title: 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.
authors.affiliation: Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, California.
authors.forename: Aishwarya
authors.initials: A
authors.lastname: Kundu
authors.affiliation: Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, California.
authors.forename: Jeanne G
authors.initials: JG
authors.lastname: Quirit
authors.affiliation: Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, California.
authors.forename: Michelle G
authors.initials: MG
authors.lastname: Khouri
authors.affiliation: Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, California.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Firestone
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D007211:Indoles / Q000494:pharmacology / Q000627:therapeutic use*; D008545:Melanoma / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D008819:Mice, Nude; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics*; D017354:Point Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012867:Skin / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D012878:Skin Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D013449:Sulfonamides / Q000494:pharmacology / Q000627:therapeutic use; D000077484:Vemurafenib
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D007211:Indoles; D051739:Microphthalmia-Associated Transcription Factor; D013449:Sulfonamides; D000077484:Vemurafenib; C016517:indole-3-carbinol; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4
keywords: BRN2; I3C; MITF-M; MITF-M promoter activity; Vemurafenib combinations; anti-proiferative signaling; human melanoma; indole-3-carbinol