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Vassiliki Poulaki, Sue Anne Chew, Bin He, Vijay Kumar Eedunuri, Martine Jager, Bert O'Malley, Nicholas Mitsiades; Microphthalmia-associated transcription factor (MITF) and Steroid Receptor Coactivator (SRC)-3 cooperate to promote proliferation, survival and metabolism in Gα-mutant uveal melanoma (UM) cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4524.
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© ARVO (1962-2015); The Authors (2016-present)
We recently reported that somatic mutations in Gαq and Gα11 (encoded by GNAQ and GNA11, respectively), found in most UMs, activate protein kinase C (PKC) and protein kinase D (PKD), which then phosphorylate and stabilize SRC-3. SRC-3 expression is critically important for UM cells, but its exact function remains unknown.
We screened 359,199 compounds in vitro for SRC-3 small molecule inhibitors (SMIs) by a high-throughput cell-based reporter assay. Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) for SRC-3 and MITF (a critical transcription factor for melanocytes) was performed in Gαq-mutant (mt) Mel202 cells. We explored the role of SRC-3 and MITF in Gα-mt UM cell lines using RNA interference (RNAi) and adenoviral-mediated gene transfer.
Our screen for SRC-3 SMIs yielded 620 hits, with significant overlap to a previous assay for MITF SMIs (406 overlapping compounds, P<0.0001, by Fisher's exact test). Representative MITF/SRC-3 SMIs exerted greater anticancer activity against Gα-mt than wild type cells, which was attenuated by SRC-3 overexpression. Co-immunoprecipitation revealed a direct physical interaction of MITF with SRC-3. Per ChIP-Seq, 96.7% of genes recruiting SRC-3 also recruit MITF. The consensus binding motifs for MITF and SRC-3 overlapped significantly, supporting their cooperative interaction. Silencing either MITF or SRC-3, by RNAi, had profound anticancer activity in Gα-mt cells, and the resulting gene expression profiles revealed significant overlap, that involved suppression of transcripts for kinases, nutrient transporters and metabolic enzymes. The promoters of those genes directly recruited SRC-3 and MITF, as documented by ChIP-Seq and confirmatory ChIP-PCR. Inhibition of the Gα/PKC/SRC3 pathway in Gα-mt cells suppressed metabolic activity and oxygen consumption and led to autophagy.
SRC-3 is stabilized post-translationally in Gα-mt UM cells by the Gα/PKC pathway and co- localizes on chromatin with MITF to cooperatively drive transcription of metabolism, proliferation and survival genes. This direct physical and functional interaction of SRC-3 with MITF highlights both of them as effectors of Gα-induced oncogenic signaling and as therapeutic targets in UM. The 406 SMIs found to target both SRC-3 and MITF represent potential drug candidates for UM.
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