Abstract 4691: Inhibition of ubiquitin specific protease Usp18 reduces cancer cell proliferation by down regulating multiple proto-oncogenes including EGFR

Abstract

Abstract A greater understanding of the mechanisms by which specific oncogenic proteins are regulated is critical to developing improved targeted therapies. To identify novel regulators of oncogenic cell surface receptors we screened for enzymes whose inhibition promoted down-regulation of these receptors. One of these screens involved exposing cancer cells to an RNAi library targeting deubiquitinase enzymes and subsequently analyzing the cell-surface levels of the proto-oncogene, epidermal growth factor receptor (EGFR) in squamous cell carcinoma cells. This screen ultimately led to the discovery that depletion of the ubiquitin specific protease Usp18 from numerous cancer cell lines dramatically reduced protein and activity levels of EGFR, by as much as 90%. Furthermore, the activity of Usp18 was critical to its ability to control EGFR protein levels. Interestingly, this study also determined that Usp18 does not directly target EGFR proteins but instead controls EGFR protein synthesis. The work to be presented here shows that Usp18 executes this function by regulating the levels of microRNA-7 (miR-7). Acute depletion (72h) of Usp18 from several cancer cell lines, including squamous cell carcinoma (SCC2) and glioma (T98; U87), leads to an increase (up to 2-fold) in miR-7. Since miR-7 has previously been shown to be inhibitory to EGFR mRNA translation we hypothesized that miR-7 functions intermediary to Usp18 regulation of EGFR. This idea was confirmed when we observed that addition of a miR-7 specific inhibitor to Usp18 siRNA experiments completely reversed EGFR down-regulation. These findings illuminate for the first time the Usp18/miR-7/EGFR pathway in cancer cells. We have also found that Usp18 depletion promotes down-regulation of the proto-oncogene cyclin D1. Thus, acute depletion of Usp18 in numerous cancer cell lines leads to the down-regulation of multiple proto-oncogenes. To determine if chronic Usp18 inhibition also promotes down-regulation of EGFR and cyclin D1 we generated clonal SCC2 and U87 cells which express inducible Usp18 shRNA via lentiviral integration. Long term Usp18 depletion (5-15 days) also promotes proto-oncogene down-regulation. Furthermore, we observed a >50% reduction in cellular proliferation, matching observations made with acute depletion. We also hypothesized that since miR-7 regulates EGFR mRNA translation at the 3’ UTR the mutational status of EGFR would not counter the capabilities of Usp18 inhibition. Indeed, depletion of Usp18 from cancer cell lines encoding mutated EGFR also leads to dramatic down-regulation of the receptor (e.g., 75% in EGFR-T790M expressing H1975 cells). Thus, Usp18 inhibition may represent a unique therapeutic opportunity, even in tumors which express a heterogenic population of EGFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4691. doi:10.1158/1538-7445.AM2011-4691