H6PD as a metabolic target of DAP5 regulating NADPH homeostasis in TNBC

Triple-negative breast cancer (TNBC) is an aggressive malignancy defined by the absence of estrogen receptor (ER), progesterone receptor, and HER2 expression. This lack of receptor expression makes TNBC particularly challenging to treat using standard targeted therapies. TNBC cell proliferation is driven in part by cellular stress responses, such as oxidative stress and nutrient deprivation, that activate adaptive mechanisms supporting survival and growth under hostile conditions. H6PD (Hexose-6-phosphate dehydrogenase) is a key enzyme of the endoplasmic reticulum (ER)-associated pentose phosphate pathway (PPP) that generates NADPH, a reducing agent crucial for managing oxidative stress, facilitating protein folding, and supporting anabolic metabolism. Although the cytosolic PPP enzyme G6PD is extensively characterized, the role of H6PD in cancer metabolism and stress adaptation remains underexplored. Its ER localization provides a unique capability to buffer redox imbalance and maintain protein quality in cells with high anabolic demands. We investigated the regulation of H6PD by DAP5/eIF3d-dependent non-canonical translation in TNBC. Our analysis indicates that H6PD is a target of this translational mechanism. Targeted metabolomic profiling and DAP5 silencing revealed significant reductions in the levels of nucleotide cofactors (ADP, ATP, CTP, GTP, and UTP) and impaired nucleotide biosynthesis, indicating disruption of ER-associated PPP activity. These results suggest that DAP5-dependent translation maintains H6PD expression to support NADPH production, which is essential for anabolic metabolism in TNBC.