IRE1 RNase controls CD95-mediated cell death.

Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling.

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.

Endoplasmic reticulum homeostasis-From molecules to organisms: Report on the 14th International Calreticulin Workshop, Saint Malo, France.

The Calreticulin Workshop, initiated in 1994 by Marek Michalak in Banff (Alberta, Canada), was first organized to be an informal scientific meeting attended by researchers working on diverse biological questions related to functions associated with the endoplasmic reticulum (ER)-resident lectin-like chaperone and applied to a wide range of biological systems and models. Since then, this workshop has broadened the range of topics to cover all ER-related functions, has become international and has been held in Canada, Chile, Denmark, Italy, Switzerland, UK, USA, Greece and this year in France. Each conference, which is organized every other year (pending world-wide pandemic), generally attracts between 50 and 100 participants, including both early career researchers and international scientific leaders to favour discussions and exchanges. Over the years, the International Calreticulin Workshop has become an important gathering of the calreticulin and ER communities as a whole. The 14th International Calreticulin Workshop occurred from May 9-12 in St-Malo, Brittany, France, and has been highlighted by its rich scientific content and open-minded discussions held in a benevolent atmosphere. The 15th International Calreticulin Workshop will be organized in 2025 in Brussels, Belgium.

A novel IRE1 kinase inhibitor for adjuvant glioblastoma treatment.

Inositol-requiring enzyme 1 (IRE1) is a major mediator of the unfolded protein response (UPR), which is activated upon endoplasmic reticulum (ER) stress. Tumor cells experience ER stress due to adverse microenvironmental cues, a stress overcome by relying on IRE1 signaling as an adaptive mechanism. Herein, we report the discovery of structurally new IRE1 inhibitors identified through the structural exploration of its kinase domain. Characterization in in vitro and in cellular models showed that they inhibit IRE1 signaling and sensitize glioblastoma (GB) cells to the standard chemotherapeutic, temozolomide (TMZ). Finally, we demonstrate that one of these inhibitors, Z4P, permeates the blood-brain barrier (BBB), inhibits GB growth, and prevents relapse in vivo when administered together with TMZ. The hit compound disclosed herein satisfies an unmet need for targeted, non-toxic IRE1 inhibitors and our results support the attractiveness of IRE1 as an adjuvant therapeutic target in GB.

A novel optinEuRin stress connection in glaucoma.

The endoplasmic reticulum (ER) is the organelle where the production and shaping of most secreted and transmembrane proteins happens. ER function is finely regulated to prevent accumulation of misfolded proteins generating ER stress. ER stress is common in both healthy and pathological situations due to multiple intrinsic and extrinsic factors including acute demand in protein synthesis, hypoxia or impaired protein folding caused by gene mutations. Sayyad et al. found that the M98K mutation of optineurin sensitizes glaucoma retinal ganglion cells to ER stress-induced cell death. This is associated with an autophagy-dependent elevation of ER stress sensor expression.

Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

A cell-based system combined with flow cytometry to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in patients with COVID-19.

This protocol describes a flow cytometry approach to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in COVID-19-positive patient sera samples without the need of specific laboratory facilities for viral infection. We developed a human-cell-based system using spike-expressing HEK293T cells that mimics membrane insertion and N-glycosylation of viral integral membrane proteins in host cells. This assay represents a powerful tool to test antibody responses against SARS-CoV-2 variants and vaccine effectiveness. For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).

SARS-CoV-2 integral membrane proteins shape the serological responses of patients with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has elicited a unique mobilization of the scientific community to develop efficient tools to understand and combat the infection. Like other coronavirae, SARS-CoV-2 hijacks host cell secretory machinery to produce viral proteins that compose the nascent virions; including spike (S), envelope (E), and membrane (M) proteins, the most exposed transmembrane proteins to the host immune system. As antibody response is part of the anti-viral immune arsenal, we investigate the immunogenic potential of S, E, and M using a human cell-based system to mimic membrane insertion and N-glycosylation. Both S and M elicit specific Ig production in patients with SARS-CoV-2. Patients with moderate and severe diseases exhibit elevated Ig responses. Finally, reduced Ig binding was observed with spike G614 compared to D614 variant. Altogether, our assay points toward an unexpected immune response against M and represents a powerful tool to test humoral responses against actively evolving SARS-CoV-2 variants and vaccine effectiveness.

The unfolded protein response as regulator of cancer stemness and differentiation: Mechanisms and implications for cancer therapy.

The unfolded protein response (UPR) is an adaptive mechanism that regulates protein and cellular homeostasis. Three endoplasmic reticulum (ER) membrane localized stress sensors, IRE1, PERK and ATF6, coordinate the UPR in order to maintain ER proteostasis and cell survival, or induce cell death when homeostasis cannot be restored. However, recent studies have identified alternative functions for the UPR in developmental biology processes and cell fate decisions under both normal and cancerous conditions. In cancer, increasing evidence points towards the involvement of the three UPR sensors in oncogenic reprogramming and the regulation of tumor cells endowed with stem cell properties, named cancer stem cells (CSCs), that are considered to be the most malignant cells in tumors. Here we review the reported roles and underlying molecular mechanisms of the three UPR sensors in regulating stemness and differentiation, particularly in solid tumor cells, processes that have a major impact on tumor aggressiveness. Mainly PERK and IRE1 branches of the UPR were found to regulate CSCs and tumor development and examples are provided for breast cancer, colon cancer and aggressive brain tumors, glioblastoma. Although the underlying mechanisms and interactions between the different UPR branches in regulating stemness in cancer need to be further elucidated, we propose that PERK and IRE1 targeted therapy could inhibit self-renewal of CSCs or induce differentiation that is predicted to have therapeutic benefit. For this, more specific UPR modulators need to be developed with favorable pharmacological properties that together with patient stratification will allow optimal evaluation in clinical studies.

Death sentence: The tale of a fallen endoplasmic reticulum.

Endoplasmic Reticulum (ER) stress signaling is an adaptive mechanism triggered when protein folding demand overcomes the folding capacity of this compartment, thereby leading to the accumulation of improperly folded proteins. This stress signaling pathway is named the Unfolded Protein Response (UPR) and aims at restoring ER homeostasis. However, if this fails, mechanisms orienting cells towards death processes are initiated. Herein, we summarize the most recent findings connecting ER stress and the UPR with identified death mechanisms including apoptosis, necrosis, pyroptosis, ferroptosis, and autophagy. We highlight new avenues that could be investigated and controlled through actionable mechanisms in physiology and pathology.

IRE1-mediated miRNA maturation in macrophage phosphoinositide signaling.

Endoplasmic reticulum (ER) stress signaling has long been associated with various pathological states in particular with the development of diseases with an underlying inflammation, such as diabetes, liver or cardiovascular dysfunctions, and cancer. ER stress signaling is mediated by three stress sensors. The most evolutionarily conserved one, the inositol-requiring enzyme 1 alpha (IRE1), transduces most of the signals through an endoribonuclease (RNase) activity toward RNAs including mRNAs and microRNAs (miRNAs). By exploring phosphoinositide signaling in human macrophages, Hamid and colleagues discovered a novel function of IRE1 RNase that through the cleavage of pre-miR-2317 generates a mature miR-2317 independently of the canonical Dicer endonuclease to yield specific biological outcomes (Hamid et al, 2020).

Local intracerebral inhibition of IRE1 by MKC8866 sensitizes glioblastoma to irradiation/chemotherapy in vivo.

Glioblastoma multiforme (GBM) is the most severe primary brain cancer. Despite an aggressive treatment comprising surgical resection and radio/chemotherapy, patient's survival post diagnosis remains short. A limitation for success in finding novel improved therapeutic options for such dismal disease partly lies in the lack of a relevant animal model that accurately recapitulates patient disease and standard of care. In the present study, we have developed an immunocompetent GBM model that includes tumor surgery and a radio/chemotherapy regimen resembling the Stupp protocol and we have used this model to test the impact of the pharmacological inhibition of the endoplasmic reticulum (ER) stress sensor IRE1, on treatment efficacy.

The integrated stress response promotes B7H6 expression.

The B7 family member, B7H6, is a ligand for the natural killer cell receptor NKp30. B7H6 is hardly expressed on normal tissues, but undergoes upregulation on different types of tumors, implicating it as an attractive target for cancer immunotherapy. The molecular mechanisms that control B7H6 expression are poorly understood. We report that in contrast to other NK cell ligands, endoplasmic reticulum (ER) stress upregulates B7H6 mRNA levels and surface expression. B7H6 induction by ER stress requires protein kinase R-like ER kinase (PERK), one of the three canonical sensors of the unfolded protein response. PERK phosphorylates eIF2α, which regulates protein synthesis and gene expression. Because eIF2α is phosphorylated by several kinases following different stress conditions, the program downstream to eIF2α phosphorylation is called the integrated stress response (ISR). Several drugs were reported to promote the ISR. Nelfinavir and lopinavir, two clinically approved HIV protease inhibitors, promote eIF2α phosphorylation by different mechanisms. We show that nelfinavir and lopinavir sustainably instigate B7H6 expression at their pharmacologically relevant concentrations. As such, ER stress and ISR conditions sensitize melanoma targets to CAR-T cells directed against B7H6. Our study highlights a novel mechanism to induce B7H6 expression and suggests a pharmacological approach to improve B7H6-directed immunotherapy. KEY MESSAGES: B7H6 is induced by ER stress in a PERK-dependent mechanism. Induction of B7H6 is obtained pharmacologically by HIV protease inhibitors. Exposure of tumor cells to the HIV protease inhibitor nelfinavir improves the recognition by B7H6-directed CAR-T.

Extracellular AGR3 regulates breast cancer cells migration via Src signaling.

Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas and are often secreted in cancer patients' specimens, which suggests a role for AGR proteins in intra and extracellular compartments. Although these proteins exhibit high sequence homology, AGR2 is predominantly described as a pro-oncogene and a potential prognostic biomarker. However, little is known about the function of AGR3. Therefore, the aim of the present study was to investigate the role of AGR3 in breast cancer. The results demonstrated that breast cancer cells secrete AGR3. Furthermore, it was revealed that extracellular AGR3 (eAGR3) regulates tumor cell adhesion and migration. The current study indicated that the pharmacological and genetic perturbation of Src kinase signaling, through treatment with Dasatinib (protein kinase inhibitor) or investigating cells that express a dominant-negative form of Src, significantly abrogated eAGR3-mediated breast cancer cell migration. Therefore, the results indicated that eAGR3 may control tumor cell migration via activation of Src kinases. The results of the present study indicated that eAGR3 may serve as a microenvironmental signaling molecule in tumor-associated processes.

Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation.

Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.

Emerging Roles of the Endoplasmic Reticulum Associated Unfolded Protein Response in Cancer Cell Migration and Invasion.

Endoplasmic reticulum (ER) proteostasis is often altered in tumor cells due to intrinsic (oncogene expression, aneuploidy) and extrinsic (environmental) challenges. ER stress triggers the activation of an adaptive response named the Unfolded Protein Response (UPR), leading to protein translation repression, and to the improvement of ER protein folding and clearance capacity. The UPR is emerging as a key player in malignant transformation and tumor growth, impacting on most hallmarks of cancer. As such, the UPR can influence cancer cells' migration and invasion properties. In this review, we overview the involvement of the UPR in cancer progression. We discuss its cross-talks with the cell migration and invasion machinery. Specific aspects will be covered including extracellular matrix (ECM) remodeling, modification of cell adhesion, chemo-attraction, epithelial-mesenchymal transition (EMT), modulation of signaling pathways associated with cell mobility, and cytoskeleton remodeling. The therapeutic potential of targeting the UPR to treat cancer will also be considered with specific emphasis in the impact on metastasis and tissue invasion.