Raf Kinase Inhibitor Protein Expression Research Articles

High Expression of the Resistant Factor YY1 and Low Expression of the RKIP Chemosensitizing Factor in Multiple Myeloma: Delineation of Several Crosstalk Pathways That Regulate the Expression of These Factors

Background: We have reported that multiple myeloma (MM) expresses high levels of the transcription (oncogenic) factor YY1, which regulates cell survival, proliferation, and resistance to both immuno- and chemotherapeutic drugs.1 Inhibition of YY1 sensitizes the tumor cells to cell death/apoptosis to cytotoxic drugs. In contrast, we have also reported that MM expresses very low levels of Raf kinase inhibitor protein (RKIP) and/or high levels of inactive phosphorylated RKIP.2 Overexpression of RKIP or de-phosphorylation of phosphorylated RKIP sensitized drug-resistant MM to drug-induced apoptosisHypothesis: The inverse relationship between YY1 and RKIP expression levels suggested that the two factors may be regulated through crosstalk pathways.Methods: Analysis of the literature revealed the presence of five crosstalk pathways that link RKIP and YY1.Results: The crosstalks consisted of the followings: (1) The NF-κB/YY1/Snail/RKIP loop. In the presence of RKIP, the NF-κB pathway is inhibited and consequently, the NF-κB targets YY1 and Snail are also inhibited. In turn, inhibition of the RKIP repressor Snail resulted in the upregulation of RKIP expression, all of which led to sensitization of drug-resistant tumor cells. (2) The p38 MAPK/RKIP/GSK3β/Snail/YY1 loop. In the presence of RKIP expression, the p38 MAPK-mediated phosphorylation of GSK3β is inhibited and results in the phosphorylation of Snail and its degradation, leading to the de-repression of RKIP expression and inhibition of YY1. (3) The RKIP/Smurf-2/YY1/Snail loop. SMAD ubiquitin regulatory factor 2 (Smurf-2) is an E3 ubiquitin ligase that degrades YY1. In tumor cells, there is low expression of Smurf-2 and, thus, little inhibition of YY1 with high Snail expression in the presence of low expression of RKIP. In the presence of high levels of Smurf-2, YY1 is inhibited, resulting in the inhibition of Snail and de-repression of RKIP. (4) The RKIP/MAPK/Myc/Let-7/HMGA2/Snail/YY1 loop. Let-7/miR98 suppresses Ras and inhibits the MAPK signaling pathway. In the absence of RKIP, the Raf/MEK1-2 pathway is activated and downstream Myc is expressed. In turn, Myc-induced Lin-28 inhibits Let-7, and leads to upregulation of HMGA2. Consequently, HMGA2 activates Snail, in addition to its regulation by YY1. In contrast, in the presence of RKIP, the Raf/MEK pathway is inhibited and there is downstream inhibition of its targets Myc, Lin-28, and HMGA2, leading to an inhibition of Snail and de-repression of RKIP. (5) The RKIP/GPCR/STAT3/miR-34/YY1 loop. In tumor cells expressing low levels of RKIP, there is no efficient inhibition of GPCR and MAKP signaling pathways. Hence, there is a constitutive activation of oncogenic STAT3 that inhibits the tumor suppressor miR-34. Thus, low miR-34 levels de-repress YY1 translation and facilitate its effects on the survival and the resistance of tumor cells to cytotoxic drugs.Conclusion and Implications: The above YY1/RKIP gene signatures might predict the tumor expanding potential and overall aggressiveness and their detriments that contribute to tumor regression. Targeting YY1 inhibition or RKIP induction via interference of the above crosstalk pathways may result in synergistic activity in combination with conventional treatments.References1 Bonavida B, Kaufhold S. Prognostic significance of YY1 protein expression and mRNA levels by bioinformatics analysis in human cancers: a therapeutic target. Pharmacol Ther. 150:149-68, 2015.2 Baritaki, S, Huerta-Yepez, S, Cabrava-Haimandez, M, Sensi, M, Canevari, S, Libra, M, Penichet, M, Chen, H, Berenson, JR, Bonavida, B. Unique Pattern of Overexpression of Raf-1 Kinase Inhibitory Protein in Its Inactivated Phosphorylated Form in Human Multiple Myeloma. Forum on Immunopathological Diseases and Therapeutics, 2: 179-188, 2011. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Drug Resistance Via the Overexpression of Phopho-RKIP (P-RKIP) in Multiple Myeloma: Dephosphorylation of P-RKIP Reverses Drug Resistance

Background: Multiple myeloma (MM) remains an incurable disease. We have reported that MM cell lines and patient-derived MM cells overexpress the gene product Raf-Kinase Inhibitor Protein (RKIP) in its inactivated phosphorylated (p-RKIP) form.1 Active RKIP inhibits both the Raf/MEK/ERK and NF-κB pathways and resulting in the inhibition of proliferation, metastasis, and sensitization to chemo and immune drugs.2Objective: Drug resistance is a common feature of patients with MM. The delineation of factors that regulate resistance and their targeting may reverse resistance and leads to prolongation of survival.Hypothesis: We hypothesized that dephosphorylation of RKIP in MM may restore the activity of RKIP and resulting in the sensitization of drug-resistant MM cells to drug-induced cytotoxicity.Experimental Design: The expression of RKIP and p-RKIP (specific anti-p-RKIP antibody) was analyzed in both MM cell lines and fresh tumor cells derived from bone marrow aspirates of MM patients using Western, flow cytometry and IHC and a specific anti-pRKIP monoclonal antibody. We also examined the effect of a PKC inhibitor, bisindolymalemide-1 (BIM) treatment in MM cell lines and their response to treatment with anti-MM drugs such as bortezomib, melphalan, and bendamustine.Findings: The analyses of MM cell lines and patient-derived MM cells revealed the overexpression of RKIP, the majority of which was in its inactive phosphorylated form, p-RKIP. Treatment of MM cell lines with the PKC inhibitor BIM resulted in the dephosphorylation of p-RKIP and the treated drug resistant MM cells were sensitized to bortezomib, melphalan, and bendamustine-induced cell death. We have also found a correlation between the RKIP/p-RKIP ratios in MM and clinical response; Kaplan-Meyer analysis demonstrated that there was a correlation between the high expression of RKIP and poor survival.Conclusions and Clinical Implication: The findings demonstrated that the overexpression of p-RKIP in MM was involved, in part, on the regulation of drug resistance since dephosphorylation of p-RKIP reversed the drug resistance. The role of p-RKIP in the regulation of drug resistance in MM is corroborated in our prior reported studies in other cancer types that demonstrated that knockout of RKIP rendered the sensitive tumor cells drug resistant, whereas in contrast, overexpression of RKIP sensitized the drug-resistant cells to drug-induced apoptosis. We conclude that the combination treatment of a PKC inhibitor and anti-MM drugs can be clinically relevant in the treatment of patients who are resistant to drug treatment. Also, we propose that the examination of the levels of p-RKIP in MM may reveal a novel prognostic biomarker and the determination of proper treatment regimens.Future studies: We propose to investigate the molecular and biochemical means by which p-RKIP is overexpressed in MM and characterizing the specific PKC that is involved in the phosphorylation of RKIP in MM.References1 Baritaki, S, Huerta-Yepez, S, Cabrava-Haimandez, M, Sensi, M, Canevari, S, Libra, M, Penichet, M, Chen, H, Berenson, JR, Bonavida, B. Unique Pattern of Overexpression of Raf-1 Kinase Inhibitory Protein in Its Inactivated Phosphorylated Form in Human Multiple Myeloma. Forum on Immunopathological Diseases and Therapeutics, 2: 179-188, 2011.2 Bonavida B. RKIP-mediated chemo-immunosensitization of resistant cancer cells via disruption of the NF-κB/Snail/YY1/RKIP resistance-driver loop. Crit Rev Oncog. 19:431-45, 2014. DisclosuresNo relevant conflicts of interest to declare.

Association of low RKIP expression with poor prognosis in non-small cell lung cancer.

3068 Background: Raf1 kinase inhibitor protein (RKIP) is able to bind Raf1 to inhibit Ras-Raf-MEK-ERK signaling, a major oncogenic pathway. It has been reported that reduced RKIP expression associates with poor prognosis in many cancers, including gastric adenocarcinoma, gliomas and bladder cancer. However, there are only several studies on its role in non-small cell lung cancer (NSCLC) and the conclusion is still controversial. Hence, we performed this study to assess the prognostic significance of RKIP in our NSCLC population. Methods: Between June 2017 and June 2020, 156 NSCLC patients treated at our hospital were included for the present study. None of the patients had received chemotherapy, radiotherapy or surgery before. Their tumor tissues and surrounding normal lung tissues were collected for immunostain and western blot analysis of RKIP expression and ERK signaling. We collected information about gender, age, histological differentiation, tumor size, TNM stage, and lymph node status. Survival curves were analyzed using the Kaplan-Meier method. Cox proportional hazards model was used to determine the prognostic value of various variables in a univariate and multivariate setting. Results: Immunostain and western blot results showed a lower RKIP expression and a higher p-ERK level in cancer tissues compared with the surrounding normal tissues. A reduced RKIP expression with high level of p-ERK was also observed in TNM stages III and IV as compared with I and II. Pearson's chi-squared test confirmed low RKIP expression associated with poorer TNM stage ( p< 0.001) and N-stage ( p< 0.05). No significant correlation was observed between RKIP expression level and gender, age, histological type or tumor size. Kaplan-Meier survival analysis revealed that patients with low RKIP expression had significantly worse overall survival than patients with high RKIP expression ( p= 0.019, log-rank). This conclusion was consistent in the stage I&II patients ( p= 0.011, log-rank) but not in the stage III&IV patients ( p= 0.711, log-rank). Univariate Cox proportional hazards regression analysis indicated Tumor size, TNM stage and RKIP expression significantly affected overall survival of the NSCLC patients. Multivariate Cox proportional hazards regression analysis confirmed RKIP expression remained a significant predictor of survival after correcting for the effects of Tumor size and TNM stage (hazard ratio = 1.730, 95% confidence interval = 1.017 – 2.942, p = 0.043). Conclusions: In this study, low RKIP expression was a poor prognostic indicator in NSCLC as it significantly correlated with poorer TNM stage, N-status, and overall survival. Our findings suggest that by inhibiting Ras-Raf-MEK-ERK pathway RKIP may play an anti-tumor role in NSCLC.

Transcriptional Repression of Raf Kinase Inhibitory Protein Gene by Metadherin during Cancer Progression.

Raf kinase inhibitory protein (RKIP), also known as a phosphatidylethanolamine-binding protein 1 (PEBP1), functions as a tumor suppressor and regulates several signaling pathways, including ERK and NF-B. RKIP is severely downregulated in human malignant cancers, indicating a functional association with cancer metastasis and poor prognosis. The transcription regulation of RKIP gene in human cancers is not well understood. In this study, we suggested a possible transcription mechanism for the regulation of RKIP in human cancer cells. We found that Metadherin (MTDH) significantly repressed the transcriptional activity of RKIP gene. An analysis of publicly available datasets showed that the knockdown of MTDH in breast and endometrial cancer cell lines induced the expression RKIP. In addition, the results obtained from qRT-PCR and ChIP analyses showed that MTDH considerably inhibited RKIP expression. In addition, the RKIP transcript levels in MTDH-knockdown or MTDH-overexpressing MCF-7 cells were likely correlated to the protein levels, suggesting that MTDH regulates RKIP expression. In conclusion, we suggest that MTDH is a novel factor that controls the RKIP transcription, which is essential for cancer progression.

Phosphatidylethanolamine binding protein 1 enhances sensitivity of gastric cancer cell to 5-fluorouracil via inhibition of cell proliferation, migration and invasion

Purpose: To determine the association between phosphatidylethanolamine binding protein 1, which is an Raf kinase inhibitor protein (RKIP), and 5-fluorouracil (5-FU) via analysis of the association between RKIP and clinical responses in individuals treated using fluorouracil-based chemotherapy.Methods: Human gastric cancer cell lines MGC-803 and SGC-7901 were used in this study. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis and migration were determined with flow cytometry and Transwell chamber assays, respectively. The mRNA and protein expressions of apoptosis-related factors were assayed using realtime polymerase chain reaction (RT-PCR) and Western blotting, respectively, while the expression of RKIP was determined by immunohistochemical staining.Results: Chemotherapeutic drug (5-FU) treatment induced low RKIP expression levels in tumorigenic GC cells, thereby sensitizing the cells to apoptosis (8.57 vs 1.25 %, p < 0.01). The highest RKIP level correlated well with initiation of apoptosis (4.20 vs 1.25 %, p < 0.01). Following in vitro downregulation of RKIP, there was increase in the viability and proliferation of RKIP-inhibited cells over time, and these changes were linked to alterations in cell cycle phases and increased optical density in MTT proliferation assay (1.55 vs 1.18, p < 0.01). In vitro Transwell assay measurement revealed an association between RKIP downregulation and enhancement of cell migration potential (652 vs 436, p < 0.01). Ectopic RKIP expression restored the apoptotic sensitivity of resistant cells (14.30 vs 1.36 %, p <0.01). This sensitization was annulled by upregulation of survival routes. Reduction of RKIP by expression of antisense and siRNA conferred resistance on cancer cells sensitive to 5-FU-mediated apoptosis (6.88 vs 2.13 %, p < 0.01).Conclusion: Thus, RKIP is a promising therapeutic strategy for improving the efficacy of clinically relevant chemotherapeutic drugs for GC.
 Keywords: Gastric cancer, Raf kinase inhibitor protein, Cell proliferation, Invasion, Apoptosis, Chemotherapy, Phosphatidylethanolamine binding protein 1

Clinical significance of raf-1 kinase inhibitor protein in oral squamous cell carcinoma and its role in cell metastasis.

To detect the expression pattern of raf-1 kinase inhibitor protein (RKIP) in oral squamous cell carcinoma (OSCC) samples and to explore its clinical significance in OSCC metastasis. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot assay were conducted to detect the expression of RKIP in OSCC tissues and cells. The relationship between RKIP expression and OSCC clinicopathological characteristics was statistically analyzed. Transwell assay, wound healing assay, and Western blot were used to detect the influence of RKIP on the metastasis ability of OSCC cells. RKIP was significantly downregulated in OSCC samples. Low expression of RKIP predicted high incidence of metastasis in OSCC patients. In vitro experiments demonstrated that overexpression of RKIP could significantly inhibit invasion and migration abilities of OSCC cells. RKIP was a novel factor involved in OSCC progression, which was a potential biomarker and therapeutic target for the patients.

The inhibitor effect of RKIP on inflammasome activation and inflammasome-dependent diseases.

Aberrant inflammasome activation contributes to the pathogenesis of various human diseases, including atherosclerosis, gout, and metabolic disorders. Elucidation of the underlying mechanism involved in the negative regulation of the inflammasome is important for developing new therapeutic targets for these diseases. Here, we showed that Raf kinase inhibitor protein (RKIP) negatively regulates the activation of the NLRP1, NLRP3, and NLRC4 inflammasomes. RKIP deficiency enhanced caspase-1 activation and IL-1β secretion via NLRP1, NLRP3, and NLRC4 inflammasome activation in primary macrophages. The overexpression of RKIP in THP-1 cells inhibited NLRP1, NLRP3, and NLRC4 inflammasome activation. RKIP-deficient mice showed increased sensitivity to Alum-induced peritonitis and Salmonella typhimurium-induced inflammation, indicating that RKIP inhibits NLRP3 and NLRC4 inflammasome activation in vivo. Mechanistically, RKIP directly binds to apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and competes with NLRP1, NLRP3, or NLRC4 to interact with ASC, thus interrupting inflammasome assembly and activation. The depletion of RKIP aggravated inflammasome-related diseases such as monosodium urate (MSU)-induced gouty arthritis and high-fat diet (HFD)-induced metabolic disorders. Furthermore, the expression of RKIP was substantially downregulated in patients with gouty arthritis or type 2 diabetes (T2D) compared to healthy controls. Collectively, our findings suggest that RKIP negatively regulates NLRP1, NLRP3, and NLRC4 inflammasome activation and is a potential therapeutic target for the treatment of inflammasome-related diseases.

Bmi-1-induced miR-27a and miR-155 promote tumor metastasis and chemoresistance by targeting RKIP in gastric cancer

BackgroundWe previously reported an inverse relationship between B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) and Raf kinase inhibitory protein (RKIP), which is associated with the prognosis of gastric cancer (GC). In this study, we further explored the microRNA (miRNA) regulatory mechanism between Bmi-1 and RKIP.MethodsMicroarray analysis was first carried out to identify miRNA profiles that were differentially expressed in cells overexpressing Bmi-1. Then, miRNAs that could regulate RKIP were identified. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to measure the expression of Bmi-1, miR-155, miR-27a and RKIP. RKIP was confirmed as a target of miR-27a and miR-155 through luciferase reporter assays, qRT-PCR and Western blotting. The effects of the Bmi-1/miR-27a/RKIP and Bmi-1/miR-155/RKIP axes on tumor growth, proliferation, migration, invasion, colony-formation ability, metastasis and chemoresistance were investigated both in vitro and in vivo.ResultsThe downregulation of RKIP by Bmi-1 occurred at the protein but not mRNA level. This indicates probable posttranscriptional regulation. miRNA expression profiles of cells with ectopic expression of Bmi-1 were analyzed and compared to those of control cells by microarray analysis. A total of 51 upregulated and 72 downregulated miRNAs were identified. Based on publicly available algorithms, miR-27a and miR-155 were predicted, selected and demonstrated to target RKIP. Bmi-1, miR-27a and miR-155 are elevated in human GC and associated with poor prognosis of GC, while RKIP is expressed at lower levels in GC and correlated with good prognosis. Then, in vitro tests shown that in addition to regulating RKIP expression via miR-27a and miR-155, Bmi-1 was also able to regulate the migration, invasion, proliferation, colony-formation ability and chemosensitivity of GC cells through the same pathway. Finally, the in vivo test showed similar results, whereby the knockdown of the Bmi-1 gene led to the inhibition of tumor growth, metastasis and chemoresistance through miR-27a and miR-155.ConclusionsBmi-1 was proven to induce the expression of miR-27a and miR-155 and thus promote tumor metastasis and chemoresistance by targeting RKIP in GC. Overall, miR-27a and miR-155 might be promising targets for the screening, diagnosis, prognosis, treatment and disease monitoring of GC.

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.

MicroRNA-18a induces epithelial-mesenchymal transition like cancer stem cell phenotype via regulating RKIP pathway in pancreatic cancer.

BackgroundPancreatic cancer is a devastating invasive disease. Understanding the molecular mechanism of metastasis of this cancer is basis for its treatment and prevention.MethodsPancreatic cancer tissues and normal adjacent tissues were collected from patients tour hospital. Western blotting and a sphere growth and invasion assay were performed to conduct analysis. Pancreatic ductal adenocarcinoma cell Line PANC-1 were cultured. To test the level of Raf-1 kinase inhibitor protein (RKIP), immunofluorescence analyses were performed.ResultsIn this study, we showed that expression of RKIP was downregulated in pancreatic cancer. RKIP can inhibit epithelial to mesenchymal transition (EMT) in PANC-1 cells. MicroRNA-181a (miR-181a) has a high expression in pancreatic cancer and can induce EMT phenotype by directly degrading RKIP in pancreatic cancer PANC-1 cells.ConclusionsWe concluded that miR-181a induces EMT phenotype through its regulation of RKIP in pancreatic cancer. MicroRNA-18a may be a novel target in the treatment of pancreatic cancer in future.

Endogenous nocciceptin/orphanin FQ affect ischemic arrhythmias in rats through Raf kinase inhibitor protein

To investigate whether endogenous nociceptin/orphanin FQ (N/OFQ) can inhibit arrhythmia and expression of β1-adrenergic receptor (β1-AR) on the surface of myocardial cell membrane in acute myocardial ischemia rats by Raf kinase inhibitory protein (RKIP). (1) Experiment one: according to random number table method, 30 adult male Sprague-Dawley (SD) rats with only 6 weeks of age were divided into Sham group (open the chest but do not ligate the coronary artery), myocardial ischemia model group (coronary ligation of left anterior descending branch), and endogenous N/OFQ antagonists UFP-101 pretreatment group (UFP-101 group, preoperative 10 minutes after tail vein injection of 1 mL/kg UFP-101), with 10 rats in each group. Arrhythmia was recorded within 15 minutes after operation. The expression of phosphorylated RKIP (p-RKIP) was detected by Western Blot. (2) Experiment two: according to the random number table method, 30 4-week-old male SD rats were divided into UFP-101 control group, RKIP over expression group and RKIP antagonism group, with 10 rats in each group. The UFP-101 control group was intraperiton eally injected with corn oil every day, while the other two groups were injected with up adjuster of RKIP (Didymin). The rats in the three groups were all ligated after 4 weeks of feeding, and UFP-101 was injected through the tail vein 10 minutes before the operation. The RKIP antagonist group received intraperitoneal injection of the RKIP-specific antagonist locostatin 2 hours before surgery. Arrhythmia results were recorded within 15 minutes after operation. Western Blot was used to detect the expression of p-RKIP in myocardial tissue and expression of β1-AR on the surface of myocardial cell membrane 15 minutes after surgery. (1) Experiment one: compared with Sham group, ventricular ectopic beat (VEB), ventricular tachycardia (VT) and ventricular fibrillation (VF) increased significantly in the model group and UFP-101 group, and arrhythmia score increased significantly. In addition, compared with the Sham group, p-RKIP expression was increased in the model group and decreased in the UFP-101 group. Compared with the model group, preconditioning with UFP-101 significantly reduced the occurrence of arrhythmia [arrhythmia score: 1.5 (0.3, 5.0) vs. 4.0 (2.0, 5.0), P < 0.05], and the expression of p-RKIP in myocardial tissue significantly decreased (p-RKIP/total RKIP: 0.20±0.11 vs. 0.43±0.11, P < 0.05). This indicated that antagonistic N/OFQ could reduce the phosphorylation of RKIP and the occurrence of arrhythmia. (2) Experiment two: compared with the UFP-101 control group, overexpression of RKIP significantly increased the occurrence of arrhythmia events, and the expression of β1-AR on the surface of the myocardial cell membrane significantly increased. And antagonism RKIP overexpression could make the occurrence of arrhythmia eased [arrhythmia score: 3.0 (2.0, 3.0) vs. 4.0 (2.0, 5.0), P < 0.05], and significantly reduce the expression of myocardial cell membrane surface β1-AR (β1-AR/Na+-K+-ATPase: 0.88±0.09 vs. 1.02±0.08, P < 0.05), while there was no significant difference in total RKIP expression (total RKIP/GAPDH: 5.40±0.21 vs. 5.36±0.19, P > 0.05). This indicated that endogenous N/OFQ affected the expression of plasma β1-AR on the surface of myocardial cell membrane and ischemic arrhythmia in rats through RKIP. Endogenous N/OFQ can affect the expression of plasma β1-AR on the membrane surface of ischemic myocardium and arrhythmia in rats via increased expression of RKIP phosphorylation.

RAF Kinase Inhibitor Protein in Myeloid Leukemogenesis.

RAF kinase inhibitor protein (RKIP) is an essential regulator of intracellular signaling. A somatic loss of RKIP expression is a frequent event in solid human cancers, and a role of RKIP as metastasis-suppressor is widely accepted nowadays. Recently, RKIP loss has been described in acute myeloid leukemia (AML) and a series of other myeloid neoplasias (MNs). Functional in vitro and in vivo experiments revealed that RKIP is an essential player within the development of these liquid tumors; however, the respective role of RKIP seems to be complex and multi-faceted. In this review, we will summarize the current knowledge about RKIP in myeloid leukemogenesis. We will initially describe its involvement in physiologic hematopoiesis, and will then proceed to discuss its role in the development of AML and other MNs. Finally, we will discuss potential therapeutic implications arising thereof.

Overexpression of Raf-1 kinase inhibitor protein inhibits cell invasion and migration in lung cancer cells through suppressing epithelial-mesenchymal transition

Background: Raf-1 kinase inhibitor protein (RKIP) is a small evolutionary conserved protein that was associated with the Raf-MEK-ERK pathway. However, whether RKIP would alter the invasion and metastasis of non-small cell lung cancer (NSCLC) and play the role through suppressing epithelial-mesenchymal transition (EMT) remains to be explored. Methods: A549 cells were transfected with RKIP-GV141 plasmid for overexpression of RKIP. Colony formation assay and MTT assay were performed to measure the effects of RKIP on the proliferation and cell viability assay of A549 cells. Transwell, Migration Assay and wound healing assay were performed to analyze the effects of RKIP on the invasion and metastasis of A549 cells. E-cadherin and vimentin were measured by Western blot to conform RKIP affects invasion and metastasis of NSCLC via inhibiting EMT. Results: RKIP is downregulated in NSCLC tissues compared to adjacent normal lung tissues by IHC. Decreased expression of RKIP contributes to poor prognosis in lung adenocarcinoma patients. Age and pTNM stage were independent prognostic factors for adenocarcinoma patients. Overexpression of RKIP reduces the cell viability and limits the proliferation, invasion, and migration of A549 cells in vitro . Wound healing assay showed the degressive ability of metastasis. High expression of E-cadherin and low expression of vimentin indicated that RKIP affects invasion and metastasis of NSCLC via inhibiting EMT. Conclusions: RKIP is decreased in NSCLC tissues. Overexpression of RKIP in A549 cells would decrease the cellular proliferation, viability, invasion ability, and metastasis ability probably via inhibiting EMT through upregulating E-cadherin expression and downregulating vimentin expression.

Loss of RAF kinase inhibitor protein is involved in myelomonocytic differentiation and aggravates RAS-driven myeloid leukemogenesis.

RAS-signaling mutations induce the myelomonocytic differentiation and proliferation of hematopoietic stem and progenitor cells. Moreover, they are important players in the development of myeloid neoplasias. RAF kinase inhibitor protein (RKIP) is a negative regulator of RAS-signaling. As RKIP loss has recently been described in RAS-mutated myelomonocytic acute myeloid leukemia, we now aimed to analyze its role in myelomonocytic differentiation and RAS-driven leukemogenesis. Therefore, we initially analyzed RKIP expression during human and murine hematopoietic differentiation and observed that it is high in hematopoietic stem and progenitor cells and lymphoid cells but decreases in cells belonging to the myeloid lineage. By employing short hairpin RNA knockdown experiments in CD34+ umbilical cord blood cells and the undifferentiated acute myeloid leukemia cell line HL-60, we show that RKIP loss is indeed functionally involved in myelomonocytic lineage commitment and drives the myelomonocytic differentiation of hematopoietic stem and progenitor cells. These results could be confirmed in vivo, where Rkip deletion induced a myelomonocytic differentiation bias in mice by amplifying the effects of granulocyte macrophage-colony-stimulating factor. We further show that RKIP is of relevance for RAS-driven myelomonocytic leukemogenesis by demonstrating that Rkip deletion aggravates the development of a myeloproliferative disease in NrasG12D-mutated mice. Mechanistically, we demonstrate that RKIP loss increases the activity of the RAS-MAPK/ERK signaling module. Finally, we prove the clinical relevance of these findings by showing that RKIP loss is a frequent event in chronic myelomonocytic leukemia, and that it co-occurs with RAS-signaling mutations. Taken together, these data establish RKIP as novel player in RAS-driven myeloid leukemogenesis.

Effect of RKIP on proliferation and migration of malignant melanoma cells and potential mechanism

Objective: To explore the effect and mechanism of Rafkinase inhibitor protein (RKIP) on proliferation and migration of malignant melanoma cells in vitro. Methods: The RKIP overexpression and down-regulated stable transfected strains of mouse malignant melanoma cell line B16 were constructed by recombinant lentiviral transfection technique and established as RKIP overexpression group and RKIP down-regulation group, the mouse malignant melanoma B16 cells without any treatment were used as a blank control group, and the proliferation activity and migration ability of each group were detected by cell counting kit-8 (CCK-8) and cell scratch test. The relative expression levels of CyclinD1, Calcium-dependent cell adhesin, Ki-67, Matrix metalloproteinase (MMP)-9, MMP-13, MMP-2 and Phosphatidylethanolamine binding protein (PEBP-1) were detected by quantitative real-time polymerase chain reaction (qPCR). Western blot was used to detect the difference of RKIP expression and the protein expression level of nuclear factor-kappa B (NF-κB) signaling pathway in each group. Results: Comparison of RKIP overexpression group and blank control group shown cell proliferation and migration were significantly inhibited in RKIP overexpression group (0.794±0.038 vs 1.200±0.081) (P<0.001). However, there was no significant difference in cell proliferation between RKIP down-regulation group and blank control group (1.077±0.084 vs 1.200±0.081) (P>0.05), and the cell migration ability of RKIP down-regulation group was significantly higher than that of the blank control group (P<0.001). In addition, there was no significant difference between the RKIP down-regulation group and the blank control group in PEBP-1 expression (P>0.05), while the expression levels of the remaining genes in the RKIP overexpression group were significantly lower than those in the blank control group, and the expression levels in the RKIP down-regulation group were significantly higher than those in the blank control group (P<0.001). Furthermore, the protein level of phosphorylated P65 (p-P65) in RKIP overexpression group was significantly lower than that in blank control group (0.080±0.000 vs 0.236±0.000), and RKIP down-regulation group was significantly higher than that in blank control group (1.139±0.001 vs 0.236±0.000) (both P<0.001). Conclusion: RKIP overexpression can inhibit the proliferation and migration of malignant melanoma cells, which may be related to the regulation of NF-κB signaling pathway-related protein p-P65.

Identifying Crosstalk between Raf Kinase Inhibitor Protein and Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which hyperactive autoantibodies attack and damage healthy tissues and organs. SLE can affect multiple organs, such as the skin, kidneys, joints, and brain. The pathogenesis of SLE is multifaceted and complex, making it difficult to develop targeted therapies to ameliorate symptoms and the onset of disease. A number of signaling pathways have been investigated and shown to be implicated in SLE; however, information regarding the specific pathways involved, at least in part, in the pathogenesis of SLE remains scarce. The role of Raf kinase inhibitor protein (RKIP) in key signaling pathways in cancer is well-studied. However, studies highlighting the role of RKIP in autoimmune diseases and the inflammatory response are emerging. Whereas the induction of RKIP in cancer is associated with improved responses and reduced resistance, the overexpression of RKIP in normal tissues inhibits inflammatory cytokines and chemokines and may also inhibit autoimmunity. In this review, we have analyzed the potential crosstalk between the signaling pathways that regulate SLE and RKIP and whether targeting the pathways that activate the expression of RKIP may prove to be a novel therapeutic tool against the pathogenesis of SLE.

Role of RKIP in human hepatic stellate cell proliferation, invasion, and metastasis.

The purpose of this study was to investigate the effect of Raf kinase inhibitor protein (RKIP) on the growth, apoptosis, invasion, and metastasis of human hepatic stellate cell line (LX-2). A recombinant plasmid (pcDNA3.1-RKIP) or RKIP-targeting small interfering RNA (siRNA) vector (siRNA-RKIP) was transfected into LX-2 cells to interfere with the RKIP expression. The results demonstrated that increased RKIP expression significantly reduced cell viability, clonogenic growth, and invasion. Further, it promoted cell apoptosis and induced cell cycle arrest in the G1 phase. Overexpression of RKIP led to inactivation of LX-2 cells, as evidenced by the decrease in the expression levels of collagen I and α-smooth muscle actin (α-SMA). In addition, increased RKIP expression significantly reduced the phosphorylation of Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), the transcriptional activity of nuclear factor-κB (NF-κB), and the levels of matrix metalloproteinases-1 and -2. In conclusion, these findings clearly demonstrate that RKIP inhibits LX-2 cell growth, metastasis, and activation, primarily by downregulating the ERK/MAPK and NF-κB signaling pathways.

Raf kinase inhibitor protein negatively regulates FcεRI-mediated mast cell activation and allergic response

The signaling cascades triggered by the cross-linkage of immunoglobulin E (IgE) with its high-affinity receptor (FcεRI) on mast cells contribute to multiple allergic disorders, such as asthma, rhinitis, and atopic dermatitis. Restraint of intracellular signals for mast cell activation is essential to restore homeostasis. In this study, we found that Raf kinase inhibitor protein (RKIP) negatively regulated mast cell activation. RKIP-deficient mast cells showed greater IgE-FcεRI-mediated activation than wild-type mast cells. Consistently, RKIP deficiency in mast cells rendered mice more sensitive to IgE-FcεRI-mediated allergic responses and ovalbumin-induced airway inflammation. Mechanistically, RKIP interacts with the p85 subunit of PI3K, prevents it from binding to GRB2-associated binding protein 2 (Gab2), and eventually inhibits the activation of the PI3K/Akt/NF-κB complex and its downstream signaling. Furthermore, the expression of RKIP was significantly down-regulated in the peripheral blood of asthma patients and in the IgE-FcεRI-stimulated mast cells. Collectively, our findings not only suggest that RKIP plays an important role in controlling mast cell-mediated allergic responses but also provide insight into therapeutic targets for mast cell-related allergic diseases.

Raf kinase inhibitor protein protects microglial cells against 1-methyl-4-phenylpyridinium-induced neuroinflammation in vitro

The Raf kinase inhibitor protein (RKIP), belonging to a member of the phosphatidylethanolamine-binding protein (PEBP) family, is involved in regulating neural development. However, the role of RKIP in microglial cells stimulated with 1-methyl-4-phenylpyridinium (MPP+) has not been determined. Thus, in the present study, we investigated the role of RKIP and its underlying mechanism in Parkinson's disease (PD). Our results showed that the expression of RKIP was significantly reduced in BV-2 cells treated with MPP+. Overexpression of RKIP markedly rescued cell viability and inhibited cell apoptosis in BV-2 cells exposed to MPP+. In addition, overexpression of RKIP inhibited MPP+-induced the production of pro-inflammatory molecules in BV-2 cells. Similar results were observed in primary microglial cells isolated from neonatal mice. Exploration of the underlying mechanisms of its action indicated that overexpression of RKIP prevented the activation of NF-κB and MEK/ERK pathways in MPP+-stimulated BV-2 cells. Taken together, these findings indicated that RKIP suppresses apoptosis and inflammation in MPP+-treated microglial cells through the inactivation of NF-κB and MEK/ERK signaling pathways. Thus, RKIP may be a promising target molecular involving in the pathogenesis of PD.

RKIP: A Key Regulator in Tumor Metastasis Initiation and Resistance to Apoptosis: Therapeutic Targeting and Impact.

RAF-kinase inhibitor protein (RKIP) is a well-established tumor suppressor that is frequently downregulated in a plethora of solid and hematological malignancies. RKIP exerts antimetastatic and pro-apoptotic properties in cancer cells, via modulation of signaling pathways and gene products involved in tumor survival and spread. Here we review the contribution of RKIP in the regulation of early metastatic steps such as epithelial–mesenchymal transition (EMT), migration, and invasion, as well as in tumor sensitivity to conventional therapeutics and immuno-mediated cytotoxicity. We further provide updated justification for targeting RKIP as a strategy to overcome tumor chemo/immuno-resistance and suppress metastasis, through the use of agents able to modulate RKIP expression in cancer cells.