Inhibition of oncogenic BRAF activity by indole-3-carbinol disrupts microphthalmia-associated transcription factor expression and arrests melanoma cell proliferation

Abstract

Indole-3-carbinol (I3C), an anti-cancer phytochemical derived from cruciferous vegetables, strongly inhibited proliferation and down-regulated protein levels of the melanocyte master regulator micropthalmia-associated transcription factor (MITF-M) in oncogenic BRAF-V600E expressing melanoma cells in culture as well as in vivo in tumor xenografted athymic nude mice. In contrast, wild type BRAF-expressing melanoma cells remained relatively insensitive to I3C anti-proliferative signaling. In BRAF-V600E-expressing melanoma cells, I3C treatment inhibited phosphorylation of MEK and ERK/MAPK, the down stream effectors of BRAF. The I3C anti-proliferative arrest was concomitant with the down-regulation of MITF-M transcripts and promoter activity, loss of endogenous BRN-2 binding to the MITF-M promoter, and was strongly attenuated by expression of exogenous MITF-M. Importantly, in vitro kinase assays using immunoprecipitated BRAF-V600E and wild type BRAF demonstrated that I3C selectively inhibited the enzymatic activity of the oncogenic BRAF-V600E but not of the wild type protein. In silico modeling predicted an I3C interaction site in the BRAF-V600E protomer distinct from where the clinically used BRAF-V600E inhibitor Vemurafenib binds to BRAF-V600E. Consistent with this prediction, combinations of I3C and Vemurafenib more potently inhibited melanoma cell proliferation and reduced MITF-M levels in BRAF-V600E expressing melanoma cells compared to the effects of each compound alone. Thus, our results demonstrate that oncogenic BRAF-V600E is a new cellular target of I3C that implicate this indolecarbinol compound as a potential candidate for novel single or combination therapies for melanoma. © 2016 Wiley Periodicals, Inc.

Keywords: BRN2; I3C; MITF-M; MITF-M promoter activity; Vemurafenib combinations; anti-proiferative signaling; human melanoma; indole-3-carbinol.

Figure 1

Effects of I3C on in vivo growth of melanoma tumor xenografts, production of MITF-M, and melanoma cell proliferation. (A) Athymic mice with G-361 cell-derived tumor xenografts were injected subcutaneously with either I3C or with DMSO vehicle control, and resulting tumor volumes were calculated as described in the Supporting Information. The micrograph insert shows tumors harvested at the end of week 4. (B) At terminal sacrifice, tumor sections were analyzed for MITF-M expression by immunofluorescence using primary antibodies to MITF-M (left panel). Cultured G361cells treated with 200 μM I3C for 72 h were similarly probed for MITF-M levels (right panel). (C) Human melanoma cell lines with distinct genotypes were treated with or without 200 μM I3C for 48 h and the effects on cell proliferation measured using a CCK-8 assay relative to the vehicle control. (D) The levels of MITF-M protein were determined in melanoma cells treated with the indicated concentrations of I3C for 48 h by western blots.

Figure 2

Role of MITF-M in I3C anti-proliferative response and I3C regulation of MITF-M gene expression. (A) G-361 cells were either transfected with pCMV-MITF expression vector, or pCMV empty vector control or left untransfected, and each set of cells were treated with or without 200 μM I3C for a submaximal time of 24 h. Levels of MITF-M, CDK2, CDK4, and HSP90 protein were determined by western blots. (B) Cell proliferation was measured using a CCK-8 assay, and results show the mean of three independent experiments ±SEM (*P<0.01) (C) MITF-M transcript expression in G-361 cells treated with or without 200 μM I3C was determined by RT-PCR analysis in comparison to the GAPDH control. (D) Cells were transfected with reporter plasmids containing either a wild type MITF-M promoter (WT), a BRN2 consensus site mutant (BRN2 Mut) or the PGL2 empty control vector. Luciferase specific activity was measured in cells treated with or without 200 μM I3C for 24 h. The bar graph shows the results of three independent experiments in triplicate ± SEM (*P<0.01). (E) ChIP assay was performed on G361 cells treated with or without 200 μM I3C for 48 h using BRN2 antibodies (IP:BRN2) or the control IgG with one percent input as the loading control. The bar graphs quantify the densitometry results from three independent experiments ± SEM (*P<0.01). (F) G-361 cells were treated with or without 200 μM I3C for 48 h and BRN2 localization was examined by immunofluorescence using anti-BRN2 antibodies. The insets show magnified portions of the larger fields.

Figure 3

Effect of I3C on BRAF signalling in cells and in vivo tumors (A) BRAF-V600E expressing G-361 and DM-738 cells as well as wild type BRAF expressing SK-MEL-2 cells were treated with or without 200 μM I3C for 24, 48, and 72 h. Western blots were performed on total cell extracts and probed with the indicated antibodies. (B) Tumor xenograft sections from I3C treated and untreated animals were analyzed for ERK-p and total ERK-1 protein by immunofluorescence. (C) G-361 cells were treated with or without 10 μM Vemurafenib BRAF inhibitor, 10 μM U0126 MEK inhibitor, or 200 μM I3C for 48 h. Western blots were probed for the indicated proteins and the results are representative of three independent experiments.

Figure 4

I3C inhibits oncogenic BRAF-V600E enzymatic activity. (A) G-361 cells were treated with 200 μM I3C, 400 μM I3C, 10 μM Vemurafenib, or with DMSO vehicle control for 48 h. BRAF was immunoprecipitated from pretreated cell extracts, and assayed for its intrinsic enzymatic activity in vitro using inactive MEK as the substrate in the presence of ATP. Immunoprecipitation with non-immune IgG was used as a negative control. The level of in vitro generated phospho-MEK was determined by western blot analysis (left panel). The lower panels show the level of BRAF remaining in the cell extracts after immunoprecipitation by western blot analysis. (B) BRAF-V600E was immunoprecipitated from untreated G-361 cells and incubated in vitro with 200 μM I3C, 400 μM I3C, 10 μM Vermurafenib, or the DMSO vehicle control. BRAF-V600E enzymatic activity was assayed using the level of detected in vitro phosphorylation of inactive MEK as described above. (C) Wild type BRAF was immunoprecipitated from SK-MEL-2 melanoma cells and BRAF enzymatic activity post I3C treatment was analyzed as described for (C). (D) I3C regulation of BRAF enzymatic activity in vitro was quantified by densitometry of MEK-P and total MEK protein levels detected by western blots and the ratio of MEK-P:total MEK determined from three independent experiments±SEM (*P<0.01).

Figure 5

In silico analysis of interactions of I3C and Vermurafenib with BRAF-V600E and combinational effects on melanoma cell proliferation and BRAF-V600E signaling. (A) The crystallographic structures of the BRAF-V600E protomers (accession number: 3OG7) are shown with the known Vemurafenib binding site (right protomer) and the predicted I3C binding site visualized using the PyMol program (left protomer). (B) G-361 or DM-738 cells were treated with the indicated combinations of I3C and Vermurafenib for a suboptimal time of 24 h and inhibition of proliferation was monitored using a CCK-8 assay. The results represent the average of three independent experiments with a mean±SEM (*P< 0.01) shown in the bar graphs. (C) G-361 cells or DM-738 cells were treated with combinations of 200 μM I3C and/or 15 μM Vermurafenib for 24 h and the levels of the indicated proteins determined by western blots on total cell extracts.