Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors.,However, a limitation to such treatment is the occurrence of resistance.,Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent.,In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results.,However, also in this case resistance inevitably occurs.,It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance.,Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D).,Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array.,Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies.,The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting.,The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays.,In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT.,Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth.,We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells.,On the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response.
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.