Epigenetic Inactivation of the Secreted Frizzled-Related Protein Gene Family in Multiple Myeloma.

Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. Previous molecular studies have largely focused on acquired genetic aberrations in MM. There is increasing evidence that in addition to genetic aberrations epigenetic processes play a major role in carcinogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene function. This epigenetic event acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function. The Wnt pathway has been recognized to be essential for normal organ development, and a role for Wnt signal transduction at several stages of lymphocyte differentiation and in the self-renewal of hematopoietic stem cells could be demonstrated. Alterations in the Wnt pathway have been shown to contribute to the pathogenesis of various human malignancies. Epigenetic silencing of the family of secreted frizzled-related proteins (SFRPs), which act as Wnt antagonists, was recently reported in several solid tumors and in acute lymphoblastic leukemia. In order to investigate the potential role of abnormal Wnt signaling in MM, we determined the methylation status of the promoter-associated CpG islands of SFRP-1, 2, 4 and 5 in the MM cell lines U266, LP-1, RPMI-8226 and OPM-2. Methylation-specific polymerase chain reaction (MSP) analysis revealed that promoter hypermethylation of the SFRP genes was a frequent event in MM cell lines (SFRP-1: 2/4, SFRP-2: 2/4, SFRP-4: 1/4 and SFRP-5: 3/4). Aberrant methylation of SFRP-1 and SFRP-2 in MM cell lines was associated with transcriptional silencing, as determined by real-time reverse transcriptase polymerase chain reaction. Treatment of cell lines that carry a hypermethylated SFRP-1 and SFRP-2 gene, respectively, with the demethylating agent 5-aza-2′-deoxycytidine resulted in gene reexpression. We then analyzed by MSP the methylation status of SFRP-1, 2, 4 and 5 in 76 specimens obtained from MM patients. The frequency of aberrant methylation among the primary patient samples was 38.2% (29/76) for SFRP-1, 59.2% (45/76) for SFRP-2, 2.6% (2/76) for SFRP-4 and 7.6% (6/76) for SFRP-5. There was a high incidence of concomitant hypermethylation of SFRP-1 and SRFP-2. We conclude that promoter hypermethylation of the SFRP genes is a novel epigenetic event in MM that may contribute to aberrant activation of the Wnt pathway. Further studies are warranted to elucidate the functional consequences of aberrant Wnt signaling by downregulation of the SFRP genes in the pathogenesis of MM. Additionally, the increasing evidence for the important role of DNA methylation changes in malignant plasma cell disorders may serve as a basis for the use of epigenetically targeted therapeutic approaches in MM in the future.