Marion Bouchecareilh

IRE1 Signaling Is Essential for Ischemia-Induced Vascular Endothelial Growth Factor-A Expression and Contributes to Angiogenesis and Tumor Growth In vivo

In solid tumors, cancer cells subjected to ischemic conditions trigger distinct signaling pathways contributing to angiogenic stimulation and tumor development. Characteristic features of tumor ischemia include hypoxia and glucose deprivation, leading to the activation of hypoxia-inducible factor-1-dependent signaling pathways and to complex signaling events known as the unfolded protein response. Here, we show that the activation of the endoplasmic reticulum stress sensor IRE1 is a common determinant linking hypoxia- and hypoglycemia-dependent responses to the up-regulation of vascular endothelial growth factor-A (VEGF-A). Tumor cells expressing a dominant-negative IRE1 transgene as well as Ire1alpha-null mouse embryonic fibroblasts were unable to trigger VEGF-A up-regulation upon either oxygen or glucose deprivation. These data correlated with a reduction of tumor angiogenesis and growth in vivo. Our results therefore suggest an essential role for IRE1-dependent signaling pathways in response to ischemia and identify this protein as a potential therapeutic target to control both the angiogenic switch and tumor development.

Inositol-requiring enzyme 1α is a key regulator of angiogenesis and invasion in malignant glioma

Inositol-requiring enzyme 1 (IRE1) is a proximal endoplasmic reticulum (ER) stress sensor and a central mediator of the unfolded protein response. In a human glioma model, inhibition of IRE1α correlated with down-regulation of prevalent proangiogenic factors such as VEGF-A, IL-1β, IL-6, and IL-8. Significant up-regulation of antiangiogenic gene transcripts was also apparent. These transcripts encode SPARC, decorin, thrombospondin-1, and other matrix proteins functionally linked to mesenchymal differentiation and glioma invasiveness. In vivo, using both the chick chorio-allantoic membrane assay and a mouse orthotopic brain model, we observed in tumors underexpressing IRE1: (i) reduction of angiogenesis and blood perfusion, (ii) a decreased growth rate, and (iii) extensive invasiveness and blood vessel cooption. This phenotypic change was consistently associated with increased overall survival in glioma-implanted recipient mice. Ectopic expression of IL-6 in IRE1-deficient tumors restored angiogenesis and neutralized vessel cooption but did not reverse the mesenchymal/infiltrative cell phenotype. The ischemia-responsive IRE1 protein is thus identified as a key regulator of tumor neovascularization and invasiveness.

Posttranscriptional Regulation of PER1 Underlies the Oncogenic Function of IREα

Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; however, the precise molecular mechanisms underlying this phenomenon remain elusive. Herein, we identified the circadian clock PER1 mRNA as a novel substrate of the endoribonuclease activity of the UPR sensor IRE1α. Analysis of the mechanism shows that IRE1α endoribonuclease activity decreased PER1 mRNA in tumor cells without affecting PER1 gene transcription. Inhibition of IRE1α signaling using either siRNA-mediated silencing or a dominant-negative strategy prevented PER1 mRNA decay, reduced tumorigenesis, and increased survival, features that were reversed upon PER1 silencing. Clinically, patients showing reduced survival have lower levels of PER1 mRNA expression and increased splicing of XBP1, a known IRE-α substrate, thereby pointing toward an increased IRE1α activity in these patients. Hence, we describe a novel mechanism connecting the UPR and circadian clock components in tumor cells, thereby highlighting the importance of this interplay in tumor development.

Modulation of the Maladaptive Stress Response to Manage Diseases of Protein Folding

This study shows how chronic stress and heat shock response exacerbate the phenotype in protein misfolding diseases by triggering a Maladaptive Stress Response; this pathway represents a promising therapeutic target for multiple genetic disorders.

Autocrine control of glioma cells adhesion and migration through IRE1α-mediated cleavage of SPARC mRNA

Summary The endoplasmic reticulum (ER) is an organelle specialized for the folding and assembly of secretory and transmembrane proteins. ER homeostasis is often perturbed in tumor cells because of dramatic changes in the microenvironment of solid tumors, thereby leading to the activation of an adaptive mechanism named the unfolded protein response (UPR). The activation of the UPR sensor IRE1&agr; has been described to play an important role in tumor progression. However, the molecular events associated with this phenotype remain poorly characterized. In the present study, we examined the effects of IRE1&agr; signaling on the adaptation of glioma cells to their microenvironment. We show that the characteristics of U87 cell migration are modified under conditions where IRE1&agr; activity is impaired (DN_IRE1). This is linked to increased stress fiber formation and enhanced RhoA activity. Gene expression profiling also revealed that loss of functional IRE1&agr; signaling mostly resulted in the upregulation of genes encoding extracellular matrix proteins. Among these genes, Sparc, whose mRNA is a direct target of IRE1&agr; endoribonuclease activity, was in part responsible for the phenotypic changes associated with IRE1&agr; inactivation. Hence, our data demonstrate that IRE1&agr; is a key regulator of SPARC expression in vitro in a glioma model. Our results also further support the crucial contribution of IRE1&agr; to tumor growth, infiltration and invasion and extend the paradigm of secretome control in tumor microenvironment conditioning.

Peptides derived from the bifunctional kinase/RNase enzyme IRE1α modulate IRE1α activity and protect cells from endoplasmic reticulum stress

Activation of the bifunctional kinase/RNase enzyme IRE1α is part of an adaptive response triggered on accumulation of misfolded proteins in the endoplasmic reticulum (ER). To facilitate recovery of ER homeostasis, IRE1α molecules oligomerize, allowing for their transautophosphorylation and endoribonuclease activation. These, in turn, induce the activation of specific transcriptional and post‐transcriptional programs. To identify novel and selective modulators of IRE1α activity, we investigated IRE1α oligomerization properties using IRE1α‐derived peptides identified through an activity‐based in vitro assay. We then used these peptides to probe IRE1α activity in vitro and in vivo using both cultured human hepatocellular carcinoma‐derived HuH7 cells and Caenorhabditis elegans experimental systems. We identified a peptide derived from the kinase domain of human IRE1α, which promoted IRE1α oligomerization in vitro, enhanced its Xbp1 mRNA cleavage activity in vitro (1.7×) in cell culture (1.8×) and in vivo (1.3×), and attenuated both ER stress‐mediated JNK activation and regulated IRE1‐dependent mRNA decay (RIDD). This was accompanied by a 2.5‐fold increase in survival on tunicamycin‐induced ER stress and reduced apoptosis by 1.4‐fold in cells expressing this peptide. Hence, targeted and selective activation of the catalytic properties of IRE1α may consequently define new strategies to protect cells from deleterious effects of ER stress signaling.—Bouchecareilh, M., Higa, A., Fribourg, S., Moenner, M., Chevet, E. Peptides derived from the bifunctional kinase/RNase enzyme IRE1α modulate IRE1α activity and protect cells from endoplasmic reticulum stress. FASEB J. 25, 3115‐3129 (2011). www.fasebj.org

Role of Pro-oncogenic Protein Disulfide Isomerase (PDI) Family Member Anterior Gradient 2 (AGR2) in the Control of Endoplasmic Reticulum Homeostasis

Background: AGR2 is a novel ER protein for which the molecular and cellular functions remain uncharacterized. Results: AGR2 associates to nascent chains in the ER, and its silencing impacts UPR and ERAD and sensitizes cells to autophagy. Conclusion: AGR2 plays an important role in the maintenance of ER homeostasis. Significance: AGR2-mediated control of ER homeostasis could be of importance for cancer development. The protein-disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) is reportedly overexpressed in numerous cancers and plays a role in cancer development. However, to date the molecular functions of AGR2 remain to be characterized. Herein we have identified AGR2 as bound to newly synthesized cargo proteins using a proteomics analysis of endoplasmic reticulum (ER) membrane-bound ribosomes. Nascent protein chains that translocate into the ER associate with specific ER luminal proteins, which in turn ensures proper folding and posttranslational modifications. Using both imaging and biochemical approaches, we confirmed that AGR2 localizes to the lumen of the ER and indirectly associates with ER membrane-bound ribosomes through nascent protein chains. We showed that AGR2 expression is controlled by the unfolded protein response and is in turn is involved in the maintenance of ER homeostasis. Remarkably, we have demonstrated that siRNA-mediated knockdown of AGR2 significantly alters the expression of components of the ER-associated degradation machinery and reduces the ability of cells to cope with acute ER stress, properties that might be relevant to the role of AGR2 in cancer development.

Histone Deacetylase Inhibitor (HDACi) Suberoylanilide Hydroxamic Acid (SAHA)-mediated Correction of α1-Antitrypsin Deficiency

Background: α1-Antitrypsin (α1AT) deficiency (α1ATD) is a consequence of defective folding, trafficking, and secretion of α1AT. Results: SAHA restores the secretion of an active form of Z-α1AT in part through a calnexin- and HDAC7-sensitive dependent mechanism(s). Conclusion: SAHA may represent a potential therapeutic approach for α1ATD. Significance: SAHA is a regulator of the proteostasis biology of Z-α1AT, favoring export of a functional form to serum. α1-Antitrypsin (α1AT) deficiency (α1ATD) is a consequence of defective folding, trafficking, and secretion of α1AT in response to a defect in its interaction with the endoplasmic reticulum proteostasis machineries. The most common and severe form of α1ATD is caused by the Z-variant and is characterized by the accumulation of α1AT polymers in the endoplasmic reticulum of the liver leading to a severe reduction (>85%) of α1AT in the serum and its anti-protease activity in the lung. In this organ α1AT is critical for ensuring tissue integrity by inhibiting neutrophil elastase, a protease that degrades elastin. Given the limited therapeutic options in α1ATD, a more detailed understanding of the folding and trafficking biology governing α1AT biogenesis and its response to small molecule regulators is required. Herein we report the correction of Z-α1AT secretion in response to treatment with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), acting in part through HDAC7 silencing and involving a calnexin-sensitive mechanism. SAHA-mediated correction restores Z-α1AT secretion and serpin activity to a level 50% that observed for wild-type α1AT. These data suggest that HDAC activity can influence Z-α1AT protein traffic and that SAHA may represent a potential therapeutic approach for α1ATD and other protein misfolding diseases.

Acute L-glutamine deprivation compromises VEGF-a upregulation in A549/8 human carcinoma cells.

Tumor ischemia participates in angiogenesis and cancer progression through cellular responses to hypoxia and nutrient deprivation. However, the contribution of amino acids limitation to this process remains poorly understood. Using serum‐free cell culture conditions, we tested the impact of L‐glutamine deprivation on metabolic and angiogenic responses in A549/8 carcinoma cells. In these cells, lowering glutamine concentration modified the cell cycle distribution and significantly induced apoptosis/necrosis. Although glutamine deprivation led to a HIF‐independent increase in VEGF‐A mRNA, the corresponding protein level remained low and correlated with the inhibition of protein synthesis and activation of the GCN2/eIF2α pathway. Limitation of glutamine availability also hampers hypoxia‐ and hypoglycemia‐induced VEGF‐A protein upregulation. Thus, glutamine deprivation may have no direct effect on VEGF‐dependent angiogenesis, compared to hypoxia or to glucose deprivation, and may instead be detrimental to cancer progression by antagonizing ischemia‐induced stresses. J. Cell. Physiol. 212: 463–472, 2007.

GTPase-Mediated Regulation of the Unfolded Protein Response in Caenorhabditis elegans Is Dependent on the AAA+ ATPase CDC-48

ABSTRACT When endoplasmic reticulum (ER) homeostasis is perturbed, an adaptive mechanism is triggered and named the unfolded protein response (UPR). Thus far, three known UPR signaling branches (IRE-1, PERK, and ATF-6) mediate the reestablishment of ER functions but can also lead to apoptosis if ER stress is not alleviated. However, the understanding of the molecular mechanisms integrating the UPR to other ER functions, such as membrane traffic or endomembrane signaling, remains incomplete. We consequently sought to identify new regulators of UPR-dependent transcriptional mechanisms and focused on a family of proteins known to mediate, among other, ER-related functions: the small GTP-binding proteins of the RAS superfamily. To this end, we used transgenic UPR reporter Caenorhabditis elegans strains as a model to specifically silence small-GTPase expression. We show that the Rho subfamily member CRP-1 is an essential component of UPR-induced transcriptional events through its physical and genetic interactions with the AAA+ ATPase CDC-48. In addition, we describe a novel signaling module involving CRP-1 and CDC-48 which may directly link the UPR to DNA remodeling and transcription control.