Complementary Cell-Based High-Throughput Screens Identify Novel Modulators of the Unfolded Protein Response

Carmen and Ann Adams Department of Pediatrics, Division of Hematology/Oncology, Wayne State University, 421 E. Canfield, Detroit, MI 48201, USA.
Journal of Biomolecular Screening (Impact Factor: 2.42). 08/2011; 16(8):825-35. DOI: 10.1177/1087057111414893
Source: PubMed


Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

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    • "Our findings that SARS-CoV and HCoV-HKU1 S proteins activate ER stress and the UPR might have implications in therapeutic intervention. Pharmaceutical modulators of ER stress and the UPR have been developed and tested for various disease conditions including viral infection [54,55]. Interestingly, whereas inhibition of PERK kinase has been found to inhibit cytomegalovirus replication [56], activation of the UPR with a small-molecule compound also has broad-spectrum antiviral activity [57]. "
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    ABSTRACT: Whereas severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is associated with severe disease, human coronavirus HKU1 (HCoV-HKU1) commonly circulates in the human populations causing generally milder illness. Spike (S) protein of SARS-CoV activates the unfolded protein response (UPR). It is not understood whether HCoV-HKU1 S protein has similar activity. In addition, the UPR-activating domain in SARS-CoV S protein remains to be identified. In this study we compared S proteins of SARS-CoV and HCoV-HKU1 for their ability to activate the UPR. Both S proteins were found in the endoplasmic reticulum. Transmembrane serine protease TMPRSS2 catalyzed the cleavage of SARS-CoV S protein, but not the counterpart in HCoV-HKU1. Both S proteins showed a similar pattern of UPR-activating activity. Through PERK kinase they activated the transcription of UPR effector genes such as Grp78, Grp94 and CHOP. N-linked glycosylation was not required for the activation of the UPR by S proteins. S1 subunit of SARS-CoV but not its counterpart in HCoV-HKU1 was capable of activating the UPR. A central region (amino acids 201--400) of SARS-CoV S1 was required for this activity. SARS-CoV and HCoV-HKU1 S proteins use distinct UPR-activating domains to exert the same modulatory effects on UPR signaling.
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    • "Activation of PERK has been implicated in a wide variety of cancers as a mediator of response to chemotherapy (Kraus et al., 2008; Lust et al., 2009; Yan et al., 2010; Fribley et al., 2011; Qiao et al., 2012; Sailaja et al., 2013). Most convincingly, small interfering RNA (siRNA) against PERK or dominant negative models can ameliorate chemotherapy-induced death in many types of cancer cells (Lai and Wong, 2008; Yacoub et al., 2008; Kahali et al., 2010; Pan et al., 2012). "
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    ABSTRACT: Increased cellular protein production places stress on the endoplasmic reticulum (ER), because many of the nascent proteins pass through the ER for folding and trafficking. Accumulation of misfolded proteins in the ER triggers the activation of three well-known pathways including IRE1 (inositol requiring kinase 1), ATF6 (activating transcription factor 6), and PERK (double stranded RNA-activated protein kinase-like ER kinase). The activity of each sensor modulates the overall ER strategy for managing protein quality control as cellular needs change due to growth, differentiation, infection, transformation, and host of other possible physiological states. Here we review the role of ER stress in multiple myeloma (MM), an incurable plasma cell neoplasm. MM is closely linked to dysregulated unfolded protein response in the ER due to the heightened production of immunoglobulin and the metabolic demands of malignant uncontrolled proliferation. Together, these forces may mean that myeloma cells have an "Achilles heel" which can be exploited as a treatment target: their ER stress response must be constitutively active at a remarkably high level to survive their unique metabolic needs. Therefore, inhibition of the ER stress response is likely to injure the cells, as is any further demand on an already over-worked system. Evidence for this vulnerability is summarized here, along with an overview of how each of the three ER stress sensors has been implicated in myeloma pathogenesis and treatment.
    Full-text · Article · Jun 2013 · Frontiers in Genetics
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    • "ER stress-induced apoptosis is an important pathogenic factor in a number of widespread diseases, including diabetes, neurodegenerative diseases, atherosclerosis, and renal disease (Tabas and Ron 2011). Because of the UPR's central role in determining cell fate, there have been multiple studies to identify small molecules modulators to exploit the UPR for therapeutic benefit (Fribley et al. 2011; Papandreou et al. 2011; Volkmann et al. 2011; Cross et al. 2012; Mimura et al. 2012). "
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    ABSTRACT: Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation. The unfolded protein response (UPR) maintains the protein-folding homeostasis within the ER, ensuring that the protein-folding capacity of the ER meets the load of client proteins. Activation of the UPR depends on three ER stress sensor proteins, Ire1, PERK, and ATF6. Although the consequences of activation are well understood, how these sensors detect ER stress remains unclear. Recent evidence suggests that yeast Ire1 directly binds to unfolded proteins, which induces its oligomerization and activation. BiP dissociation from Ire1 regulates this oligomeric equilibrium, ultimately modulating Ire1's sensitivity and duration of activation. The mechanistic principles of ER stress sensing are the focus of this review.
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