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author:("rosai, Ze'ev")
1.  Genetic inactivation or pharmacological inhibition of Pdk1 delays development and inhibits metastasis of BrafV600E::Pten−/− melanoma 
Oncogene  2013;33(34):4330-4339.
Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and 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, due to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI 3-K). Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten−/− or BrafV600E::Cdkn2a−/−::Pten−/− mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-HT. 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 BrafV600E::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 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.
doi:10.1038/onc.2013.383
PMCID: PMC3955742  PMID: 24037523
PDK1; FOXO3a; melanoma; Braf; Pten; GSK2334470
2.  Siah2 regulates tight junction integrity and cell polarity through control of ASPP2 stability 
Oncogene  2013;33(15):2004-2010.
Changes in cell adhesion and polarity are closely associated with epithelial cell transformation and metastatic capacity. The tumor suppressor protein ASPP2 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 Siah2 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 TJ integrity and polarized architecture in 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.
doi:10.1038/onc.2013.149
PMCID: PMC3917971  PMID: 23644657
ASPP2; Siah2; epithelial polarity; tight junction; hypoxia
3.  Inhibition of Melanoma Growth by Small Molecules that Promote the Mitochondrial Localization of ATF2 
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 ATF2T52E 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.
doi:10.1158/1078-0432.CCR-12-2689
PMCID: PMC3690798  PMID: 23589174
ATF2; PKCε; melanoma; nuclear translocation; high content screen
4.  Degradation of Newly Synthesized Polypeptides by Ribosome-Associated RACK1/c-Jun N-Terminal Kinase/Eukaryotic Elongation Factor 1A2 Complex 
Molecular and Cellular Biology  2013;33(13):2510-2526.
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 the 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 an 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.
doi:10.1128/MCB.01362-12
PMCID: PMC3700114  PMID: 23608534
5.  RACK1 Function in Cell Motility and Protein Synthesis 
Genes & Cancer  2013;4(9-10):369-377.
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.
doi:10.1177/1947601913486348
PMCID: PMC3863339  PMID: 24349634
RACK1; translation; ribosomes; stress response; cell migration; cancer
6.  PKCε regulates ATF2 availability to alter mitochondrial permeability following genotoxic stress 
Cell  2012;148(3):543-555.
Summary
The transcription factor ATF2 elicits oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin cancer. Here we identify that ATF2 tumor suppressor function is determined by its ability to localize at the mitochondria, where it alters membrane permeability following genotoxic stress. The ability of ATF2 to reach the mitochondria is determined by PKCε, which directs ATF2 nuclear localization. Genotoxic stress attenuates PKCε effect on ATF2, enables ATF2 nuclear export and localization at the mitochondria, where it perturbs the HK1-VDAC1 complex, increases mitochondrial permeability and promotes apoptosis. Significantly, high levels of PKCε, as seen in melanoma cells, block ATF2 nuclear export and function at the mitochondria, thereby attenuating apoptosis following exposure to genotoxic stress. In melanoma tumor samples, high PKCε levels associates with poor prognosis. Overall, our findings provide the framework for understanding how subcellular localization enables ATF2 oncogenic or tumor suppressor functions.
doi:10.1016/j.cell.2012.01.016
PMCID: PMC3615433  PMID: 22304920
7.  Siah2-dependent concerted activity of HIF & FoxA2 regulates formation of neuroendocrine phenotype & neuroendocrine prostate tumors 
Cancer cell  2010;18(1):23-38.
Summary
Neuroendocrine (NE) phenotype, seen in >30% of prostate adenocarcinomas (PCa), and NE prostate tumors are implicated in aggressive prostate cancer. Formation of NE prostate tumors in the TRAMP mouse model was suppressed in mice lacking the ubiquitin ligase Siah2, which regulates HIF-1α availability. Cooperation between HIF-1α and FoxA2, a transcription factor expressed in NE tissue, promotes recruitment of p300 to transactivate select HIF-regulated genes, Hes6, Sox9 and Jmjd1a. These HIF-regulated genes are highly expressed in metastatic PCa and required for hypoxia-mediated NE phenotype, metastasis in PCa and the formation of NE tumors. Tissue-specific expression of FoxA2 combined with Siah2-dependent HIF-1α availability enables a transcriptional program required for NE prostate tumor development and NE phenotype in PCa.
doi:10.1016/j.ccr.2010.05.024
PMCID: PMC2919332  PMID: 20609350
8.  Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis 
Pigment cell & melanoma research  2009;22(6):799-808.
Summary
The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-κB signaling pathways. Both Ras/mitogen-activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high-throughput electro-chemiluninescent-based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self-ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF-1α and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah-KO cells, pointing to its Siah-dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS-independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.
doi:10.1111/j.1755-148X.2009.00628.x
PMCID: PMC2863310  PMID: 19712206
Siah2; ubiquitin ligase; Meso-scale; melanoma; hypoxia; Ras
9.  Increased Expression of the E3 Ubiquitin Ligase RNF5 Is Associated with Decreased Survival in Breast Cancer 
Cancer research  2007;67(17):8172-8179.
The selective ubiquitination of proteins by ubiquitin E3 ligases plays an important regulatory role in control of cell differentiation, growth, and transformation and their dysregulation is often associated with pathologic outcomes, including tumorigenesis. RNF5 is an E3 ubiquitin ligase that has been implicated in motility and endoplasmic reticulum stress response. Here, we show that RNF5 expression is upregulated in breast cancer tumors and related cell lines. Elevated expression of RNF5 was seen in breast cancer cell lines that became more sensitive to cytochalasin D– and paclitaxel-induced apoptosis following its knockdown with specific short interfering RNA. Inhibition of RNF5 expression markedly decreased cell proliferation and caused a reorganization of the actin cytoskeleton in response to stress in MCF-7 but not in p53 mutant breast cancer cells, suggesting a p53- dependent function. Significantly, high levels of RNF5 were associated with decreased survival in human breast cancer specimens. Similarly, RNF5 levels were higher in metastatic melanoma specimens and in melanoma, leukemia, ovarian, and renal tumor-derived cell lines, suggesting that increased RNF5 expression may be a common event during tumor progression. These results indicate that RNF5 is a novel regulator of breast cancer progression through its effect on actin cytoskeletal alterations, which also affect sensitivity of breast cancer cells to cytoskeletal targeting antineoplastic agents.
doi:10.1158/0008-5472.CAN-07-0045
PMCID: PMC2962863  PMID: 17804730
10.  The Siah2-HIF-FoxA2 axis in prostate cancer – new markers and therapeutic opportunities 
Oncotarget  2010;1(5):379-385.
Recent studies indicate the importance of the ubiquitin ligase Siah2 in control of more aggressive prostate tumors – namely, neuroendocrine (NE) prostate tumors and prostate adenocarcinoma (PCa) harboring neuroendocrine lesions. Siah2-dependent expression and activity of HIF-1α regulate its availability to form a transcriptional complex with FoxA2, resulting in expression of specific target genes, including Hes6, Sox9 and Jmjd1a, whose co-expression is sufficient for formation of NE tumors and NE lesions in PCa. These studies provide novel markers to diagnose and monitor formation of NE lesions and NE tumors. Furthermore, defining the regulatory axis consisting of Siah2 and HIF-1α/FoxA2 cooperation suggests novel therapeutic modalities to treat these most aggressive forms of prostate cancer. Here we review current understanding of Siah role in control of hypoxia and prostate tumor development and highlight potential approaches for targeting components along Siah-regulated pathways.
PMCID: PMC2964873  PMID: 21037926
11.  Radiation Sensitivity and Tumor Susceptibility in ATM Phospho-Mutant ATF2 Mice 
Genes & cancer  2010;1(4):316-330.
The transcription factor ATF2 was previously shown to be an ATM substrate. Upon phosphorylation by ATM, ATF2 exhibits a transcription-independent function in the DNA damage response through localization to DNA repair foci and control of cell cycle arrest. To assess the physiological significance of this phosphorylation, we generated ATF2 mutant mice in which the ATM phosphoacceptor sites (S472/S480) were mutated (ATF2KI). ATF2KI mice are more sensitive to ionizing radiation (IR) than wild-type (ATF2 WT) mice: following IR, ATF2KI mice exhibited higher levels of apoptosis in the intestinal crypt cells and impaired hepatic steatosis. Molecular analysis identified impaired activation of the cell cycle regulatory protein p21Cip/Waf1 in cells and tissues of IR-treated ATF2KI mice, which was p53 independent. Analysis of tumor development in p53KO crossed with ATF2KI mice indicated a marked decrease in amount of time required for tumor development. Further, when subjected to two-stage skin carcinogenesis process, ATF2KI mice developed skin tumors faster and with higher incidence, which also progressed to the more malignant carcinomas, compared with the control mice. Using 3 mouse models, we establish the importance of ATF2 phosphorylation by ATM in the acute cellular response to DNA damage and maintenance of genomic stability.
doi:10.1177/1947601910370700
PMCID: PMC2926982  PMID: 20740050
ATF2; tumorigenesis; p53; radiation; DNA damage
12.  Preclinical Studies of Celastrol and Acetyl Isogambogic Acid in Melanoma 
Purpose
Sensitize melanomas to apoptosis and inhibit their growth and metastatic potential by compounds that mimic the activities of activating transcription factor 2 (ATF2)-driven peptides.
Experimental Design
Small-molecule chemical library consisting of 3,280 compounds was screened to identify compounds that elicit properties identified for ATF2 peptide, including (a) sensitization of melanoma cells to apoptosis, (b) inhibition of ATF2 transcriptional activity, (c) activation of c-Jun NH2-terminal kinase (JNK) and c-Jun transcriptional activity, and (d) inhibition of melanoma growth and metastasis in mouse models.
Results
Two compounds, celastrol (CSL) and acetyl isogambogic acid, could, within a low micromolar range, efficiently elicit cell death inmelanoma cells. Both compounds efficiently inhibit ATF2 transcriptional activities, activate JNK, and increase c-Jun transcriptional activities. Similar to the ATF2 peptide, both compounds require JNK activity for their ability to inhibit melanoma cell viability. Derivatives of CSL were identified as potent inducers of cell death in mouse and human melanomas. CSL and a derivative (CA19) could also efficiently inhibit growth of human and mouse melanoma tumors and reduce the number of lung metastases in syngeneic and xenograft mouse models.
Conclusions
These studies show for the first time the effect of CSL and acetyl isogambogic acid on melanoma. These compounds elicit activities that resemble the well-characterized ATF2 peptide and may therefore offer new approaches for the treatment of this tumor type.
doi:10.1158/1078-0432.CCR-07-1536
PMCID: PMC2874065  PMID: 18006779
13.  Understanding Signaling Cascades in Melanoma† 
Photochemistry and photobiology  2007;84(2):289-306.
Understanding regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now appreciated that melanoma is the result of complex changes in multiple signaling pathways that affect growth control, metabolism, motility and the ability to escape cell death programs. Here we review the major signaling pathways currently known to be deregulated in melanoma with an implication to its development and progression. Among these pathways are Ras, B-Raf, MEK, PTEN, phosphatidylinositol-3 kinase (PI3Ks) and Akt which are constitutively activated in a significant number of melanoma tumors, in most cases due to genomic change. Other pathways discussed in this review include the [Janus kinase/signal transducer and activator of transcription (JAK/STAT), transforming growth factor-β pathways which are also activated in melanoma, although the underlying mechanism is not yet clear. As a paradigm for remodeled signaling pathways, melanoma also offers a unique opportunity for targeted drug development.
doi:10.1111/j.1751-1097.2007.00254.x
PMCID: PMC2874067  PMID: 18086245
14.  BI-69A11-mediated inhibition of AKT leads to effective regression of xenograft melanoma 
Pigment cell & melanoma research  2009;22(2):187-195.
Summary
The AKT/PKB pathway plays a central role in tumor development and progression and is often up-regulated in different tumor types, including melanomas. We have recently reported on the in silico approach to identify putative inhibitors for AKT/PKB. Of the reported hits, we selected BI-69A11, a compound which was shown to inhibit AKT activity in in vitro kinase assays. Analysis of BI-69A11 was performed in melanoma cells, a tumor type that commonly exhibits up-regulation of AKT. Treatment of the UACC903 human melanoma cells, har-boring the PTEN mutation, with BI-69A11 caused efficient inhibition of AKT S473 phosphorylation with concomitant inhibition of AKT phosphorylation of PRAS40. Treatment of melanoma cells with BI-69A11 also reduced AKT protein expression, which coincided with inhibition of AKT association with HSP-90. BI-69A11 treatment not only caused cell death of melanoma, but also prostate tumor cell lines. Notably, the effect of BI-69A11 on cell death was more pronounced in cells that express an active form of AKT. Significantly, intra-peritoneal injection of BI-69A11 caused effective regression of melanoma tumor xenografts, which coincided with elevated levels of cell death. These findings identify BI-69A11 as a potent inhibitor of AKT that is capable of eliciting effective regression of xenograft melanoma tumors.
doi:10.1111/j.1755-148X.2009.00544.x
PMCID: PMC2860277  PMID: 19175524
AKT; melanoma; BI-69A11; HSP90; Pten; PI3K
15.  Radiation Sensitivity and Tumor Susceptibility in ATM Phospho-Mutant ATF2 Mice 
Genes & Cancer  2010;1(4):316-330.
The transcription factor ATF2 was previously shown to be an ATM substrate. Upon phosphorylation by ATM, ATF2 exhibits a transcription-independent function in the DNA damage response through localization to DNA repair foci and control of cell cycle arrest. To assess the physiological significance of this phosphorylation, we generated ATF2 mutant mice in which the ATM phosphoacceptor sites (S472/S480) were mutated (ATF2KI). ATF2KI mice are more sensitive to ionizing radiation (IR) than wild-type (ATF2 WT) mice: following IR, ATF2KI mice exhibited higher levels of apoptosis in the intestinal crypt cells and impaired hepatic steatosis. Molecular analysis identified impaired activation of the cell cycle regulatory protein p21Cip/Waf1 in cells and tissues of IR-treated ATF2KI mice, which was p53 independent. Analysis of tumor development in p53KO crossed with ATF2KI mice indicated a marked decrease in amount of time required for tumor development. Further, when subjected to two-stage skin carcinogenesis process, ATF2KI mice developed skin tumors faster and with higher incidence, which also progressed to the more malignant carcinomas, compared with the control mice. Using 3 mouse models, we establish the importance of ATF2 phosphorylation by ATM in the acute cellular response to DNA damage and maintenance of genomic stability.
doi:10.1177/1947601910370700
PMCID: PMC2926982  PMID: 20740050
ATF2; tumorigenesis; p53; radiation; DNA damage
16.  JAMP Optimizes ERAD to Protect Cells from Unfolded Proteins 
Molecular Biology of the Cell  2008;19(11):5019-5028.
Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER.
doi:10.1091/mbc.E08-08-0839
PMCID: PMC2575167  PMID: 18784250
17.  Requirements for PKC-augmented JNK activation by MKK4/7 
The c-Jun N-terminal kinases (JNKs) are activated in response to stress, DNA damage, and cytokines by MKK4 and MKK7. We recently demonstrated that PKC can augment the degree of JNK activation by phosphorylating JNK, which requires the adaptor protein RACK1. Here we report on the conditions required for PKC-dependent JNK activation. In vitro kinase assays reveal that PKC phosphorylation of JNK is not sufficient for its activation but rather augments JNK activation by canonical JNK upstream kinases MKK4 or MKK7 alone or in combination. Further, to enhance JNK activity, PKC phosphorylation of JNK should precede its phosphorylation by MKK4/7. Inhibition of PKC phosphorylation of JNK affects both early and late phases of JNK activation following UV-irradiation and reduces the apoptotic response mediated by JNK. These data provide important insight into the requirements for PKC activation of JNK signaling.
doi:10.1016/j.biocel.2007.11.011
PMCID: PMC2327215  PMID: 18182317
JNK; MKK4; MKK7; PKC; RACK1
18.  The LIM domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans 
The Journal of Cell Biology  2004;165(6):857-867.
Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/β-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle–muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.
doi:10.1083/jcb.200401133
PMCID: PMC2172400  PMID: 15210732
UNC-95; RNF-5; LIM; RING; E3 ligase
19.  An ATF2-derived peptide sensitizes melanomas to apoptosis and inhibits their growth and metastasis 
Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high capacity to metastasize. ATF2 is among transcription factors implicated in the progression of melanoma and its resistance to treatment. Here we demonstrate that the expression of a peptide spanning amino acids 50–100 of ATF2 (ATF250–100) reduces ATF2 transcriptional activities while increasing the expression and activity of c-Jun. Altering the balance of Jun/ATF2 transcriptional activities sensitized melanoma cells to apoptosis, an effect that could be attenuated by inhibiting c-Jun. Inhibition of ATF2 via RNA interference likewise increased c-Jun expression and primed melanoma cells to undergo apoptosis. Growth and metastasis of SW1 and B16F10 mouse melanomas were inhibited by ATF250–100 to varying degrees up to a complete regression, depending on the mode (inducible, constitutive, or adenoviral delivery) of its expression.
doi:10.1172/JCI16081
PMCID: PMC151112  PMID: 12208865
20.  Synthesis and SAR studies of dual AKT/NF-κB inhibitors against melanoma 
Chemical biology & drug design  2013;82(5):520-533.
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.
doi:10.1111/cbdd.12177
PMCID: PMC3966491  PMID: 23790042
21.  Regulators and effectors of Siah ubiquitin ligases 
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 Siah2.
doi:10.1007/s12013-013-9636-2
PMCID: PMC3758783  PMID: 23700162
22.  Emerging roles of E3 ubiquitin ligases in autophagy 
Trends in biochemical sciences  2013;38(9):453-460.
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 the current understanding of the role of ubiquitylation in the control of autophagy.
doi:10.1016/j.tibs.2013.06.008
PMCID: PMC3771342  PMID: 23870665
Ubiquitin ligase; Proteasome; Autophagy; Parkin; RNF5
23.  Structure Based Design of Covalent Siah Inhibitors 
Chemistry & biology  2013;20(8):973-982.
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 novel 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 cell. The strategy proposed may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions.
doi:10.1016/j.chembiol.2013.06.008
PMCID: PMC3763817  PMID: 23891150
24.  Translational Homeostasis via the mRNA Cap-Binding Protein, eIF4E 
Molecular cell  2012;46(6):847-858.
Summary
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.
doi:10.1016/j.molcel.2012.04.004
PMCID: PMC4085128  PMID: 22578813
25.  Fine Tuning of the UPR by the Ubiquitin Ligases Siah1/2 
PLoS Genetics  2014;10(5):e1004348.
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.
Author Summary
Maintaining a balanced level of stress (protein folding, reactive oxygen radicals) is important for keeping cellular homeostasis (the ability of a cell to maintain internal equilibrium by adjusting its physiological processes). The accumulation of stress (external or internal) will trigger a well-orchestrated machinery that attempts to restore homeostasis, namely, the unfolded protein response (UPR). The UPR either restores balance to the cells or induces a cell death program, which clears the damaged cell. How this machinery activates cell survival versus cell death is not entirely clear. Here we identify a new layer in the regulation of the UPR, which determines the magnitude of this response. We demonstrate the importance of this newly identified regulatory component for cell death commitments, in response to the more severe conditions (ischemia, lack of oxygen and nutrients). Our findings highlight an undisclosed mechanism that is important for the cell death decision following severe stress conditions, while pointing to the ability to fine tune cellular response to stress.
doi:10.1371/journal.pgen.1004348
PMCID: PMC4014425  PMID: 24809345

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