Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. The AAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.

Novel roles of the unfolded protein response in the control of tumor development and aggressiveness.

The hallmarks of cancer currently define the molecular mechanisms responsible for conferring specific tumor phenotypes. Recently, these characteristics were also connected to the status of the secretory pathway, thereby linking the functionality of this cellular machinery to the acquisition of cancer cell features. The secretory pathway ensures the biogenesis of proteins that are membrane-bound or secreted into the extracellular milieu and can control its own homeostasis through an adaptive signaling pathway named the unfolded protein response (UPR). In the present review, we discuss the specific features of the UPR in various tumor types and the impact of the selective activation of this pathway on cell transformation, tumor development and aggressiveness.

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.

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.

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation.

BACKGROUND: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. METHODS: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. RESULTS: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. CONCLUSIONS: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes.

Dual IRE1 RNase functions dictate glioblastoma development.

Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1α (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1-dependent decay (RIDD) of RNA Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1-dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression.

Adaptation of the secretory pathway in cancer through IRE1 signaling.

The unfolded protein response (UPR) was originally identified as a signaling network coordinating adaptive and apoptotic responses to accumulation of unfolded proteins in the endoplasmic reticulum (ER). More recent work has shown that UPR signaling can be triggered by a multitude of cellular events and that the UPR plays a critical role in the prevention of cell transformation but also in tumor development. This has been particularly well illustrated with studies on one of the three major ER stress sensors, IRE1. This ER resident type I transmembrane protein senses luminal ER stress and transduce signals through its cytosolic RNase activity. IRE1 signaling has been shown to contribute to the progression of solid tumors through pro-angiogenic mechanisms. Herein, we expose the methodologies for investigating IRE1 signaling in tumor cells and in tumors. Moreover, we show that selective pharmacological inhibition of IRE1 RNase activity sensitizes tumor cells to ER stress.