title: Deubiquitylating enzyme, USP9X, regulates proliferation of cells of head and neck cancer lines.
abstract: OBJECTIVE
  Truncating mutations in USP9X have been identified in oral squamous cell carcinoma patients. The aim of this study was to determine USP9X's functional role, if any, in head and neck cancer cells.
  
  
  METHODS
  USP9X was depleted/overexpressed in head and neck cancer cell line: SCC15 (tongue), CAL27 (tongue), FaDu (pharynx) and Detroit 562 (pharynx). Cell proliferation was monitored using the CyQUANT assay, and cell cycle distribution was determined by flow cytometry. Immunoblot assays were conducted to assess protein levels. RT-qPCR was performed to determine Notch and Wnt pathway target gene expression.
  
  
  RESULTS
  Our data showed a direct correlation between USP9X protein levels and proliferation, as well as Notch pathway activity in head and neck cancer cells. However, at least in FaDu, USP9X did not appear to regulate proliferation through the Notch pathway. Immunoblotting revealed a dramatic reduction in downstream targets of mTOR complex 1, namely total ribosomal protein (S6) and its phosphorylated form (pS6), when USP9X was depleted in FaDu cells. In contrast, in immortalized but non-tumorigenic HaCaT keratinocytes, USP9X depletion led to increase in cell proliferation, maintaining direct regulation of Notch activity.
  
  
  CONCLUSIONS
  The functional role of USP9X was found to be context dependent. USP9X possibly promotes head and neck cancer cell proliferation through the mTOR pathway.
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Nathan, Qld, Australia.
authors.forename: D M
authors.initials: DM
authors.lastname: Nanayakkara
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Nathan, Qld, Australia.
authors.forename: M N
authors.initials: MN
authors.lastname: Nguyen
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Nathan, Qld, Australia.
authors.forename: S A
authors.initials: SA
authors.lastname: Wood
mesh_terms: D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D051880:Receptors, Notch / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*
chemical_list: D051880:Receptors, Notch; C506234:USP9X protein, human; D058570:TOR Serine-Threonine Kinases; D043222:Ubiquitin Thiolesterase