ATF2 in Synovial Sarcoma
Le Su, T Michael Underhill, Torsten O Nielsen. University of British Columbia, Vancouver, BC, Canada
Background: The SS18-SSX fusion oncoprotein in synovial sarcoma has no DNA binding domain and its mechanism of transcriptional dysregulation has been unclear. Using mass spectrometry, reciprocal immunoprecipitation (IP), siRNA and chromatin IP studies in synovial sarcoma cell lines, we were able to identify Activating Transcription Factor 2 as its probable DNA binding partner, abnormally bridged to the TLE1 corepressor by SS18-SSX. We sought to extend this study to other model systems and patient specimens, characterize target genes and survey ATF2 expression in sarcomas.
Design: Models: 2 synovial sarcoma cell lines, HEK293 cells engineered to express tagged SS18 or SS18SSX, mouse model synovial sarcoma cells. Patient specimens: 11 frozen primary synovial sarcomas including SSX1 & 2 variants, and tissue microarrays representing 57 synovial sarcomas and 219 other tumors. Methods: reciprocal IP, glycerol gradient fractionation, cell viability & apoptosis assays, expression profile analysis, chromatin IP and immunohistochemistry on tissue arrays
Results: ATF2, SS18-SSX and TLE1 reciprocally immunoprecipitate in patient specimens (SSX1 and SSX2), as do their murine homologues in mouse synovial sarcomas generated by conditional expression of SS18-SSX2. ATF2 binds SS18 but cofractionates with the repressor TLE1 only in the presence of SS18-SSX. Both ATF2 and TLE1 knockdowns induce apoptosis in synovial sarcoma. Seven target genes of ATF2, bearing its CRE consensus binding sequence in their promoters, were identified and all appear to be abnormally repressed in patient synovial sarcoma specimens. Chromatin IP assay demonstrates ATF2, SS18-SSX and TLE are all resident at the CRE elements in these target genes, true also of murine homologues in the mouse model. Histone deacetylase inhibitor drugs disrupt this complex, restore target gene expression and lead to cell death. Tissue microarrays reveal consistent and high ATF2 nuclear expression in synovial sarcoma, with only solitary fibrous tumor showing higher levels. However, its expression is not as specific as TLE1 as relatively high levels are also seen in melanoma/clear cell sarcoma, Ewing tumors and MPNST. Levels in other mesencymal tumors and carinomas are significantly lower.
Conclusions: The ATF2-SS18SSX-TLE1 complex is present in patient synovial sarcoma specimens. ATF2 directs SS18-SSX to target genes in synovial sarcoma, which become repressed instead of activated. While not as useful a diagnostic marker as TLE1, the combined presence of ATF2 and TLE1 in a complex explains a mechanism of action for SS18SSX, and suggests means of therapeutic intervention for which clinical trials have recently opened.
Category: Bone & Soft Tissue
Tuesday, March 20, 2012 8:00 AM
Platform Session: Section G, Tuesday Morning