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.

Figure 1. Treatment with MEK inhibitor RG7167 or rapamycin activates AKT pathway in NIH3T3 vector control clones but not in BRAFV600E clones.

(A) Western blot analysis of AKT, MEK, and ERK phosphorylation in the isogenic pair of NIH3T3 clones 4 hours post treatment with MEK inhibitor RG7167 at indicated concentration. (B) Western blot analysis of AKT substrates (FOXO1, GSK3α/β, PRAS40) phosphorylation in the isogenic pair of NIH3T3 cells 4 hours post treatment with either MEK inhibitor RG7167 or rapamycin at indicated concentrations. (C) Western blot analysis of MEK and AKT phosphorylation in the isogenic pair of NIH3T3 cells 24 hours after CRAF or BRAF knock-down and 4 hours post RG7167 treatment. (D) Schematic presentation of a model of the cross-talk between MAPK and AKT pathways in engineered NIH3T3 clones. The left panel - MEK inhibitor induces pAKT by suppressing ERK-dependent negative feedback loop in control NIH3T3 cells. The right panel – although MEK inhibitor should induce pAKT through alleviating ERK-dependent feedback loop, the presence of BRAFV600E imparts a negative impact on pAKT induction.

Figure 2. Melanoma cell lines with different genetic backgrounds respond differently to treatment-induced AKT phosphorylation.

Western blot analysis of the phosphorylation of AKT, ERK and S6 ribosome proteins in melanoma cell lines 4 hours after treatment with MEK inhibitor RG7167 or rapamycin.

Figure 3. BRAFV600E is required and sufficient to suppress AKT phosphorylation in a subset of melanoma cells.

(A) Western blot analysis of AKT phosphorylation 24 hours after BRAF or CRAF knock-down in A375 melanoma cell line 4 hour post RG7167 treatment. (B) Western blot analysis of AKT substrates (FOXO1, GSK3α/β, PRAS40) phosphorylation 24 hours after BRAF knock-down in A375 melanoma cell line. (C) Western blot analysis of AKT phosphorylation 24 hours after BRAF knock-down in LOX melanoma cell line. (D) Western blot analysis of AKT, MEK, and ERK phosphorylation in CHL1 melanoma cell line 24 hours after transient transfection (wild-type BRAF or BRAFV600E) and treated with RG7167 for 4 hours.

Figure 4. BRAFV600E suppresses AKT phosphorylation through rictor, but independent of MEK, ERK and its kinase activity.

(A) Western blot analysis of AKT phosphorylation and PTEN in A375 melanoma cell line 24 hours after knock-down of BRAF, MEK1/2, ERK1/2, rictor, raptor, or combined knock-down. (B) Western blot analysis of AKT and ERK phosphorylation in CHL1, A375 and LOX melanoma cell lines treated with RG7167 or vemurafenib for 4 hours.

Figure 5. BRAFV600E interacts with rictor complex (mTORC2) and impairs the enzymatic activity.

(A) Western blot analysis of BRAF and rictor or raptor complex in respective rictor and raptor immunoprecipitation in A375. (B) Western blot analysis of in vitro phosphorylation of recombinant AKT by rictor complex purified from NIH3T3 isogenic pair or CHL1 and A375 melanoma cell lines. (C) Left panel: Western blot analysis of AKT phosphorylation in A375 melanoma cell line 24 hours after control or BRAF siRNA treatment. Right panel: Western blot analysis of in vitro phosphorylation of recombinant AKT by rictor complex purified from A375 melanoma cell line 24 hours after control or BRAF siRNA treatment.

Figure 6. Schematic representations of the cross-talk between MAPK and AKT pathways in cells with different genetic backgrounds.

(A) In cells with “wild-type” AKT signaling, the presence of BRAFV600E exerts a negative impact through mTORC2 on AKT phosphorylation and pathway activation. Suppression of AKT activity leads to a de-repression of AKT substrates PRAS40, FOXO and GSK3β due to the reduction of AKT-mediated phosphorylation. (B) In cells where AKT signaling is aberrantly activated (by PTEN loss, PI3K activation, or RTK amplification), the negative impact of BRAFV600E is countered by a dominant input upstream of AKT. Consequently, activated AKT phosphorylates substrates PRAS40, FOXO and GSK3β and suppresses their activity.