IntroductionBasal-like breast cancers (BLBC) frequently overexpress the epidermal growth factor receptor (EGFR) and subsequently have high levels of signaling through the MAP kinase pathway, which is thought to contribute to their aggressive behavior. While we have previously reported the expression of Y-box binding protein-1 (YB-1) in 73% of BLBC, it is unclear whether it can be regulated by a component of the MAP kinase signaling pathway. Phosphorylation of YB-1 at the serine 102 residue is required for transcriptional activation of growth-enhancing genes, such as EGFR. Using Motifscan we identified p90 ribosomal S6 kinase (RSK) as a potential candidate for activating YB-1.MethodsInhibition of RSK1 and RSK2 was achieved using siRNA and the small molecule SL0101. RSK1, RSK2, activated RSK and kinase-dead RSK were expressed in HCC1937 cells. Kinase assays were performed to illustrate direct phosphorylation of YB-1 by RSK. The impact of inhibiting RSK on YB-1 function was measured by luciferase assays and chromatin immunoprecipitation.ResultsUsing an in vitro kinase assay, RSK1 and RSK2 were shown to directly phosphorylate YB-1. Interestingly, they were more effective activators of YB-1 than AKT or another novel YB-1 kinase, PKCα. Phosphorylation of YB-1 (serine 102 residue) is blocked by inhibition of the MAP kinase pathway or by perturbing RSK1/RSK2 with siRNA or SL0101. In immortalized breast epithelial cells where RSK is active yet AKT is not, YB-1 is phosphorylated. Supporting this observation, RSK2-/- mouse embryo fibroblasts lose the ability to phosphorylate YB-1 in response to epidermal growth factor. This subsequently interfered with the ability of YB-1 to regulate the expression of EGFR. The RSK inhibitor SL0101 decreased the ability of YB-1 to bind the promoter, transactivate and ultimately reduce EGFR expression. In concordance with these results the expression of constitutively active RSK1 increased YB-1 phosphorylation, yet the kinase-dead RSK did not.ConclusionsWe therefore conclude that RSK1/RSK2 are novel activators of YB-1, able to phosphorylate the serine 102 residue. This provides a newly described mechanism whereby YB-1 is activated in breast cancer. This implicates the EGFR/RSK/YB-1 pathway as an important component of BLBC, providing an important opportunity for therapeutic intervention.

Synovial sarcoma (SS) is a soft tissue cancer characterized by the fusion of the SS18 gene, which encodes for a subunit of the BAF ATP-dependent chromatin remodeling complex, with either SSX1, SSX2, or SSX4 genes. The resulting SS18-SSX fusion protein is incorporated into the BAF complex, where it evicts wild-type SMARCB1 (BAF47) and retargets the complex to polycomb-repressed chromatin domains, thus activating oncogenic gene expression to drive tumor progression. Traditional means of diagnosis of SS include immunohistochemical staining, including that for the biomarker TLE1, but TLE1 is not overexpressed in all tumors and cases ultimately need to be confirmed by either FISH or PCR assays to detect the SS18-SSX fusion. We have developed the first highly specific, recombinant monoclonal antibodies for the detection of SS18-SSX fusion proteins by immunohistochemistry and for the capture of SS18-SSX-containing BAF complexes in cells, with downstream utility in biochemistry and genomics-centered approaches such as ChIP-seq. The E9 × 9V clone directly targets the highly conserved fusion breakpoint, while the E5A2C clone detects the C-terminus of the SSX protein family and is capable of recognizing alternative breakpoints. Taken together, our data show that these antibodies can accurately and reproducibly detect the SS18-SSX fusion protein in SS cells and tissues. Citation Format: Curtis Desilets, Fang Chen, Christopher Comeau, Katrina Costa, Katherine Crosby, Michelle Ferrante, Christopher Grange, Maggie Patrikas, Matthew McBride, Esther Baranov, Jason Hornick, Cigall Kadoch. Development and characterization of antibodies specific for the SS18-SSX fusion protein in synovial sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 290.