Raf Kinase Inhibitor Protein Protein Research Articles

RKIP Regulates Differentiation-Related Features in Melanocytic Cells.

Raf Kinase Inhibitor Protein (RKIP) has been extensively reported as an inhibitor of key signaling pathways involved in the aggressive tumor phenotype and shows decreased expression in several types of cancers. However, little is known about RKIP in melanoma or regarding its function in normal cells. We examined the role of RKIP in both primary melanocytes and malignant melanoma cells and evaluated its diagnostic and prognostic value. IHC analysis revealed a significantly higher expression of RKIP in nevi compared with early-stage (stage I–II, AJCC 8th) melanoma biopsies. Proliferation, wound healing, and collagen-coated transwell assays uncovered the implication of RKIP on the motility but not on the proliferative capacity of melanoma cells as RKIP protein levels were inversely correlated with the migration capacity of both primary and metastatic melanoma cells but did not alter other parameters. As shown by RNA sequencing, endogenous RKIP knockdown in primary melanocytes triggered the deregulation of cellular differentiation-related processes, including genes (i.e., ZEB1, THY-1) closely related to the EMT. Interestingly, NANOG was identified as a putative transcriptional regulator of many of the deregulated genes, and RKIP was able to decrease the activation of the NANOG promoter. As a whole, our data support the utility of RKIP as a diagnostic marker for early-stage melanomas. In addition, these findings indicate its participation in the maintenance of a differentiated state of melanocytic cells by modulating genes intimately linked to the cellular motility and explain the progressive decrease of RKIP often described in tumors.

Increased Expression of miR-23a Mediates a Loss of Expression in the RAF Kinase Inhibitor Protein RKIP.

RAF kinase inhibitor protein (RKIP) is a seminal regulator of intracellular signaling and exhibits both antimetastatic and antitumorigenic properties. Decreased expression of RKIP has been described in several human malignancies, including acute myelogenous leukemia (AML). As the mechanisms leading to RKIP loss in AML are still unclear, we aimed to analyze the potential involvement of miRNAs within this study. miRNA microarray and qPCR data of more than 400 AML patient specimens revealed correlation between decreased expression of RKIP and increased expression of miR-23a, a member of the miR-23a/27a/24-2 cluster. In functional experiments, overexpression of miR-23a decreased RKIP mRNA and protein expression, whereas miR-23a inhibition caused the opposite effect. By using an RKIP 3'-untranslated region luciferase reporter construct with and without mutation or deletion of the putative miR-23a-binding site, we could show that RKIP modulation by miR-23a is mediated via direct binding to this region. Importantly, miR-23a overexpression induced a significant increase of proliferation in hematopoietic cells. Simultaneous transfection of an RKIP expression construct lacking the miR-23a-binding sites reversed this phenotype, indicating that this effect is truly mediated via downregulation of RKIP. Finally, by analyzing more than 4,300 primary patient specimens via database retrieval from The Cancer Genome Atlas, we could highlight the importance of the miR-23a/RKIP axis in a broad range of human cancer entities. In conclusion, we have identified miR-23a as a negative regulator of RKIP expression in AML and have provided data that suggest the importance of our observation beyond this tumor entity. Cancer Res; 76(12); 3644-54. ©2016 AACR.

Raf kinase inhibitor protein inhibits cholangiocarcinoma cell metastasis by downregulating matrix metalloproteinase 9 and upregulating tissue inhibitor of metalloproteinase 4 expression.

Cholangiocarcinoma cells originate in the biliary epithelium. The cells easily metastasize and cause relapse. The effect of Raf kinase inhibitor protein (RKIP) on the biological behavior of cholangiocarcinoma cells is not yet clear. In the present study, RKIP and cytokeratin 19 expression was detected in the extrahepatic tissues of cholangiocarcinoma patients by immunohistochemistry. RKIP small interfering (si)RNA or an RKIP-overexpressing adenoviral vector were used to infect the human cholangiocarcinoma RBE cell line. RKIP protein or gene expression was analyzed by western blotting or reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. The cells were assayed for proliferation, apoptosis, invasion and migration. Matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 4 (TIMP-4) mRNA was assayed by RT-qPCR. RKIP expression was reduced in the extrahepatic cholangiocarcinoma tumor compared with the adjacent uninvolved peritumoral tissues. The current study revealed that RKIP expression was positively correlated with cell differentiation, but negatively correlated with lymph node or distant metastasis (P<0.05). RKIP siRNA treatment promoted RBE cell invasion, but RKIP overexpression prevented cell invasion. In the pDC316-siRNA recombinant vector group, the cells migrated more quickly compared with the siRNA-negative control group, and in the RKIP-expressing adenoviral vector group, the cells migrated more slowly compared with the adenoviral negative control group. RKIP inhibited the invasive and metastatic ability of the cholangiocarcinoma cell line, RBE, by downregulating MMP-9 and upregulating TIMP-4 mRNA expression. RKIP is negatively associated with cholangiocarcinoma distant metastasis and prevents cholangiocarcinoma cell metastasis through downregulating MMP-9 expression and upregulating TIMP-4 expression.

RKIP suppresses gastric cancer cell proliferation and invasion and enhances apoptosis regulated by microRNA-224.

The purposes of this study were to determine the expression profile of Raf kinase inhibitor protein (RKIP) in human gastric cancer cells and its effect on the biological characteristics of SGC-7901 cell lines, to examine the modulatory effect of microRNA-224 (miR-224) on RKIP. The research will provide novel strategies for gastric cancer treatment in the future. Quantitative real-time reverse transcription PCR (qRT-PCR) was employed to determine the expression profile of RKIP in gastric cancer cell lines (SGC-7901, MGC80-3, and MKN45). A eukaryotic expression vector, pcDNA3.1-RKIP, was constructed and transfected into SGC-7901 cells. Changes in RKIP protein expression were examined by Western blot assays, and the effect of RKIP overexpression on SCG-7901 cell viability was determined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assays. The effect of RKIP overexpression on SGC-7901 cell proliferation and apoptosis was analyzed by flow cytometry and that on the migration of SGC-7901 cells was investigated by Transwell migration assays. RKIP was identified to be a regulatory target gene of miR-224 using a luciferase reporter gene system, and the effect of miR-224 on intracellular RKIP protein expression was examined by Western blot assays. The regulatory effect of miR-224 on the biological characteristics of RKIP was investigated by MTT, flow cytometry, and Transwell invasion chamber assays. The expression of RKIP in gastric cancer cells was decreased significantly in comparison to that of normal gastric mucosal epithelial cells (GES-1) (p < 0.01), as demonstrated by qRT-PCR assays. Compared with the control group, the up-regulation of RKIP intracellular expression was observed in SGC-7901 cells after transfection of pcDNA3.1-RKIP for 48 h (p < 0.01). There were significant decreases in cell viability and the S-phase fraction (p < 0.05), concomitant with a significant increase in apoptosis (p < 0.01), as well as a significant reduction in cells migrating through Transwell chambers (p < 0.05), as shown by MTT, flow cytometry, and Transwell invasion chamber assays. A significant decrease in luciferase activities in cells transfected with a miR-224 mimic was observed compared with that of the control group (p < 0.05), as suggested by the luciferase reporter gene system. As shown by Western blot assays, there was a significant decrease in RKIP expression in SGC-7901 cells transfected with the miR-224 mimic for 48 h compared with the control group (p < 0.05). As shown by MTT, flow cytometry, and Transwell invasion chamber assays, the changes in biological characteristics induced by RKIP overexpression could be suppressed in SGC-7901 cells after transfection of the miR-224 mimic. In conclusion, the down-regulation of RKIP expression was observed in human gastric cell lines, and miR-224 could negatively regulate the expression and biological characteristics of RKIP, contributing to suppress the proliferation and invasion of gastric cells.

Raf kinase inhibitor protein (RKIP) and phospho-RKIP expression in melanomas

Melanoma, a cancer notorious for its high potential to metastasize, arises from melanocytes, cells dedicated to melanin production and located in the basal layer of the epidermis. Raf-1 kinase inhibitor protein (RKIP) is an inhibitory molecule that down-regulates the effects of the Ras/Raf/MEK/ERK signaling pathway. The aim of this study was to examine the expression of RKIP and pRKIP in melanomas at different stages. We evaluated the RKIP and pRKIP protein by immunohistochemistry in control skin, pigmented nevi and melanomas, and through Western blotting in human normal melanocytes and in four different melanoma-derived cell lines (WM35, A375, M14, and A2058). Our results demonstrated a correlation between the expression of RKIP and pRKIP, and metastatic ability in melanoma cells. This raises the possibility to analyze both RKIP and pRKIP in all melanomas. Down-regulation of both RKIP and pRKIP expression could represent a useful marker of metastatic melanoma. On the contrary for non-metastatic melanoma, especially in Clark I and II, low RKIP and high pRKIP expression could be indicative. In conclusion, the observed negative correlation of the RKIP and pRKIP expression in metastatic melanomas indicates that expression of these proteins may become a prognostic marker for the progression of human cutaneous melanoma. We propose that the investigation of both RKIP and pRKIP may provide a useful tool indicative for metastatic or non-metastatic melanoma in different Clark's level melanomas. Further studies are required to verify the molecular background of the observed RKIP and pRKIP variations.

Expression and Significance of RKIP and E-cadherin in Lung Squamous Cell Carcinoma

The purpose of this study was to investigate the expression of Raf kinase inhibitor protein (RKIP) and epithelial cadherin (E-cadherin) in lung squamous cell carcinoma tissue and its correlation with the clinical pathology of lung squamous cell carcinoma. RKIP and E-cadherin mRNA (by RT-PCR) and protein (by western blotting) levels were monitored in carcinoma tissues and surrounding normal tissues from 86 lung squamous cell carcinoma cases, and their positive rates were calculated. The rates of positive RKIP and E-cadherin mRNA expression were significantly lower in lung squamous cell carcinoma than in the surrounding normal tissues (P < 0.05). The positive expression rates were significantly lower in those with lymph node metastasis than in those without (P < 0.05). The lower the degree of tumor differentiation, the lower the E-cadherin mRNA positive expression rate (P < 0.05). The rates of positive RKIP and E-cadherin mRNA expression were significantly lower in patients at advanced (III, IV) stages than in patients at early (I, II) stages (p < 0.05); this rate, however, was independent of gender, age, and tumor size (P > 0.05). The protein levels of RKIP and E-cadherin were significantly lower in lung squamous cell carcinoma than in the surrounding normal tissues (P < 0.05). The levels were significantly lower in patients with lymph node metastasis than in those without it (P < 0.05). The lower the degree of tumor differentiation, the lower the protein level of E-cadherin (P < 0.05). Both RKIP and E-cadherin are tumor suppressors, their low expression levels may be associated with initiation, invasion and/or metastasis, as well as with the inhibition of lung squamous cell carcinoma differentiation.

RKIP expression associated with gastric cancer cell invasion and metastasis

The purpose of this study was to analyze Raf kinase inhibitor protein (RKIP) expression in gastric cancer tissue, its correlation with gastric cancer clinical pathology, and its role in gastric cancer invasion and metastasis in order to provide experimental evidence for the potential biological therapy of this disease. Both immunohistochemistry and western blot analyses were used to test for RKIP expression in 55 cases of gastric cancer tissue and the adjacent gastric mucous membrane tissue. The correlations of RKIP expression with the onset, development, and clinical pathology of gastric cancer were analyzed. After transiently transfecting the human gastric cancer cell line MKN45 with a eukaryotic expression vector containing the full length RKIP cDNA, the changes in MKN45 cell invasiveness and metastatic ability were studied. Immunohistochemistry and western blot results revealed that the quantity of RKIP protein expressed in the gastric cancer tissues was significantly lower than that of the adjacent normal gastric mucous membrane tissues (p < 0.05). The quantity of RKIP protein expression was reduced (p < 0.05) as the gastric cancer cells' differentiation decreased, the TNM stage increased, and the extent of invasion expanded. However, the expression of RKIP in the gastric cancer tissues was not associated with the patients' age or gender (p > 0.05). By overexpressing RKIP in the human gastric cancer cell line MKN45 and through the use of a Transwell invasion chamber, we determined that RKIP overexpression significantly reduced both the invasiveness and metastatic ability of MKN45 cells (p < 0.05). Low or absent RKIP expression may be associated with the onset and development of gastric cancer and its ability to invade and metastasize.

The Role of Raf Kinase Inhibitor Protein in Rheumatoid Fibroblast-like Synoviocytes Invasiveness and Cytokine and Matrix Metalloproteinase Expression

Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis. Raf kinase inhibitor protein (RKIP) negatively regulates the Raf/MEK/ERK and NF-κB pathway. The role of RKIP in rheumatoid FLS is unknown. The purpose of the present study was to investigate the function of RKIP in rheumatoid FLS. Rheumatoid FLS were transfected with either RKIP-expressing plasmids or RKIP small interfering RNA (siRNA). RKIP protein was detected in rheumatoid synovial tissue (ST) and FLS. RKIP overexpression significantly decreased IL-6 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP overexpression also showed a decreased trend in IL-8, MMP-1, and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing resulted in significantly increased MMP-1 and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing also increased IL-6 and IL-8 mRNA expression in TNF-α-stimulated rheumatoid FLS, but this increase did not reach statistical significance. TNF-α-induced ERK and NF-κB activation was suppressed in FLS with RKIP overexpression. RKIP silencing resulted in a significantly higher invasion index in TNF-α-stimulated rheumatoid FLS compared to controls. These results suggest that RKIP might be a potential therapeutic target for rheumatoid arthritis.

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.

Activation of Raf-1 through the Depletion of RKIP by Bcr-Abl Promotes CML Cell Proliferation

AbstractAbstract 5105 [Background and Aims]RKIP (Raf kinase inhibitor protein) regulates cell proliferation, apoptosis, and cell cycle through the inhibition of Raf-1, and it has been reported that the suppression of the RKIP expression promotes the proliferation of tumor cells. However, its function is little known in CML. We found that the expression of RKIP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the RKIP in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562, Meg01, and SHG3 cells. Primary CML cells (ALDHhicells) were obtained from the bone marrow of CML (CP) patients (n=12). Human normal ALDHhi cells were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of RKIP mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Erk1/2 phosphorylation in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl, RKIP siRNA, or RKIP cDNA respectively. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with RKIP siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines treated with Abl kinase inhibitors or transfection with Bcr-Abl siRNA, the expressions of RKIP mRNA and protein were significantly increased more than untreated cells. Moreover, in CML cells transfected with the RKIP siRNA, the cell proliferation inhibition by treatment of the Abl kinase inhibitors was weakened compared to RKIP siRNA-untransfected cells, and the phosphorylation levels of Erk1/2 and Raf-1 were markedly increased compared to theRKIP siRNA-untransfected cells. On the other hands, the overexpression of RKIP induced G1 cell cycle arrest through p27 and p21 accumulation, decreased the phosphorylation levels of ERK1/2 and Raf-1, and inhibited the proliferation in CML cells. In CML stem/progenitor cells obtained from patients with CML, the expression of RKIP mRNA was suppressed in 10/10 (100 %) of CML. The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of RKIP mRNA and protein, and the overexpression of RKIP decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of RKIP, the depletion of RKIP induced Raf-1 activation, and induced the proliferation of CML cells through ERK1/2 phosphorylation. Moreover, in CML, the induction of RKIP expression inhibited the proliferation of CML stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Abstract 5161: Activation of Raf-1 through the depletion of RKIP by Bcr-Abl promotes CML cell proliferation

Abstract Activation of Raf-1 through the depletion of RKIP by Bcr-Abl promotes CML cell proliferation [Background and Aims] RKIP (Raf kinase inhibitor protein) regulates cell proliferation, apoptosis, and cell cycle through the inhibition of Raf-1, and it has been reported that the suppression of the RKIP expression promotes the proliferation of tumor cells. However, its function is little known in CML. We found that the expression of RKIP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the RKIP in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML cells were obtained from the bone marrow of CML (CP) patients. Human normal mononuclear cells (MNCs) were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of RKIP mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Erk1/2 phosphorylation in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl, RKIP siRNA, or RKIP cDNA respectively. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with RKIP siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines, the expressions of RKIP mRNA and protein were significantly increased more than normal MNCs by treatment with Abl kinase inhibitors or transfection with Bcr-Abl siRNA. Moreover, in CML cells transfected with the RKIP siRNA, it is shown that the phosphorylation levels of Erk1/2 and Raf-1 were markedly increased compared to the untransfected cells. On the other hands, the overexpression of RKIP induced G1 cell cycle arrest through p27 and p21 accumulation, decreased the phosphorylation levels of Erk1/2 and Raf-1, and inhibited the proliferation in CML cells. In CML stem/progenitor cells obtained from patients with CML, the expression of RKIP mRNA was suppressed in 10/10 (100 %) of CML. The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of RKIP mRNA and protein, and decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of RKIP, the depletion of RKIP induced Raf-1 activation, and induced the proliferation of CML cells through Erk1/2 phosphorylation. Moreover, induction of RKIP expression might regulate the proliferation of CML stem/progenitor cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5161.

Relationship between raf kinase inhibitor protein and metastasis of ovarian carcinoma

To investigate the relationship between raf kinase inhibitor protein (RKIP), a novel metastasis suppressor gene, and metastasis of ovarian carcinoma. Immunohistochemistry, RT-PCR, and western blot analysis were performed to examine the expression of RKIP in clinical samples of ovarian tumors and five human ovarian carcinoma cell lines. Stable cell lines over-expressed or deleted of RKIP were cloned to investigate the function of RKIP in ovarian cancer cells. The recombinant plasmids expressing sense (ss) or antisense (as) RKIP cDNA or empty vector was transfected into ovarian cancer cell line SKOV3 by lipofectamine. The expression level of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in ovarian cancer cells were detected by western blot analysis. Assays of cell proliferation, soft-agar colony formation, cell adhesion, and cell invasion in vitro were used to examine the malignant phenotypes of the transfected cells. Flow cytometric analysis was performed to observe the effect of RKIP on cell cycle distribution before and after transfection. (1) The expression levels of RKIP protein in ovarian carcinoma tissues from patients were found to be reduced than those in ovarian benign tumor and borderline tumor. SKOV3 clones stably expressing full-length recombinant ssRKIP, asRKIP, and their respective empty vector were obtained. (2) RKIP was able to block basal levels of MEK and ERK in ovarian cancer cells. The expression level of phosphorylation MEK in ssRKIP#1 and ssRKIP#4 cells were 0.35, 0.34; while the expression level of phosphorylation ERK in ssRKIP #1 and ssRKIP #4 cells were 0. 48 and 0.46. (3) Abilities of cell proliferation in the ssRKIP vector-transfected cells were decreased compared with that in the non-transfected cells (P < 0.01). (4)Anchorage-independent growth in the ssRKIP#1 and ssRKIP#4 cells (83.7 +/- 5.7, 106.0 +/- 9.2) were decreased compared with that in the empty vector-transfected cells (158.3 +/- 14.6, P < 0.01). (5)Cell adhesion in the ssRKIP#1 and ssRKIP#4 cells [(68.3 +/- 0.8)%, (64.1 +/- 0.9)%] were decreased compared with that in the non-transfected cells [(100.0 +/- 1.1)%, P < 0.01]. (6) Cell invasion in the ssRKIP#1 and ssRKIP#4 cells (24 +/- 5, 25 +/- 4) were decreased compared with that in the non-transfected cells (68 +/- 5, P < 0.01). (7) ssRKIP cells had a significant increase in the G1 phase and decrease in the G2 + S phase. RKIP could inhibits the metastasis, but also the growth of ovarian cancer cells. patients were found to be reduced than those in ovarian benign tumor and borderline tumor. SKOV3 clones stably expressing full-length recombinant ssRKIP, asRKIP, and their respective empty vector were obtained. (2) RKIP was able to block basal levels of MEK and ERK in ovarian cancer cells. The expression level of phosphorylation MEK in ssRKIP#1 and ssRKIP#4 cells were 0.35, 0.34; while the expression level of phosphorylation ERK in ssRKIP #1 and ssRKIP #4 cells were 0.48 and 0.46. (3) Abilities of cell proliferation in the ssRKIP vector-transfected cells were decreased compared with that in the non-transfected cells (P < 0.01). (4) Anchorage-independent growth in the ssRKIP#1 and ssRKIP#4 cells (83.7 +/- 5.7, 106.0 +/- 9.2) were decreased compared with that in the empty vector-transfected cells (158.3 +/- 14.6, P < 0.01). (5) Cell adhesion in the ssRKIP#1 and ssRKIP#4 cells [(68.3 +/- 0.8)%, (64.1 +/- 0.9)%] were decreased compared with that in the non-transfected cells [(100.0 +/- 1.1)%, P < 0.01]. (6) Cell invasion in the ssRKIP#1 and ssRKIP#4 cells (24 +/- 5, 25 +/- 4) were decreased compared with that in the nontransfected cells (68 +/- 5, P < 0.01). (7) ssRKIP cells had a significant increase in the G1 phase and decrease in the G2 + S phase. RKIP could inhibits the metastasis, but also the growth of ovarian cancer cells.

Raf kinase inhibitor protein inhibits cell proliferation but promotes cell migration in rat hepatic stellate cells

Hepatic stellate cells (HSCs) play an important role in the pathogenesis of liver fibrosis and cirrhosis. Raf kinase inhibitor protein (RKIP), an inhibitor of extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway, has been proved to suppress tumor metastasis. Interestingly, RKIP promotes cell migration in Madin-Darby canine kidney epithelial cells. However, the effects of RKIP on HSC behaviours are unknown. The purpose of the present study is to investigate the role of RKIP in HSC proliferation, apoptosis and migration. Two types of cells, freshly isolated HSC and HSC-T6 cell line, were used in this study. The amount of RKIP, the phosphorylation of RKIP, Raf and ERK (pRKIP, pRaf and pERK) were analysed in quiescent and activated HSCs by Western blots. HSC-T6 cells were transfected with RKIP-expressing plasmid or treated with locostatin, a RKIP inhibitor. HSC proliferation, apoptosis and migration were evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) staining and Transwell cell migration assay respectively. In activated HSCs, RKIP protein expression was downregulated whereas pRKIP, pRaf and pERK were upregulated. RKIP overexpression significantly mitigated the phosphorylation of RKIP, Raf and ERK. This in turn inhibited HSC proliferation. Locostatin not only inhibited RKIP protein expression but also, to some extent, reversed the RKIP-inhibited phosphorylation of RKIP, Raf and ERK. RKIP augmented HSC migration and enhanced wound closure. Locostatin reversed the effects of RKIP. Raf kinase inhibitor protein inhibits ERK/MAPK signalling and this inhibition impedes HSC proliferation. RKIP promotes HSC migration and wound closure.

Effects of Raf Kinase Inhibitor Protein Expression on Metastasis and Progression of Human Epithelial Ovarian Cancer

Loss of function of metastasis suppressor genes is an important step in the progression to a malignant tumor type. Studies in cell culture and animal models have suggested a role of Raf kinase inhibitor protein (RKIP) in suppressing the metastatic spread of prostate cancer, breast cancer, and melanoma cells. However, the function of RKIP in ovarian cancer (OVCA) has not been reported. To explore the potential role of RKIP in epithelial OVCA metastasis, we detected the expression levels of RKIP protein in tissue samples from patients with epithelial OVCA. Consequently, the expression of RKIP is reduced in the poorly differentiated OVCA than in the well-differentiated and moderately differentiated OVCA. In addition, in vitro cell invasion assay indicated that the RKIP expression was inversely associated with the invasiveness of five OVCA cell lines. Consistent with this result, the cell proliferation, anchorage-independent growth, cell adhesion, and invasion were decreased in RKIP overexpressed cells but increased in RKIP down-regulated cells. Further investigation indicated that RKIP inhibited OVCA cell proliferation by altering cell cycle progression rather than promoting apoptosis. Furthermore, the overexpression of RKIP suppressed the ability of human OVCA cells to metastasize when the tumor cells were transplanted into nude mice. Our data show the effect of RKIP on the proliferation, migration, or adhesion of OVCA cells. These results indicate that RKIP is also a metastasis suppressor gene of human epithelial OVCA.

Nitric Oxide Sensitizes B-NHL Cells to TRAIL-Mediated Apoptosis through Induction of RKIP, Inhibition of YY1 and Upregulation of DR5.

A better knowledge of the mechanisms of survival and escape from apoptosis of B-cell lymphoma cells has led to the proposal of new drugs that selectively interfere at the different steps of these cascades. Among the novel agents that have recently emerged for the sensitization of several resistant tumor cells to chemo- or immuno-mediated cell death are agents that induce nitric oxide (NO) (Bonavida B et al., Drug Resistance Update, 2006). The molecular mechanisms which are implicated in the NO-induced sensitization of tumor cells to apoptosis are not clearly elucidated. Recent findings in our laboratory have reported that treatment of various cancer cell lines with the NO donor, DETA/NONOate, sensitizes tumor cells to FasL- and TRAIL-mediated apoptosis. Sensitization is mediated through inhibition of NF-κB and YY1 activities and upregulation of death receptors. The objective of the present study was to examine whether DETA/NONOate is also able to sensitize B-NHL cells to TRAIL-mediated apoptosis and to identify additional molecular targets which could be involved in the NO-mediated chemo- and immuno-sensitization of tumor cells. Contrary to YY1, RKIP (Raf kinase inhibitor protein) has been shown to play a pivotal role in suppression of mitogenesis through inhibition of Raf/MEK/ERK and NF-κB survival signaling pathways (Yeung et al., Molecular Cellular Biology ; 21:7207, 2001). Since both NO and RKIP inhibit NF-κB, we hypothesized that NO may induce RKIP expression and may participate in NO-mediated effects. The TRAIL-resistant B-NHL cell line, Ramos, was treated with DETA/NONOate and examined for changes in YY1 and RKIP protein and m-RNA levels, as well as for sensitization to TRAIL-mediated apoptosis. The findings demonstrate that treatment of Ramos cells with DETA/NONOate results in slight induction of RKIP m-RNA levels followed by significantly elevated RKIP protein expression. In contrast, both YY1 transcript and protein levels were found remarkably reduced after DETA/NONOate treatment. Since YY1 has been recently shown to negatively regulate DR5 transcription, NO-induced suppression of YY1 was able to sensitize Ramos cells to TRAIL-mediated apoptosis through significant DR5 upregulation, as assessed by flow cytometry. However, DR4 surface protein expression remained unchanged. Noteworthy, the rituximab resistant Ramos clone (RR1) was not sensitized by NO, while no changes were observed in both DR4 and DR5 levels, suggesting different regulation of cell sensitivity to TRAIL. The above findings support the role of NO as an immuno-sensitizing agent via inhibition of YY1 and suggest, for the first time, the pivotal role of RKIP as one potential NO target for apoptosis induction in tumor cells. Further experimental studies will clarify the interlink between RKIP and YY1 involved in NO-based B-cell lymphoma sensitization to apoptosis and validation with patient derived tissues.

Over Expression of YY1 and RKIP in Multiple Myeloma (Cell Lines and MM Tissues).

There is a need to identify underlying molecular mechanisms and gene products (targets for intervention and biomarkers) involved in MM chemo- or immuno-resistance. Recent findings from our laboratory have reported high expression of the transcription factor Yin Yang 1 (YY1) in several tumor types and have demonstrated its fundamental role in tumor cell chemo/immuno-resistance (Gordon, S., et al., Oncogene 25:1125, 2006). In contrast, low levels of the Raf kinase inhibitor protein (RKIP) has been shown to play a pivotal role in the negative regulation of cell survival signaling pathways, and has been considered a metastasis tumor suppressor. The overall objective of our studies is to examine whether MM cell lines and tissues derived from MM patients present deregulated expression patterns of RKIP and YY1, and to demonstrate the roles of YY1 and RKIP in MM pathogenesis and response to various therapies. In the present study, the myeloma cell lines RPMI-8226 and MM-1S were screened for RKIP and YY1 expression at the protein and m-RNA levels. In addition, fresh tissues from MM patients were also examined for RKIP and YY1 expression by IHC and their patterns compared with those observed in normal bone marrow cells. The findings demonstrate that both MM cell lines express remarkably higher levels of RKIP protein and transcripts compared to other tumor cell lines examined (lymphomas and prostate cancer), as assessed by western, RT-PCR and IHC. There was significantly higher expression of RKIP in MM patient's samples compared to normal bone marrow cells. YY1 protein and m-RNA levels were detected in the MM cell lines; however, they were lower compared to Ramos and PC-3 cells. YY1 expression in MM tissues was significantly elevated compared to normal bone marrow cells. RKIP was basically present in the cytoplasm while YY1 was mainly accumulated in the nucleus. In contrast, normal bone marrow cells presented primarily cytoplasmic YY1 and RKIP expression. The YY1 expressing population was the CD38+/CD138− cell subset, which has been reported to be more susceptible to apoptosis (Mitsiadis, S.C., et al., Blood 98:795, 2001). Overall, the present findings support the potential involvement of two new gene products, such as YY1 and RKIP, in MM pathogenesis and their implication in regulating MM resistance to conventional therapeutics. Moreover, these gene products suggest their potential use as new therapeutic targets and/or biomarkers in multiple myeloma. Future studies with large cohorts of tissues will elucidate the importance of the above findings and will give new insights in the unexpected RKIP over expression and activity in multiple myeloma.

Metastasis suppressor gene Raf kinase inhibitor protein (RKIP) is a novel prognostic marker in prostate cancer

Diminished expression of Raf kinase inhibitor protein (RKIP), an inhibitor of the Raf signaling cascade, promotes prostate cancer (PCa) metastasis in a murine model, suggesting that it is a metastasis suppressor gene. However, the prognostic significance of RKIP expression and its association with metastasis in PCa patients is unknown. To investigate RKIP protein expression is a prognostic marker in PCa we performed immunohistochemical staining for RKIP expression in tissue microarrays consisting of 758 non-neoplastic prostate tissues, primary tumors and metastases from 134 PCa patients. The Cox proportional-hazards model was used to adjust for covariates including Gleason score, tumor volume, tumor weight, clinical stage, digital rectal exam findings, serum PSA level and surgical margins. RKIP expression was low in approximately 5%, 48%, and 89% of non-neoplastic prostate, primary tumors and metastases, respectively. Low RKIP expression in primary tumors was a strong positive predictive factor for PCa recurrence based on PSA levels. In patients whose primary tumors expressed high RKIP levels, the 7-year PSA recurrence rate was <10%; whereas in patients with tumors with low RKIP expression the recurrence rate was 50% (P<0.001). Multivariate analysis revealed RKIP was an independent prognostic factor (P<0.001). In contrast to increased expression of pro-tumorigenic genes, these results demonstrate decreased protein expression of a gene, for example, RKIP, can serve as a prognostic marker in PCa patients.

Metastasis suppressor genes: a role for raf kinase inhibitor protein (RKIP)

The metastatic cascade is a complicated process that involves many steps from gain of the metastatic phenotype in the primary tumor cells through establishment of macroscopic tumor at the distant target organ. A group of genes, termed metastasis suppressor genes (MSG), encode for proteins that inhibit various steps of the metastatic cascade. Accordingly, loss of MSG promotes the metastatic phenotype. Although several MSG have been identified, the mechanisms through which they enhance metastasis are not clearly defined. Gene array analysis of a low metastatic LNCaP prostate cancer cell line compared to its highly metastatic derivative C4-2B prostate cancer cell line revealed decreased expression of raf kinase inhibitor protein (RKIP) in the C4-2B cell line. RKIP blocks the activation of several signaling pathways including MEK, G-proteins and NFkappaB. Immunohistochemical analysis of prostate cancer primary tumors and metastases revealed that RKIP protein expression was decreased in metastases. Restoration of RKIP expression in the C4-2B cell line diminished metastasis in a murine model. These results demonstrate that RKIP is a MSG. Loss of RKIP enhanced both angiogenesis and vascular invasion, and protected against apoptosis. These findings suggest that targeting the RKIP pathway may diminish the metastatic cascade. However, challenges exist as to the best method to target RKIP expression. Restoration of RKIP expression in all cancer cells in vivo is challenging. A plausible strategy is to use small molecules that target proteins in signaling pathways that are dysregulated due to loss of RKIP.