title: GSK-3 directly regulates phospho-4EBP1 in renal cell carcinoma cell-line: an intrinsic subcellular mechanism for resistance to mTORC1 inhibition.
abstract: The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin 1 (mTORC1) signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated glycogen synthase kinase-3β (GSK-3β) positively regulated RCC proliferation. The aim of this study was to evaluate the role of GSK-3 in the PI3K/Akt/mTORC1 pathway and regulation of the downstream substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), ribosomal protein S6 kinase (S6K), and ribosomal protein S6 (S6RP).
  
  
  
  We used human RCC cell lines (ACHN, Caki1, and A498) and, as normal controls, human renal proximal tubular epithelial cell (HRPTEpC) and non-tumorous kidney tissues that were obtained surgically for treatment of RCC patients. Rapamycin-resistant ACHN (ACHN/RR) cells were generated with chronic exposure of ACHN to rapamycin ranging from 1nM finally to 1 μM. Cell viability, cell cycling and direct interaction between GSK-3β and 4EBP1 were evaluated with MTS assay, flowcytometry and in vitro kinase assay with recombinant GSK-3β and 4EBP1products, respectively. Protein expression and phosphorylation of molecules associated with the PI3K/Akt/mTORC1 pathway were examined by immunoblotting. Effects of drug combination were determined as the combination index with CompuSyn software.
  
  
  
  Overexpression and phosphorylation of 4EBP1 and S6RP together with GSK-3 activation were observed in RCC cell lines, but not in human normal kidney cells and tissues. Cell proliferation, p4EBP1 and pS6RP were strongly suppressed by GSK-3 inhibition. Rapamycin and LY294002 sufficiently decreased pS6RP, but only moderately p4EBP1. In vitro kinase assays showed that recombinant GSK-3β phosphorylated recombinant 4EBP1, and the effect was blocked by GSK-3 inhibitors. Different from rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 and pS6RP in ACHN and ACHN/RR (in 30 min to 1 h). AR- A014418 and rapamycin combination showed additivity at lower concentrations, but antagonism at higher concentrations.
  
  
  
  GSK-3β could directly phosphorylate 4EBP1 and activate the mTORC1 downstream signaling cascades to enhance protein biosynthesis and cell proliferation in RCC cell lines independent of rapamycin sensitivity. The direct GSK-3β/4EBP1 pathway might be an important subcellular mechanism as an inherent equipment for RCC cells to acquire clinical chemoresistance to mTORC1 inhibitors.
authors.affiliation: Department of Urology, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata, 990-9585, Japan. ito.hiromi@med.id.yamagata-u.ac.jp.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ito
authors.affiliation: Department of Urology, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata, 990-9585, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Ichiyanagi
authors.affiliation: Department of Urology, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata, 990-9585, Japan.
authors.forename: Sei
authors.initials: S
authors.lastname: Naito
authors.affiliation: Division of Urology, Department of Regenerative and Transplant Medicine, Niigata Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori, Chuo-ku, Niigata, 951-8510, Japan.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Bilim
authors.affiliation: Division of Urology, Department of Regenerative and Transplant Medicine, Niigata Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori, Chuo-ku, Niigata, 951-8510, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Tomita
authors.affiliation: Department of Urology, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata, 990-9585, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Kato
authors.affiliation: Department of Urology, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata, 990-9585, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Nagaoka
authors.affiliation: Department of Urology, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata, 990-9585, Japan.
authors.forename: Norihiko
authors.initials: N
authors.lastname: Tsuchiya
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D002867:Chromones / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D000071679:Glycogen Synthase Kinase 3 beta / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics / Q000378:metabolism*; D000076222:Mechanistic Target of Rapamycin Complex 1; D009025:Morpholines / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000037:antagonists & inhibitors; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D038601:Ribosomal Protein S6 / Q000235:genetics / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002867:Chromones; C087000:EIF4EBP1 protein, human; D009025:Morpholines; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D038601:Ribosomal Protein S6; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D000076222:Mechanistic Target of Rapamycin Complex 1; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus
keywords: 4EBP1; Akt; Combination index; GSK-3β; Rapamycin resistance; Renal cell carcinoma; mTORC1