LAUSR

Triple negative breast cancer (TNBC) cells overexpress enhancer of zeste homolog 2 (EZH2) the catalytic subunit of polycomb repressive complex 2 (PRC2). EZH2 is a potential driver of TNBC metastasis, and its high expression strongly associates with the TNBC phenotype as compared with other molecular subtypes of breast cancer. Furthermore, several studies have also shown the involvement of the neurotransmitter, dopamine, in proliferation, apoptosis, tumor angiogenesis, and drug resistance among different cancers, including the breast. Dopamine D1 receptor (D1R) activation in TNBC cell line induces apoptosis, autophagy, inhibit the invasion, and regress mammary tumors. The oncogenic nature of EZH2 in driving aggressiveness in TNBC cells led us to simultaneously target D1R and EZH2 to completely ablate tumor growth and metastasis. Conventional in vitro models utilize monolayers to investigate the effect of drugs on cancer cells but do not accurately recapitulate the in vivo microenvironment as the cells are subjected to homogeneous growth conditions. Microgels are miniature compartmentalized hydrogels that yield isotropic cell culture conditions unbiased by gravitational or hydrodynamic forces. As such, microgels lend suitable microenvironments conducive to the growth of tumor spheroids, which impose radial-dependent growth conditions as observed in vivo. While non-transformed epithelial cells with reconstituted basement membrane form hollow, growth-arrested, polarized three-dimensional (3D) structures, tumorigenic cells form large, solid, proliferating and invasive structures characteristic of in vivo tumor, which are recapitulated by tumor spheroids. As such, tumor spheroids become an ideal model to study the sensitivity of drugs combination, which led us to investigate the efficacy of EZH2 inhibitor and D1R agonist on TNBC tumor spheroids. Tumor spheroids were formed in monodisperse calcium-alginate microgels generated via droplet-based microfluidics, which mimic the viscoelastic properties of decellularized ECM and induce aggregation by removing integrin-binding sites. Tumor spheroids formed after 2 days were subjected to single and combination therapies of GSK126, an EZH2 inhibitor, and A77636, a dopamine agonist over the span of 4 days. Single doses demonstrated a dose-dependent decrease in tumor spheroid area. Furthermore, the combination therapy demonstrated a regression in growth, synergistic decrease in area (CI < 0.6), and the induction of necrosis. Furthermore, knockout of EZH2 in the TNBC cells resulted in inability for tumor spheroid formation and a sensitivity to A77636. Our findings imply that EZH2 is critical for spheroid formation and growth in TNBC and suggest that targeting the EZH2 alongside D1R presents a novel strategy for enhancing EZH2 inhibition (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: Xilal Y. Rima, Gautham Sarathy, Lara Rizzotto, Anagh Kulkarni, Dario Palmieri, Meisam Naeimi Kararoudi, Eduardo Reátegui, Bhuvaneswari Ramaswamy, Eswar Shankar. Simultaneous inhibition of EZH2 and activation of dopamine D1 in an isotropic triple-negative breast cancer microgel model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 332.