Activation of the Unfolded Protein Response Contributes toward the Antitumor Activity of Vorinostat 1

Radiation Oncology, H. Lee Moffitt Cancer Center, Tampa, FL 33612, USA.
Neoplasia (New York, N.Y.) (Impact Factor: 4.25). 01/2010; 12(1):80-6. DOI: 10.1593/neo.91422
Source: PubMed


Histone deacetylase (HDAC) inhibitors represent an emerging class of anticancer agents progressing through clinical trials. Although their primary target is thought to involve acetylation of core histones, several nonhistone substrates have been identified, including heat shock protein (HSP) 90, which may contribute towards their antitumor activity. Glucose-regulated protein 78 (GRP78) is a member of the HSP family of molecular chaperones and plays a central role in regulating the unfolded protein response (UPR). Emerging data suggest that GRP78 is critical in cellular adaptation and survival associated with oncogenesis and may serve as a cancer-specific therapeutic target. On the basis of shared homology with HSP family proteins, we sought to determine whether GRP78 could serve as a molecular target of the HDAC inhibitor vorinostat. Vorinostat treatment led to GRP78 acetylation, dissociation, and subsequent activation of its client protein double-stranded RNA-activated protein-like endoplasmic reticulum kinase (PERK). Investigations in a panel of cancer cell lines identified that UPR activation after vorinostat exposure is specific to certain lines. Mass spectrometry performed on immunoprecipitated GRP78 identified lysine-585 as a specific vorinostat-induced acetylation site of GRP78. Downstream activation of the UPR was confirmed, including eukaryotic initiating factor 2alpha phosphorylation and increase in ATF4 and C/EBP homologous protein expression. To determine the biologic relevance of UPR activation after vorinostat, RNA interference of PERK was performed, demonstrating significantly decreased sensitivity to vorinostat-induced cytotoxicity. Collectively, these findings indicate that GRP78 is a biologic target of vorinostat, and activation of the UPR through PERK phosphorylation contributes toward its antitumor activity.

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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). It is therefore perhaps unsurprising that this effect has also been seen in myeloma cells, which already have baseline ER stress and may not be able to tolerate perturbations to the system. "
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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.
    Frontiers in Genetics 06/2013; 4:109. DOI:10.3389/fgene.2013.00109
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    • "M344 induced expression of ATF3 was completely abolished in ATF4 (-/-) MEFs implicating an ISR dependent mechanism downstream of ATF4. In accordance with this finding, the endoplasmic reticulum chaperone protein glucose-regulated protein 78 (GRP78) was recently identified as a non-histone target of SAHA, whose action leads to dissociation of GRP78 and its client protein, double-stranded RNA-activated protein-like ER kinase (PERK), and subsequent activation of the ISR through the induction of the endoplasmic reticulum stress response including activation of ATF4 [39]. Since ATF3 is a known effector of the ISR pathway downstream of ATF4 [36], our finding that M344 induces ATF3 may be mediated by HDAC inhibitor mediated acetylation of GRP78. "
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    ABSTRACT: Activating Transcription Factor (ATF) 3 is a key regulator of the cellular integrated stress response whose expression has also been correlated with pro-apoptotic activities in tumour cell models. Combination treatments with chemotherapeutic drugs, such as cisplatin, and histone deacetylase (HDAC) inhibitors have been demonstrated to enhance tumour cell cytotoxicity. We recently demonstrated a role for ATF3 in regulating cisplatin-induced apoptosis and others have shown that HDAC inhibition can also induce cellular stress. In this study, we evaluated the role of ATF3 in regulating the co-operative cytotoxicity of cisplatin in combination with an HDAC inhibitor. The HDAC inhibitor M344 induced ATF3 expression at the protein and mRNA level in a panel of human derived cancer cell lines as determined by Western blot and quantitative RT-PCR analyses. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatments also enhanced the cytotoxic effects of cisplatin in these cancer cell lines. The mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways and dependent on ATF4, a known regulator of ATF3 expression. ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) as well as chromatin immunoprecipitation (ChIP) assays were utilized in determining the mechanistic induction of ATF3 by M344. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.
    Cancer Cell International 09/2010; 10(1):32. DOI:10.1186/1475-2867-10-32 · 2.77 Impact Factor
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