title: Morphoproteomic and pharmacoproteomic rationale for mTOR effectors as therapeutic targets in head and neck squamous cell carcinoma.
abstract: Head and neck squamous cell carcinoma (HNSCC) has a relatively high mortality rate and poor prognosis. Recently, we showed that overexpression of phosphorylated (p) nuclear factor-kappaB (NF-kappaB) in squamous cell carcinoma of the tonsil (SCCT) and high grade dysplasia is associated with a poor prognosis. Because the mammalian target of the rapamycin (mTOR) pathway contributes to the activation of NF-kappaB through immunophilin/mTOR signaling, we investigated: (a) the immunohistochemical expression and state of activation and potential clinical significance of components of the mTOR signal transduction pathway in SCCT patients (morphoproteomics); and (b) the inhibitory effects of rapamycin on the growth and state of activation of mTOR in 2 HNSCC cell lines (pharmacoproteomics). Archival biopsy materials from 39 patients with SCCT were studied by immunohistochemistry for the expression of p-mTOR (Ser 2448), and p-p70S6K (Thr 389), and/or cyclin D1. Results for SCCT were compared with adjacent non-neoplastic epithelium, when present, and with normal tonsillar epithelium from approximately age-matched controls; clinical outcomes were also assessed. SCCT showed mTOR (Ser 2448) expression in 93% (30/32 cases) with 2+ or 3+ plasmalemmal and/or cytoplasmic intensity in 84% vs 42% in surface epithelium from normal tonsils (p <0.001). The mean combined expression score (signal intensity x percentage of positive cells) for p-p70S6K was significantly greater in the SCCT group vs adjacent non-neoplastic squamous epithelium and normal tonsillar epithelium of the control group (p <0.05). A relationship existed between higher p-p70S6K expression levels in the non-neoplastic squamous epithelium adjacent to the SCCT and increased risk of death from disease (hazard ratio = 7.9; 95% confidence interval (CI) = 2.1 to 29.9; p = 0.002). There was also a relationship between nuclear expression of cyclin D1 in SCCT and shortened recurrence-free survival (p = 0.015). Two human HNSCC cell lines, SCC-15 and FaDu, were incubated with and without rapamycin to assess its impact on growth and on the expression of p-mTOR. Rapamycin in a dose-dependent fashion inhibited growth more in SCC-15, which correlated with a greater reduction in constitutively activated p-mTOR (Ser 2448) as shown by Western blotting. In conclusion, these morphoproteomic and pharmacoproteomic data collectively provide a rationale for selecting mTOR effectors as therapeutic targets in HNSCC.
authors.affiliation: Department of Pathology, University of Texas Houston Medical School, 6431 Fannin Street, Room 2.286, Houston, TX 77030, USA. robert.brown@uth.tmc.edu
authors.forename: Robert E
authors.initials: RE
authors.lastname: Brown
authors.forename: Ping L
authors.initials: PL
authors.lastname: Zhang
authors.forename: Mingyue
authors.initials: M
authors.lastname: Lun
authors.forename: Shaobo
authors.initials: S
authors.lastname: Zhu
authors.forename: Phillip K
authors.initials: PK
authors.lastname: Pellitteri
authors.forename: Waldemar
authors.initials: W
authors.lastname: Riefkohl
authors.forename: Amy
authors.initials: A
authors.lastname: Law
authors.forename: G Craig
authors.initials: GC
authors.lastname: Wood
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Kennedy
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D018307:Neoplasms, Squamous Cell / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000627:therapeutic use; D058570:TOR Serine-Threonine Kinases; D014067:Tonsillar Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology
chemical_list: D000970:Antineoplastic Agents; D019938:Cyclin D1; D011494:Protein Kinases; C546842:MTOR protein, human; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus