KRAS Promotes GLI2-Dependent Transcription during Pancreatic Carcinogenesis.

Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth-promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.

Gradual ER calcium depletion induces a progressive and reversible UPR signaling.

The unfolded protein response (UPR) is a widespread signal transduction pathway triggered by endoplasmic reticulum (ER) stress. Because calcium (Ca2+) is a key factor in the maintenance of ER homeostasis, massive Ca2+ depletion of the ER is a potent inducer of ER stress. Although moderate changes in ER Ca2+ drive the ubiquitous Ca2+ signaling pathways, a possible incremental relationship between UPR activation and Ca2+ changes has yet to be described. Here, we determine the sensitivity and time-dependency of activation of the three ER stress sensors, inositol-requiring protein 1 alpha (IRE1α), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 alpha (ATF6α) in response to controlled changes in the concentration of ER Ca2+ in human cultured cells. Combining Ca2+ imaging, fluorescence recovery after photobleaching experiments, biochemical analyses, and mathematical modeling, we uncover a nonlinear rate of activation of the IRE1α branch of UPR, as compared to the PERK and ATF6α branches that become activated gradually with time and are sensitive to more important ER Ca2+ depletions. However, the three arms are all activated within a 1 h timescale. The model predicted the deactivation of PERK and IRE1α upon refilling the ER with Ca2+. Accordingly, we showed that ER Ca2+ replenishment leads to the complete reversion of IRE1α and PERK phosphorylation in less than 15 min, thus revealing the highly plastic character of the activation of the upstream UPR sensors. In conclusion, our results reveal a dynamic and dose-sensitive Ca2+-dependent activation/deactivation cycle of UPR induction, which could tightly control cell fate upon acute and/or chronic stress.

ER stress signaling at the interphase between MASH and HCC.

HCC is the most frequent primary liver cancer with an extremely poor prognosis and often develops on preset of chronic liver diseases. Major risk factors for HCC include metabolic dysfunction-associated steatohepatitis, a complex multifactorial condition associated with abnormal endoplasmic reticulum (ER) proteostasis. To cope with ER stress, the unfolded protein response engages adaptive reactions to restore the secretory capacity of the cell. Recent advances revealed that ER stress signaling plays a critical role in HCC progression. Here, we propose that chronic ER stress is a common transversal factor contributing to the transition from liver disease (risk factor) to HCC. Interventional strategies to target the unfolded protein response in HCC, such as cancer therapy, are also discussed.

Exploring the IRE1 interactome: From canonical signaling functions to unexpected roles.

The unfolded protein response is a mechanism aiming at restoring endoplasmic reticulum (ER) homeostasis and is likely involved in other adaptive pathways. The unfolded protein response is transduced by three proteins acting as sensors and triggering downstream signaling pathways. Among them, inositol-requiring enzyme 1 alpha (IRE1α) (referred to as IRE1 hereafter), an endoplasmic reticulum-resident type I transmembrane protein, exerts its function through both kinase and endoribonuclease activities, resulting in both X-box binding protein 1 mRNA splicing and RNA degradation (regulated ire1 dependent decay). An increasing number of studies have reported protein-protein interactions as regulators of these signaling mechanisms, and additionally, driving other noncanonical functions. In this review, we deliver evolutive and structural insights on IRE1 and further describe how this protein interaction network (interactome) regulates IRE1 signaling abilities or mediates other cellular processes through catalytic-independent mechanisms. Moreover, we focus on newly discovered targets of IRE1 kinase activity and discuss potentially novel IRE1 functions based on the nature of the interactome, thereby identifying new fields to explore regarding this protein's biological roles.

Protein homeostasis imprinting across evolution.

Protein homeostasis (a.k.a. proteostasis) is associated with the primary functions of life, and therefore with evolution. However, it is unclear how cellular proteostasis machines have evolved to adjust protein biogenesis needs to environmental constraints. Herein, we describe a novel computational approach, based on semantic network analysis, to evaluate proteostasis plasticity during evolution. We show that the molecular components of the proteostasis network (PN) are reliable metrics to deconvolute the life forms into Archaea, Bacteria and Eukarya and to assess the evolution rates among species. Semantic graphs were used as new criteria to evaluate PN complexity in 93 Eukarya, 250 Bacteria and 62 Archaea, thus representing a novel strategy for taxonomic classification, which provided information about species divergence. Kingdom-specific PN components were identified, suggesting that PN complexity may correlate with evolution. We found that the gains that occurred throughout PN evolution revealed a dichotomy within both the PN conserved modules and within kingdom-specific modules. Additionally, many of these components contribute to the evolutionary imprinting of other conserved mechanisms. Finally, the current study suggests a new way to exploit the genomic annotation of biomedical ontologies, deriving new knowledge from the semantic comparison of different biological systems.

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.

Modulation of Protein Disulfide Isomerase Functions by Localization: The Example of the Anterior Gradient Family.

Significance: Oxidative folding within the endoplasmic reticulum (ER) introduces disulfide bonds into nascent polypeptides, ensuring proteins' stability and proper functioning. Consequently, this process is critical for maintaining proteome integrity and overall health. The productive folding of thousands of secretory proteins requires stringent quality control measures, such as the unfolded protein response (UPR) and ER-Associated Degradation (ERAD), which contribute significantly to maintaining ER homeostasis. ER-localized protein disulfide isomerases (PDIs) play an essential role in each of these processes, thereby contributing to various aspects of ER homeostasis, including maintaining redox balance, proper protein folding, and signaling from the ER to the nucleus. Recent Advances: Over the years, there have been increasing reports of the (re)localization of PDI family members and other ER-localized proteins to various compartments. A prime example is the anterior gradient (AGR) family of PDI proteins, which have been reported to relocate to the cytosol or the extracellular environment, acquiring gain of functions that intersect with various cellular signaling pathways. Critical Issues: Here, we summarize the functions of PDIs and their gain or loss of functions in non-ER locations. We will focus on the activity, localization, and function of the AGR proteins: AGR1, AGR2, and AGR3. Future Directions: Targeting PDIs in general and AGRs in particular is a promising strategy in different human diseases. Thus, there is a need for innovative strategies and tools aimed at targeting PDIs; those strategies should integrate the specific localization and newly acquired functions of these PDIs rather than solely focusing on their canonical roles.

Different binding modalities of quercetin to inositol-requiring enzyme 1 of S. cerevisiae and human lead to opposite regulation.

The flavonoid Quercetin (Qe) was identified as an activator of Inositol-requiring enzyme 1 (IRE1) in S. cerevisiae (scIre1p), but its impact on human IRE1 (hIRE1) remains controversial due to the absence of a conserved Qe binding site. We have explored the binding modes and effect of Qe on both scIre1p and hIRE1 dimers using in silico and in vitro approaches. The activation site in scIre1p stably accommodates both Qe and its derivative Quercitrin (Qi), thus enhancing the stability of the RNase pocket. However, the corresponding region in hIRE1 does not bind any of the two molecules. Instead, we show that both Qe and Qi block the RNase activity of hIRE1 in vitro, with sub-micromolar IC50 values. Our results provide a rationale for why Qe is an activator in scIre1p but a potent inhibitor in hIRE1. The identification of a new allosteric site in hIRE1 opens a promising window for drug development and UPR modulation.

UFMylation: a ubiquitin-like modification.

Post-translational modifications (PTMs) add a major degree of complexity to the proteome and are essential controllers of protein homeostasis. Amongst the hundreds of PTMs identified, ubiquitin and ubiquitin-like (UBL) modifications are recognized as key regulators of cellular processes through their ability to affect protein-protein interactions, protein stability, and thus the functions of their protein targets. Here, we focus on the most recently identified UBL, ubiquitin-fold modifier 1 (UFM1), and the machinery responsible for its transfer to substrates (UFMylation) or its removal (deUFMylation). We first highlight the biochemical peculiarities of these processes, then we develop on how UFMylation and its machinery control various intertwined cellular processes and we highlight some of the outstanding research questions in this emerging field.

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.

Dynamic tandem proximity-based proteomics-Protein trafficking at the proteome-scale.

TransitID is a new methodology based on proximity labeling allowing for the study of protein trafficking a the proteome scale.

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.

GLI1, a novel target of the ER stress regulator p97/VCP, promotes ATF6f-mediated activation of XBP1.

Upon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The induction of stress genes is a sine qua non condition for effective adaptive UPR. Although this requirement has been extensively described, the mechanisms underlying this process remain in part uncharacterized. Here, we show that p97/VCP, an AAA+ ATPase known to contribute to ER stress-induced gene expression, regulates the transcription factor GLI1, a primary effector of Hedgehog (Hh) signaling. Under basal (non-ER stress) conditions, GLI1 is repressed by a p97/VCP-HDAC1 complex while upon ER stress GLI1 is induced through a mechanism requiring both USF2 binding and increase histone acetylation at its promoter. Interestingly, the induction of GLI1 was independent of ligand-regulated Hh signaling. Further analysis showed that GLI1 cooperates with ATF6f to induce promoter activity and expression of XBP1, a key transcription factor driving UPR. Overall, our work demonstrates a novel role for GLI1 in the regulation of ER stress gene expression and defines the interplay between p97/VCP, HDAC1 and USF2 as essential players in this process.

Metalloprotease-mediated cleavage of CD95 ligand.

CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or nonapoptotic signals. CD95L can exist as both membrane-anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modelling, we confirmed data from the literature regarding CD95L cleavage by MMP-3 and MMP-7. Moreover, we found that MMP-2 and MMP-12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyse the cleavage of CD95L, which until now had only been poorly described.

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.

IRE1α overexpression in malignant cells limits tumor progression by inducing an anti-cancer immune response.

IRE1α is one of the three ER transmembrane transducers of the Unfolded Protein Response (UPR) activated under endoplasmic reticulum (ER) stress. IRE1α activation has a dual role in cancer as it may be either pro- or anti-tumoral depending on the studied models. Here, we describe the discovery that exogenous expression of IRE1α, resulting in IRE1α auto-activation, did not affect cancer cell proliferation in vitro but resulted in a tumor-suppressive phenotype in syngeneic immunocompetent mice. We found that exogenous expression of IRE1α in murine colorectal and Lewis lung carcinoma cells impaired tumor growth when syngeneic tumor cells were subcutaneously implanted in immunocompetent mice but not in immunodeficient mice. Mechanistically, the in vivo tumor-suppressive effect of overexpressing IRE1α in tumor cells was associated with IRE1α RNAse activity driving both XBP1 mRNA splicing and regulated IRE1-dependent decay of RNA (RIDD). We showed that the tumor-suppressive phenotype upon IRE1α overexpression was characterized by the induction of apoptosis in tumor cells along with an enhanced adaptive anti-cancer immunosurveillance. Hence, our work indicates that IRE1α overexpression and/or activation in tumor cells can limit tumor growth in immunocompetent mice. This finding might point toward the need of adjusting the use of IRE1α inhibitors in cancer treatments based on the predominant outcome of the RNAse activity of IRE1α.