SCCs. Gains of 1, 3q, and 20 were characteristic for CIN2/3 lesions with chromosomal signatures resembling carcinomas. In addition, dense promoter methylation of the CADM1 gene was significantly more frequent in these CIN2/3 lesions (P = 0.004). No chromosomal alterations were detected in six CIN1 lesions, five of which were completely p16(INK4a) immunonegative. These findings suggest that biomarkers associated with gains at chromosomes 1, 3q, and 20 are potential hallmarks of advanced p16(INK4a)-positive CIN2/3 lesions with a high short-term risk of progression.

Methylation-mediated transcriptional repression of microRNAs during cervical carcinogenesis

Deregulated expression of microRNAs (miRNAs) is common and biologically relevant in cervical carcinogenesis and appears only partly related to chromosomal changes. We recently identified 32 miRNAs showing decreased expression in high-grade cervical intraepithelial neoplasia (CIN) and carcinomas not associated with a chromosomal loss, 6 of which were located within a CpG island. This study aimed to investigate to what extent these miRNAs are subject to DNA methylation-mediated transcriptional repression in cervical carcinogenesis. Methylation-specific PCR (MSP) analysis on a cell line panel representing different stages of human papillomavirus (HPV) induced transformation revealed an increase in methylation of hsa-miR-149, -203 and -375 with progression to malignancy, whereas expression of these miRNAs was restored upon treatment with a demethylating agent. All three miRNAs showed significantly increased levels of methylation in cervical carcinomas, whereas methylation levels of hsa-miR-203 and -375 were also significantly increased in high-grade CIN. A pilot analysis showed that increased hsa-miR-203 methylation was also detectable in HPV-positive cervical scrapes of women with high-grade CIN compared with controls. Similar to recent findings on hsa-miR-375, ectopic expression of hsa-miR-203 in cervical cancer cells decreased both the proliferation rate and anchorage independent growth. We found evidence for methylation-mediated transcriptional repression of hsa-miR-149, -203 and -375 in cervical cancer. Methylation of the latter two was already apparent in precancerous lesions and represent functionally relevant events in HPV-mediated transformation. Increased hsa-miR-203 methylation was detectable in scrapes of women with high-grade CIN, indicating that methylated miRNAs may provide putative markers to assess the presence of (pre)cancerous lesions.