Epigenetic regulation of CD44in Hodgkin and non-Hodgkin lymphoma

Abstract

BackgroundEpigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a hallmark of cancer. To assay its extent in human lymphoma, methylation of 24 TSG was analyzed in lymphoma-derived cell lines as well as in patient samples.MethodsWe screened for TSG methylation using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in 40 lymphoma-derived cell lines representing anaplastic large cell lymphoma, Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma and mantle cell lymphoma (MCL) as well as in 50 primary lymphoma samples. The methylation status of differentially methylated CD44 was verified by methylation-specific PCR and bisulfite sequencing. Gene expression of CD44 and its reactivation by DNA demethylation was determined by quantitative real-time PCR and on the protein level by flow cytometry. Induction of apoptosis by anti-CD44 antibody was analyzed by annexin-V/PI staining and flow cytometry.ResultsOn average 8 ± 2.8 of 24 TSG were methylated per lymphoma cell line and 2.4 ± 2 of 24 TSG in primary lymphomas, whereas 0/24 TSG were methylated in tonsils and blood mononuclear cells from healthy donors. Notably, we identified that CD44 was hypermethylated and transcriptionally silenced in all BL and most FL and DLBCL cell lines, but was usually unmethylated and expressed in MCL cell lines. Concordant results were obtained from primary lymphoma material: CD44 was not methylated in MCL patients (0/11) whereas CD44 was frequently hypermethylated in BL patients (18/29). In cell lines with CD44 hypermethylation, expression was re-inducible at mRNA and protein levels by treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine, confirming epigenetic regulation of CD44. CD44 ligation assays with a monoclonal anti-CD44 antibody showed that CD44 can mediate apoptosis in CD44+ lymphoma cells. CD44 hypermethylated, CD44- lymphoma cell lines were consistently resistant towards anti-CD44 induced apoptosis.ConclusionOur data show that CD44 is epigenetically regulated in lymphoma and undergoes de novo methylation in distinct lymphoma subtypes like BL. Thus CD44 may be a promising new epigenetic marker for diagnosis and a potential therapeutic target for the treatment of specific lymphoma subtypes.

Characteristic DNA methylation profiles in peripheral blood monocytes are associated with inflammatory phenotypes of asthma

“…The interactions of these receptors with hyaluronan within ECM facilitate cell motility and adhesion. Promoter methylation is known to regulate CD44 expression, where hypermethylation results in transcriptional inactivation 40 ; however, epigenetic regulation of HMMR is largely unknown.…”

Cancer cells in epithelial-to-mesenchymal transition and tumor-propagating–cancer stem cells: distinct, overlapping or same populations

“…It is identified as a senescence-induced cell adhesion gene responsible for monocyte recruitment (Mun and Boo, 2010). In lymphoma and lymphoma cell lines, it is an epigenetically suppressed inducer of apoptosis (Eberth et al, 2010). On the other hand, CD44 expression enhances growth, self renewal and tumorosphere formation in breast carcinoma cell lines .…”

“…Other examples abound. In a panel of human breast cancer cell lines, there is a parallelism between an EMT mRNA or other marker signatures and the CSC-TPC (CD44 þ /CD24 þ /À ) phenotype (Mani et al, 2008;Blick et al, 2010;Eberth et al, 2010). The two programs EMT and CSC-TPC are induced by activation of Ras (Morel et al, 2008) and TGF-b actors.…”

Method to detect differentially methylated loci with case-control designs using Illumina arrays

“…This is often associated with gene silencing [Jones and Laird, 1999] and is an important mechanism for the inactivation of tumor suppressor genes. In more recent studies on DNA methylation and cancers, Eberth et al [2010] found that CD44 may be a promising new epigenetic marker for diagnosis and a potential therapeutic target for the treatment of specific lymphoma subtypes as it is found to be epigenetically regulated in lymphoma and undergoes de novo methylation in distinct lymphoma subtypes; Bediaga et al [2010] found that DNA methylation profiles may enable breast cancer subtype prediction; Christensen et al [2010] found that breast cancer prognostic characteristics and risk-related exposures appear to be associated with gene-specific tumor methylation and overall methylation pattern; and Lugthart et al [2011] found aberrant DNA hypermethylation in acute myeloid leukemia among many other studies. Apart from the importance in DNA methylation with cancer development, what makes DNA methylation even more interesting is, unlike genetic alterations, DNA methylation is reversible.…”