Epigenetic Silencing of Novel Tumour Suppressor Genes in Medulloblastoma.

摘要

Medulloblastomas (MB) are the most common pediatric nervous system malignancy. Known mutations account for only a subset of MB cases. We hypothesized that CpG island methylation-mediated tumour suppressor gene (TSG) silencing contributes to MB pathogenesis, either alone, or in combination with genetic events such as loss of heterozygosity (LOH). We performed a microarray-based genome-wide screen of MB cell lines treated with 5-aza-2'deoxycytidine, identifying genes exhibiting increased expression following treatment. Using this strategy, we identified inhibitors of WNT signalling (SFRP1, SFRP2, and SFRP3) and an inhibitor of the HGF/MET signalling pathway (SPINT2) as putative TSGs silenced by promoter region methylation in MB.;Methylation of the WNT signalling inhibitors SFRP1, SFRP2, and SFRP3 was identified using bisulfite sequencing and methylation-specific PCR (MSP). Stable re-expression of SFRP1, SFRP2, and SFRP3 reduced proliferation, impaired anchorage-independent growth, and limited WNT signalling activity. SFRP1 re-expression reduced tumour growth in vivo in xenograft models. Aberrant WNT signalling plays a role in the pathogenesis of a subset of sporadic human MB, as well as MB in cases of Turcot syndrome with germline mutations of APC. Activating mutations of β-catenin are also implicated in a subset of MB. We have identified for the first time an additional mechanism – loss of normal pathway inhibition by SFRP gene silencing – that contributes to MB pathogenesis.;SPINT2 methylation was confirmed with bisulfite sequencing and MSP. Stable re-expression of SPINT2 reduced proliferation, impaired cell migratory ability, and decreased the capacity for anchorage-independent growth. In vivo, re-expression of SPINT2 reduced tumour formation in xenograft models. This study identified for the first time SPINT2 as a putative TSG in human MB, and further implicated aberrant HGF/MET oncogenic signalling in the pathogenesis of this disease.;The efficacy of targeting the HGF/MET pathway as a novel therapeutic strategy was tested in vitro using the small molecule MET kinase inhibitor PHA665752. Treatment of MB cell lines with PHA665752 reduced cell proliferation, anchorage-independent growth, migration, and limited downstream signalling via the MAPK and PI3K/AKT pathways.