Promoter Hypermethylation of SASH1 Supports a Tumor Suppressor Role in Diffuse Large B-Cell Lymphomas
T Zhang, K Nie, R Shaknovich, DM Knowles, W Tam. Weill Cornell Medical College, New York, NY
Background: The gene SASH1 (SAM- and SH3-domain containing 1) has been identified as a candidate tumor suppressor gene (TSG) in breast and colon cancers. Previously, deletion mapping of chromosome 6q identified a minimal deletion region around D6S311 in Reed-Sternberg cells of classical Hodgkin lymphoma (cHL). Sequence comparison showed that D6S311 fell within the SASH1 gene located in 6q24.3. Morevoer, SASH1 is located within the frequently deleted 6q region of non-Hodgkin B-cell lymphomas. The goal of this study was to evaluate the presence of genetic and epigenetic alterations in this putative TSG in lymphomas.
Design: The entire SASH1 coding region was sequenced in L1236, which has a 6q14.3-qter deletion. Methylation status of 94 CpG in the promoter and exon 1 of SASH1 was assessed by bisulfite sequencing in 20 primary diffuse large B-cell lymphoma (DLBCL) clinical cases and 12 cell lines: 3 cHL (L1236, L428 and KMH2), 2 Burkitt lymphoma (Daudi, Raji), 2 germinal-center B-cell (GCB) type DLBCL (OCI-Ly1, SUDHL6), 4 non-GCB type DLBCL (OCI-Ly3, OCI-Ly10, SUDHL2 and U2932), and 1 myeloma (U266). Normal naïve B cells and GCB cells were analyzed as a control. SASH1 expressions in the cell lines, as well as normal B cells and plasma cells, were measured by quantitative RT-PCR. To determine if there is a causal relationship between promoter hypermethylation and SASH1 transcription repression, DLBCL and cHL cell lines were treated with 5-aza-2-deoxycytidine (5-aza).
Results: No SASH1 mutation was detected in L1236 cells. Two CG islands spanning the SASH1 promoter and first exon were identified. While these are rarely methylated in normal B cells, they are hypermethylated in all primary DLBCL and cell lines tested. The extent of SASH1 hypermethylation in each CpG varies from 10 to 90%. SASH1 is expressed at low levels in naïve and GCB cells, but its expression is 5 to10 fold higher in plasma cells. All cell lines demonstrated lower SASH1 mRNA levels relative to plasma cells. Inhibition of DNA methylation by 5-aza resulted in >100 fold increase in SASH1 mRNA in DLBCL and Hodgkin cell lines.
Conclusions: SASH1 is hypermethylated in primary DLBCL and B-lymphoma cell lines, resulting in its transcription inhibition. These results implicate SASH1 as a TSG in B-cell lymphomas. In addition, its up-regulation in plasma cells suggests a role in normal B cell differentiation. Methylation analysis of additional clinical samples is ongoing to further confirm its tumor suppressor role in DLBCL and other lymphomas.
Monday, March 22, 2010 9:15 AM
Platform Session: Section B, Monday Morning