IRE1 endoribonuclease signaling promotes myeloid cell infiltration in glioblastoma.

BACKGROUND: Intrinsic or environmental stresses trigger the accumulation of improperly folded proteins in the endoplasmic reticulum (ER), leading to ER stress. To cope with this, cells have evolved an adaptive mechanism named the unfolded protein response (UPR) which is hijacked by tumor cells to develop malignant features. Glioblastoma (GB), the most aggressive and lethal primary brain tumor, relies on UPR to sustain growth. We recently showed that IRE1 alpha (referred to IRE1 hereafter), 1 of the UPR transducers, promotes GB invasion, angiogenesis, and infiltration by macrophage. Hence, high tumor IRE1 activity in tumor cells predicts a worse outcome. Herein, we characterized the IRE1-dependent signaling that shapes the immune microenvironment toward monocytes/macrophages and neutrophils. METHODS: We used human and mouse cellular models in which IRE1 was genetically or pharmacologically invalidated and which were tested in vivo. Publicly available datasets from GB patients were also analyzed to confirm our findings. RESULTS: We showed that IRE1 signaling, through both the transcription factor XBP1s and the regulated IRE1-dependent decay controls the expression of the ubiquitin-conjugating E2 enzyme UBE2D3. In turn, UBE2D3 activates the NFκB pathway, resulting in chemokine production and myeloid infiltration in tumors. CONCLUSIONS: Our work identifies a novel IRE1/UBE2D3 proinflammatory axis that plays an instrumental role in GB immune regulation.

The PD-L1/PD-1 Axis Blocks Neutrophil Cytotoxicity in Cancer.

The PD-L1/PD-1 axis mediates immune tolerance and promotes tumor growth and progression via the inhibition of anti-tumor immunity. Blocking the interaction between PD-L1 and PD-1 was clinically shown to be beneficial in maintaining the anti-tumor functions of the adaptive immune system. Still, the consequences of blocking the PD-L1/PD-1 axis on innate immune responses remain largely unexplored. In this context, neutrophils were shown to consist of distinct subpopulations, which possess either pro- or anti-tumor properties. PD-L1-expressing neutrophils are considered pro-tumor as they are able to suppress cytotoxic T cells and are propagated with disease progression. That said, we found that PD-L1 expression is not limited to tumor promoting neutrophils, but is also evident in anti-tumor neutrophils. We show that neutrophil cytotoxicity is effectively and efficiently blocked by tumor cell-expressed PD-1. Furthermore, the blocking of either neutrophil PD-L1 or tumor cell PD-1 maintains neutrophil cytotoxicity. Importantly, we show that tumor cell PD-1 blocks neutrophil cytotoxicity and promotes tumor growth via a mechanism independent of adaptive immunity. Taken together, these findings highlight the therapeutic potential of enhancing anti-tumor innate immune responses via blocking of the PD-L1/PD-1 axis.

Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression.

Fusobacterium nucleatum is an oral anaerobe recently found to be prevalent in human colorectal cancer (CRC) where it is associated with poor treatment outcome. In mice, hematogenous F. nucleatum can colonize CRC tissue using its lectin Fap2, which attaches to tumor-displayed Gal-GalNAc. Here, we show that Gal-GalNAc levels increase as human breast cancer progresses, and that occurrence of F. nucleatum gDNA in breast cancer samples correlates with high Gal-GalNAc levels. We demonstrate Fap2-dependent binding of the bacterium to breast cancer samples, which is inhibited by GalNAc. Intravascularly inoculated Fap2-expressing F. nucleatum ATCC 23726 specifically colonize mice mammary tumors, whereas Fap2-deficient bacteria are impaired in tumor colonization. Inoculation with F. nucleatum suppresses accumulation of tumor infiltrating T cells and promotes tumor growth and metastatic progression, the latter two of which can be counteracted by antibiotic treatment. Thus, targeting F. nucleatum or Fap2 might be beneficial during treatment of breast cancer.

Neutrophil Cathepsin G and Tumor Cell RAGE Facilitate Neutrophil Anti-Tumor Cytotoxicity.

Neutrophils are a heterogeneous population of myeloid cells which may either promote or hinder tumor growth and progression. Anti-tumor neutrophils have the capacity to kill tumor cells in a contact-dependent manner. However, the molecular mechanisms underlying tumor cell recognition by neutrophils remained unexplored. Tumor cells were shown to express aberrant glycosylation patterns and neutrophils are equipped with receptors capable of recognizing such glycosylations. Accordingly, we hypothesized that the receptor for advanced glycation end products (RAGE) may facilitate neutrophil recognition of tumor cells. Indeed, RAGE decoy receptors and RAGE-specific blocking antibodies dramatically reduce tumor cell susceptibility to neutrophil cytotoxicity. Unexpectedly, we found that tumor cell RAGE rather than neutrophil RAGE is important for the killing process. We further identified neutrophil Cathepsin G as the neutrophil component interacting with tumor cell RAGE. Cathepsin G-deficient neutrophils show impaired ability to kill tumor cells, suggesting that RAGE-Cathepsin G interaction is required for neutrophil cytotoxicity. These data unravel new aspects of neutrophil anti-tumor activity and identify a novel role for RAGE and Cathepsin G in neutrophil-mediated cytotoxicity.

TRPM2 modulates neutrophil attraction to murine tumor cells by regulating CXCL2 expression.

In recent years, immune cells were shown to play critical roles in tumor growth and metastatic progression. In this context, neutrophils were shown to possess both pro- and anti-tumor properties. To exert their anti-tumor effect, neutrophils need to migrate towards, and form physical contact with tumor cells. Neutrophils secrete H2O2 in a contact-dependent mechanism, thereby inducing a lethal Ca2+ influx via the activation of the H2O2-dependent TRPM2 Ca2+ channel. Here, we explored the mechanism regulating neutrophil chemoattraction to tumor cells. Interestingly, we found that TRPM2 plays a role in this context as well, since it regulates the expression of potent neutrophil chemoattractants. Consequently, cells expressing reduced levels of TRPM2 are not approached by neutrophils. Together, these observations demonstrate how tumor cells expressing reduced levels of TRPM2 evade neutrophil cytotoxicity in two interrelated mechanisms-downregulation of neutrophil chemoattractants and blocking of the apoptotic Ca2+-dependent cascade. These observations demonstrate a critical role for TRPM2 in neutrophil-mediated immunosurveillance and identify cells expressing low levels of TRPM2, as a potential target for cancer therapy.

Microenvironmental Cues Determine Tumor Cell Susceptibility to Neutrophil Cytotoxicity.

We have recently shown that neutrophil antitumor cytotoxicity is Ca2+ dependent and is mediated by TRPM2, an H2O2-dependent Ca2+ channel. However, neutrophil antitumor activity is dependent on context and is manifested in the premetastatic niche, but not at the primary site. We therefore hypothesized that expression of TRPM2 and the consequent susceptibility to neutrophil cytotoxicity may be associated with the epithelial/mesenchymal cellular state. We found that TRPM2 expression was upregulated during epithelial-to-mesenchymal transition (EMT), and mesenchymal cells were more susceptible to neutrophil cytotoxicity. Conversely, cells undergoing mesenchymal-to-epithelial transition (MET) expressed reduced levels of TRPM2, rendering them resistant to neutrophil cytotoxicity. Cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung. These data identify TRPM2 as the link between environmental cues at the primary tumor site, tumor cell susceptibility to neutrophil cytotoxicity, and disease progression. Furthermore, these data identify EMT as a process enhancing tumor-cell immune susceptibility and, by contrast, MET as a novel mode of immune evasion.Significance: EMT is required for metastatic spread and concomitantly enhances tumor cell susceptibility to neutrophil cytotoxicity. Cancer Res; 78(17); 5050-9. ©2018 AACR.

TRPM2 Mediates Neutrophil Killing of Disseminated Tumor Cells.

Neutrophils play a critical role in cancer, with both protumor and antitumor neutrophil subpopulations reported. The antitumor neutrophil subpopulation has the capacity to kill tumor cells and limit metastatic spread, yet not all tumor cells are equally susceptible to neutrophil cytotoxicity. Because cells that evade neutrophils have greater chances of forming metastases, we explored the mechanism neutrophils use to kill tumor cells. Neutrophil cytotoxicity was previously shown to be mediated by secretion of H2O2 We report here that neutrophil cytotoxicity is Ca2+ dependent and is mediated by TRPM2, a ubiquitously expressed H2O2-dependent Ca2+ channel. Perturbing TRPM2 expression limited tumor cell proliferation, leading to attenuated tumor growth. Concomitantly, cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung.Significance: These findings identify the mechanism utilized by neutrophils to kill disseminated tumor cells and to limit metastatic spread. Cancer Res; 78(10); 2680-90. ©2018 AACR.

Hyperglycemia Impairs Neutrophil Mobilization Leading to Enhanced Metastatic Seeding.

Preexisting diabetes is a risk factor for the development of multiple types of cancer. Additionally, diabetic patients face a poorer prognosis when diagnosed with cancer. To gain insight into the effects of hyperglycemia, a hallmark of diabetes, on tumor growth and metastatic progression, we combined mouse models of cancer and hyperglycemia. We show that while hyperglycemia attenuates primary tumor growth, it concomitantly increases metastatic seeding in a distant organ. We further show that the increase in metastatic seeding is due to impaired secretion of granulocyte colony-stimulating factor (G-CSF) and impaired neutrophil mobilization. Normalizing blood glucose levels using insulin rescues neutrophil recruitment and tumor growth and concomitantly reduces metastatic seeding. These results provide links among hyperglycemia-induced changes in neutrophil mobilization, primary tumor growth, and metastatic progression. Furthermore, our observations highlight the importance of normalizing blood glucose levels in hyperglycemic cancer patients.