Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling
Ayush Kumar, Hira Lal Goel, Christi A. Silva, Tao Wang, Yansong Geng, Mengdie Wang, Shivam Goel, Kai Hu, Rui Li, Lihua J. Zhu, Jennifer L. Clark, Lindsay M. Ferreira, Michael A. Brehm, Thomas J. FitzGerald, Arthur M. Mercurio
Ayush Kumar, Hira Lal Goel, Christi A. Silva, Tao Wang, Yansong Geng, Mengdie Wang, Shivam Goel, Kai Hu, Rui Li, Lihua J. Zhu, Jennifer L. Clark, Lindsay M. Ferreira, Michael A. Brehm, Thomas J. FitzGerald, Arthur M. Mercurio
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Research Article Cell biology Oncology

Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

Abstract

The high rate of recurrence after radiation therapy in triple-negative breast cancer (TNBC) indicates that novel approaches and targets are needed to enhance radiosensitivity. Here, we report that neuropilin-2 (NRP2), a receptor for vascular endothelial growth factor (VEGF) that is enriched on subpopulations of TNBC cells with stem cell properties, is an effective therapeutic target for sensitizing TNBC to radiotherapy. Specifically, VEGF/NRP2 signaling induces nitric oxide synthase 2 (NOS2) transcription by a mechanism dependent on Gli1. NRP2-expressing tumor cells serve as a hub to produce nitric oxide (NO), an autocrine and paracrine signaling metabolite, which promotes cysteine-nitrosylation of Kelch-like ECH-associated protein 1 (KEAP1) and, consequently, nuclear factor erythroid 2-related factor 2–mediated (NFE2L2-mediated) transcription of antioxidant response genes. Inhibiting VEGF binding to NRP2, using a humanized mAb, results in NFE2L2 degradation via KEAP1, rendering cell lines and organoids vulnerable to irradiation. Importantly, treatment of patient-derived xenografts with the NRP2 mAb and radiation resulted in significant tumor necrosis and regression compared with radiation alone. Together, these findings reveal a targetable mechanism of radioresistance, and they support the use of NRP2 mAb as an effective radiosensitizer in TNBC.

Authors

Ayush Kumar, Hira Lal Goel, Christi A. Silva, Tao Wang, Yansong Geng, Mengdie Wang, Shivam Goel, Kai Hu, Rui Li, Lihua J. Zhu, Jennifer L. Clark, Lindsay M. Ferreira, Michael A. Brehm, Thomas J. FitzGerald, Arthur M. Mercurio

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Figure 4

NOS2 transcription is dependent on Gli1.

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NOS2 transcription is dependent on Gli1. We evaluated the Gli1 mRNA exp...
We evaluated the Gli1 mRNA expression in (A) BT549 shCtrl and shNRP2 cells (n = 3), (B) 4T1-RR cells that had been treated with either IgG or aNRP2-10 for 24 hours (n = 3), and (C) BT549 cells given a combined treatment of radiation (0, 5, and 10 Gy) with antibody for 24 hours (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) NOS2 mRNA expression was quantified in BT549 cells that had been treated with either DMSO or GANT61 (10 μM) for 24 hours (n = 3). ***P < 0.001. (E) Gli1 and NOS2 mRNA expression was quantified in BT549 shCtrl and shGli1 cells (n = 3). ***P < 0.001; ****P < 0.0001. (F) NOS2 mRNA expression in BT549 shNRP2 cells that had been transfected with either empty vector or a Gli1-HA construct (n = 3). The immunoblot shows the protein expression of NOS2, Gli1, and GAPDH in the same cells. ***P < 0.001; ****P < 0.0001. (G) Binding of Gli1 on the NOS2 promoter was analyzed using ChIP-qPCR in BT549 cells (n = 2, representative image). **P < 0.01. (H) NOS2 expression of CRISPR-generated mutations of the Gli1-binding site (Gli1-bind KO1 and KO2) compared with control (n = 3). (I) Clonogenic assay of control (sgCtrl), Gli1-bind KO1, and Gli1-bind KO2 cells that had been irradiated (0–6 Gy; n = 2, representative image). *P < 0.05 Data are presented as means ± SD (A–G, and I). Statistical analysis was performed using 2-tailed Student’s t test (B, D, and F), 1-way ANOVA multiple comparisons (A, C, and E), or 2-way ANOVA multiple comparisons (I).