title: BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.
abstract: Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.
authors.affiliation: Discovery Oncology, Hoffmann-La Roche Inc, Nutley, New Jersey, United States of America.
authors.forename: Brenden
authors.initials: B
authors.lastname: Chen
authors.forename: Christine
authors.initials: C
authors.lastname: Tardell
authors.forename: Brian
authors.initials: B
authors.lastname: Higgins
authors.forename: Kathryn
authors.initials: K
authors.lastname: Packman
authors.forename: John F
authors.initials: JF
authors.lastname: Boylan
authors.forename: Huifeng
authors.initials: H
authors.lastname: Niu
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation / Q000235:genetics*; D041681:NIH 3T3 Cells; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D020123:Sirolimus / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D047428:Protein Kinase Inhibitors; C492571:RICTOR protein, human; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D020123:Sirolimus