CCAAT/Enhancer-Binding Protein Homologous (CHOP) Protein Promotes Carcinogenesis in the DEN-Induced Hepatocellular Carcinoma Model

Background and Aims C/EBP homologous protein (CHOP) plays pro-apoptotic roles in the integrated stress response. Recently, a tumor suppressive role for CHOP was demonstrated in lung cancer via regulation of tumor metabolism. To explore the role of CHOP in hepatocarcinogenesis, we induced hepatocellular carcinoma (HCC) in wild type (wt) and CHOP knockout (KO) mice using the carcinogen N-diethylnitrosamine (DEN). Results: Analysis of tumor development showed reduced tumor load, with markedly smaller tumor nodules in the CHOP KO animals, suggesting oncogenic roles of CHOP in carcinogen-induced HCC. In wt tumors, CHOP was exclusively expressed in tumor tissue, with minimal expression in normal parenchyma. Analysis of human adenocarcinomas of various origins demonstrated scattered expression of CHOP in the tumors, pointing to relevance in human pathology. Characterization of pathways that may contribute to preferential expression of CHOP in the tumor identified ATF6 as a potential candidate. ATF6, a key member of the endoplasmic reticulum stress signaling machinery, exhibited a similar pattern of expression as CHOP and strong activation in wt but not CHOP KO tumors. Because HCC is induced by chronic inflammation, we assessed whether CHOP deficiency affects tumor-immune system crosstalk. We found that the number of macrophages and levels of IFNγ and CCL4 mRNA were markedly reduced in tumors from CHOP KO relative to wt mice, suggesting a role for CHOP in modulating tumor microenvironment and macrophage recruitment to the tumor. Conclusion: Our data highlights a role for CHOP as a positive regulator of carcinogen-induced HCC progression through a complex mechanism that involves the immune system and modulation of stress signaling pathways

The second-generation antipsychotic drug aripiprazole modulates the serotonergic system in pancreatic islets and induces beta cell dysfunction in female mice

[Aims/hypothesis]: Second-generation antipsychotic (SGA) drugs have been associated with the development of type 2 diabetes and the metabolic syndrome in patients with schizophrenia. In this study, we aimed to investigate the effects of two different SGA drugs, olanzapine and aripiprazole, on metabolic state and islet function and plasticity. [Methods]: We analysed the functional adaptation of beta cells in 12-week-old B6;129 female mice fed an olanzapine- or aripiprazole-supplemented diet (5.5–6.0 mg kg−1 day−1) for 6 months. Glucose and insulin tolerance tests, in vivo glucose-stimulated insulin secretion and indirect calorimetry were performed at the end of the study. The effects of SGAs on beta cell plasticity and islet serotonin levels were assessed by transcriptomic analysis and immunofluorescence. Insulin secretion was assessed by static incubations and Ca2+ fluxes by imaging techniques. [Results]: Treatment of female mice with olanzapine or aripiprazole for 6 months induced weight gain (p<0.01 and p<0.05, respectively), glucose intolerance (p<0.01) and impaired insulin secretion (p<0.05) vs mice fed a control chow diet. Aripiprazole, but not olanzapine, induced serotonin production in beta cells vs controls, likely by increasing tryptophan hydroxylase 1 (TPH1) expression, and inhibited Ca2+ flux. Of note, aripiprazole increased beta cell size (p<0.05) and mass (p<0.01) vs mice fed a control chow diet, along with activation of mechanistic target of rapamycin complex 1 (mTORC1)/S6 signalling, without preventing beta cell dysfunction. [Conclusions/interpretation]: Both SGAs induced weight gain and beta cell dysfunction, leading to glucose intolerance; however, aripiprazole had a more potent effect in terms of metabolic alterations, which was likely a result of its ability to modulate the serotonergic system. The deleterious metabolic effects of SGAs on islet function should be considered while treating patients as these drugs may increase the risk for development of the metabolic syndrome and diabetes.This work was funded by H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement 721236, European Commission). We also acknowledge grants RTI2018-094052-B-100/ AEI/10.13039/501100011033 (Ministerio de Ciencia e Innovación y Fondo Europeo de Desarrollo Regional [FEDER]) and S2017/BMD-3684 (Comunidad de Madrid, Spain), and grants from Fundación Ramón Areces (Spain) and CIBERDEM (ISCIII, Spain)

HCV Causes Chronic Endoplasmic Reticulum Stress Leading to Adaptation and Interference with the Unfolded Protein Response

BACKGROUND: The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance. METHODS AND FINDINGS: The HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2-5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells. CONCLUSIONS: Our study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways

Author response: Repression of viral gene expression and replication by the unfolded protein response effector XBP1u

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Delayed kinetics of MCMV protein expression in XBP-1 KO MEFs.

<p>Wild type or XBP-1 KO MEFs were infected with MCMV at MOI of 0.1 (A) or 1 (B). Culture supernatants were collected at indicated time points and titers were determined using standard plaque assay. (C) Flow cytometry analysis of MEFs, infected with MCMV-GFP at MOI = 1. GFP positive cells were gated and counted as infected cells. (D) The expression of intrinsic viral proteins was measured by Western blot analysis, using monoclonal antibodies against Immediate Early 1 (IE1), Early 1 (E1) and a 48 kDa late protein. Cellular p97 served as loading control. (E) mRNA was extracted from mock infected MEFs, and the ratio XBP-1s to total XBP-1 mRNAs was calculated from quantitative RT-PCR data. (F) Western blot analysis of phosphorylated and total eIF2α of mock and MCMV infected WT or XBP-1 KO MEFs. Blot was quantified using ImageJ and the ratio between phosphorylated and total eIF2α was calculated. Presented data is from one experiment out of three preformed (C, D, F). Results are shown as mean ±SEM of at least three replicates. * P<0.05, ** P<0.01.</p

Viral induced deletion of XBP-1 impairs viral DNA replication and gene expression in vivo.

<p>XBP-1^WT/WT and XBP-1^f/f mice infected with 10∧5 PFU of MCMV-CRE. Mice were sacrificed 4 dpi and livers were extracted. (A) Viral genome copy number in mg tissue was determined using quantitative RT-PCR analysis of viral M55(gB) and the host gene pThrp (n = 3 (XBP-1^f/f), n = 4 (XBP-1^WT/WT)). The expression of representative viral RNAs (IE1, E1 and M55(gB)) in liver was measured by RT-PCR analysis and calculated relative to WT animals. Results are shown as mean ±SEM. * P<0.05, ** P<0.01.</p

XBP-1 does not affect the accumulation of viral DNA in host nuclei, but is required for optimal transcription of viral genes.

<p>Primary MEFs derived from RERT/XBP-1^f/f mice were treated with vehicle (EtOH) or 4-OHT for 48 h and infected with MCMV-GFP at MO1 = 1. DNA was purified from nuclear extracts of infected cells at indicated time points. The number of MCMV genome copies in the nucleus was calculated from qPCR data (A). Samples were loaded on 2% agarose gel (B). Untreated RERT/XBP-1^f/f MEFs were also infected with MCMV or MCMV-CRE and genome copy number was analyzed the same way (C, D). The transcription levels of representative MCMV genes were analyzed by quantitative RT-PCR and were calculated relative to expression levels in EtOH treated cells at 12 hpi (E) or MCMV infected cells (F). Results are shown as mean ±SEM of two (A, C) or three (E, F) replicates. * P<0.05, ** P<0.01.</p