Abstract PS2-12-20: The Oncogenic Role of MitoNEET in Breast Cancer

Abstract Background: Aberrant glucose and energy metabolism of cancer cells, a phenomenon referred as the Warburg Effect in which most cancer cells produce energy predominantly through aerobic glycolysis in the cytosol, has long been considered as the major metabolic process for energy production and anabolic growth of cancer cells and is documented to promote breast cancer (BC) development and progression. However, how dysregulated glycolysis promotes BC carcinogenesis largely remains undefined. We discovered that mitoNEET or CISD1, a mitochondrial outer membrane protein, has a novel function as a redox enzyme. We believe mitoNEET constitutes a previously unknown cytosolic route of NADH oxidation to enlarge the cytosolic NAD+ pool, leading to aberrantly increased glycolysis and ATP/energy production in the cytosol, rendering mitoNEET a potential energy metabolism regulator. This study aims to determine whether mitoNEET acts as an oncogene to promote breast cancer growth by increasing glycolysis via an increase in the cytosolic pool of NAD+, promoting the Warburg Effect, and to determine the conditions in which mitoNEET promotes BC growth. Methods: Pan-cancer mitoNEET mRNA expression levels were determined using the TCGA database and analyzed using GraphPad Prism. mitoNEET expression was determined via Western Blotting (Cell Signaling #D5M4C). Colony formations were performed via staining with 0.5% Crystal Violet. mitoNEET knockout was achieved utilizing mitoNEET CRISPR sgRNA lentivectors (ABM LA116200). mitoNEET overexpression was achieved using mitoNEET Lentiviral Vector (ABM LC116200). Cell Viability was determined using 0.5% Crystal Violet staining with absorbance measured at 590nm using Promega’s GloMax Detection System (E9032). Inhibition of glycolysis was achieved using 2-Deoxy-D-glucose (2DG) (Sigma #D8375-1G). OCR, ECAR, and ATP production rates were determined using the Aglient Seahorse XF Analyzer. Conclusions: mitoNEET is highly expressed in the majority of BC cell lines screened. Its expression is also upregulated in oncogene-transformed mammary epithelial cell lines compared to normal or immortalized controls. Overexpresion of mitoNEET in immortalized mammary epithelial cells significantly increased growth compared to control. Suppression of mitoNEET via CRISPR-CAS9 led to significantly reduced growth of BC cell lines. Stable overexpression mitoNEET in BC cell lines led to significantly increased growth. Control BC cells were found to be more sensitive to 2DG treatment than mitoNEET-KO BC cells. Funding: LSU Health Sciences Center - New Orleans: Startup FundLSU: LSU Interinstitutional Cancer Research Funding Initiative Seed Grant, LCRC: Louisiana Cancer Research Center Strategic Investment In Translational Research Award Citation Format: C. Kent, Z. Liang, G. Vontz, C. Li, H. Ding, Q. Shen. The Oncogenic Role of MitoNEET in Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-12-20.