Abstract 4821: Investigating FOXC2-dependent mechanisms regulating stem cell Identity in breast cancer

Abstract Stem cells maintain tissue homeostasis by balancing self-renewal and differentiation, a process shaped by how fate determinants are stabilized and partitioned during mitosis. Disruption of these mechanisms can either generate cancer stem cells (CSCs) that drive tumor progression or deplete CSCs that lead to the elimination of the tumor. In triple-negative breast cancer (TNBC), the embryonic transcription factor FOXC2 is aberrantly reactivated, leading to induction of EMT, the gain of CSC properties, and poor patient outcomes. Although our lab has shown that FOXC2 levels fluctuate across the cell cycle, the cues governing this regulation remain unclear. Since PLK1 is a key mitotic kinase and a targetable vulnerability in aggressive TNBC, we investigated whether FOXC2 is regulated by PLK1-mediated phosphorylation. Pharmacologic PLK1 inhibition (Volasertib) reduces FOXC2 protein levels, and this loss is reversed by the proteasome inhibitor MG132, suggesting that PLK1-dependent phosphorylation stabilizes FOXC2. Using SUM159 cells expressing phospho-ablated (S125A, T465A) and phospho-mimetic (S125E, T465E) FOXC2 mutants, we evaluated its CSC-related function, including mammosphere assays, and found that phosphorylation influences FOXC2’s role in maintaining CSC traits. Biochemical pulldown, immunoblotting, and imaging assays support a potential interaction and role for FOXC2 and PLK1. Promoter analysis of the PLK1 promoter identified FOXC2 binding sites near the transcription start site, suggesting possible reciprocal control. Live-cell imaging further indicates that FOXC2 is spatially regulated during mitosis and that phosphorylation may affect its chromatin association and inheritance. Collectively, our findings suggest PLK1-dependent phosphorylation of FOXC2 may stabilize the FOXC2 protein and influence its function during mitosis and regulate stem cell identity in daughter cells during cell division. Citation Format: Joanna Joyce Maddela, Petra den Hollander, Nick Allen Kuburich, Maria Castaneda, Mika Pietila, Mercy Adewumi, Sendurai A. Mani. Investigating FOXC2-dependent mechanisms regulating stem cell Identity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4821.