NAXE supports hypoxia-associated triple-negative breast cancer progression in association with HIF1α and PI3K/AKT signaling

Hypoxia contributes to triple-negative breast cancer (TNBC) progression, but the metabolic factors associated with sustained hypoxia-inducible factor 1α (HIF1α) signaling remain incompletely defined. NAD(P)HX epimerase (NAXE) repairs damaged NAD(P)H metabolites, and its role in TNBC has not been established. NAXE expression was examined in public transcriptomic datasets, an exploratory series of three paired TNBC specimens, and TNBC cell lines. NAXE loss-of-function with two independent siRNAs, HIF1α rescue, co-immunoprecipitation, cycloheximide-chase, MG132 treatment, ubiquitination, immunofluorescence, functional assays, and an MDA-MB-468 xenograft model were used to evaluate the relationship between NAXE and HIF1α-associated signaling under hypoxia. NAXE expression was higher in TNBC datasets and in MDA-MB-468 and BT-549 cells than in the corresponding non-tumor controls. The three-patient tissue series showed concordant higher NAXE staining in tumor tissue but was considered exploratory. Transient NAXE depletion reduced cell viability, wound closure, and invasion, and the phenotypes were reproduced with a second siRNA. NAXE and HIF1α were detected in the same immunoprecipitated complexes. NAXE depletion accelerated HIF1α loss during cycloheximide treatment, increased HIF1α-associated total and K48-linked ubiquitin, and the reduction in HIF1α was attenuated by MG132. HIF1α overexpression partially restored the cellular phenotypes and PI3K/AKT-associated protein signals reduced by NAXE depletion. Stable NAXE knockdown reduced xenograft growth, Ki67 and HIF1α staining, and PI3K/AKT-associated protein abundance, while increasing TUNEL positivity. The findings support a preclinical model in which NAXE contributes to hypoxia-associated TNBC phenotypes and maintenance of HIF1α protein. They do not establish direct NAXE-HIF1α binding, a requirement for NAXE enzymatic activity, or clinical efficacy.