Begell House Inc.
Onco Therapeutics
OT
2694-4642
2
1
2011
FIRST INTERNATIONAL WORKSHOP ON "PROGNOSTIC AND THERAPEUTIC APPLICATIONS OF RKIP IN CANCER"Held in los angeles, caliFornia, March 19−20, 2010GUEST EDITORS: BENJAMIN BONAVIDA, FAHD AL-MULLA, & KAM C. YEUNG
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10.1615/ForumImmunDisTher.v2.i1.10
The first International Workshop on "Prognostic and Therapeutic Applications of RKIP in Cancer" was held at the University of California at Los Angeles, March 19−20, 2010. This workshop was focused on the current status of the various functions assigned to RKIP and its pivotal role in the regulation of cancer cells’ survival and response to therapies. The workshop was attended by national and international experts. The topics presented included the role of RKIP in the regulation of cell survival, cell proliferation, cell response to growth factors, metastasis, and sensitivity to chemotherapy and immunotherapy, as well as its prognostic significance and its role as a new target for therapeutic intervention. The proceedings of this workshop are published in volume 2. This volume is the first to publish manuscripts and reviews on RKIP and serves as an important reference for scientists, clinicians, and students.
Phosphatidylenthanolamine Binding Protein aka Raf Kinase Inhibitor Protein: A Brief History of Its Discovery and the Remarkable Diversity of Biological Functions
1-12
10.1615/ForumImmunDisTher.v2.i1.20
John M.
Sedivy
Department of Molecular Biology, Cell Biology and Biochemistry, Laboratories for Molecular Medicine, Brown University, USA
cancer
signal transduction
metastasis
tumor suppression
phosphorylation
apoptosis
MAP kinase pathway
NF-kB pathway
G protein–coupled receptor pathway
Phosphatidylethanolamine-binding protein (PEBP) was identified almost three decades ago as an abundant protein in bovine brain. PEBP is the prototype of a highly conserved family of proteins represented in all three major phylogenetic divisions, eukaryota, bacteria, and archaea, with no significant sequence homology to other proteins. PEBP proteins have been studied in many species. The most thoroughly explored biological role of PEBP is that of a modulator of intracellular signaling pathways, which is mediated by its ability to bind and inhibit a number of protein kinases. The first such interaction that came to light was with the Raf1 kinase, and PEBP is thus widely referred to in the literature under its alternate name RKIP (Raf kinase inhibitory protein). The activity of RKIP itself is subject to regulation by phosphorylation. Intriguingly, PEBP has also been reported to possess additional, and diverse, biological functions unrelated to protein kinase networks that remain to be investigated in detail. Recent findings that RKIP may function as a suppressor of cancer metastasis are of great interest and importance. Prognostic and therapeutic applications of RKIP in human cancer were the subject of the first international workshop on RKIP that was held at the University of California, Los Angeles, in March 2010. This paper was presented at the workshop as a summary of the history of this still small but rapidly evolving field.
Raf Kinase Inhibitory Protein: A Signal Transduction Modulator and Metastasis Suppressor
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10.1615/ForumImmunDisTher.v2.i1.30
Eva M.
Eves
Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois, USA
Marsha Rich
Rosner
Ben May Department for Cancer Research, University of Chicago, Gordon Center for Integrative Sciences, Chicago, Illinois
Raf kinase inhibitory protein (RKIP)
signal transduction
metastasis suppressor
modulator
genomic stability
Cells have a multitude of controls to maintain their integrity and prevent random switching from one biological state to another. Raf Kinase Inhibitory protein (RKIP or PEBP1), a member of the phosphatidylethanolamine-binding protein family, is representative of a new class of modulators of signaling cascades that functions to maintain the balance of biological systems. RKIP inhibits MAP kinase (Raf-MEK-ERK), G protein–coupled receptor (GPCR), and NF-κB-signaling cascades. RKIP targets different kinases dependent on its phosphorylation state, and integrates cross talk initiated by multiple environmental stimuli. We have shown that RKIP is a unique kinase inhibitor and substrate that uses a flexible pocket to integrate ligand-binding and phosphorylation-dependent interactions to modulate the MAPK signaling pathway. This mechanism is likely conserved among RKIP homologues in eukaryotes. RKIP also functions as a suppressor of metastasis. We have identified two mechanisms leading to altered cellular signaling and potentiation of tumorigenesis and metastasis. First, loss or depletion of RKIP results in chromosomal abnormalities and genomic instability via disregulation of the spindle checkpoint. Second, RKIP inhibits a signaling cascade involving MAPK, Myc, LIN28, let-7, and downstream let-7 targets that promotes invasion and metastasis of breast cancer cells. Our results highlight the importance of RKIP as a key metastasis suppressor and potential therapeutic agent.
Dual Role of RKIP in NF-κB Signaling Pathways
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10.1615/ForumImmunDisTher.v2.i1.40
Kam C.
Yeung
Department of Biochemistry & Cancer Biology, College of Medicine, University of Toledo, Toledo, Ohio
Sungdae
Park
Department of Biochemistry & Cancer Biology, College of Medicine, University of Toledo, Health Science Campus, Toledo, Ohio, USA
Huihui
Tang
Department of Biochemistry & Cancer Biology, College of Medicine, University of Toledo, Health Science Campus, Toledo, Ohio, USA
RKIP
NF-kB
scaffold protein
protein ubiquitination
auto-regulatory feedback loop
Raf kinase inhibitor protein (RKIP) was first identified as an inhibitor of Raf-1 kinase in the Raf-MEK-ERK signaling pathway. It inhibits the activation phosphorylation of MEK by Raf-1 by competing with MEK for Raf-1 binding. RKIP was also found to play an important role in regulating the NF-κB pathway. Our studies showed that RKIP interacted with multiple components of both canonical and non-canonical pathways. Genetic and biochemical studies demonstrated that RKIP functioned as a scaffold protein facilitating the phos-
phorylation of IkκB and NF-κB2/p100 by upstream kinases. However, contrary to what one would expect of a scaffold protein, our results show that RKIP has an overall inhibitory effect on NF-κB transcriptional activities.
The regulation of the expression of NF-κB target genes is subject to negative regulation involving the optimal induction of negative regulators, including IkB and p100. Our data thus support a hypothesis that RKIP inhibits the NF-κB activity via an auto-regulatory feedback loop by rapidly inducing the expression and synthesis of inhibitors of NF-κB activation. This mode of action is, therefore, very different from the mechanisms by which RKIP inhibits the Raf-MEK-ERK pathway.
Inhibition of STAT3 Activation by RKIP in Colon Cancer
35-45
10.1615/ForumImmunDisTher.v2.i1.50
Devasis
Chatterjee
Rhode Island Hospital and The Alpert Medical School of Brown University, Providence, Rhode Island
Erika L.
Moen
Department of Medicine, Rhode Island Hospital and The Alpert Medical School of Brown University, Providence, Rhode Island, USA
Y. Eugene
Chin
Department of Surgery, Rhode Island Hospital and The Alpert Medical School of Brown University, Providence, Rhode Island, USA
RKIP
STAT3
colon cancer
Colorectal cancer is the fourth leading cause of cancer-related deaths worldwide. Combination chemotherapy, including camptothecin (CPT) derivatives, is considered one of the standard first-line treatments. We found that in CPT-sensitive colon cancer cell lines, Raf kinase inhibitor protein (RKIP) was robustly induced following CPT treatment. Furthermore, ectopic RKIP is able to reverse resistance to CPT in human tumor cell lines thus making RKIP an important determinant of CPT sensitivity. It has been shown that elevated RKIP expression inhibits a number of cell-signaling pathways, including signal transducer and activator of transcription3 (STAT3). Given that STAT3 activation leads to metastasis in colon cancer and suppression of STAT3 signaling leads to the demise of tumor cells, a close relationship between uncontrolled STAT3 activation and the cancerous phenotype has been established. In colorectal cancer, RKIP expression is reduced in lymph node metastases, suggesting that high RKIP expression may prevent patient disease progression. Our results indicate that over-expression of RKIP inhibits interleukin-6 (IL-6)-, JAK-, and c-Src-mediated STAT3 activation and phosphorylation. From these data, we hypothesize that overexpression of RKIP may be predictive of disease outcome and provide a useful target for therapeutic intervention. Specifically, we predict that elevated RKIP levels, through the inhibition of STAT3 activation, decreases the potential for metastasis of human colon cancer.
Locostatin Disrupts Association of Raf Kinase Inhibitor Protein With Binding Proteins by Modifying a Conserved Histidine Residue in the Ligand-Binding Pocket
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10.1615/ForumImmunDisTher.v2.i1.60
Anwar B.
Beshir
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
Christian E.
Argueta
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
Lochana C.
Menikarachchi
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
Jose A.
Gascon
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
Gabriel
Fenteany
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
RKIP
locostatin
mechanism of action
binding
Raf-1
GRK2
IKKa
TAK1
Raf kinase inhibitor protein (RKIP) interacts with a number of different proteins and regulates multiple signaling pathways. Here, we show that locostatin, a small molecule that covalently binds RKIP, not only disrupts interactions of RKIP with Raf-1 kinase, but also with G protein-coupled receptor kinase 2. In contrast, we found that locostatin does not disrupt binding of RKIP to two other proteins: inhibitor of κB kinase α and transforming growth factor β-activated kinase 1. These results thus imply that different proteins interact with different regions of RKIP. Locostatin’s mechanism of action involves modification of a nucleophilic residue on RKIP. We observed that after binding RKIP, part of locostatin is slowly hydrolyzed, leaving a smaller RKIP-butyrate adduct. We identified the residue alkylated by locostatin as His86, a highly conserved residue in RKIP’s ligand-binding pocket. Computational modeling of the binding of locostatin to RKIP suggested that the recognition interaction between small molecule and protein ensures that locostatin’s electrophilic site is poised to react with His86. Furthermore, binding of locostatin would sterically hinder binding of other ligands in the pocket. These data provide a basis for understanding how locostatin disrupts particular interactions of RKIP with RKIP-binding proteins and demonstrate its utility as a probe of specific RKIP interactions and functions.
Phosphotyrosine Recognition by the Raf Kinase Inhibitor Protein
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10.1615/ForumImmunDisTher.v2.i1.70
Philip C.
Simister
Department of Biochemistry, University of Bristol, Bristol, United Kingdom; and Cell Signalling Group, Cancer Research UK, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK
Nicholas M.
Burton
Department of Biochemistry, University of Bristol, Bristol, United Kingdom
R. Leo
Brady
Department of Biochemistry, University of Bristol, Bristol, United Kingdom
PEBP
phosphotyrosine
RKIP
X-ray crystallography
Raf-1
c-Raf
Raf kinase inhibitor protein (RKIP) negatively regulates the mitogen-activated protein kinase (MAPK) signaling cascade by direct interaction with Raf-1. RKIP belongs to a family of proteins for which a wide range of structures have now been described; all are highly homologous and share a distinctive anion-binding pocket. Using X-ray crystallography, we show that phosphotyrosine can be neatly accommodated within this pocket, and suggest that RKIP proteins may therefore form a novel phosphotyrosine recognition motif. As studies have previously indicated that phosphorylation of the S338SYY341 region of Raf-1 enhances binding to RKIP leading to suppression of MEK activation, we also propose a model of the Raf-1 DS338SYpY341W motif bound to RKIP based on the RKIP-pTyr crystal structure. Consistent with reported experimental data, this model suggests phosphorylation of Ser338 may additionally stabilize this complex. Together, these results imply a mechanism for RKIP/Raf-1 interaction in which RKIP regulates Raf-1 activity via direct steric inhibition of the Tyr341 region.
RKIP Enhances Angiotensin II - Stimulated Signaling
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10.1615/ForumImmunDisTher.v2.i1.80
Yasser
el Faramawy
Molecular Pharmacology Unit, Swiss Federal Institute of Technology, and University of Zurich, Zurich, Switzerland
Xuebin
Fu
Molecular Pharmacology Unit, Swiss Federal Institute of Technology, and University of Zurich, Zurich, Switzerland
Sara H.
Agwa
Medical Research Center (MRC), Ain Shams University Hospital, Cairo, Egypt
Andreas
Langer
Molecular Pharmacology Unit, Swiss Federal Institute of Technology, and University of Zurich, Zurich, Switzerland
Sherif S.
Elzahwy
Medical Research Center (MRC), Ain Shams University Hospital, Cairo, Egypt
Ursula
Quitterer
Molecular Pharmacology Unit, Swiss Federal Institute of Technology, and University of Zurich, Zurich, Switzerland
RKIP
PEBP
raf kinase
GRK2
betaARK1
angiotensin II AT1 receptor
G protein–coupled receptor
cardiomyocytes
Protein kinase C–mediated phosphorylation converts the raf kinase inhibitor protein (RKIP) into an inhibitor of the G protein–coupled receptor kinase 2 (GRK2). As a result of GRK2 inhibition, RKIP prevents receptor desensitization and enhances signaling stimulated by the classical substrate of GRK2, i.e., the Gs-coupled β-adrenergic receptor. The Gq/11-coupled angiotensin II AT1 receptor is another prototypic substrate of GRK2 in the cardiovascular system. However, the role of RKIP in Gq/11-coupled receptor signaling is not clear. Here, we show that overexpression of RKIP in kidney cells led to a significant increase of the AT1-stimulated calcium signal. In contrast, a phosphorylation-deficient mutant of RKIP that cannot act as GRK2 inhibitor had no effect. Analogously to the signal sensitization of kidney cells, RKIP increased the angiotensin II–stimulated hypertrophic response of cardiomyocytes. RNA interference studies revealed that endogenous RKIP levels of kidney cells and cardiomyocytes were sufficient to produce signal enhancement of AT1. Thus, RKIP acts as a physiological enhancer of angiotensin II–stimulated signaling in kidney cells and cardiomyocytes.
RKIP-1 and the Cell Cycle: Novel Insight Into Genomic Instability
79-87
10.1615/ForumImmunDisTher.v2.i1.90
Fahd
Al-Mulla
Faculty of Medicine, Department of Pathology, Kuwait University Health Sciences Centre, Safat, Kuwait
RKIP-1
PEBP
cancer
cell cycle
genetic instability
homozygosity
Aurora
NEK 6
HEK-293
RKIP-1 has emerged as a significant metastasis suppressor in various cancers. Loss or reduced RKIP-1 expression has been well documented in aggressive and malignant cancers. In addition, recent data have stipulated a potential prognostication utility for the RKIP-1 expression level in colorectal, prostate, and gastrointestinal stromal tumors. This repressor and scaffold protein appear to be involved in a wide variety of intracellular signaling cascades ranging from the inhibition of Raf-1/MEK/ERK to the activation of the G protein–coupled receptor signaling pathway. However, its role in cellular growth, the cell cycle, and genomic instability has received little attention, especially in primary and immortalized (noncancerous) cells. We demonstrated that RKIP-1 depletion in HEK-293 cells caused a dramatic fast-forwarding or acceleration in the cell cycle at the S- and G2M phases, which may be a consequence to alteration in key cell cycle checkpoint proteins, such as Aurora B and NEK 6. Our data also demonstrate an increased trend for homozygosity accumulation after long-term culture of RKIP-1–depleted HEK-293 cells. This novel finding might have important ramifications to cancer development, particularly in the inactivation of tumor suppressor genes.
Role of Raf Kinase Inhibitor Protein in Pathophysiology of Prostate Cancer
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10.1615/ForumImmunDisTher.v2.i1.100
Evan T.
Keller
Department of Urology, School of Medicine, University of Michigan, 1500 E. Medical Center Dr., Room 5308 CCGCB, Ann Arbor, MI 48105, USA; Center for Translational Medicine, Guangxi Medical University, Nanning, China
protein kinase
metastasis
ERK
MAP kinase
Raf kinase inhibitor protein (RKIP) is a small, cytosolic protein named for its ability to block Raf-mediated activation of MAPK and ERK. It also block G-protein signaling and NF-κB activation. An in vitro screen to identify genes that regulate prostate cancer (PCa) metastasis revealed that expression of RKIP was decreased in high versus low metastatic PCa cells. Modulation of RKIP expression revealed that it inhibited invasion and loss of RKIP promoted in vitro invasion. Animal studies were used to demonstrate that RKIP could inhibit PCa metastasis from orthotopically injected tumor cells without an effect on primary tumor growth. Taken together, these results indicated RKIP acted as a PCa metastasis suppressor gene. Evaluation of RKIP expression in clinical cases of PCa revealed that RKIP expression was moderate to high in non-neoplastic prostate, low in 50% of primary prostate cancers, and absent to low in the majority of metastases. Furthermore, low RKIP expression in primary prostate tumors was predictive of early tumor recurrence. Loss of RKIP was shown to induce resistance to radiation in PCa cells in vitro and in an in vivo murine model. Taken together, these studies indicate that RKIP plays multiple roles in PCa pathophysiology, suggesting that a method to increase RKIP expression in PCa may have therapeutic benefits.
Dual Roles of Raf-1 Kinase Inhibitor Protein in the Regulation of Both Tumor Cell Resistance to Apoptotic Stimuli and Epithelial to Mesenchymal Transition
95-109
10.1615/ForumImmunDisTher.v2.i1.110
Stavroula
Baritaki
Center for Systems Biomedicine, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA
Kam C.
Yeung
Department of Biochemistry & Cancer Biology, College of Medicine, University of Toledo, Toledo, Ohio
Benjamin
Bonavida
Department of Microbiology, Immunology, &
Molecular Genetics, David Geffen School of Medicine at UCLA, Johnson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, CA 90025-1747, USA
apoptosis
epithelial to mesenchymal transition (EMT)
NF-κB
Raf-1 inhibitory protein (RKIP)
Snail
Most tumors develop several strategies to resist conventional chemotherapy as well as to escape from the host immunosurveillance mechanisms. These result in tumor persistence and spread, through activation of the epithelial to mesenchymal transition (EMT) program. The molecular bases of tumor resistance to apoptosis and EMT are thought to be due, in part, to the constitutive activation of survival signaling pathways including NF-κB. RKIP is a member of the PEBP family and it has been implicated in the negative regulation of the NF-κB and MAPK survival pathways. RKIP expression has been found significantly diminished in several tumors and almost absent in metastatic tissues, thus suggesting that RKIP may serve as a metastasis suppressor gene product. We have reported that RKIP overexpression reverses the tumor cell resistance to both chemotherapy and immunotherapy, as well as inhibits the mesenchymal cell phenotype (EMT) in metastatic tumor cell lines. In contrast, RKIP silencing by RKIP siRNA-inhibited TRAIL- or drug-induced apoptosis and induced EMT. RKIP overexpression was paralleled with upregulation of the TRAIL and Fas receptors DR5 and Fas, respectively, through inhibition of their transcriptional repressor Yin Yang 1 (YY1). YY1 siRNA resensitized the cells to TRAIL/FasL and drug apoptosis. RKIP transcription is directly repressed by Snail, a transcriptional target of NF-κB that serves as an EMT inducer through repression of E-cadherin. RKIP overexpression reduced Snail expression through NF-κB inhibition, while Snail silencing reversed both tumor resistance and EMT. Novel therapeutics such as the proteasome inhibitor NPI-0052 and the NO donor DETANONOate upregulated RKIP expression and led to tumor cell resensitization to TRAIL and inhibition of EMT. These findings demonstrate that RKIP regulates tumor cell sensitivity to apoptotic stimuli, and inhibits EMT via modulation of the dysregulated NF-κB/YY1/Snail circuitry. We propose that RKIP is a therapeutic target for intervention in the treatment of resistant and metastatic cancers.