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Cooperative antiproliferative signaling by aspirin and indole-3-carbinol targets microphthalmia-associated transcription factor gene expression and promoter activity in human melanoma cells

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Abstract

Antiproliferative signaling of combinations of the nonsteroidal anti-inflammatory drug acetylsalicylic acid (aspirin) and indole-3-carbinol (I3C), a natural indolecarbinol compound derived from cruciferous vegetables, was investigated in human melanoma cells. Melanoma cell lines with distinct mutational profiles were sensitive to different extents to the antiproliferative response of aspirin, with oncogenic BRAF-expressing G361 cells and wild-type BRAF-expressing SK-MEL-30 cells being the most responsive. I3C triggered a strong proliferative arrest of G361 melanoma cells and caused only a modest decrease in the proliferation of SK-MEL-30 cells. In both cell lines, combinations of aspirin and I3C cooperatively arrested cell proliferation and induced a G1 cell cycle arrest, and nearly ablated protein and transcript levels of the melanocyte master regulator microphthalmia-associated transcription factor isoform M (MITF-M). In melanoma cells transfected with a −333/+120-bp MITF-M promoter-luciferase reporter plasmid, treatment with aspirin and I3C cooperatively disrupted MITF-M promoter activity, which accounted for the loss of MITF-M gene products. Mutational analysis revealed that the aspirin required the LEF1 binding site, whereas I3C required the BRN2 binding site to mediate their combined and individual effects on MITF-M promoter activity. Consistent with LEF1 being a downstream effector of Wnt signaling, aspirin, but not I3C, downregulated protein levels of the Wnt co-receptor LDL receptor-related protein-6 and β-catenin and upregulated the β-catenin destruction complex component Axin. Taken together, our results demonstrate that aspirin-regulated Wnt signaling and I3C-targeted signaling pathways converge at distinct DNA elements in the MITF-M promoter to cooperatively disrupt MITF-M expression and melanoma cell proliferation.

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Abbreviations

BRN2:

Brain-2

COX:

Cyclooxygenase

LEF1:

Lymphoid enhancer-binding factor 1

I3C:

Indole-3-carbinol

MITF-M:

Microphthalmia-associated transcription factor isoform M

NEDD4-1:

Neural precursor cell expressed developmentally downregulated 4

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Acknowledgments

The pGL2-333/+120-MITF-M, MITF promoter-luciferase reporter plasmid was a kind gift from Dr. Richard Marais (Cancer Research UK Centre of Cell and Molecular Biology, London, UK) (Wellbrock et al. 2008). We would like to thank Dr. Aishwarya Kundu for her outstanding advice and suggestions during the course of this work as well as Lorena Hardle for her help during the preliminary stages of the project. This study was supported by National Institutes of Health Public Service grant CA164095 awarded from the National Cancer Institute.

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  1. Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, 591 LSA, Berkeley, CA, 94720-3200, USA

    Kevin M. Poindexter, Susanne Matthew, Ida Aronchik & Gary L. Firestone

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  2. Susanne Matthew
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Poindexter, K.M., Matthew, S., Aronchik, I. et al. Cooperative antiproliferative signaling by aspirin and indole-3-carbinol targets microphthalmia-associated transcription factor gene expression and promoter activity in human melanoma cells. Cell Biol Toxicol 32, 103–119 (2016). https://doi.org/10.1007/s10565-016-9321-5

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