AKT2 Counteracts Hypoxia-Induced Suppression of Homologous Recombination in Triple-Negative Breast Cancer: A Targeted Approach to Sensitize Tumors to PARP Inhibition.

Triple-negative breast cancer (TNBC) is characterized by hypoxia and impaired fidelity to DNA repair and the activation of survival pathways. However, the role of specific AKT isoforms, particularly AKT2, in regulating residual homologous recombination (HR) activity and PARP inhibitor sensitivity under hypoxic stress remains unclear. This study aimed to establish the effect of AKT2 on hypoxia-induced defects in HR repair, DNA damage accretion, and therapeutic outcomes of PARP inhibition in TNBC. TNBC cells were cultured under chronic hypoxia (1% O2) or normoxia. Western blotting was used to measure protein expression and activation of HR- and hypoxia-related markers, including, pAKT (Ser473), RAD51, BRCA1, γH2AX, HIF-1α, and CHK1/CHK2. Immunofluorescence was used to assess the presence of RAD51 foci following 4 Gy of irradiation as a functional measure of HR activity. A cell viability assay was used to determine the sensitivity to olaparib. In vitro findings were confirmed in orthotopic xenograft models with stable AKT2 knockdown. Hypoxia in tumors was measured through pimonidazole staining and HIF-1α expression. Hypoxia increases HIF-1α expression and activates AKT signaling. In our model, chronic hypoxia suppressed RAD51, p-BRCA1, and checkpoint signaling, whereas total BRCA1 did not change and RAD51 foci decreased, indicating impaired and context-dependent disruption of HR. Only AKT2 silencing reduced RAD51 and p-BRCA1 under hypoxia, indicating an isoform-specific role for AKT2 in HR maintenance. In AKT2-deficient hypoxic cells, there was accumulation of persistent γH2AX and a corresponding repair defect. Functionally, AKT2 loss significantly enhanced olaparib sensitivity, resulting in marked RAD51 loss, robust γH2AX accumulation, and decreased viability. Knockdown of AKT2 in vivo increased the efficacy of olaparib, decreasing tumor weight and hypoxia and increasing the extent of DNA damage and suppression of HR markers, demonstrating a synergistic disruption of hypoxia-driven survival pathways. AKT2 plays a vital role in the regulation of the HR repair and hypoxia-driven survival in TNBC. Although hypoxia impaired HR through suppression of RAD51, p-BRCA1, and checkpoint signaling, AKT2 preserved residual HR activity required for tumor cell survival under hypoxic stress. Its loss disrupts HR components, enhances sustained DNA damage, inhibits tumor hypoxia and sensitizes hypoxic tumors dramatically to PARP inhibition. These findings support a mechanistic model in which AKT2 enables hypoxic TNBC cells to tolerate HR dysfunction and survive genotoxic stress, thereby contributing to therapeutic resistance. These results establish AKT2 as a therapeutic target to counteract resistance in hypoxia and increase the efficacy of PARP inhibitors in TNBC. Although AKT2-selective inhibitors have been reported, they remain largely preclinical. Accordingly, our findings support AKT2 as a promising therapeutic target and warrant further validation using AKT2-selective pharmacologic inhibition.