SS18-SSX drives CREB activation in synovial sarcoma.

ABSTRACT

PURPOSE: Synovial sarcoma (SySa) is a rare soft tissue tumor characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein represents the major driver of the disease, acting as aberrant transcriptional dysregulator. Oncogenic mechanisms whereby SS18-SSX mediates sarcomagenesis are incompletely understood, and strategies to selectively target SySa cells remain elusive. Based on results of Phospho-Kinase screening arrays, we here investigate the functional and therapeutic relevance of the transcription factor CREB in SySa tumorigenesis. METHODS: Immunohistochemistry of phosphorylated CREB and its downstream targets (Rb, Cyclin D1, PCNA, Bcl-xL and Bcl-2) was performed in a large cohort of SySa. Functional aspects of CREB activity, including SS18-SSX driven circuits involved in CREB activation, were analyzed in vitro employing five SySa cell lines and a mesenchymal stem cell model. CREB mediated transcriptional activity was modulated by RNAi-mediated knockdown and small molecule inhibitors (666-15, KG-501, NASTRp and Ro 31-8220). Anti-proliferative effects of the CREB inhibitor 666-15 were tested in SySa avian chorioallantoic membrane and murine xenograft models in vivo. RESULTS: We show that CREB is phosphorylated and activated in SySa, accompanied by downstream target expression. Human mesenchymal stem cells engineered to express SS18-SSX promote CREB expression and phosphorylation. Conversely, RNAi-mediated knockdown of SS18-SSX impairs CREB phosphorylation in SySa cells. Inhibition of CREB activity reduces downstream target expression, accompanied by suppression of SySa cell proliferation and induction of apoptosis in vitro and in vivo. CONCLUSION: In conclusion, our data underline an essential role of CREB in SySa tumorigenesis and provides evidence for molecular targeted therapies.