Integrated gene expression profiling and in silico epigenomic scanning to identify genes involved in microsatellite instability status of colorectal carcinoma

Abstract

Background: Microsatellite instability (MSI) status is currently defined as either microsatellitestable (MSS), low-frequency MSI (MSI-L), or high-frequency MSI (MS1-H). Hypermethylation of CpG islands in the promoter regions of several genes has been associated with MSI status in colorectal cancers. In order to identify novel methylation sites unique to MSI-H or MS1L tumors in an unbused fashion, we conducted an expression profiling-based methylation target search. Methods: 41 primary colorectal cancers (12 MSI-H, 15 MSI-S and 14 MSI-L) were studied with high-density eDNA microarrays, and the bioinformatics methods principal components analysis (PC.A) and significance analysis of microarrays (SAM) were used to identify differentially expressed genes, which were in turn scanned for CpG islands in their putative promoter regions. Results: SAM and PCA identified 166 genes showing significantly different expression levels in MSI-H vs. non-H or in MSI-L vs. MSS colon tumors. 95 of these genes were decreased in mRNA expression in either MSI-H or MSI-L tumors. Genomic chtahase screening revealed that of these 95 genes, 48 had CpG islands within their putative promoter regions. These 48 genes were scanned for expression in normal tissue and for likely cancer-related putative protein functions. This combined approach identified 19 genes vath both CpG islands and significantly differential expression levels between MSI categories. 10 genes were excluded because of pro-carcinogenic (oncogenic) functions. Among the remaining 9 genes, 2 were underexpressed in MSI-L vs. MSS tumors: RBM8A (a binding partner of potential tumor suppressor gene OVCA1) and OAS3 (an IFN-induced protein required to degrade RNA). 7 were underexpressed in MSI-H vs. non-H tumors: RAB32 (ras family gene), PTPRO (a receptor-protein tyrosine phnsphatase), SDBCAG84 (breast cancer anugen 84), HDACll (histone deacetylase), ITPR2 (inositol triphosphate receptor), N33 (putative prostate cancer tumor suppressor), and TSC22 (TGFI~ stimulated transcription regulating factor). Conclusions: The anti-carcinogenic functions of these genes suggest that they are potential targets of tumor-specific methylation. In support of this hypothesis, one gene (N33) is a known tumor suppressor reactivated by promoter hypermethylation in multiple cancers. These results suggest that our approach will identify novel genes involved in the MSI status of colorectal carcinoma.