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 7

Conventional fractionation with aNRP2 mitigates tumor growth.

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Conventional fractionation with aNRP2 mitigates tumor growth. (A) 4T1 ce...
(A) 4T1 cells (5 × 105) were injected into the mammary fat pads of BALB/c mice. Once the tumor volume reached approximately 100 mm3, the mice were divided into 4 groups of 5 mice each (mouse IgG, 2Gyx5; mouse IgG, 2Gyx5; aNRP2, 2Gyx5; aNRP2, 2Gyx5). The mice were given i.p. injections of the specified antibody (25 mg/kg) every 48 hours starting 1 day prior to irradiation for 2 weeks. Tumor volumes were measured with calipers every 2 days and are shown as means ± SEM. Tumors were extracted on day 16 and were used for histological and molecular profiling. **P < 0.01. (B) Necrotic areas of tissue sections of tumors were measured after H&E staining by finding the fraction of the area that is necrotic compared with the area of the tumor (n = 5).*P < 0.05. (C) Immunoblot showing γ-H2AX protein levels in irradiated tumors that had been treated with either mIgG or aNRP2-28. (D) NOS2 mRNA and protein levels were quantified for each treatment group using qPCR and immunoblotting (n = 3). *P < 0.05. (E) mRNA expression of NFE2L2 target genes (SLC7A11 and HMOX1) was measured for each treatment group using qPCR (n = 3). *P < 0.05; **P < 0.01. Data are presented as means ± SD (B, D, and E). Statistical analysis was performed using 2-tailed Student’s t test (B, D, and E) or 2-way ANOVA multiple comparisons (A).