Raf Kinase Inhibitor Protein Research Articles

Etoh-Induced Sensitization of CCK Signaling in Pancreatic Acinar Cells: Role of Raf Kinase Inhibitory Protein (RKIP)

Etoh-Induced Sensitization of CCK Signaling in Pancreatic Acinar Cells: Role of Raf Kinase Inhibitory Protein (RKIP)

Abstract 2162: Inhibition of the metastasis suppressor RKIP by EZH2 and YY1

Abstract The Raf kinase inhibitory protein (RKIP) acts as an activator of apoptosis and inhibitor of metastasis in prostate cancer. Diminished expression of RKIP has been reported in several cancers including prostate carcinomas; however, the underlying mechanism of RKIP regulation remains largely unknown. RKIP transcription is inhibited by the EMT inducer Snail which also represses the expression of the metastasis suppressor E-cadherin. E-cadherin and RKIP are co-expressed in many tumors. E-cadherin transcription is also suppressed by the polycomp repressive complex (PRP) transcription repressor, enhancer of zeste homolog 2 (EZH2), which mediates an H3 lysine 27 trimethylation in target promoters. EZH2 is highly expressed in several metastatic tumors including prostate cancer and its levels are inversely correlated with E-cadherin and RKIP expressions. The zinc-finger transcription factor Yin-Yang 1 (YY1) is also overexpressed in prostate tumors and has been associated with tumor progression. Silencing of YY1 by siRNA reverses the EMT phenotype of metastatic prostate cancer cell lines by induction of E-cadherin and RKIP. Here, we explored the direct roles of YY1 and EZH2 in the transcriptional regulation of RKIP in prostate cancer. We used as experimental models the metastatic and non-metastatic prostate cancer cell lines DU145 and LNCaP, respectively, which differ in the expression levels of EZH2, YY1 and RKIP. DU145 cells express low RKIP and high EZH2 and YY1 levels, whereas LNCaP cells express the opposite. Putative binding sites were identified for both YY1 and EZH2 (proximal E-boxes) within the RKIP promoter. CHiP analyses showed that both factors can physically associate with the RKIP promoter. Loss and gain of function reporter approaches showed that ectopic expression of EZH2 in LNCaP cells inhibits the wild type, but not the E-box mutant RKIP basal promoter activity. Similarly, a mutated EZH2 vector that lacks efficient binding to the target promoter couldn't inhibit the RKIP promoter activity. EZH2-mediated inhibition of RKIP promoter activity could also be inhibited by HDAC inhibitors, suggesting that HDAC recruitment and deacetylation of histone H3 is perquisite for EZH2 suppressive activity. Silencing of EZH2 or YY1 in DU145 also resulted in induction of RKIP promoter activity. The negative effect of YY1 on RKIP promoter activity was corroborated by inhibition of RKIP mRNA and protein levels. The above observations were further validated in prostate cancer tissue microarrays whereby YY1 and RKIP protein expressions were found to be inversely correlated. The present findings support the roles of EZH2 and YY1 as transcription repressors of RKIP in prostate cancer and suggest that their overexpression may be associated, in part, with the high cell metastatic potential through downregulation of RKIP. We propose that EZH2 and YY1 might be potential prognostic markers of prostate cancer progression and targets for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2162. doi:10.1158/1538-7445.AM2011-2162

Abstract 1457: Raf kinase inhibitor protein (RKIP) negatively regulates melanoma differentiation associated gene-9/syntenin-mediated melanoma metastasis

Abstract Melanoma differentiation associated gene-9 (mda-9/syntenin) functions as a positive regulator of melanoma progression and metastasis by physically interacting with c-Src and promoting the formation of an active FAK/c-Src signaling complex leading to NFκB and matrix metalloproteinase (MMP) activation. In contrast, Raf Kinase Inhibitor Protein (RKIP), an inhibitor of RAF-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEKK), is strongly downregulated in metastatic melanoma cells suggesting a possible inverse relationship between expression of mda-9/syntenin and RKIP. We have now genetically modulated expression of mda-9/syntenin and RKIP in melanocytes and melanoma cells and evaluated biological consequences using invasion, anchorage-independent growth, Western blotting, immunoprecipitation and in vivo confocal imaging assays. These studies confirm an inverse correlation between mda-9/syntenin and RKIP expression. This reciprocal relationship was also documented in vivo using a chick embryo chorioallantoic membrane (CAM) model, which monitors spontaneous intravasation of cells from the upper to the lower layer by polymerase chain reaction (PCR) amplification of human Alu genomic DNA. Using tissue microarrays and a series of cell lines we confirmed a higher expression of MDA-9/syntenin and absence of RKIP in both malignant melanoma tumor sections and cell lines. We also established through gain-of-function and loss-of-function experiments that mda-9/syntenin transcriptionally downregulates RKIP, providing evidence for crosstalk between these two proteins. Moreover, transient overexpression of RKIP in metastatic melanoma cells overrides mda-9/syntenin-mediated signaling resulting in decreased cell invasion, anchorage-independent growth and spontaneous tumor cell dissemination in the CAM assay. RKIP through its RAF1 binding site (amino acids 77-108) physically interacts with MDA-9/syntenin and this interaction inversely correlates with downregulation of FAK and c-Src phosphorylation, crucial steps necessary for forming a stable FAK/c-Src complex and initiating a signaling cascade leading to an MDA-9/syntenin-mediated metastatic phenotype in melanoma cells. These provocative findings highlight a potential therapeutic role of RKIP in melanoma metastasis through its interaction with MDA-9/syntenin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1457. doi:10.1158/1538-7445.AM2011-1457

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The expression of RKIP, RhoGDI, galectin, c‐Myc and p53 in gastrointestinal system of Cr(VI)‐exposed rats

Hexavalent chromium (CrVI) is considered to be a risk factor in the formation of human cancer. Raf kinase inhibitor protein (RKIP), Rho-GDIα, galectin, c-Myc and p53 play important roles in cancer formation. The purpose of this study was to determine if Cr(VI) induces the formation of gastrointestinal cancer. We explored the expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 in the colon and stomach in rats exposed to chromium (CrVI). Thirty Wistar rats were divided into six groups which were chronically fed with 250, 500, 750, 1000 and 1250 ppm Na(2) Cr(2) O(7) and water for 60 days. The level of Cr(VI) was determined by electrothermal atomic absorption spectrometry. The expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 of stomach and colon was measured by western blot. The gene expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 of the stomach and colon was determined by RT-PCR. The results showed that the expression of p53 and Rho-GDIα was decreased in the stomach and colon of rats with Cr(VI) treatment. The expression of RKIP was decreased in the stomach and colon of rats treated with high-dose Cr(VI). The expression of c-Myc and gelectin-1 was increased in the stomach and colon of rats with Cr(VI) treatment. We concluded that the anomalous expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 might be a dangerous index of cancer formation in the stomach and colon of rats with Cr(VI) exposure.

The expression and significance of RKIP in lung squamous cell carcinoma tissues

背景与目的Raf激酶抑制蛋白（Raf kinase inhibitor protein, RKIP）属于磷脂酰乙醇胺结合蛋白家族的成员，可干扰Raf-1-MEK1/2-ERK1/2信号通路，抑制NF-κB和G蛋白偶联受体激酶等信号传导过程，且RKIP的表达减弱或丢失与多种肿瘤的发生发展以及侵润转移相关。本研究旨在探讨RKIP在肺鳞癌组织中的表达及其与肺鳞癌临床病理的相关性。方法应用RT-PCR方法检测56例肺鳞癌组织及其癌旁正常组织中RKIP基因的表达，计算其阳性率；并用Western blot方法检测肺鳞癌组织及其癌旁正常组织中RKIP蛋白的表达变化。结果RKIP mRNA在肺鳞癌组织中表达的阳性率明显低于癌旁正常组织（P < 0.05），在有淋巴结转移者中的表达阳性率明显低于无淋巴结转移者（P < 0.05），但与患者的性别、年龄、肿瘤的大小及分化程度无明显关系（P>0.05）。RKIP蛋白在肺鳞癌组织中表达明显低于癌旁正常组织（P < 0.05）。结论RKIP的低表达可能与肺鳞癌的发生及侵袭转移有关。

Cilia movement regulates expression of the Raf-1 kinase inhibitor protein.

Renal epithelial cell primary cilia act as mechanosensors in response to changes in luminal fluid flow. To determine the role of cilia bending in the mechanosensory function of cilia, we performed proteomic analysis of collecting duct cell lines with or without cilia that were kept stationary or rotated to stimulate cilia bending. Expression of the Raf-1 kinase inhibitor protein (RKIP), an inhibitor of the MAPK pathway, was significantly elevated in rotated cilia (+) cells. This was compared with RKIP levels in cilia (-) cells that were stationary or rotated as well as in cilia (+) cells that were stationary. This result was confirmed in cilia knockout adult mice that had lower renal RKIP levels compared with adult mice with cilia. Downstream of RKIP, expression of phosphorylated ERK was decreased only in cells that had cilia and were subjected to constant cilia bending. Furthermore, elevated RKIP levels were associated with reduced cell proliferation. Blockade of PKC abrogated ciliary bending-induced increases in RKIP. In summary, we found that ciliary movement may help control the expression of the Raf-1 kinase inhibitor protein and thus maintain cell differentiation. In terms of polycystic kidney disease, loss of cilia and therefore sensitivity to flow may lead to reduced RKIP levels, activation of the MAPK pathway, and contribute to the formation of cysts.

Frequent Alteration of the Yin Yang 1/Raf-1 Kinase Inhibitory Protein Ratio in Hepatocellular Carcinoma

The transcription factor Yin Yang 1 (YY1) can favor several aspects of tumorigenesis. In turn, Raf-1 Kinase Inhibitor Protein (RKIP) inhibits the oncogenic activities of MAPK and NF-κB pathways and promotes drug-induced apoptosis. Mutual influences between YY1 and RKIP may exist, and there are already separate evidences that relevant increases in YY1 and reductions in RKIP occur in hepatocellular carcinoma (HCC). However, the levels of the two factors have never been concomitantly examined in HCC. We evaluated by RT-PCR the mRNA levels of YY1, YY1AP, RKIP, and survivin in 35 clinical HCCs (91% HCV-related), in their adjacent cirrhotic tissues and in 6 healthy livers. Immunohistochemical analyses were also performed. The ratio of YY1 to RKIP mRNA was constantly profoundly inverted in the tumors compared with the adjacent nontumoral tissues. A similar result occurred frequently at protein level. Hyperactivation of YY1 in tumors was corroborated by its nuclear localization and the finding that in the tumors there were also increases in YY1AP, a YY1 coactivator not expressed in normal liver, and in survivin, as a possible target of YY1. The frequent alteration in the YY1-RKIP balance might represent a marker of malignant progression and be exploited for therapeutic interventions in HCC.

Immunohistochemical Detection of Raf Kinase Inhibitor Protein in Normal Cervical Tissue and Cervical Cancer Tissue

Raf kinase inhibitor protein (RKIP) may be a suppressor of metastasis: RKIP levels are high in normal tissues, low in primary cancers and lowest or absent in metastatic cancers. This immunohisto chemistry study investigated RKIP protein levels in 250 clinical specimens of human cervical tissue and lymph node metastases (LNM) from 210 patients with normal cervical tissue, cervical intra-epithelial neoplasia (CIN), or cervical cancer with/without LNM. Thirty-nine (86.7%) of the 45 normal-tissue samples were RKIP-positive, six (13.3%) were RKIP-negative; 48/60 (80.0%) CIN samples were positive, 12 (20.0%) were negative; 47/105 (44.8%) cervical cancer tissue samples were positive, 58 (55.2%) were negative; only 7/40 (17.5%) LNM tissue samples were positive, 33 (82.5%) were negative. There was no significant correlation between RKIP positivity and clinical stage, microscopic subtype or pathological differentiation grade. RKIP positivity correlated inversely with LNM. RKIP may play a role in cervical-cancer genesis and metastasis; RKIP down-regulation was associated with metastatic disease in human cervical cancer.

Raf Kinase Inhibitory Protein (RKIP) Mediates EtOH-induced Sensitization of Secretagogue Signaling in Pancreatic Acinar Cells

Raf Kinase Inhibitory Protein (RKIP) Mediates EtOH-induced Sensitization of Secretagogue Signaling in Pancreatic Acinar Cells

Locostatin Disrupts Association of Raf Kinase Inhibitor Protein With Binding Proteins by Modifying a Conserved Histidine Residue in the Ligand-Binding Pocket

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.

RAF kinase inhibitory protein (RKIP) modulates cell cycle kinetics and motility

RKIP-1 is a metastasis suppressor that is frequently downregulated in aggressive cancers. However, the consequences of RKIP loss in primary or immortalized cells have not yet been explored. Using HEK-293 RKIP depleted (termed HEK-499) and Flp-In T-Rex-293 RKIP inducible cell lines combined with whole transcriptome analysis, we show that RKIP-1 silencing accelerates DNA synthesis and G1/S transition entry by inducing the expression of cdc6, MCM 2, 4, 6, 7, cdc45L, cyclin D2, cyclin E2, cyclin D1, SKP2 and the downregulation of p21(cip1). Moreover, RKIP depletion accelerates the time from nuclear envelop breakdown (NEB) to anaphase markedly, while the upregulation of RKIP shortened the NEB to anaphase time. We show that RKIP depletion induces the expression of NEK6, a molecule known to enhance G2/M transition, and down-regulates G2/M checkpoint molecules like Aurora B, cyclin G1 and sertuin that slow the G2/M transition time. These subtle changes in the kinetics of the cell cycle culminate in a higher proliferation rate of HEK-499 compared to control cells. Finally, we show that RKIP depletion enhances cellular motility by inducing the expression/stabilization of β-catenin, vimentin, MET and PAK1. Overall, our data suggest that modulation of the cell cycle checkpoints and motility by RKIP may be fundamental to its metastasis suppressive function in cancer and that RKIP role in a cell is more intricate and diverse than previously thought.

Bcr-Abl-mediated Raf kinase inhibitor protein suppression promotes chronic myeloid leukemia progenitor cells proliferation

The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl transformed cells, and Ras activity enhances the oncogenic ability of Bcr-Abl. On the hand, Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction, resulting in blocking the MAP kinase pathway. Moreover, Raf-1 has been reported to regulate cell cycle progression. However, the mechanism by which Bcr-Abl promotes the cell cycle progression through Raf-1 is not completely understood. In the present study, we found that the expression of RKIP was suppressed in CML cells, and investigated the interaction between RKIP and Bcr-Abl in CML cells. In aldehyde dehydrogenase (ALDH)hi/CD34+ cells derived from CML patients, the inhibition of Bcr-Abl induced RKIP expression and reduced the phosphorylated-FOXM1 (pFOXM1) status, resulting in inhibited colony formation of Bcr-Abl+ progenitor cells. Moreover, overexpression of RKIP significantly decreased the colony numbers, reduced the pFOXM1 status, and reduced pFOXM-1 target genes such as Skp2, Cdc25B and KIS, and induced the expression of p27Kip1a and p21Cip1. Thus, Bcr-Abl represses the expression of RKIP, and continuously activates FOXM1, resulting in the proliferation of CML progenitor cells through the cell cycle modulation.

Phosphatidylenthanolamine Binding Protein aka Raf Kinase Inhibitor Protein: A Brief History of Its Discovery and the Remarkable Diversity of Biological Functions

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.

Phosphotyrosine Recognition by the Raf Kinase Inhibitor Protein

Phosphotyrosine Recognition by the Raf Kinase Inhibitor Protein

Prognostic and Predictive Role of Raf Kinase Inhibitor Protein (RKIP) Within the RAS/ MAP Signaling Pathway in Colorectal Cancer

Prognostic and Predictive Role of Raf Kinase Inhibitor Protein (RKIP) Within the RAS/ MAP Signaling Pathway in Colorectal Cancer

The Novel Role of Yin Yang 1 in the Regulation of Epithelial to Mesenchymal Transition in Cancer Via the Dysregulated NF-κB/Snail/YY1/RKIP/PTEN Circuitry

The majority of cancers develop genetic and molecular strategies to resist conventional cytotoxic therapies as well as escape from the host's immune surveillance. These events lead to tumor persistence and spread through activation of the epithelial to mesenchymal transition (EMT) program and metastasis. Expression profiling analysis has revealed various tumor metastasis-inducing and metastasis-suppressing genes that regulate the metastatic process and maintain the microenvironment of the tumor cells. EMT in cancer entails the molecular reprogramming and phenotypic changes that characterize the conversion of immobile cancer epithelial cells to motile mesenchymal cells. A hallmark of EMT is the loss of E-cadherin, the epithelial homotypic adhesion molecule, and gain of mesenchymal markers such as vimentin and fibronectin. The molecular mechanisms underlying the initiation of EMT consist, in part, in the constitutive activation of survival signaling pathways such as the nuclear factor (NF)-κB pathway. The NF-κB pathway has been implicated directly in the regulation of EMT and indirectly through the transcription and expression of several gene products that participate in the EMT cascade, such as Snail, the metastasis-inducer and E-cadherin suppressor transcription factor. In turn, Snail represses the metastasis-suppressor gene product Raf-kinase inhibitor protein (RKIP) that inhibits both the Raf-1/MEK/ERK and NF-κB survival pathways implicated in EMT. Consequently, tumor cells normally exhibit a dysregulated NF-κB/Snail/RKIP circuitry that is intimately involved in the initiation of EMT and maintenance of drug resistance. Additional deregulated gene products in this circuit, such as the metastasis-suppressor phosphatase and tensin homologue (PTEN; repressed by Snail) and the putative-metastasis inducer Yin Yang (YY) 1 (target of NF-κB) also have been associated in the regulation of EMT. We recently have investigated the direct role of YY1 in the regulation of EMT. This review discusses the molecular regulation of EMT in cancer cells through the activity of the dysregulated NF-κB/Snail/ YY1/PTEN/RKIP circuitry. In addition, we propose YY1 as a potential novel prognostic biomarker for EMT and a therapeutic target for both the prevention of metastasis and the reversal of resistance.

Unique Pattern of Overexpression of Raf-1 Kinase Inhibitory Protein in Its Inactivated Phosphorylated Form in Human Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological and incurable malignancy of plasma cells with low proliferative activity in the bone marrow. MM patients initially respond to conventional therapy, however, many develop resistance and recurrences occur. We have identified RKIP as a novel gene product that is differentially overexpressed in MM cell lines and MM tissues compared to other studied tumors and normal bone marrow. This overexpression consisted, in large part, of a phosphorylated inactive form of RKIP at Ser153 (p-Ser153 RKIP). In contrast to RKIP, p-Ser153 RKIP lacks its ability to inhibit the MAPK signaling pathway. The overexpression of p-Ser153 RKIP in MM cell lines and MM tissues was further validated in a mouse model carrying a human MM xenograft, namely, LAGλ-1B. Bioinformatic analyses from databases support the presence of increased RKIP mRNA expression in MM compared to normal plasma cells. In these databases, high RKIP levels in MM are also correlated with the nonhyperdiploid status and the presence of IgH translocations, parameters that generally display more aggressive clinical features and shorter patients' survival irrespective of the treatment. Since RKIP expression regulates both the NF-κB and MAPK survival pathways, the overexpression of "inactive" p-Ser153 RKIP in MM might contribute positively to the overall cell survival/antiapoptotic phenotype and drug resistance of MM through the constitutive activation of survival pathways and downstream the transcription of anti-apoptotic gene products. The overexpression of RKIP and p-Ser153 RKIP in MM is the first demonstration in the literature, since in most tumor tissues the expression of RKIP is very low and the expression of p-Ser153 RKIP is much lower. The relationship between the levels of active RKIP and inactive p-Ser153 RKIP in MM may be of prognostic significance, and the regulation of RKIP activity may be a target for therapeutic intervention.

Dual Roles of Raf-1 Kinase Inhibitor Protein in the Regulation of Both Tumor Cell Resistance to Apoptotic Stimuli and Epithelial to Mesenchymal Transition

Dual Roles of Raf-1 Kinase Inhibitor Protein in the Regulation of Both Tumor Cell Resistance to Apoptotic Stimuli and Epithelial to Mesenchymal Transition

Role of Raf Kinase Inhibitor Protein in Pathophysiology of Prostate Cancer

Role of Raf Kinase Inhibitor Protein in Pathophysiology of Prostate Cancer