Nucleotide metabolism in cancer cells fuels a UDP-driven macrophage cross-talk, promoting immunosuppression and immunotherapy resistance.

Many individuals with cancer are resistant to immunotherapies. Here, we identify the gene encoding the pyrimidine salvage pathway enzyme cytidine deaminase (CDA) among the top upregulated metabolic genes in several immunotherapy-resistant tumors. We show that CDA in cancer cells contributes to the uridine diphosphate (UDP) pool. Extracellular UDP hijacks immunosuppressive tumor-associated macrophages (TAMs) through its receptor P2Y6. Pharmacologic or genetic inhibition of CDA in cancer cells (or P2Y6 in TAMs) disrupts TAM-mediated immunosuppression, promoting cytotoxic T cell entry and susceptibility to anti-programmed cell death protein 1 (anti-PD-1) treatment in resistant pancreatic ductal adenocarcinoma (PDAC) and melanoma models. Conversely, CDA overexpression in CDA-depleted PDACs or anti-PD-1-responsive colorectal tumors or systemic UDP administration (re)establishes resistance. In individuals with PDAC, high CDA levels in cancer cells correlate with increased TAMs, lower cytotoxic T cells and possibly anti-PD-1 resistance. In a pan-cancer single-cell atlas, CDAhigh cancer cells match with T cell cytotoxicity dysfunction and P2RY6high TAMs. Overall, we suggest CDA and P2Y6 as potential targets for cancer immunotherapy.

pH-dependent modulation of TRPV1 by modality-selective antagonists.

BACKGROUND AND PURPOSE: Antagonists of TRPV1 that inhibit all activation modes cause hyperthermia, hampering their medical use as novel analgesics. TRPV1 antagonists that do not (fully) inhibit responses to low pH do not cause hyperthermia, but it remains incompletely understood how such antagonists affect channel gating. We tested the hypothesis that pH-sparing antagonists act in a modality-selective manner on TRPV1, differentially affecting channel activation by protons and capsaicin. EXPERIMENTAL APPROACH: Using whole-cell patch-clamp and calcium imaging to measure channel activity in cells expressing wild type human TRPV1 or the pH-insensitive mutant F660A. Responses to protons and capsaicin were measured at different pH values in the presence of antagonists that reportedly partially spare (A-1165442) or potentiate (AMG7905) acid-evoked channel activation. KEY RESULTS: At pH 5.5, A-1165442 was equipotent at blocking acid- and capsaicin-evoked responses of wild type TRPV1. Its potency to inhibit acid-evoked responses was attenuated at pH ≤ 5.0. AMG7905, at a concentration (1 µM) that fully inhibits capsaicin-evoked responses, potentiated proton-evoked (pH 5.5) responses of wild type TRPV1. In the F660A mutant, the inhibitory efficacy of A-1165442 and AMG7905 towards capsaicin-evoked responses was reduced at lower pH values and AMG7905 acted as a partial agonist. CONCLUSION AND IMPLICATIONS: Our findings show that A-1165442 and AMG7905 interact in a pH-dependent manner with TRPV1, but this pH dependence is not strictly modality-selective. Reduced TRPV1 antagonism at acidic pH may limit analgesic efficacy in injured tissue and needs to be considered in models explaining the effects of antagonists on core body temperature.

Inhibition of TRPM8 by the urinary tract analgesic drug phenazopyridine.

BACKGROUND: and purpose: Phenazopyridine (PAP) is an over-the-counter drug widely used to provide symptomatic relief of bladder pain in conditions such as cystitis or bladder pain syndrome (BPS). Whereas the analgesic effect of PAP has been attributed to a local effect on the mucosa of the lower urinary tract (LUT), the molecular targets of PAP remain unknown. We investigated the effect of PAP on pain-related Transient Receptor Potential (TRP) channels expressed in sensory neurons that innervate the bladder wall. EXPERIMENTAL APPROACH: The effects of PAP on the relevant TRP channels (TRPV1, TRPA1, TRPM8, TRPM3) expressed in HEK293 or CHO cells was investigated using Fura-2-based calcium measurements and whole-cell patch-clamp recordings. Activity of PAP on TRPM8 was further analysed using Fura-2-based calcium imaging on sensory neurons isolated from lumbosacral dorsal root ganglia (DRG) of mice. KEY RESULTS: PAP rapidly and reversibly inhibits responses of TRPM8 expressed in HEK293 cells to cold and menthol, with IC50 values between 2 and 10 µM. It acts by shifting the voltage dependence of channel activation towards positive potentials, opposite to the effect of menthol. PAP also inhibits TRPM8-mediated, menthol-evoked calcium responses in lumbosacral DRG neurons. At a concentration of 10 µM, PAP did not significantly affect TRPA1, TRPV1, or TRPM3. CONCLUSION AND IMPLICATIONS: PAP inhibits TRPM8 in a concentration range consistent with PAP levels in the urine of treated patients. Since TRPM8 is expressed in bladder afferent neurons and upregulated in patients with painful bladder disorders, TRPM8 inhibition may underlie the analgesic activity of PAP.

Regulation of the Mitochondrial BKCa Channel by the Citrus Flavonoid Naringenin as a Potential Means of Preventing Cell Damage.

Naringenin, a flavanone obtained from citrus fruits and present in many traditional Chinese herbal medicines, has been shown to have various beneficial effects on cells both in vitro and in vivo. Although the antioxidant activity of naringenin has long been believed to be crucial for its effects on cells, mitochondrial pathways (including mitochondrial ion channels) are emerging as potential targets for the specific pharmacological action of naringenin in cardioprotective strategies. In the present study, we describe interactions between the mitochondrial large-conductance calcium-regulated potassium channel (mitoBKCa channel) and naringenin. Using the patch-clamp method, we showed that 10 µM naringenin activated the mitoBKCa channel present in endothelial cells. In the presence of 30 µM Ca2+, the increase in the mitoBKCa channel probability of opening from approximately 0.25 to 0.50 at -40 mV was observed. In addition, regulation of the mitoBKCa channel by naringenin was dependent on the concentration of calcium ions. To confirm our data, physiological studies on the mitochondria were performed. An increase in oxygen consumption and a decrease in membrane potential was observed after naringenin treatment. In addition, contributions of the mitoBKCa channel to apoptosis and necrosis were investigated. Naringenin protected cells against damage induced by tumor necrosis factor (TNF-) in combination with cycloheximide. In this study, we demonstrated that the flavonoid naringenin can activate the mitoBKCa channel present in the inner mitochondrial membrane of endothelial cells. Our studies describing the regulation of the mitoBKCa channel by this natural, plant-derived substance may help to elucidate flavonoid-induced cytoprotective mechanisms.