Abstract 6649: Pharmacological inhibition of HuR deregulates mitochondrial function and reduces mitochondrial activity in cancer cells

Abstract The RNA-binding protein, human antigen R (HuR), is overexpressed in many cancer types, and regulates genes linked to tumor growth, angiogenesis, cell cycle, and chemoresistance. HuR overexpression correlates with increased tumor size, invasiveness, and poor prognosis. Inhibiting HuR through pharmacological or RNA interference (RNAi) methods leads to antitumor activity both in vitro and in vivo against a broad spectrum of human cancers. Molecular mechanisms of antitumor activity include cell cycle arrest, increased ROS production, and enhanced DNA damage culminating in apoptotic cell death. However, the role of HuR in cancer metabolism and mitochondrial dynamics remains largely unexplored. In the present study we investigated the effects of the HuR inhibitor, CMLD-2, on glycolysis, mitochondrial function, ROS production, and mitophagy in human breast (MDA-MB-231) and lung (H1299) cancer cell lines. Both cell lines were treated with vehicle (DMSO) or CMLD-2 (20 µM or 30 µM) and analyzed at 24 or 48 hours post treatment. CMLD-2 treated cells showed a dose- and time-dependent reduction in markers of glycolysis and oxidative phosphorylation (GLUT1, GLUT4, HK2, LDHA), and a decrease in OXPHOS complex proteins. CMLD-2 treatment also led to a significant reduction in the rate of glycolysis (ECAR) and mitochondrial oxidative phosphorylation (OCR) as measured by Seahorse metabolism assays. Transmission electron microscopy (TEM) analysis showed CMLD-2 treatment increased autophagosome formation, mitochondrial fragmentation, and reduced mitochondrial count. Additionally, CMLD-2 treatment increased ROS production, with a 1.3- and 1.6-fold rise in MDA-MB-231 cells at 20 µM and 30 µM, respectively, and a 1.1- and 1.3-fold increase in H1299 cells at 20 µM and 30 µM, as measured by DCFDA fluorescence. Immunoblotting of AMPK-dependent autophagy signaling showed a time- and dose-dependent increase in LKB1, phospho-AMPK (Thr172), phospho-ULK (Ser555), phospho-FOXO3 (Ser413), p62, and LC3-I cleavage in CMLD-2-treated MDA-MB-231 cells. In H1299 cells, CMLD-2 treatment increased the above-mentioned markers except for phospho-ULK (Ser555), suggesting ULK-independent autophagy activation. Confocal microscopy of mitochondria (using Mitotracker Red CMX Ros) and LC3 (using LC3-GFP expression plasmid) in CMLD-2 treated cells, revealed colocalization of mitochondria with autophagosomes in both cell lines, indicating mitophagy. Our study demonstrates HuR inhibition results in mitochondrial dysfunction and significant metabolic changes in cancer cells, providing insights into the potential of HuR-targeted therapies for cancer treatment. Funding: The study was supported in part by a grant received from the National Institutes of Health (R01 CA 282735-01) and American Cancer Society (ACS-DICR: INTR-23-1253708-01-DICR INTR). Citation Format: Amber M. Vu, Meghna Mehta, Suriya Jayaraman, Megan Fuller, Rajagopal Ramesh, Anupama Munshi. Pharmacological inhibition of HuR deregulates mitochondrial function and reduces mitochondrial activity in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6649.