MON-319 - Rarα Functions as A Lineage Stabilizer in Breast Cancer Are Disrupted by the Corepressor PRAME

Retinoic acid receptor alpha (RAR) cooperates with estrogen receptor alpha (ER) to act as a luminal lineage-stabilizer in mammary gland morphogenesis, functions which appear disrupted in breast cancer. To reveal how RAR functions are altered we firstly undertook analyses of the TCGA-BRCA cohort, which revealed that RAR was highly expressed in tumors with a high luminal score and RAR corepressors were down-regulated including PRAME/MAPE. To explore the impact of PRAME on RAR we stably modulated expression using CRISPR activation (A) and repression (R) in luminal A (MCF-7, T47-D) ), HER2 (AU565) and TNBC (MDA-MB-231) breast cancer cell lines. We measured RAR ligand-independent and dependent effects in PRAME-A and PRAME-R cells treated with either vehicle control, or ATRA, or AM580 (RAR selective) ligands, and undertook proliferation and colony formation assays, RNA-Seq (8 h) and CUT&RUN for RAR and H3K27ac (6 h).
PRAME-A in MCF-7, AU565, and MDA-MB-231 cells led to significantly increased proliferation, independent of ligand, and also attenuated the antiproliferative responses towards ligand. The effects on colony formation were more subtle. At the transcriptomic level PRAME-A in both luminal A and TNBC cells supported a corepressor function with more genes down-regulated at the basal level, and suppressed ATRA and AM580-dependent gene expression patterns. For example in MCF-7 cells PRAME-A significantly repressed induction of BTG2 and other RAR-canonical target genes, and many of these effects were the opposite with PRAME-R. Gene set enrichment analyses also supported a role for PRAME to uniquely shape RAR signaling by increasing inflammatory networks (MCF-7 and AU565) and decreasing estrogen (MCF-7) and TNFA (MDA-MB-231) signaling. In HER2 positive AU565 the basal and ligand-dependent responses suggested PRAME exerts a broader transcriptional integrator function. Next, we examined the effect of PRAME on the H3K27ac and RAR cistromes. PRAME-A in MCF-7 cells profoundly reduced RAR basal binding, and PRAME-R increased this to more than 5000 binding sites. Both PRAME-A and PRAME-R also significantly impacted H3K27ac levels, including shifting levels at enhancer and promoter regions. Motif enrichment indicated that PRAME-dependent H3K27ac regions lost enrichment of luminal lineage motifs. In MCF-7 mock cells the basal and ligand treated H3K27ac cistromes were enriched for FOXA2 and TFAP2C motifs, both of which are associated with luminal lineage commitment. In PRAME-A cells enrichment of these motifs was reduced, further supporting a role for PRAME to gate-keep luminal commitment programs both at the basal level and in the context of RAR activation. Together these findings support a model of RAR promoting luminal commitment, and that the corepressor PRAME suppresses these functions and limits luminal phenotypes and may associate with more aggressive disease states.

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