Abstract B023: Ectopic Expression of the Obscurin PH-domain in Aggressive Breast Cancer Cells Modulates PI3K/Akt Activity and Inhibits Cellular Migration and Invasion

Abstract New therapies targeting metastatic spread are greatly needed to improve breast cancer patient survival. Recently, obscurin, an ~800kDa protein localized to the cell membrane, has risen to the spotlight as an overlooked metastasis suppressor commonly lost in breast cancer. Specifically, breast epithelial cells lacking obscurin undergo epithelial-mesenchymal transition (EMT) and demonstrate increased migration and invasion. Biochemical studies in our lab have linked breast epithelial obscurin loss to increased activation of the pro-metastatic PI3K/Akt signaling pathway. Mechanistically, the obscurin pleckstrin homology (PH)-domain directly binds the SH3-domain of the p85 regulatory subunit of PI3K. Thus far, due to obscurin’s massive size, gain of function/rescue experiments of obscurin have not been feasible. To combat this challenge, we have generated a mini-obscurin protein construct composed of an obscurin core functional unit: a myristilated obscurin PH-domain (Myr-PH). We hypothesized that expression of Myr-PH in obscurin-low breast cancer cells will reduce PI3K/Akt pathway activity and suppress metastasis. Remarkably, we report that ectopic expression of Myr-PH in the obscurin-low MDA-MB-231 (triple negative) and SKBr3 (HER2 positive) breast cancer cell lines diminishes PI3K/Akt pathway activity. Furthermore, Myr-PH sequesters p85 to the cell membrane, anchoring actin at the cell periphery. Functionally, we demonstrate that expression of Myr-PH in both cell line receptor subtypes: 1) reduces cellular migration collectively through decreased scratch assay wound closure and decreased transwell assay 8um pore migration or at the single cell level, through reduced 3um diameter microchannel migration, 2) diminishes cell seeding on multiple extracellular matrix (ECM) substrates known to populate metastatic microenvironments, and 3) decreases cell dissemination and tumorsphere invasion into a 3D collagen-1 matrix. Underlying these functional changes, we show that Myr-PH inhibits actin-dependent filopodia and invadopodia formation and decreases the expression of downstream PI3K/Akt target matrix metalloproteinases, all of which are potent drivers of metastatic dissemination. To this end, we are currently delivering the obscurin-PH domain in animals via lipid nanoparticles, aiming to examine its anti-metastatic efficacy in vivo. If successful, this work would pioneer the first gene therapy-based, targeted use of a single metastasis suppressor protein domain to block PI3K signaling and inhibit breast cancer metastasis. Citation Format: Matthew K Eason, Se-Jong Lee, Poornima Dubey, Anthony Kim, Konstantinos Konstantopoulos, Aikaterini Kontrogianni-Konstantopoulos. Ectopic Expression of the Obscurin PH-domain in Aggressive Breast Cancer Cells Modulates PI3K/Akt Activity and Inhibits Cellular Migration and Invasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B023.