Mitochondrial matrix protein RTN4IP1 promotes the progression and metastasis of triple-negative breast cancer through metabolic reprogramming

BACKGROUND: Distant organ metastasis is the leading cause of mortality in breast cancer (BC), particularly in aggressive subtypes such as triple-negative breast cancer (TNBC). Metabolic reprogramming is a key driver of TNBC metastasis and is closely linked to remodeling of the mitochondrial proteome. Reticulon 4-interacting protein 1 (RTN4IP1) is a mitochondrial matrix protein that has recently emerged as a regulator of cellular metabolism. However, whether RTN4IP1 promotes BC metastasis through metabolic reprogramming remains unclear. METHODS: We performed integrative bioinformatics analysis to assess the association between RTN4IP1 expression and BC prognosis. RTN4IP1 was knocked down in TNBC cell lines to evaluate its effects on cell proliferation, invasion, and migration in vitro, as well as on metastatic colonization in an experimental lung metastasis model using tail vein injection in vivo. Metabolomic profiling and targeted metabolic assays, including measurement of the nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD+/NADH ratio), were performed to characterize metabolic alterations. The functional interaction between RTN4IP1 and mitochondrial adenylate kinase 4 (AK4) was examined using co-immunoprecipitation and genetic rescue experiments. RESULTS: Elevated RTN4IP1 expression was associated with poorer survival in patients with BC, with the strongest prognostic impact observed in TNBC. RTN4IP1 knockdown suppressed TNBC cell invasion and migration in vitro and significantly suppressed lung metastatic outgrowth in vivo. Mechanistically, RTN4IP1 knockdown depleted the intracellular NAD+/NADH pool, creating a dual metabolic constraint that impaired both oxidative phosphorylation and glycolysis. Notably, AK4 was identified as a direct and essential downstream effector of RTN4IP1. RTN4IP1 physically interacted with AK4, and AK4 knockdown abrogated the prometastatic phenotypes driven by RTN4IP1. CONCLUSION: This study identifies RTN4IP1 as a critical mitochondrial regulator that promotes TNBC metastasis by coordinating NAD+ metabolism and cellular bioenergetics through an AK4-dependent mechanism. Targeting the RTN4IP1-AK4 axis may represent a promising therapeutic strategy to inhibit metastatic progression in TNBC.