Peter Chhoy

Understanding how cancer cells resist ferroptosis is a significant problem that impacts ongoing efforts to stimulate ferroptosis as a therapeutic strategy. We reported that prominin2 is induced by ferroptotic stimuli and functions to resist ferroptotic death. Although this finding has significant implications for therapy, specific prominin2 inhibitors are not available. We rationalized that the mechanism by which prominin2 expression is induced by ferroptotic stress could be targeted, expanding the range of options to overcome ferroptosis resistance. Here, we show that that 4-hydroxynonenal (4HNE), a specific lipid metabolite formed from the products of lipid peroxidation stimulates PROM2 transcription by a mechanism that involves p38 MAP kinase-mediated activation of HSF1 and HSF1-dependent transcription of PROM2. HSF1 inhibitors sensitize a wide variety of resistant cancer cells to drugs that induce ferroptosis. Importantly, the combination of a ferroptosis-inducing drug and an HSF1 inhibitor causes the cytostasis of established tumors in mice, although neither treatment alone is effective. These data reveal a novel approach for the therapeutic induction of ferroptosis in cancer.

Extracellular vesicles (EVs) play key roles in transporting key molecular constituents as cargo for extracellular trafficking. While several approaches have been developed to extract EVs from mammalian cells, the specific method of EV isolation can have a profound effect on membrane integrity and yield. Here, we describe a step-by-step procedure to separate EVs from adherent epithelial cells using differential ultracentrifugation. Separated EVs can be further analyzed by immunoblotting, mass spectrometry, and transmission electron microscopy to derive EV yield and morphology. For complete details on the use and execution of this protocol, please refer to Brown et al. (2019).

Prostate cancers are largely unresponsive to immune checkpoint inhibitors (ICIs), and there is strong evidence that programmed death-ligand 1 (PD-L1) expression itself must be inhibited to activate antitumor immunity. Here, we report that neuropilin-2 (NRP2), which functions as a vascular endothelial growth factor (VEGF) receptor on tumor cells, is an attractive target to activate antitumor immunity in prostate cancer because VEGF-NRP2 signaling sustains PD-L1 expression. NRP2 depletion increased T cell activation in vitro. In a syngeneic model of prostate cancer that is resistant to ICI, inhibition of the binding of VEGF to NRP2 using a mouse-specific anti-NRP2 monoclonal antibody (mAb) resulted in necrosis and tumor regression compared with both an anti–PD-L1 mAb and control immunoglobulin G. This therapy also decreased tumor PD-L1 expression and increased immune cell infiltration. We observed that the NRP2 , VEGFA , and VEGFC genes are amplified in metastatic castration-resistant and neuroendocrine prostate cancer. We also found that individuals with NRP2 High PD-L1 High metastatic tumors had lower androgen receptor expression and higher neuroendocrine prostate cancer scores than other individuals with prostate cancer. In organoids derived from patients with neuroendocrine prostate cancer, therapeutic inhibition of VEGF binding to NRP2 using a high-affinity humanized mAb suitable for clinical use also diminished PD-L1 expression and caused a substantial increase in immune-mediated tumor cell killing, consistent with the animal studies. These findings provide justification for the initiation of clinical trials using this function-blocking NRP2 mAb in prostate cancer, especially for patients with aggressive disease.