Targeting VEGF/Neuropilin-2 as a Novel Approach to Induce Radiosensitivity in Triple Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is an aggressive phenotype with patients having more limited survival compared with other breast cancer subtypes. The high rate of locoregional/distant recurrence after primary management implies that additional treatment is necessary to optimize the sensitivity of TNBC tumors to therapy including radiation. Our lab studies the role of VEGF/Neuropilin-2 (NRP2) in mediating cancer stem cell properties such as self-renewal and therapy resistance. We sought to identify a new therapeutic approach that can induce radiosensitization in TNBC. Cell viability in response to radiotherapy in patient-derived organoids (PDO) and xenografts (PDX) was determined using CellTiter-Glo. The VEGF/NRP2 pathway was disrupted in human BT549 and murine 4T1 models using RNA interference and a function blocking antibody (aNRP2-10) provided by aTyr Pharma. Nitrite levels, a surrogate marker for NOS2 activity, were measured using Measure-IT Nitrite Assay Kit. H2DCFDA was used to measure intracellular reactive oxygen species (ROS). Clonogenic survival assays were used to calculate radiosensitivity enhancement ratios (rER) in TNBC cell lines. For our in vivo models, 4T1 xenografts were given antibody treatment every 48 hours starting a day prior to 10Gy irradiation. We identified a novel radioresistant population expressing high levels of NRP2 and NOS2 and observed that NOS2 expression was dependent on VEGF/NRP2 via Gli1 transcription. Interestingly, reducing NRP2 expression increased radiation-induced ROS because of reduced NOS2/NO levels. The downstream consequence of higher NOS2/NO was increased Nrf2 activation via KEAP1 S-nitrosylation; thus, inducing expression of antioxidant response elements. TNBC cells were radiosensitized by knocking down NRP2 (rER 1.19 - 1.34). We saw similar results when using aNRP2-10 vs IgG (rER 1.30 - 1.37). Ectopic expression of NOS2 and constitutively active Nrf2 rescued the radioresistance phenotype in the NRP2 knock-down cells (rER 0.73 - 0.82). Consistent with the cell line data, the clinically relevant models (PDOs and PDXs) had a synergistic reduction in cell viability in response to aNRP2-10 prior to radiotherapy. We also observed a significant reduction in tumor volume and increase in necrosis in vivo in response to VEGF/NRP2 inhibition during radiotherapy. Inhibiting VEGF/NRP2 induces radiosensitization of TNBC by decreasing NOS2/NO levels and, consequently, Nrf2-mediated antioxidant gene expression. The role of Gli1 as a transcription factor for NOS2 is a novel finding that provides an unexpected link between VEGF/NRP2 and redox homeostasis in response to radiation. Current Nrf2 inhibitors are non-specific and have high toxicity; thus, making VEGF/NRP2 inhibition more favorable. Our data provide a novel therapeutic strategy for targeting TNBC breast cancer.