Epigenetic-neurotransmitter crosstalk between EZH2 and dopamine D1 receptor signaling in triple-negative breast cancer

Triple-negative breast cancer (TNBC) remains a clinically intractable subtype defined by the absence of ER, PR, and HER2 and characterized by aggressive progression and limited therapeutic vulnerabilities. EZH2, the catalytic subunit of PRC2, is a known oncogenic driver in TNBC through deposition of H3K27me3 and repression of tumor-suppressive programs. In contrast, dopamine D1 receptor (DRD1) signaling has recently emerged as an unexpected tumor-suppressive axis. However, the mechanistic interplay between EZH2-mediated epigenetic repression and DRD1-mediated neurotransmitter signaling is unknown, representing a critical gap in understanding neuro-epigenetic integration in cancer. We hypothesized that coordinated dysregulation of EZH2 and DRD1 reprograms are transcriptional states that govern TNBC aggressiveness. CRISPR-engineered EZH2 and DRD1 knockouts (KO) were generated in MDA-MB-231 cells and validated by immunoblotting. DRD1 KO significantly increased proliferation and migration, whereas EZH2 KO diminished growth and disrupted cytoskeletal architecture. Notably, DRD1 ablation enhanced EZH2 activity, indicating a previously unrecognized feedback loop linking dopaminergic signaling to epigenetic repression. RNA-seq profiling (n=4/group) revealed extensive remodeling of transcriptional networks with discrete EZH2- and DRD1-dependent signatures (FDR < 0.05, |log2FC| > 2). EZH2 KO selectively induced cytokine activity, calcium ion binding, MHC class II interactions, and biological processes regulating adhesion, cell-cell communication, leukocyte activation, and axon/projection guidance, collectively reflecting cytoskeletal restructuring and immune-modulatory reprogramming. In contrast, DRD1 KO enriched signaling receptor binding, regulator/activator activity, receptor-ligand interactions, and pathways governing adhesion, migration, and IL-10-mediated inflammatory signaling, consistent with a pro-invasive, pro-inflammatory phenotype. Together, these data delineate a coordinated epigenetic-dopaminergic axis in TNBC. DRD1 loss potentiates EZH2-driven inflammatory and migratory programs, while EZH2 depletion activates adhesion and immune-regulatory pathways that constrain tumor aggressiveness. Targeting this dual regulatory node may represent a novel strategy to disrupt pro-tumor transcriptional circuitry and improve therapeutic outcomes in TNBC (Supported by DOD: W81XWH2010065, Eswar Shankar).