title: AKT/mTOR substrate P70S6K is frequently phosphorylated in gallbladder cancer tissue and cell lines.
abstract: BACKGROUND
  Gallbladder carcinoma is a highly malignant tumor and a public health problem in some parts of the world. It is characterized by a poor prognosis and its resistance to radio and chemotherapy. There is an urgent need to develop novel therapeutic alternatives for the treatment of gallbladder carcinoma. The mammalian target of the rapamycin (mTOR) signaling pathway is activated in about 50% of human malignancies, and its role in gallbladder carcinoma has previously been suggested. In the present study, we investigated the phosphorylation status of the mTOR substrate p70S6K in preneoplastic and neoplastic gallbladder tissues and evaluated the effect of three mTOR inhibitors on cell growth and migration in gallbladder carcinoma cell lines.
  
  
  METHODS
  Immunohistochemical staining of phospho-p70S6K was analyzed in 181 gallbladder carcinoma cases, classified according to lesion type as dysplasia, early carcinoma, or advanced carcinoma. Protein expression of AKT/mTOR members was also evaluated in eight gallbladder carcinoma cell lines by Western blot analysis. We selected two gallbladder carcinoma cell lines (G415 and TGBC-2TKB) to evaluate the effect of rapamycin, RAD001, and AZD8055 on cell viability, cell migration, and protein expression.
  
  
  RESULTS
  Our results showed that phospho-p70S6K is highly expressed in dysplasia (66.7%, 12/18), early cancer (84.6%, 22/26), and advanced cancer (88.3%, 121/137). No statistical correlation was observed between phospho-p70S6K status and any clinical or pathological features, including age, gender, ethnicity, wall infiltration level, or histological differentiation (P < 0.05). In vitro treatment with rapamycin, RAD001, and AZD8055 reduced cell growth, cell migration, and phospho-p70S6K expression significantly in G-415 and TGBC-2TKB cancer cells (P < 0.001).
  
  
  CONCLUSIONS
  Our findings confirm the upregulation of this signaling pathway in gallbladder carcinoma and provide a rationale for the potential use of mTOR inhibitors as a therapeutic strategy for human gallbladder carcinoma.
authors.affiliation: Department of Pathology, Universidad de La Frontera, Center of Genetical and Immunological Studies-Scientific and Technological Bioresource Nucleus, Temuco, Santiago, Chile.
authors.forename: Pamela
authors.initials: P
authors.lastname: Leal
authors.forename: Patricia
authors.initials: P
authors.lastname: Garcia
authors.forename: Alejandra
authors.initials: A
authors.lastname: Sandoval
authors.forename: Kurt
authors.initials: K
authors.lastname: Buchegger
authors.forename: Helga
authors.initials: H
authors.lastname: Weber
authors.forename: Oscar
authors.initials: O
authors.lastname: Tapia
authors.forename: Juan C
authors.initials: JC
authors.lastname: Roa
keywords: AKT; cell line; gallbladder cancer; mTOR inhibitor; migration; p70S6K