Eric Chevet

Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Here, we performed high-resolution copy-number analysis on 125 HCC tumors and whole-exome sequencing on 24 of these tumors. We identified 135 homozygous deletions and 994 somatic mutations of genes with predicted functional consequences. We found new recurrent alterations in four genes (ARID1A, RPS6KA3, NFE2L2 and IRF2) not previously described in HCC. Functional analyses showed tumor suppressor properties for IRF2, whose inactivation, exclusively found in hepatitis B virus (HBV)-related tumors, led to impaired TP53 function. In contrast, inactivation of chromatin remodelers was frequent and predominant in alcohol-related tumors. Moreover, association of mutations in specific genes (RPS6KA3-AXIN1 and NFE2L2-CTNNB1) suggested that Wnt/β-catenin signaling might cooperate in liver carcinogenesis with both oxidative stress metabolism and Ras/mitogen-activated protein kinase (MAPK) pathways. This study provides insight into the somatic mutational landscape in HCC and identifies interactions between mutations in oncogene and tumor suppressor gene mutations related to specific risk factors.

Endoplasmic Reticulum-Mediated Phagocytosis Is a Mechanism of Entry into Macrophages

Phagocytosis is a key aspect of our innate ability to fight infectious diseases. In this study, we have found that fusion of the endoplasmic reticulum (ER) with the macrophage plasmalemma, underneath phagocytic cups, is a source of membrane for phagosome formation in macrophages. Successive waves of ER become associated with maturing phagosomes during phagolysosome biogenesis. Thus, the ER appears to possess unexpectedly pluripotent fusion properties. ER-mediated phagocytosis is regulated in part by phosphatidylinositol 3-kinase and used for the internalization of inert particles and intracellular pathogens, regardless of their final trafficking in the host. In neutrophils, where pathogens are rapidly killed, the ER is not used as a major source of membrane for phagocytosis. We propose that intracellular pathogens have evolved to adapt and exploit ER-mediated phagocytosis to avoid destruction in host cells.

Targeting the unfolded protein response in disease

Stress induced by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a feature of specialized secretory cells and is also observed in many diseases, including cancer, diabetes, autoimmune conditions, liver disorders, obesity and neurodegenerative disorders. Cellular adaptation to ER stress is achieved by the activation of the unfolded protein response, which is an integrated signal transduction pathway that modulates many aspects of ER physiology. When these mechanisms of adaptation are insufficient to handle the unfolded protein load, cells undergo apoptosis. Here, we discuss recent advances in the design of novel compounds and therapeutic strategies to manipulate levels of ER stress in disease.

Mitogen-Activated Protein (MAP) Kinase/MAP Kinase Phosphatase Regulation: Roles in Cell Growth, Death, and Cancer

Mitogen-activated protein kinase dual-specificity phosphatase-1 (also called MKP-1, DUSP1, ERP, CL100, HVH1, PTPN10, and 3CH134) is a member of the threonine-tyrosine dual-specificity phosphatases, one of more than 100 protein tyrosine phosphatases. It was first identified approximately 20 years ago, and since that time extensive investigations into both mkp-1 mRNA and protein regulation and function in different cells, tissues, and organs have been conducted. However, no general review on the topic of MKP-1 exists. As the subject matter pertaining to MKP-1 encompasses many branches of the biomedical field, we focus on the role of this protein in cancer development and progression, highlighting the potential role of the mitogen-activated protein kinase (MAPK) family. Section II of this article elucidates the MAPK family cross-talk. Section III reviews the structure of the mkp-1 encoding gene, and the known mechanisms regulating the expression and activity of the protein. Section IV is an overview of the MAPK-specific dual-specificity phosphatases and their role in cancer. In sections V and VI, mkp-1 mRNA and protein are examined in relation to cancer biology, therapeutics, and clinical studies, including a discussion of the potential role of the MAPK family. We conclude by proposing an integrated scheme for MKP-1 and MAPK in cancer.

Proteomics Characterization of Abundant Golgi Membrane Proteins

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and α2P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Proteostasis control by the unfolded protein response

Stress induced by accumulation of misfolded proteins in the endoplasmic reticulum is observed in many physiological and pathological conditions. To cope with endoplasmic reticulum stress, cells activate the unfolded protein response, a dynamic signalling network that orchestrates the recovery of homeostasis or triggers apoptosis, depending on the level of damage. Here we provide an overview of recent insights into the mechanisms that cells employ to maintain proteostasis and how the unfolded protein response determines cell fate under endoplasmic reticulum stress.

Getting RIDD of RNA: IRE1 in cell fate regulation.

Inositol-requiring enzyme 1 (IRE1) is the most conserved transducer of the unfolded protein response (UPR), a homeostatic response that preserves proteostasis. Intriguingly, via its endoribonuclease activity, IRE1 produces either adaptive or death signals. This occurs through both unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay of mRNA (RIDD). Whereas XBP1 mRNA splicing is cytoprotective in response to endoplasmic reticulum (ER) stress, RIDD has revealed many unexpected features. For instance, RIDD cleaves RNA at an XBP1-like consensus site but with an activity divergent from XBP1 mRNA splicing and can either preserve ER homeostasis or induce cell death. Here we review recent findings on RIDD and propose a model of how IRE1 RNase activity might control cell fate decisions.

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.

Protein-tyrosine Phosphatase 1B Potentiates IRE1 Signaling during Endoplasmic Reticulum Stress

Protein-tyrosine phosphatase 1B (PTP-1B) is the prototypic tyrosine phosphatase whose function in insulin signaling and metabolism is well established. Although the role of PTP-1B in dephosphorylating various cell surface receptor tyrosine kinases is clear, the mechanisms by which it modulates receptor function from the endoplasmic reticulum (ER) remains an enigma. Here, we provide evidence that PTP-1B has an essential function in regulating the unfolded protein response in the ER compartment. The absence of PTP-1B caused impaired ER stress-induced IRE1 signaling. More specifically, JNK activation, XBP-1 splicing, and EDEM (ER degradation-enhancing α-mannosidase-like protein) gene induction, as well as ER stress-induced apoptosis, were attenuated in PTP-1B knock-out mouse embryonic fibroblasts in response to two ER stressors, tunicamycin and azetidine-2 carboxylic acid. We demonstrate that PTP-1B is not just a passive resident of the ER but on the contrary has an essential role in potentiating IRE1-mediated ER stress signaling pathways.

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.