[Show abstract][Hide abstract] ABSTRACT: The endoplasmic reticulum (ER) responds to changes in intracellular homeostasis through activation of the unfolded protein response (UPR). Yet, it is not known how UPR-signaling coordinates adaptation versus cell death. Previous studies suggested that signaling through PERK/ATF4 is required for cell death. We show that high levels of ER stress (i.e., ischemia-like conditions) induce transcription of the ubiquitin ligases Siah1/2 through the UPR transducers PERK/ATF4 and IRE1/sXBP1. In turn, Siah1/2 attenuates proline hydroxylation of ATF4, resulting in its stabilization, thereby augmenting ER stress output. Conversely, ATF4 activation is reduced upon Siah1/2 KD in cultured cells, which attenuates ER stress-induced cell death. Notably, Siah1a+/-::Siah2-/- mice subjected to neuronal ischemia exhibited smaller infarct volume and were protected from ischemia-induced death, compared with the wild type (WT) mice. In all, Siah1/2 constitutes an obligatory fine-tuning mechanism that predisposes cells to death under severe ER stress conditions.
[Show abstract][Hide abstract] ABSTRACT: Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5) mediates reactive oxygen species (ROS)-induced cell death. When activated by ROS, ASK1 ultimately becomes ubiquitinated and degraded by the proteasome, a process that is antagonized by the ubiquitin-specific protease USP9X. Using a functional siRNA (small interfering RNA) screen in HeLa cells, we identified Roquin-2 (also called RC3H2) as an E3 ubiquitin ligase required for ROS-induced ubiquitination and degradation of ASK1. In cells treated with H2O2, knockdown of Roquin-2 promoted sustained activation of ASK1 and the downstream stress-responsive kinases JNK (c-Jun amino-terminal kinase) and p38 MAPK (mitogen-activated protein kinase), and led to cell death. The nematode Caenorhabditis elegans produces ROS as a defense mechanism in response to bacterial infection. In C. elegans, mutation of the gene encoding the Roquin-2 ortholog RLE-1 promoted accumulation of the activated form of the ASK1 ortholog NSY-1 and conferred resistance to infection by the bacteria Pseudomonas aeruginosa. Thus, these data suggest that degradation of ASK1 mediated by Roquin-2 is an evolutionarily conserved mechanism required for the appropriate regulation of stress responses, including pathogen resistance and cell death.
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and protein kinase C (PKC), and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, owing to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI3K). Selective inactivation of Pdk1 in the melanocytes of Braf(V600E)::Pten(-/-) or Braf(V600E)::Cdkn2a(-/-)::Pten(-/-) mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-hydroxytamoxifen. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in Braf(V600E)::Pten(-/-) mice. Pdk1(-/-) melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten wild-type (WT) genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes, and inhibition of FOXO3a restored proliferation and colony formation of Pdk1(-/-) melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.Oncogene advance online publication, 16 September 2013; doi:10.1038/onc.2013.383.
[Show abstract][Hide abstract] ABSTRACT: The receptor for activated C kinase 1 (RACK1) serves as an adaptor for a number of proteins along the MAPK, protein kinase C, and Src signaling pathways. The abundance and near ubiquitous expression of RACK1 reflect its role in coordinating signaling molecules for many critical biological processes, from mRNA translation to cell motility to cell survival and death. Complete deficiency of Rack1 is embryonic lethal, but the recent development of genetic Rack1 hypomorphic mice has highlighted the central role that RACK1 plays in cell movement and protein synthesis. This review focuses on the importance of RACK1 in these processes and places the recent work in the larger context of understanding RACK1 function.
[Show abstract][Hide abstract] ABSTRACT: The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of β-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to β-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of β-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of β-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to β-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the β-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of β-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.
[Show abstract][Hide abstract] ABSTRACT: The E3 ubiquitin ligase Siah regulates key cellular events that are central to cancer development and progression. A promising route to Siah inhibition is disrupting its interactions with adaptor proteins. However, typical of protein-protein interactions, traditional unbiased approaches to ligand discovery did not produce viable hits against this target, despite considerable effort and a multitude of approaches. Ultimately, a rational structure-based design strategy was successful for the identification of Siah inhibitors in which peptide binding drives specific covalent bond formation with the target. X-ray crystallography, mass spectrometry, and functional data demonstrate that these peptide mimetics are efficient covalent inhibitors of Siah and antagonize Siah-dependent regulation of Erk and Hif signaling in the cell. The proposed strategy may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is an evolutionarily conserved intracellular catabolic process that delivers cytoplasmic components to lysosomes for degradation and recycling. Although originally considered to be a non-selective pathway, it is now recognized that autophagy is involved in selective processes, including the turnover of organelles, removal of protein aggregates, and elimination of intracellular pathogens. This specificity implies that cargo recognition and processing by the autophagy machinery are tightly regulated processes. In support of this, various forms of post-translational modification have been implicated in the regulation of autophagy, one of which is the ubiquitin-proteasome system. Here we review current understanding of the role of ubiquitylation in the control of autophagy.
Trends in Biochemical Sciences 07/2013; · 13.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The AKT and NF-κB pathways are central regulators of cellular signaling events at the basis of tumor development and progression. Both pathways are often up-regulated in different tumor types including melanoma. We recently reported the identification of compound 1 (BI-69A11) as inhibitor of the AKT and the NF-κB pathways. Here we describe SAR studies that led to novel fluorinated derivatives with increased cellular potency, reflected in efficient inhibition of AKT and IKKs. Selected compounds demonstrated effective toxicity on melanoma, breast and prostate cell lines. Finally, a representative derivative showed promising efficacy in an in vivo melanoma xenograft model. This article is protected by copyright. All rights reserved.
Chemical Biology & Drug Design 06/2013; · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Siah ubiquitin ligases are members of the RING finger E3 ligases. The Siah E3s are conserved from fly to mammals. Primarily implicated in cellular stress responses, Siah ligases play a key role in hypoxia, through the regulation of HIF-1α transcription stability and activity. Concomitantly, physiological conditions associated with varying oxygen tension often highlight the importance of Siah, as seen in cancer and neurodegenerative disorders. Notably, recent studies also point to the role of these ligases in fundamental processes including DNA damage response, cellular organization and polarity. This review summarizes the current understanding of upstream regulators and downstream effectors of Siah.
Cell biochemistry and biophysics 05/2013; · 3.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Changes in cell adhesion and polarity are closely associated with epithelial cell transformation and metastatic capacity. The tumor suppressor protein ASPP (Apoptosis-Stimulating Proteins of p53) 2 has been implicated in control of cell adhesion and polarity through its effect on the PAR complex. Here we demonstrate that under hypoxic conditions, the ubiquitin ligase Siah (seven in absentia homolog)2 controls ASPP2 availability, with concomitant effect on epithelial cell polarity. LC-MS/MS analysis identified ASPP2 and ASPP1 as Siah2-interacting proteins. Biochemical analysis confirmed this interaction and mapped degron motifs within ASPP2, which are required for Siah2-mediated ubiquitination and proteasomal-dependent degradation. Inhibition of Siah2 expression increases ASPP2 levels and enhances ASPP2-dependent maintenance of tight junction (TJ) integrity, and polarized architecture in three dimensional (3D) organotypic culture. Conversely, increase of Siah2 expression under hypoxia decreases ASPP2 levels and the formation of apical polarity in 3D culture. In all, our studies demonstrate the role of Siah2 in regulation of TJ integrity and cell polarity under hypoxia, through its regulation of ASPP2 stability.Oncogene advance online publication, 6 May 2013; doi:10.1038/onc.2013.149.
[Show abstract][Hide abstract] ABSTRACT: Folding of newly synthesized polypeptides (NSPs) into functional proteins is a highly regulated process. Rigorous quality control ensures that NSPs attain their native fold, during or shortly after completion of translation. Nonetheless, signaling pathways that govern degradation of NSPs in mammals remain elusive. We demonstrate that the stress-induced c-Jun N-terminal kinase (JNK) is recruited to ribosomes by the receptor for activated protein C kinase 1 (RACK1). RACK1 is an integral component of the 40S ribosome and adaptor for protein kinases. Ribosome-associated JNK phosphorylates the eukaryotic translation elongation factor 1A isoform 2 (eEF1A2) on serines 205 and 358 to promote degradation of NSPs by the proteasome. These findings establish a role for a RACK1/JNK/eEF1A2 complex in the quality control of NSPs in response to stress.
Molecular and cellular biology 04/2013; · 6.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: Effective therapy for malignant melanoma, the leading cause of death from skin cancer, remains an area of significant unmet need in oncology. The elevated expression of PKCε in advanced metastatic melanoma results in the increased phosphorylation of the transcription factor ATF2 on threonine 52, which causes its nuclear localization and confers its oncogenic activities. The nuclear-to-mitochondrial translocation of ATF2 following genotoxic stress promotes apoptosis, a function that is largely lost in melanoma cells, due to its confined nuclear localization. Therefore, promoting the nuclear export of ATF2, which sensitizes melanoma cells to apoptosis, represents a novel therapeutic modality. EXPERIMENTAL DESIGN: We conducted a pilot high-throughput screen of 3,800 compounds to identify small molecules that promote melanoma cell death by inducing the cytoplasmic localization of ATF2. The imaging-based ATF2 translocation assay was performed using UACC903 melanoma cells that stably express doxycycline-inducible GFP-ATF2. RESULTS: We identified 2 compounds (SBI-0089410 and SBI-0087702) that promoted the cytoplasmic localization of ATF2, reduced cell viability, inhibited colony formation, cell motility, anchorage-free growth, and increased mitochondrial membrane permeability. SBI-0089410 inhibited the TPA-induced membrane tranlocation of PKC isoforms, whereas both compounds decreased ATF2 phosphorylation by PKCε and ATF2 transcriptional activity. Overexpression of either constitutively active PKCε or phosphomimic mutant ATF2(T52E) attenuated the cellular effects of the compounds. CONCLUSION: The imaging-based high-throughput screen provides a proof-of-concept for the identification of small molecules that block the oncogenic addiction to PKCε signaling by promoting ATF2 nuclear export, resulting in mitochondrial membrane leakage and melanoma cell death.
Clinical Cancer Research 04/2013; · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Understanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.
Cancer cell 03/2013; 23(3):332-46. · 25.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with-and regulation of its ubiquitination and stability by-RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5(-/-) MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5(-/-) using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5(-/-) mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5(-/-) macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.
[Show abstract][Hide abstract] ABSTRACT: An increasing number of transcription factors have been shown to elicit oncogenic and tumor suppressor activities, depending on the tissue and cell context. Activating transcription factor 2 (ATF2; also known as cAMP-dependent transcription factor ATF-2) has oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin tumors and breast cancer. Recent work has shown that the opposing functions of ATF2 are associated with its subcellular localization. In the nucleus, ATF2 contributes to global transcription and the DNA damage response, in addition to specific transcriptional activities that are related to cell development, proliferation and death. ATF2 can also translocate to the cytosol, primarily following exposure to severe genotoxic stress, where it impairs mitochondrial membrane potential and promotes mitochondrial-based cell death. Notably, phosphorylation of ATF2 by the epsilon isoform of protein kinase C (PKCε) is the master switch that controls its subcellular localization and function. Here, we summarize our current understanding of the regulation and function of ATF2 in both subcellular compartments. This mechanism of control of a non-genetically modified transcription factor represents a novel paradigm for 'oncogene addiction'.
[Show abstract][Hide abstract] ABSTRACT: Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.
[Show abstract][Hide abstract] ABSTRACT: The tricarboxylic acid (TCA) cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoint glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability.
[Show abstract][Hide abstract] ABSTRACT: Tumor hypoxia is associated with resistance to antiangiogenic therapy and poor prognosis. The Siah E3 ubiquitin ligases regulate the hypoxic response pathway by modulating the turnover of the master proangiogenic transcription factor hypoxia-inducible factor-1α (Hif-1α). In this study, we show that genetic deficiency in the Siah family member Siah2 results in vascular normalization and delayed tumor growth in an established transgenic model of aggressive breast cancer. Tumors arising in a Siah2(-/-) genetic background showed increased perfusion and pericyte-associated vasculature, similar to that occurring with antiangiogenic therapy. In support of the role of Siah2 in regulating levels of Hif-1α, expression of angiogenic factors was decreased in Siah2(-/-) tumors. Blood vessel normalization in Siah2(-/-) tumors resulted in an increased response to chemotherapy and prolonged survival. Together, our findings offer a preclinical proof of concept that targeting Siah2 is sufficient to attenuate Hif-1α-mediated angiogenesis and hypoxia signaling, thereby improving responses to chemotherapy.
Cancer Research 02/2012; 72(7):1694-704. · 9.28 Impact Factor