Members of the sFRP gene family are frequently methylated in multiple human cancers

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Background: The Secreted Frizzled-Related Proteins (sFRPs) are glycoproteins that structurally resemble the Frizzled receptors of the Wnt pathway. Five different genes, sFRP-1 through sFRP-5 have been cloned. Dysregulation of other proteins involved in the Wnt pathway (β-catenin and APC) has been shown to result in tumorigenesis. Given the oncogenic potential of the constitutive Wnt pathway signaling, sFRPs have been postulated to work as tumor suppressor genes. Methylation of the promoter region is a well-known event leading to inactivation of potential tumor suppressor genes. The goal of this study was to determine the methylation status of the sFRP genes in several common human cancers. Materials and Methods: Using Methylation Specific PCR (MSP), we studied the methylation status of the promoter region of four of the sFRP genes, sFRP-1, sFRP-2, sFRP-4 and sFRP-5 in frozen and paraffin-fixed tumor samples from multiple human cancers. Cell lines and corresponding normal tissues representative of this group of human cancers were also tested for aberrant methylation of the sFRP gene family. Samples from brain tumors (glioblastomas), lung cancers (SCLC and NCSL), melanoma, pancreatic cancer, esophageal cancer, colon cancer, renal cell carcinoma, breast and ovarian cancer were tested for aberrant methylation in this gene family. RNA expression in cell lines was determine before and after treatment with the demethylating agents DAC and TSA. Clinical and pathological data was also evaluated for correlation between prognosis, disease stage and methylation status. Results: Our data shows overall aberrant methylation of the sFRP gene family in > 80% of all the tumor samples tested. sFRP1 methylation was seen in > 85% of tumor samples, sFRP2 methylation in > 90%, sFRP4 in > 70%, and sFRP5 in > 50%. In cell lines, promoter hypermethylation of the sFRP gene family correlated with lack of gene expression. Gene expression was reactivated after treatment with a demethylating agent. Some tumor such as lung cancer, glioblastomas and colon cancer expressed the highest levels of methylation, while, breast and ovarian the lowest. Aberrant methylation was also seen in up to 40% of “adjacent normal” lung and brain tissue. Methylation was seen very rarely in non-affected normal tissues. There was no correlation between disease stage or prognosis and methylation status for any of the tested sFRP genes. Conclusions: Aberrant methylation of the sFRP-1, sFRP-2, sFRP-4 and sFRP-5 is a common epigenetic event in human cancers, but methylation frequency appears to differ by tumor site. Moderate levels of methyaltion were seen in adjacent normal tissues in lung and brain tumors but were rarely seen in other non-affected normal tissues.