Triple‐negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca²⁺ uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca²⁺ uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU‐silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia‐inducible factor‐1α (HIF‐1α) is reduced, suggesting a signaling role for mROS and HIF‐1α, downstream of mitochondrial Ca²⁺. Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF‐1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca²⁺ uptake is a potential novel therapeutic target for clinical intervention. Synopsis MCU is the highly selective channel responsible for mitochondrial Ca²⁺ uptake. MCU expression is shown here to correlate with breast cancer progression. Depletion of MCU reduces tumor growth and metastasis formation via suppression of ROS production and inhibition of HIF‐1α signaling. MCU expression correlates with breast tumor size and lymph node infiltration MCU silencing reduces TNBC cell migration and invasion MCU deletion reduces in vivo tumor growth and metastasis formation MCU silencing inhibits ROS–HIF‐1α signaling route

Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H₂O₂ produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H₂O₂ production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca²⁺ uptake.

Redox sensitivity of T cells decreases through ORAI Ca ²⁺ channel subunit switching during T cell differentiation.