Usp18 Regulates Epidermal Growth Factor (EGF) Receptor Expression and Cancer Cell Survival via MicroRNA-7

Abstract

Epidermal growth factor receptor (EGFR) is involved in development and progression of many human cancers. We have previously demonstrated that the ubiquitin-specific peptidase Usp18 (Ubp43) is a potent regulator of EGFR protein expression. Here we report that the 3-untranslated region (3-UTR) of the EGFR message modulates RNA translation following cell treatment with Usp18 siRNA, suggesting microRNA as a possible mediator. Given earlier evidence of EGFR regulation by the microRNA miR-7, we assessed whether miR-7 mediates Usp18 siRNA effects. We found that Usp18 depletion elevates miR-7 levels in several cancer cell lines because of a transcriptional activation and/or mRNA stabilization of miR-7 host genes and that miR-7 acts downstream of Usp18 to regulate EGFR mRNA translation via the 3-UTR. Also, depletion of Usp18 led to a decrease in protein levels of other known oncogenic targets of miR-7, reduced cell proliferation and soft agar colony formation, and increased apoptosis. Notably, all of these phenotypes were reversed by a specific inhibitor of miR-7. Thus, our findings support a model in which Usp18 inhibition promotes upregulation of miR-7, which in turn inhibits EGFR expression and the tumorigenic activity of cancer cells.Despite advances in cancer treatments, improvement of overall patient survival remains poor. One of the primary reasons for this low success is the inherent complexity of oncogenic pathways such as those driven by the epidermal growth factor (EGF) 5 receptor (EGFR). EGFR is a member of the receptor tyrosine kinase (RTK) family. Upon binding of ligand, EGFR autophosphorylates at tyrosine residues and triggers an intracellular signal transduction cascade, which ultimately promotes cellular survival and division (1, 2). Dysregulation of EGFR and downstream signaling events is found in a multitude of tumor types (3-6). Therefore, targeting the tyrosine kinase activity of EGFR with small molecule inhibitors or targeting EGFR with antibodies has been a focus in the treatment of several tumors, including brain (glioblastoma), cervical, lung, and head and neck (squamous cell carcinoma). However, this strategy has resulted in minimal success. A major limitation of these approaches is that tumor cells eventually develop resistance to the current therapeutics. The resistance develops through increased ligand expression, additional somatic mutations in the EGFR tyrosine kinase domain, and increased heterodimerization with other RTKs (3, 7-9).As an alternative to developing approaches to directly inhibit EGFR signaling, our recent efforts focused on identifying allosteric modulators of EGFR protein levels. Inhibition of these modulators has the potential to significantly decrease EGFR protein levels irrespective of ligand levels or EGFR mutational status. Using a library of small interfering RNAs (siRNAs) that target deubiquitinase enzymes (DUBs), a class of proteins known to regulate receptor trafficking and expression (10 -12), we identified a number of candidate proteins which regulate EGFR prote...

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