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.

MicroRNA-1291-mediated silencing of IRE1α enhances Glypican-3 expression.

MicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3' untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5' UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3' UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α.

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

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 IRE1α 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 IRE1α signaling on the adaptation of glioma cells to their microenvironment. We show that the characteristics of U87 cell migration are modified under conditions where IRE1α 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 IRE1α signaling mostly resulted in the upregulation of genes encoding extracellular matrix proteins. Among these genes, Sparc, whose mRNA is a direct target of IRE1α endoribonuclease activity, was in part responsible for the phenotypic changes associated with IRE1α inactivation. Hence, our data demonstrate that IRE1α is a key regulator of SPARC expression in vitro in a glioma model. Our results also further support the crucial contribution of IRE1α to tumor growth, infiltration and invasion and extend the paradigm of secretome control in tumor microenvironment conditioning.

Ascorbate/menadione-induced oxidative stress kills cancer cells that express normal or mutated forms of the oncogenic protein Bcr-Abl. An in vitro and in vivo mechanistic study.

Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

Menadione reduction by pharmacological doses of ascorbate induces an oxidative stress that kills breast cancer cells.

Oxidative stress generated by ascorbate-driven menadione redox cycling kills MCF7 cells by a concerted mechanism including glycolysis inhibition, loss of calcium homeostasis, DNA damage and changes in mitogen activated protein kinases (MAPK) activities. Cell death is mediated by necrosis rather than apoptosis or macroautophagy. Neither 3-methyladenine nor Z-VAD affects cytotoxicity by ascorbate/menadione (Asc/Men). BAPTA-AM, by restoring cellular capacity to reduce MTT, underlines the role of calcium in the necrotic process. Oxidative stress-mediated cell death is shown by the opposite effects of N-acetylcysteine and 3-aminotriazole. Moreover, oxidative stress induces DNA damage (protein poly-ADP-ribosylation and gamma-H2AX phosphorylation) and inhibits glycolysis. Asc/Men deactivates extracellular signal-regulated kinase (ERK) while activating p38, suggesting an additional mechanism to kill MCF7 cells. Since ascorbate is taken up by cancer cells and, due to their antioxidant enzyme deficiency, oxidative stress should affect cancer cells to a greater extent than normal cells. This differential sensitivity may have clinical applications.

Evaluation of Potential Mechanisms Controlling the Catalase Expression in Breast Cancer Cells.

Development of cancer cell resistance against prooxidant drugs limits its potential clinical use. MCF-7 breast cancer cells chronically exposed to ascorbate/menadione became resistant (Resox cells) by increasing mainly catalase activity. Since catalase appears as an anticancer target, the elucidation of mechanisms regulating its expression is an important issue. In MCF-7 and Resox cells, karyotype analysis showed that chromosome 11 is not altered compared to healthy mammary epithelial cells. The genomic gain of catalase locus observed in MCF-7 and Resox cells cannot explain the differential catalase expression. Since ROS cause DNA lesions, the activation of DNA damage signaling pathways may influence catalase expression. However, none of the related proteins (i.e., p53, ChK) was activated in Resox cells compared to MCF-7. The c-abl kinase may lead to catalase protein degradation via posttranslational modifications, but neither ubiquitination nor phosphorylation of catalase was detected after catalase immunoprecipitation. Catalase mRNA levels did not decrease after actinomycin D treatment in both cell lines. DNMT inhibitor (5-aza-2'-deoxycytidine) increased catalase protein level in MCF-7 and its resistance to prooxidant drugs. In line with our previous report, chromatin remodeling appears as the main regulator of catalase expression in breast cancer after chronic exposure to an oxidative stress.

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.

Control of the Unfolded Protein Response in Health and Disease.

The unfolded protein response (UPR) is an integrated, adaptive biochemical process that is inextricably linked with cell homeostasis and paramount to maintenance of normal physiological function. Prolonged accumulation of improperly folded proteins in the endoplasmic reticulum (ER) leads to stress. This is the driving stimulus behind the UPR. As such, prolonged ER stress can push the UPR past beneficial functions such as reduced protein production and increased folding and clearance to apoptotic signaling. The UPR is thus contributory to the commencement, maintenance, and exacerbation of a multitude of disease states, making it an attractive global target to tackle conditions sorely in need of novel therapeutic intervention. The accumulation of information of screening tools, readily available therapies, and potential pathways to drug development is the cornerstone of informed clinical research and clinical trial design. Here, we review the UPR's involvement in health and disease and, beyond providing an in-depth description of the molecules found to target the three UPR arms, we compile all the tools available to screen for and develop novel therapeutic agents that modulate the UPR with the scope of future disease intervention.

Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) and genomic gain of the NQO1 locus modulates breast cancer cell sensitivity to quinones.

AIMS: Alterations in the expression of antioxidant enzymes are associated with changes in cancer cell sensitivity to chemotherapeutic drugs (menadione and ß-lapachone). Mechanisms of acquisition of resistance to pro-oxidant drugs were investigated using a model of oxidative stress-resistant MCF-7 breast cancer cells (Resox cells). MAIN METHODS: FISH experiments were performed in tumor biopsy and breast cancer cells to characterize the pattern of the NQO1 gene. SNP-arrays were conducted to detect chromosomal imbalances. Finally, the importance of NQO1 overexpression in the putative acquisition of either drug resistance or an increased sensitivity to quinones by cancer cells was investigated by immunoblotting and cytotoxicity assays. KEY FINDINGS: Genomic gain of the chromosomal band 16q22 was detected in Resox cells compared to parental breast cancer MCF-7 cells and normal human mammary epithelial 250MK cells. This genomic gain was associated with amplification of the NQO1 gene in one tumor biopsy as well as in breast cancer cell lines. Using different breast cell models, we found that NQO1 overexpression was a main determinant for a potential chemotherapy resistance or an increased sensitivity to quinone-bearing compounds. SIGNIFICANCE: Because NQO1 is frequently modified in tumors at genomic and transcriptomic levels, the impact of NQO1 modulation on breast cancer cell sensitivity places NQO1 as a potential link between cancer redox alterations and resistance to chemotherapy. Thus, the NQO1 gene copy number and NQO1 activity should be considered when quinone-bearing molecules are being utilized as potential drugs against breast tumors.

Chromatin remodeling regulates catalase expression during cancer cells adaptation to chronic oxidative stress.

Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H2O2 (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at -1518/-1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H2O2 in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells.

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.

Watching the clock: endoplasmic reticulum-mediated control of circadian rhythms in cancer.

In the past 20 years both the circadian clock and endoplasmic reticulum (ER) stress signaling have emerged as major players in oncogenesis and cancer development. Although several lines of evidence have established functional links between these two molecular pathways, their interconnection and the subsequent functional implications in cancer development remain to be fully characterized. Herein, we provide an extensive review of the literature depicting the molecular connectivity linking ER stress signaling and the circadian clock and elaborate on the potential use of these functional interactions in cancer therapeutics.

Addicted to secrete - novel concepts and targets in cancer therapy.

The unfolded protein response (UPR) mediates the adaptation of the secretory pathway (SP) to fluctuations in cellular protein demand or to environmental variations. Recently, drug screenings have confirmed the therapeutic potential of targeting the UPR in cancer models. However, the UPR may not be the only druggable target of the SP. Moreover, recent studies have revealed other contributions of the SP to cancer development. This article does not intend to describe the well-established implication of UPR signaling pathways in cancer cell life and cell decision, but rather aims at defining the concept of 'tumor cell secretory addiction', from molecular, cellular, and therapeutic perspectives. Furthermore, the implication of UPR modulations in this context will be discussed.

AICAR induces Nrf2 activation by an AMPK-independent mechanism in hepatocarcinoma cells.

Hepatocellular carcinoma is one of the most frequent tumor types worldwide and oxidative stress represents a major risk factor in pathogenesis of liver diseases leading to HCC. Nuclear factor erythroid 2-related factor (Nrf2) is a transcription factor activated by oxidative stress that governs the expression of many genes which constitute the antioxidant defenses of the cell. In addition, oxidative stress activates AMP-activated protein kinase (AMPK), which has emerged in recent years as a kinase that controls the redox-state of the cell. Since both AMPK and Nrf2 are involved in redox homeostasis, we investigated whether there was a crosstalk between the both signaling systems in hepatocarcinoma cells. Here, we demonstrated that AMPK activator AICAR, in contrary to the A769662 allosteric activator, induces Nrf2 activation and concomitantly modulates the basal redox state of the hepatocarcinoma cells. When the expression of Nrf2 is knocked down, AICAR failed to induce its effect on redox state. These data highlight a major role of Nrf2 signaling pathway in mediating the AICAR effect on basal oxidative state. Furthermore, we demonstrated that AICAR metabolization by the cell is required to induce Nrf2 activation while, the silencing of AMPK does not have any effect on Nrf2 activation. This suggests that AICAR-induced Nrf2 activation is independent of AMPK activity. In conclusion, we identified AICAR as a potent modulator of the redox state of human hepatocarcinoma cells, via the Nrf2 signaling pathway and in an AMPK-independent mechanism.

Catalase expression in MCF-7 breast cancer cells is mainly controlled by PI3K/Akt/mTor signaling pathway.

Catalase is an antioxidant enzyme that catalyzes mainly the transformation of hydrogen peroxide into water and oxygen. Although catalase is frequently down-regulated in tumors the underlying mechanism remains unclear. Few transcription factors have been reported to directly bind the human catalase promoter. Among them FoxO3a has been proposed as a positive regulator of catalase expression. Therefore, we decided to study the role of the transcription factor FoxO3a and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, which regulates FoxO3a, in the expression of catalase. To this end, we developed an experimental model of mammary breast MCF-7 cancer cells that acquire resistance to oxidative stress, the so-called Resox cells, in which catalase is overexpressed as compared with MCF-7 parental cell line. In Resox cells, Akt expression is decreased but its phosphorylation is enhanced when compared with MCF-7 cells. A similar profile is observed for FoxO3a, with less total protein but more phosphorylated FoxO3a in Resox cells, correlating with its higher Akt activity. The modulation of FoxO3a expression by knockdown and overexpression strategies did not affect catalase expression, neither in MCF-7 nor in Resox cells. Inhibition of PI3K and mTOR by LY295002 and rapamycin, respectively, decreases the phosphorylation of downstream targets (i.e. GSK3ß and p70S6K) and leads to an increase of catalase expression only in MCF-7 but not in Resox cells. In conclusion, FoxO3a does not appear to play a critical role in the regulation of catalase expression in both cancer cells. Only MCF-7 cells are sensitive and dependent on PI3K/Akt/mTOR signaling.

Effect of a high-fat challenge on the proteome of human postprandial plasma.

BACKGROUND & AIMS: Postprandial lipemia has been associated with inflammation, oxidative stress and vascular dysfunction. This metabolic disturbance represents a complex process only partly understood. The purpose of this study was to identify variations in plasma proteome after a high-fat challenge in healthy middle-aged men. METHODS: Two-dimensional electrophoresis was used to compare plasma from seven subjects, drawn before and 4 h after a high-fat challenge. RESULTS: Among the 231 spots detected and analyzed, 22 were present at different levels in postprandial hyperlipemic plasma compared to preprandial plasma. For 10 of them, corresponding proteins were identified by mass spectrometry. Some of them are related to the hemostatic system (tetranectin and fibrinogen) or the complement system (complement component 3 and 4 and ficollin-3) and have been previously associated to atherothrombosis. CONCLUSION: These results provide new perspectives and broaden our understanding of the biological processes of postprandial metabolic stress, as well as its links with the development of atherosclerosis.

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.

Overexpression of GRP94 in breast cancer cells resistant to oxidative stress promotes high levels of cancer cell proliferation and migration: implications for tumor recurrence.

Targeting the altered redox status of cancer cells is emerging as an interesting approach to potentiate chemotherapy. However, to maximize the effectiveness of this strategy and define the correct chemotherapeutic associations, it is important to understand the biological consequences of chronically exposing cancer cells to reactive oxygen species (ROS). Using an H(2)O(2)-generating system, we selected a ROS-resistant MCF-7 breast cancer cell line, namely Resox cells. By exploring different survival pathways that are usually induced during oxidative stress, we identified a constitutive overexpression of the endoplasmic reticulum chaperone, GRP94, in these cells, whereas levels of its cytoplasmic homolog HSP90, or GRP78, were not modified. This overexpression was not mediated by constitutive unfolded protein response (UPR) activation. The increase in GRP94 is tightly linked to an increase in cell proliferation and migration capacities, as shown by GRP94-silencing experiments. Interestingly, we also observed that GRP94 silencing inhibits migration and proliferation of the highly aggressive MDA-MB-231 cells. By immunohistochemistry, we showed that GRP94 expression was higher in recurrent human breast cancers than in their paired primary neoplasias. Similar to the situation in the Resox cells, this increase was not associated with an increase in UPR activation in recurrent tumors. In conclusion, this study suggests that GRP94 overexpression may be a hallmark of aggressiveness and recurrence in breast cancers.