title: Evaluating rational non-cross-resistant combination therapy in advanced clear cell renal cell carcinoma: combined mTOR and AKT inhibitor therapy.
abstract: OBJECTIVE
  Inhibition of the mammalian target of rapamycin (mTOR), a regulator of hypoxia inducible factor (HIF), is an established therapy for advanced renal cell cancer (RCC). Inhibition of mTOR results in compensatory AKT activation, a likely resistance mechanism. We evaluated whether addition of the Akt inhibitor perifosine to the mTOR inhibitor rapamycin would synergistically inhibit RCC.
  
  
  METHODS
  Select RCC cell lines were studied [786-O, A498 (VHL mutant), CAKI-1 (VHL wild type), and 769-P (VHL methylated)] with single agent and combination therapy. Growth inhibition was assessed by MTT and cell cycling by flow cytometry. Phospho-AKT (S473) and HIF-2α were assessed by Western blot. Total RNA was isolated from 786-O cells subjected to single agent and combination treatments. In these cells, genome-wide expression profiles were assessed, and real-time PCR was used to confirm a limited set of expression results.
  
  
  RESULTS
  Three out of four cell lines (CAKI-1, 769-P, and 786-O) were sensitive to single-agent perifosine with 50% inhibitory concentrations ranging from 5 to 10 μM. Perifosine blocked phosphorylation of AKT induced by rapamycin and inhibited HIF-2α expression in 786-O and CAKI-1. Combined treatment resulted in sub-additive growth inhibition. GeneChip analysis and pathway modeling revealed inhibition of the IL-8 pathway by these agents, concomitant with up-regulation of the KLF2 gene, a known suppressor of HIF1α.
  
  
  CONCLUSIONS
  Perifosine is active in select RCC lines, abrogating the induction of AKT phosphorylation mediated by mTOR inhibition. Combined mTOR and AKT inhibition resulted in the modulation of pro-angiogenesis pathways, providing a basis for future investigations.
authors.affiliation: University of California, Davis Cancer Center, 4501 X Street, Suite, 3016, Sacramento, CA 95817, USA.
authors.forename: William S
authors.initials: WS
authors.lastname: Holland
authors.forename: Clifford G
authors.initials: CG
authors.lastname: Tepper
authors.forename: Jose E
authors.initials: JE
authors.lastname: Pietri
authors.forename: Danielle C
authors.initials: DC
authors.lastname: Chinn
authors.forename: David R
authors.initials: DR
authors.lastname: Gandara
authors.forename: Philip C
authors.initials: PC
authors.lastname: Mack
authors.forename: Primo N
authors.initials: PN
authors.lastname: Lara
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage / Q000494:pharmacology*; D002292:Carcinoma, Renal Cell / Q000188:drug therapy* / Q000473:pathology; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D016209:Interleukin-8 / Q000187:drug effects / Q000235:genetics; D007680:Kidney Neoplasms / Q000188:drug therapy* / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation / Q000187:drug effects; D010767:Phosphorylcholine / Q000008:administration & dosage / Q000031:analogs & derivatives; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors; D020123:Sirolimus / Q000008:administration & dosage; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors
chemical_list: D016209:Interleukin-8; D010767:Phosphorylcholine; C105905:perifosine; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus