title: Combined targeting of AKT and mTOR synergistically inhibits proliferation of hepatocellular carcinoma cells.
abstract: BACKGROUND
  Due to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients.
  
  
  METHODS
  The activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells.
  
  
  RESULTS
  AKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo. Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient.
  
  
  CONCLUSIONS
  Our results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.
authors.affiliation: Center for Experimental Medicine, Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistr. 52, Hamburg 20246, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Grabinski
authors.forename: Florian
authors.initials: F
authors.lastname: Ewald
authors.forename: Bianca T
authors.initials: BT
authors.lastname: Hofmann
authors.forename: Katharina
authors.initials: K
authors.lastname: Staufer
authors.forename: Udo
authors.initials: U
authors.lastname: Schumacher
authors.forename: Björn
authors.initials: B
authors.lastname: Nashan
authors.forename: Manfred
authors.initials: M
authors.lastname: Jücker
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000737:chemistry / Q000188:drug therapy* / Q000201:enzymology / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016503:Drug Delivery Systems; D004357:Drug Synergism; D000068338:Everolimus; D025461:Feedback, Physiological; D005260:Female; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006575:Heterocyclic Compounds, 3-Ring / Q000008:administration & dosage / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008113:Liver Neoplasms / Q000737:chemistry / Q000188:drug therapy* / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D020123:Sirolimus / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000068338:Everolimus; D006575:Heterocyclic Compounds, 3-Ring; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; C548887:MK 2206; C546842:MTOR protein, human