Hypoxia-mediated regulation of DDX5 through decreased chromatin accessibility and post-translational targeting restricts R-loop accumulation.

Local hypoxia occurs in most solid tumors and is associated with aggressive disease and therapy resistance. Widespread changes in gene expression play a critical role in the biological response to hypoxia. However, most research has focused on hypoxia-inducible genes as opposed to those that are decreased in hypoxia. We demonstrate that chromatin accessibility is decreased in hypoxia, predominantly at gene promoters and specific pathways are impacted including DNA repair, splicing, and the R-loop interactome. One of the genes with decreased chromatin accessibility in hypoxia was DDX5, encoding the RNA helicase, DDX5, which showed reduced expression in various cancer cell lines in hypoxic conditions, tumor xenografts, and in patient samples with hypoxic tumors. Most interestingly, we found that when DDX5 is rescued in hypoxia, replication stress and R-loop levels accumulate further, demonstrating that hypoxia-mediated repression of DDX5 restricts R-loop accumulation. Together these data support the hypothesis that a critical part of the biological response to hypoxia is the repression of multiple R-loop processing factors; however, as shown for DDX5, their role is specific and distinct.

Ionizing Radiation Drives Key Regulators of Antigen Presentation and a Global Expansion of the Immunopeptidome.

Little is known about the pathways regulating MHC antigen presentation and the identity of treatment-specific T cell antigens induced by ionizing radiation. For this reason, we investigated the radiation-specific changes in the colorectal tumor cell proteome. We found an increase in DDX58 and ZBP1 protein expression, two nucleic acid sensing molecules likely involved in induction of the dominant interferon response signature observed after genotoxic insult. We further observed treatment-induced changes in key regulators and effector proteins of the antigen processing and presentation machinery. Differential regulation of MHC allele expression was further driving the presentation of a significantly broader MHC-associated peptidome postirradiation, defining a radiation-specific peptide repertoire. Interestingly, treatment-induced peptides originated predominantly from proteins involved in catecholamine synthesis and metabolic pathways. A nuanced relationship between protein expression and antigen presentation was observed where radiation-induced changes in proteins do not correlate with increased presentation of associated peptides. Finally, we detected an increase in the presentation of a tumor-specific neoantigen derived from Mtch1. This study provides new insights into how radiation enhances antigen processing and presentation that could be suitable for the development of combinatorial therapies. Data are available via ProteomeXchange with identifier PXD032003.

Secretion of Acid Sphingomyelinase and Ceramide by Endothelial Cells Contributes to Radiation-Induced Intestinal Toxicity.

Ceramide-induced endothelial cell apoptosis boosts intestinal stem cell radiosensitivity. However, the molecular connection between these two cellular compartments has not been clearly elucidated. Here we report that ceramide and its related enzyme acid sphingomyelinase (ASM) are secreted by irradiated endothelial cells and act as bystander factors to enhance the radiotoxicity of intestinal epithelium. Ceramide and the two isoforms of ASM were acutely secreted in the blood serum of wild-type mice after 15 Gy radiation dose, inducing a gastrointestinal syndrome. Interestingly, serum ceramide was not enhanced in irradiated ASMKO mice, which are unable to develop intestinal failure injury. Because ASM/ceramide were secreted by primary endothelial cells, their contribution was studied in intestinal epithelium dysfunction using coculture of primary endothelial cells and intestinal T84 cells. Adding exogenous ASM or ceramide enhanced epithelial cell growth arrest and death. Conversely, blocking their secretion by endothelial cells using genetic, pharmacologic, or immunologic approaches abolished intestinal T84 cell radiosensitivity. Use of enteroid models revealed ASM and ceramide-mediated deleterious mode-of-action: when ceramide reduced the number of intestinal crypt-forming enteroids without affecting their structure, ASM induced a significant decrease of enteroid growth without affecting their number. Identification of specific and different roles for ceramide and ASM secreted by irradiated endothelial cells opens new perspectives in the understanding of intestinal epithelial dysfunction after radiation and defines a new class of potential therapeutic radiomitigators. SIGNIFICANCE: This study identifies secreted ASM and ceramide as paracrine factors enhancing intestinal epithelial dysfunction, revealing a previously unknown class of mediators of radiosensitivity.

Caveolin-1 regulates the ASMase/ceramide-mediated radiation response of endothelial cells in the context of tumor-stroma interactions.

The integral membrane protein caveolin-1 (CAV1) plays a central role in radioresistance-mediating tumor-stroma interactions of advanced prostate cancer (PCa). Among the tumor-stroma, endothelial cells (EC) evolved as critical determinants of the radiation response. CAV1 deficiency in angiogenic EC was already shown to account for increased apoptosis rates of irradiated EC. This study explores the potential impact of differential CAV1 levels in EC on the acid sphingomyelinase (ASMase)/ceramide pathway as a key player in the regulation of EC apoptosis upon irradiation and cancer cell radioresistance. Enhanced apoptosis sensitivity of CAV1-deficient EC was associated with increased ASMase activity, ceramide generation, formation of large lipid platforms, and finally an altered p38 mitogen-activated protein kinase (MAPK)/heat-shock protein 27 (HSP27)/AKT (protein kinase B, PKB) signaling. CAV1-deficient EC increased the growth delay of LNCaP and PC3 PCa cells upon radiation treatment in direct 3D spheroid co-cultures. Exogenous C6 and C16 ceramide treatment in parallel increased the growth delay of PCa spheroids and induced PCa cell apoptosis. Analysis of the respective ceramide species in PCa cells with increased CAV1 levels like those typically found in radio-resistant advanced prostate tumors further revealed an upregulation of unsaturated C24:1 ceramide that might scavenge the effects of EC-derived apoptosis-inducing C16 ceramide. Higher ASMase as well as ceramide levels could be confirmed by immunohistochemistry in human advanced prostate cancer specimen bearing characteristic CAV1 tumor-stroma alterations. Conclusively, CAV1 critically regulates the generation of ceramide-dependent (re-)organization of the plasma membrane that in turn affects the radiation response of EC and adjacent PCa cells. Understanding the CAV1-dependent crosstalk between tumor cells and the host-derived tumor microvasculature and its impact on radiosensitivity may allow to define a rational strategy for overcoming tumor radiation resistance improving clinical outcomes by targeting CAV1.

Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.

Oxidative stress is a leading cause of endothelial dysfunction. The p38 MAPK pathway plays a determinant role in allowing cells to cope with oxidative stress and is tightly regulated by a balanced interaction between p38 protein and its interacting partners. By using a proteomic approach, we identified nucleophosmin (NPM) as a new partner of p38 in HUVECs. Coimmunoprecipitation and microscopic analyses confirmed the existence of a cytosolic nucleophosmin (NPM)/p38 interaction in basal condition. Oxidative stress, which was generated by exposure to 500 µM H2O2, induces a rapid dephosphorylation of NPM at T199 that depends on phosphatase PP2A, another partner of the NPM/p38 complex. Blocking PP2A activity leads to accumulation of NPM-pT199 and to an increased association of NPM with p38. Concomitantly to its dephosphorylation, oxidative stress promotes translocation of NPM to the nucleus to affect the DNA damage response. Dephosphorylated NPM impairs the signaling of oxidative stress-induced DNA damage via inhibition of the phosphorylation of ataxia-telangiectasia mutated and DNA-dependent protein kinase catalytic subunit. Overall, these results suggest that the p38/NPM/PP2A complex acts as a dynamic sensor, allowing endothelial cells to react rapidly to acute oxidative stress.-Guillonneau, M., Paris, F., Dutoit, S., Estephan, H., Bénéteau, E., Huot, J., Corre, I. Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.