Abstract A053: Estrogen-related receptor beta2 as a regulator of the glioblastoma cytoskeleton and macrophage polarization

Glioblastoma (GBM) is a high-grade malignant glioma, the aggressive nature of which can be attributed in part to the dynamics of its actin cytoskeleton. The GBM cytoskeleton elicits invasion of tumor cells into nearby brain parenchyma, and these disseminated cells are a major cause of recurrence and mortality. Beyond tumor cell motility, actin cytoskeleton rearrangement is important for vesicle transport between the Golgi and plasma membrane that leads to the release of extracellular vesicles. GBM cells and tumor-associated macrophages (TAMs), which are 30-50% of the tumor mass, reciprocally communicate with each other through extracellular vesicles. Our overall goal is to define cytoskeletal targets that could ultimately be targeted to inhibit GBM recurrence. We published that the beta2 isoform of the orphan nuclear receptor estrogen-related receptor beta is a cytoplasmic protein that displays anti-tumor behavior when activated by the synthetic agonist DY131 in GBM cells. Beta2 co-immunoprecipitates (co-IPs) with the cytoskeletal regulatory protein cortactin, an actin nucleation-promoting factor that synergizes with the Arp2/3 complex and other NPFs from the Wiskott-Aldrich Syndrome protein (WASP) family to support actin branching. Beta2 contains a proline-rich region predicted to bind to the SH3 domain of cortactin, which should in turn disrupt cortactin·Arp2/3·N-WASP complex formation. We hypothesize that beta2 inhibits actin branching in GBM cells by disrupting the cortactin·Arp2/3·N-WASP complex, hindering motility and vesicle formation. To test this, we performed in silico modeling of the cortactin·beta2 interaction, coupled with immunofluorescence, co-IP, and western blot assays. Molecular docking shows the beta2 F-domain is in close proximity to cortactin’s SH3 domain. Co-IP confirms that beta2 interacts with SH3-wild-type cortactin in GBM cells, while the interaction with SH3-mutant cortactin is markedly diminished. Overexpression of beta2 disrupts interaction of endogenous cortactin with its binding partners N-WASP and Arp2 in GBM cells. DY131-activation of endogenous beta2 in GBM cells enhances interaction with cortactin, and disrupts actin cytoskeletal dynamics imperative for actin branching by simultaneously translocating cortactin, Arp2, and N-WASP from the cell periphery to the cell body, reducing lamellipodia, and increasing F-actin stress fibers. DY131-treated GBM cells also exhibit impaired Golgi structure and a reduction in the number of early endosomes, suggestive of impaired vesicle trafficking. To test if these alterations have an impact on cell-cell communication, we performed conditioned media experiments. Treating THP-1 human monocytes with GBM cell-conditioned media pre-treated with DY131 reduces polarization of M2, pro-tumor TAMs and induces M1, anti-tumor TAMs. Together these data support the conclusion that beta2 hinders GBM motility by disrupting cortactin’s function in cell migration and vesicle trafficking, and that this may also shift TAM polarization towards an anti-tumor M1 phenotype. Citation Format: Angela Appiah-Kubi, Deanna Tiek, Rebecca Riggins. Estrogen-related receptor beta2 as a regulator of the glioblastoma cytoskeleton and macrophage polarization [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr A053.