Secondary Bortezomib-Resistance Is Characterized by Upregulation of Proteasomal Subunits and Their Insufficient Inhibition by Bortezomib, Which Can Be Overcome by Alternative Proteasome Inhibitors Combined with Induction of the ER Stress Response.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2583-2583
Author(s):  
Thomas Ruckrich ◽  
Jeannette Gogel ◽  
Marianne Kraus ◽  
Huib Ovaa ◽  
Christoph Driessen
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Proteasome Inhibitors ◽  
Cytotoxic Drugs ◽  
Proteasome Activity ◽  
Intact Cells ◽  
Secondary Resistance ◽  
Er Stress Response ◽  
Proteolytic Pathways

Abstract Resistance towards proteasome inhibition by Bortezomib (Velcade®) represents a challenge for myeloma therapy. Its biology has not yet been characterized in detail. We have demonstrated that Bortezomib-sensitive malignant haematopoetic cells can acquire secondary resistance to Bortezomib in vitro. We here present the first analysis of proteasome biology and activity, alternative proteolytic pathways, ubiquitin-specific proteases (USP) and the ER stress response (unfolded protein response, UPR) upstream of the proteasome, as well as in vitro cytotoxicity of conventional cytotoxic drugs, alternative proteasome inhibitors and agents that target the UPR in Bortezomib-resistant (BR) cells, compared to wild type (WT) controls. BR cells had higher activities of all subunits of the constitutive and the immunoproteasome, as deferred from turnover of fluorogenic substrates as well as affinity-labelling of active proteasome subunits in intact cells. This was mirrored by increased levels of proteasomal β1 and β2, but especially β5 polypeptides, implicating a homeostatic system that senses and corrects low proteasome activity in cells chronically exposed to Bortezomib. While the vinylsulfone-type proteasome inhibitor NLVS abrogated detectable proteasome activity in both BR and WT cells, Bortezomib at therapeutic concentrations eliminated proteasomal β1 and β5-type activity only in WT cells, while BR cells retained residual activity. These changes in proteasome biology appear to be the molecular hallmark of required Bortezomib resistance, since no changes were observed between WT and BR cells in alternative cytosolic or lysosomal proteolytic pathways, UPR activity as well as the gross activity pattern of USP. As expected, this translated into sensitivity against cytotoxic drugs in vitro: BR cells were less sensitive towards alternative proteasome inhibitors. However, while the IC50 for pan-proteasome inhibitors was only roughly doubled in BR cells, it was nearly tenfold elevated for the β5-preferring vinylsulfone inhibitor NLVS. By contrast, sensitivity towards anthracyclines or cytotoxicity induced by ER stressors as well as the synergy between proteasome inhibitors and UPR-activators remained unaffected in BR cells. Based on our data, proteasome inhibitors with activity profiles different from that of Bortezomib, alone or in combination with induction of the UPR, may represent an appropriate concept to overcome secondary Bortezomib resistance.

scholarly journals Liver-specific deletion of protein tyrosine phosphatase (PTP) 1B improves obesity- and pharmacologically induced endoplasmic reticulum stress

2011 ◽  
Vol 438 (2) ◽  
pp. 369-378 ◽  
Author(s):  
Abdelali Agouni ◽  
Nimesh Mody ◽  
Carl Owen ◽  
Alicja Czopek ◽  
Derek Zimmer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Insulin Sensitivity ◽  
Er Stress ◽  
Protein Tyrosine Phosphatase ◽  
Mouse Liver ◽  
Tyrosine Phosphatase ◽  
Er Stress Response

Obesity is associated with induction of the ER (endoplasmic reticulum)-stress response signalling and insulin resistance. PTP1B (protein tyrosine phosphatase 1B) is a major regulator of adiposity and insulin sensitivity. The aim of the present study was to investigate the role of L-PTP1B (liver-specific PTP1B) in chronically HFD (high-fat diet) and pharmacologically induced (tunicamycin and thapsigargin) ER-stress response signalling in vitro and in vivo. We assessed the effects of ER-stress response induction on hepatic PTP1B expression, and consequences of hepatic-PTP1B deficiency, in cells and mouse liver, on components of ER-stress response signalling. We found that PTP1B protein and mRNA expression levels were up-regulated in response to acute and/or chronic ER stress, in vitro and in vivo. Silencing PTP1B in hepatic cell lines or mouse liver (L-PTP1B−/−) protected against induction of pharmacologically induced and/or obesity-induced ER stress. The HFD-induced increase in CHOP (CCAAT/enhancer-binding protein homologous protein) and BIP (binding immunoglobulin protein) mRNA levels were partially inhibited, whereas ATF4 (activated transcription factor 4), GADD34 (growth-arrest and DNA-damage-inducible protein 34), GRP94 (glucose-regulated protein 94), ERDJ4 (ER-localized DnaJ homologue) mRNAs and ATF6 protein cleavage were completely suppressed in L-PTP1B−/− mice relative to control littermates. L-PTP1B−/− mice also had increased nuclear translocation of spliced XBP-1 (X box-binding protein-1) via increased p85α binding. We demonstrate that the ER-stress response and L-PTP1B expression are interlinked in obesity- and pharmacologically induced ER stress and this may be one of the mechanisms behind improved insulin sensitivity and lower lipid accumulation in L-PTP1B−/− mice.

Abstract 4475: In vitro efficacy profiling of ONC201 in cancer cells reveals sensitivity pattern that is consistent with ER stress response

2015 ◽  
Author(s):  
Joshua E. Allen ◽  
Jo Ishizawa ◽  
Wafik S. El-Deiry ◽  
Michael Andreeff ◽  
Mathew Garnett ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Cancer Cells ◽  
Er Stress Response ◽  
Sensitivity Pattern

NRF2 at the Interface of Oxidative and Endoplasmic Reticulum Stress Pathways Is a Critical Integrator of Bortezomib Response in Mantle Cell Lymphoma in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 727-727 ◽  
Author(s):  
Marc A Weniger ◽  
Edgar Gil Rizzatti ◽  
Patricia Perez Galan ◽  
Delong Liu ◽  
Peter J Munson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Er Stress ◽  
Tumor Cells ◽  
Cell Lines ◽  
Target Genes ◽  
Resistant Cell ◽  
Er Stress Response

Abstract Abstract 727 The proteasome inhibitor bortezomib (BZM) is effective as single-agent in relapsed and refractory mantle cell lymphoma (MCL) but more than half of patients remain insensitive to BZM. Suggested mechanisms of action include activation of Noxa, p53, oxidative, and endoplasmic reticulum (ER) stress. To define mechanisms relevant for BZM-induced cytotoxicity we pursued two approaches: first, we characterized gene expression changes in 10 MCL cell lines exposed for 24h to 10nM BZM, a concentration that kills >50% of sensitive, but <20% of resistant cells. Secondly, we analyzed gene expression changes in tumor cells of patients with leukemic MCL undergoing BZM treatment in a clinical trial. RNA was extracted from MCL cell lines at early (1h and 3h), intermediate (6h) and late (24h) time points. Virtually no changes in gene expression were detectable at 1 and 3h of drug exposure and only about 100 genes changed by 6h. After 24h of treatment 524 genes were significantly changed in sensitive and 271 genes in resistant cell lines respectively. The delayed onset of gene expression changes is consistent with the reversibility of BZM toxicity for up to 8 hours. Using Ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA) we identified two dominant responses induced by BZM: 1) an oxidative stress response mediated by NRF2 and related transcription factors, and 2) an ER stress/ubiquitin proteasome response (FDR by GSEA <0.1). Both responses were primarily apparent in sensitive cell lines. A set of 20 experimentally validated NRF2 target genes was used as a core NRF2 signature: this signature was increased 15-fold on average in sensitive cell lines but only 2-fold in resistant cell lines (P=0.006). Similarly, an XBP1 and ATF6 signature, reflecting activation of the ER stress response, was stronger induced in sensitive than in resistant cell lines (average 1.9-fold vs 1.3-fold; P=0.003). Activation of these stress pathways upon BZM treatment was confirmed by demonstrating accumulation of nuclear NRF2 in sensitive Jeko1 but not in resistant Mino cells. Also markers of ER stress such as phosphorylation of ER resident nuclease Ire1 that splices the transcription factor XBP1 and activation of ATF3, ATF4, and CHOP downstream of PERK were readily detected in Jeko1 but not in Mino cells. Finally, Noxa, the BH3-only protein primarily responsible for BZM-induced apoptosis, was only induced in sensitive Jeko1 cells. We next analyzed the effect of BZM on purified tumor cells from five patients with leukemic MCL treated with BZM (1.5mg/m2, day 1, 4, 8 and 11). Two patients showed a >50% reduction in circulating tumor cells after 2 injections of drug (day 8) and >75% reduction after 4 injections (day 2, sensitive), while in three patients there was no change or an intermediate response (resistant). Western blotting demonstrated Noxa up-regulation in circulating tumor cells of sensitive but not resistant samples. This is consistent with the demonstrated importance of Noxa for induction of apoptosis in response to BZM in cell line studies. Next we performed gene expression profiling immediately before, at 6h, and 24h after the first and 24h after the second dose of BZM. Using IPA and GSEA up-regulation of the ubiquitin/proteasome pathway and the NRF2-mediated oxidative stress response was again prominent, but an ER stress response was less apparent. XBP1 splicing was not detected in tumor cells from sensitive samples indicating that an ER stress response was not fully activated by BZM in vivo. Consistent with in vitro data the NRF2 signature was induced 2.3-fold on average in sensitive but not in resistant samples (P<0.05). Intriguingly, baseline expression of the NRF2 signature genes was significantly higher in resistant than in sensitive cells (P=0.0007). In summary, we identify NRF2 as critical integrator of different stress pathways in response to BZM in MCL. Thus, rapid induction of NRF2 target genes might be a useful biomarker of BZM-induced cellular stress and predict clinical response. Our data suggest a more complex function of NRF2 than previously appreciated. NRF2-regulated genes serve primarily homeostatic roles and enable cells to deal with oxidative and xenobiotic insults; a function that may come to play in BZM resistant cells with higher baseline expression of NRF2 target genes. On the other hand, our data suggest a possible pro-apoptotic role of acute induction of high levels of NRF2 that is currently under investigation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Overexpression of TCL1 activates the endoplasmic reticulum stress response: a novel mechanism of leukemic progression in mice

Blood ◽  
2012 ◽  
Vol 120 (5) ◽  
pp. 1027-1038 ◽  
Author(s):  
Crystina L. Kriss ◽  
Javier A. Pinilla-Ibarz ◽  
Adam W. Mailloux ◽  
John J. Powers ◽  
Chih-Hang Anthony Tang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Er Stress ◽  
Cytidine Deaminase ◽  
Lymphocytic Leukemia ◽  
Endoplasmic Reticulum Stress Response ◽  
Transgenic Expression ◽  
Er Stress Response ◽  
Adult Leukemia

Abstract Chronic lymphocytic leukemia (CLL) represents 30% of adult leukemia. TCL1 is expressed in ∼ 90% of human CLL. Transgenic expression of TCL1 in murine B cells (Eμ-TCL1) results in mouse CLL. Here we show for the first time that the previously unexplored endoplasmic reticulum (ER) stress response is aberrantly activated in Eμ-TCL1 mouse and human CLL. This includes activation of the IRE-1/XBP-1 pathway and the transcriptionally up-regulated expression of Derlin-1, Derlin-2, BiP, GRP94, and PDI. TCL1 associates with the XBP-1 transcription factor, and causes the dysregulated expression of the transcription factors, Pax5, IRF4, and Blimp-1, and of the activation-induced cytidine deaminase. In addition, TCL1-overexpressing CLL cells manufacture a distinctly different BCR, as we detected increased expression of membrane-bound IgM and altered N-linked glycosylation of Igα and Igβ, which account for the hyperactive BCR in malignant CLL. To demonstrate that the ER stress-response pathway is a novel molecular target for the treatment of CLL, we blocked the IRE-1/XBP-1 pathway using a novel inhibitor, and observed apoptosis and significantly stalled growth of CLL cells in vitro and in mice. These studies reveal an important role of TCL1 in activating the ER stress response in support for malignant progression of CLL.

Targeted regulation of lymphocytic ER stress response with an overall immunosuppression to alleviate allograft rejection

Biomaterials ◽  
2021 ◽  
pp. 120757
Author(s):  
Yingying Shi ◽  
Yichao Lu ◽  
Chunqi Zhu ◽  
Zhenyu Luo ◽  
Xiang Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Allograft Rejection ◽  
Er Stress Response

scholarly journals Highly Specialized Ubiquitin-Like Modifications: Shedding Light into the UFM1 Enigma

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 255
Author(s):  
Katharina F. Witting ◽  
Monique P.C. Mulder
Keyword(s):  
Dna Repair ◽  
Stress Response ◽  
Embryonic Development ◽  
Er Stress ◽  
Unmet Needs ◽  
Biological Function ◽  
Post Translational Modification ◽  
Er Stress Response ◽  
Cellular Events ◽  
Complex Signaling

Post-translational modification with Ubiquitin-like proteins represents a complex signaling language regulating virtually every cellular process. Among these post-translational modifiers is Ubiquitin-fold modifier (UFM1), which is covalently attached to its substrates through the orchestrated action of a dedicated enzymatic cascade. Originally identified to be involved embryonic development, its biological function remains enigmatic. Recent research reveals that UFM1 regulates a variety of cellular events ranging from DNA repair to autophagy and ER stress response implicating its involvement in a variety of diseases. Given the contribution of UFM1 to numerous pathologies, the enzymes of the UFM1 cascade represent attractive targets for pharmacological inhibition. Here we discuss the current understanding of this cryptic post-translational modification especially its contribution to disease as well as expand on the unmet needs of developing chemical and biochemical tools to dissect its role.

Abstract 292: The Role of the ATF6 Branch of the ER Stress Response in the Endocrine Heart

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Erik A Blackwood ◽  
Christopher C Glembotski
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Knockout Mice ◽  
Ex Vivo ◽  
Proteolytic Cleavage ◽  
Isolated Perfused Heart ◽  
Wild Type ◽  
Atrial Myocytes ◽  
Perfused Heart ◽  
Er Stress Response

Rationale: Atrial natriuretic peptide (ANP) is stored in the heart in large dense core granules of atrial myocytes as a biologically inactive precursor, pro-ANP. Hemodynamic stress and atrial stretch stimulate coordinate secretion and proteolytic cleavage of pro-ANP to its bioactive form, ANP, which promotes renal salt excretion and vasodilation, which, together contribute to decreasing blood pressure. While the ATF6 branch of the ER stress response has been studied in ventricular tissue mouse models of myocardial ischemia and pathological hypertrophy, roles for ATF6 and ER stress on the endocrine function of atrial myocytes have not been studied. Objective/Methods: To address this gap in our knowledge, we knocked down ATF6 in primary cultured neonatal rat atrial myocytes (NRAMs) using a chemical inhibitor of the proteolytic cleavage site enabling ATF6 activation and siRNA and measured ANP expression and secretion basally and in response to alpha- adrenergic agonist stimulation using phenylephrine. We also compared the ANP secretion from wild- type mice and ATF6 knockout mice in an ex vivo Langendorff model of the isolated perfused heart. Results: ATF6 knockdown in NRAMs significantly impaired basal and phenylephrine-stimulated ANP secretion. ATF6 knockout mice displayed lower levels of ANP in atrial tissue at baseline as well as after phenylephrine treatment. Similarly, in the ex vivo isolated perfused heart model, less ANP was detected in effluent of ATF6 knockout hearts compared to wild-type hearts. Conclusions: The ATF6 branch of the ER stress response is necessary for efficient co-secretional processing of pro-ANP to ANP and for agonist-stimulated ANP secretion from atrial myocytes. As ANP is secreted in a regulated manner in response to a stimulus and pro-ANP is synthesized and packaged through the classical secretory pathway, we posit that ATF6 is required for adequate expression, folding, trafficking, processing and secretion of biologically active ANP from the endocrine heart.

Abstract 415: Cardioprotective Effects of Interleukin-15 in Immortalized Human Ventricular Cardiomyocytes

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Marin Jane McBride ◽  
Kristina Durham ◽  
Bernardo L Trigatti
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Interleukin 2 ◽  
Cell Types ◽  
Human Cardiomyocytes ◽  
Er Stress Response ◽  
Distinct Cell ◽  
Receptor Chain ◽  
Interleukin 15 ◽  
Receptor Beta

Interleukin-15 (IL-15) is a pleotropic cytokine that has a profound effect on the proliferation, survival and differentiation of many distinct cell types. The IL-15 receptor complex has 3 subunits: the unique receptor chain IL-15 receptor alpha (IL-15Rα), and two receptor chains shared with interleukin-2 (IL-2) and/or other cytokines, referred to as IL-2 receptor beta (IL-2Rβ) and IL-2 receptor gamma/gamma common chain (IL-2Rγ/γc), respectively. To our knowledge, this is the first study to examine the effects of IL-15 in immortalized human cardiomyocytes. Data collected by RT-PCR shows mRNA expression of IL-15Rα, IL-2Rβ and IL-2 Rγ/γc in these cells. Additionally, western blotting for IL-15Rα, IL-2Rβ and IL-2 Rγ/γc confirms the presence of all three IL-15 receptors. Early experiments examining the effect of IL-15 on cardiomyocyte cell survival show a statistically significant protective effect of IL-15 on the survival of cells exposed to tunicamycin, a pharamacological endoplasmic reticulum (ER) stress inducing agent. These findings suggest that IL-15 signaling may be an important cardioprotective pathway that is involved in the cardiac ER stress response. As ER stress is a major component of multiple different cardiac pathologies, such as myocardial infarction, heart failure and diabetes, uncovering the molecular mechanism by which IL-15 protects the heart will allow for deeper understanding of the cardiac ER stress response.

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