Supplementary MaterialsDocument S1

Supplementary MaterialsDocument S1. transforming activity in a xenograft model (Nagai et?al., 2001). Notably, mice conditionally expressing the SS18-SSX fusion gene in certain cell lineages develop tumors that are pathologically indistinguishable from and molecularly consistent with synovial sarcoma in humans (Haldar et?al., 2007), thus confirming Nutlin 3b the Rabbit Polyclonal to PTPN22 crucial role for SS18-SSX in the pathogenesis of synovial sarcoma. Fundamental progress has been made in understanding how the SS18-SSX fusion protein promotes tumorigenesis, which indeed involves multiple parallel mechanisms, such as epigenetic remodeling (Su et?al., 2012, Kadoch and Crabtree, 2013, Banito et?al., 2018, McBride et?al., 2018), cellular adhesion (Eid et?al., 2000), mesenchymal-to-epithelial transition (Saito et?al., 2006, Barrott et?al., 2015), protein translocation (Pretto et?al., 2006), and microRNA regulation (Hisaoka et?al., 2011, Minami et?al., 2014). Such complexities in SS18-SSX action make the development of targeted therapies for synovial sarcoma extremely challenging. Despite the lack of effective treatment options, several lines of evidence have shown that human synovial sarcoma cells are highly sensitive to histone deacetylase (HDAC) inhibitors in cell cultures and in a cell-line-based xenograft model (Ito et?al., 2005, Laporte et?al., 2017a). One well-supported explanation for the action of HDAC inhibitors is usually histone acetylation through which key tumor suppressor genes become epigenetically reactivated (Lubieniecka et?al., Nutlin 3b 2008, Su et?al., 2010, Su et?al., 2012, Laporte et?al., 2017b). In the present study, we propose an additional, transcription-independent mechanism whereby HDAC inhibition facilitates proteasomal degradation of the SS18-SSX fusion protein. This action relies mostly on a novel combination of HDAC2 and MDM2 activities in concert with the MULE E3 ligase function. Our findings connect HDAC2 activity to oncogenic protein stabilization via a series of post-translational events, which constitute an acetylation-dependent ubiquitin pathway that may serve as a common therapeutic target in human cancers. Results HDAC Inhibitor Treatment Reduces SS18-SSX Levels through the Ubiquitin System To assess the efficacy of HDAC inhibition in synovial sarcoma, we generated transgenic mice expressing human fusion oncogene within the myogenic factor 5 (Myf5) lineage (Haldar et?al., 2007). Treatment with the HDAC inhibitor FK228 on a weekly basis significantly reduced growth of mouse synovial sarcomas (Figures S1A and Nutlin 3b S1B), associated with amazing cytoreductive activity (Figures S1CCS1H). In addition to the histological observations, we noticed that SS18-SSX2 protein abundance was substantially decreased in FK228-treated tumors (Figures 1A and 1B). Considering the fusion oncogene dependency in synovial sarcoma, we decided to examine the molecular mechanism of SS18-SSX downregulation upon HDAC inhibition. To this end, we first developed a CRISPR/Cas9-based genome editing approach for FLAG epitope tagging of endogenous SS18-SSX2 fusion oncoprotein in patient-derived SYO-1 cells (Figures S1ICS1K). Anti-FLAG western blots revealed that SS18-SSX levels remained constant through the early time points of FK228 treatment, but fell drastically after overnight stimulation (Physique?S1L). Comparable results were obtained in cells treated with other structurally different HDAC inhibitors, such as SB939 and PCI-24781 (Physique?1C). We also tested this in SS18-SSX1-associated synovial sarcoma cells (Yamato-SS) and found that treatment with the HDAC inhibitors FK228 and SB939 led to a marked reduction of SS18-SSX protein levels, coupled with impaired tumor cell growth (Figures 1D and 1E). Importantly, the Nutlin 3b mRNA levels of SS18-SSX remained unchanged (Physique?S1M), whereas its protein stability was significantly reduced (Figures 1F and 1G). This effect was efficiently blocked by the proteasome inhibitor MG-132 (Physique?1C), and Nutlin 3b restoration of SS18-SSX levels correlated positively.