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.