RKIP Expression Research Articles

Absence of RKIP expression is an independent prognostic biomarker for gastric cancer patients

Gastric cancer is a leading cause of cancer-related mortality, and the presence of lymph node metastasis an important prognostic factor. Downregulation of RKIP has been associated with tumor progression and metastasis in several types of neoplasms, being currently categorized as a metastasis suppressor gene. Our aim was to determine the expression levels of RKIP in gastric tissues and to evaluate its impact in the clinical outcome of gastric carcinoma patients. RKIP expression levels were studied by immunohistochemistry in a series of gastric tissues. Overall, we analysed 222 non-neoplastic gastric tissues, 152 primary tumors and 42 lymph node metastasis samples. We observed that RKIP was highly expressed in ~83% of non-neoplastic tissues (including normal tissue and metaplasia), was lost in ~56% of primary tumors and in ~90% of lymph node metastasis samples. Loss of RKIP expression was significantly associated with several markers of poor clinical outcome, including the presence of lymph node metastasis. Furthermore, the absence of RKIP protein constitutes an independent prognostic marker for these patients. In conclusion, RKIP expression is significantly lost during gastric carcinoma progression being almost absent in lymph node metastasis samples. Of note, we showed that the absence of RKIP expression is associated with poor outcome features of gastric cancer patients, this being also an independent prognostic marker.

Raf Kinase Inhibitor RKIP Inhibits MDA-9/Syntenin-Mediated Metastasis in Melanoma

Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, functions as a positive regulator of melanoma progression and metastasis. In contrast, the Raf kinase inhibitor, RKIP, a negative modulator of RAF-stimulated MEKK activation, is strongly downregulated in metastatic melanoma cells. In this study, we explored a hypothesized inverse relationship between MDA-9 and RKIP in melanoma. Tumor array and cell line analyses confirmed an inverse relationship between expression of MDA-9 and RKIP during melanoma progression. We found that MDA-9 transcriptionally downregulated RKIP in support of a suggested cross-talk between these two proteins. Furthermore, MDA-9 and RKIP physically interacted in a manner that correlated with a suppression of FAK and c-Src phosphorylation, crucial steps necessary for MDA-9 to promote FAK/c-Src complex formation and initiate signaling cascades that drive the MDA-9-mediated metastatic phenotype. Finally, ectopic RKIP expression in melanoma cells overrode MDA-9-mediated signaling, inhibiting cell invasion, anchorage-independent growth, and in vivo dissemination of tumor cells. Taken together, these findings establish RKIP as an inhibitor of MDA-9-dependent melanoma metastasis, with potential implications for targeting this process therapeutically.

Prognostic Value of Raf Kinase Inhibitor Protein in Esophageal Squamous Cell Carcinoma

Raf kinase inhibitory protein (RKIP, also PEBP1) is involved in regulation of multiple cellular signaling processes and suppressing metastasis in animal models. Downregulation of RKIP expression has been shown to promote tumor progression in a variety of human cancers. However, its role and clinical significance in resectable esophageal squamous cell carcinoma (ESCC) is still scanty. The purpose of this study was to investigate the prognostic significance of RKIP expression by immunohistochemistry in a group of patients with ESCC treated with surgical resection. RKIP expression in 233 surgically resected ESCC specimens and 49 cases of adjacent normal tissues was detected by using immunohistochemical staining. The clinical and prognostic significance of RKIP expression was statistically analyzed. Kaplan-Meier analysis was used to compare the postoperative survival between groups. Significant downregulation was noted for RKIP protein in ESCCs, compared to adjacent normal tissues (p < 0.001). A lower disease-free survival and overall survival of ESCC was found in patients whose tissues had low RKIP expression (both P < 0.001). In addition, RKIP expression could stratify the patient survival (disease-free survival/overall survival) in stage II (P = 0.01 and 0.02, repectively). The Cox proportionate hazard regression model also established that low expression of RKIP was significantly correlated with increased risk (RR = 3.572) of recurrence compared with high RKIP expression (P < 0.001). Furthermore, the results of multivariate analysis suggested that RKIP expression (P < 0.001) was an independent factor that affected overall survival. These findings suggest that the low expression of RKIP be associated with poor survival in resectable ESCC patients.

Role of NF-κB/Snail/RKIP loop in the response of tumor cells to photodynamic therapy

Photodynamic therapy (PDT) is a therapeutic modality whose efficacy depends on several factors including type of photosensitizer, light fluence and cellular response. Cell recurrence is one of the problems still unsolved in PDT. In this work we found that in B78-H1 murine amelanotic melanoma cells there is a correlation between cell recurrence and the NF-κB/Snail/RKIP loop. Proliferation and migration of surviving cells were analyzed by MTT and wound-scratch assays. The levels of ROS/NO in B78-H1 melanoma cells treated with pheophorbide a (Pba) and light (Pba/PDT) were measured by FACS, while expression of NF-κB, Snail and RKIP were determined by Western blots. The mechanism of cell death was investigated by caspase and microscopy assays. Our data show that after a low-dose Pba/PDT treatment, B78-H1 cells are able to recover. This correlates with a low level of NO production, which blocks apoptosis via NF-κB pathway. Western blot analyses showed that a low-dose Pba/PDT increases the expression of NF-κB and anti-apoptotic Snail, but reduces the expression of pro-apoptotic RKIP. The role played by NF-κB in the modulation of Snail and RKIP was investigated using DHMEQ: a NF-κB inhibitor which behaves as NO donor. DHMEQ caused a decrease of Snail and an increase of RKIP expression. When B78-H1 cells were treated with a low dose Pba/PDT and DHMEQ, the NO level strongly increased, with the result that Snail was down-regulated and RKIP was upregulated, as observed with a high-dose Pba/PDT. One major problem in PDT is the cellular rescue occurring in tissue regions receiving a low-dose PDT. To minimize this problem and sensitize cancer cells to PDT we propose a combined treatment in which the photosensitizer is delivered with a donor of NO acting on the NF-κB/Snail/RKIP loop.

Expression of phosphorylated raf kinase inhibitor protein (pRKIP) is a predictor of lung cancer survival

BackgroundRaf-1 kinase inhibitor protein (RKIP) has been reported to negatively regulate signal kinases of major survival pathways. RKIP activity is modulated in part by phosphorylation on Serine 153 by protein kinase C, which leads to dissociation of RKIP from Raf-1. RKIP expression is low in many human cancers and represents an indicator of poor prognosis and/or induction of metastasis. The prognostic power has typically been based on total RKIP expression and has not considered the significance of phospho-RKIP.MethodsThe present study examined the expression levels of both RKIP and phospho-RKIP in human lung cancer tissue microarray proteomics technology.ResultsTotal RKIP and phospho-RKIP expression levels were similar in normal and cancerous tissues. phospho-RKIP levels slightly decreased in metastatic lesions. However, the expression levels of phospho-RKIP, in contrast to total RKIP, displayed significant predictive power for outcome with normal expression of phospho-RKIP predicting a more favorable survival compared to lower levels (P = 0.0118); this was even more pronounced in more senior individuals and in those with early stage lung cancer.ConclusionsThis study examines for the first time, the expression profile of RKIP and phospho-RKIP in lung cancer. Significantly, we found that phospho-RKIP was a predictive indicator of survival.

Prognostic Significance of TWEAK Expression in Colorectal Cancer and Effect of Its Inhibition on Invasion

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been implicated in tumor development and progression. The aim of this study was to investigate the role of TWEAK in colorectal cancer (CRC) progression. To investigate the involvement of TWEAK in the progression of human CRC, normal, and tumor specimens from 174 patients were analyzed immunohistochemically for the expression of TWEAK. TWEAK recombinant protein treatment, transfection of expression plasmids, and small interfering RNA to knockdown TWEAK expression were performed to test invasive ability with a Boyden chamber. The mRNA expression profile in recombinant TWEAK treatment was compared to a control group by microarray analysis. To identify downstream effectors, Raf kinase inhibitor (RKIP) and its correlation with TWEAK in vitro and in vivo were examined by quantitative real-time polymerase chain reaction and invasion assays. CRC patients whose tumors displayed high TWEAK expression had a statistically significantly higher overall survival and a disease-free advantage over those with a low TWEAK expression. In in vitro invasion assays, alterations in TWEAK expression in CRC cell lines inversely modulated their invasive ability. By means of integrated genomics, we identified RKIP as a downstream effector in TWEAK-mediated invasion inhibition. Knockout of RKIP expression in HCT116 cells by short hairpin RNA (shRKIP) resulted in increased invasiveness. Clinically, RKIP and TWEAK mRNA expression showed strong positive correlations in CRC patient samples. Our results implicate TWEAK as a key regulator of CRC invasion, and it appears to be a useful prognostic factor for patients with CRC.

Expression of Raf kinase inhibitor protein and its significance in invasion and metastasis of hepatocellular carcinoma

To investigate the expression of RKIP, p65 and pERK in hepatocellular carcinoma (HCC) and theIr correlation with invasion and metastasis of HCC. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of RKIP mRNA. The expression levels of RKIP, p65 and pERK proteins in HCC tumor and peritumoral tissues were determined by immunohistochemistry and Western blot analysis. Statistical analysis was performed to determine the relationship between their expression and clinicopathological parameters. RKIP protein expression level (RKIP/actin) was 0.579 ± 0.380 in HCCs, 1.178 ± 0.659 in peritumoral tissues and 1.115 ± 0.442 in normal liver tissues. The pERK protein level was 1.023 ± 0.478, 0.605 ± 0.367 and 0.461 ± 0.293, p65 protein level was 0.83 ± 0.376, 0.63 ± 0.337 and 0.466 ± 0.345, respectively. Immunohistochemistry analysis showed that the RKIP positive rates in HCCs, peritumoral tissues and normal liver tissues, were 22.2%, 86.0%, and 93.8%, positive rates of p65 were 73.6%, 56.0% and 37.5%, positive rates of pERK were 65.3%, 38.0% and 31.3%, respectively. Statistical analysis revealed that there was a significant difference in RKIP protein expression levels (P < 0.05), but no significant difference in RKIP mRNA expression levels (P > 0.05) among HCC tumors, peritumoral tissues and normal liver tissues. The p65-positive and pERK-positive rates were higher in tumor tissues than that in peritumoral tissues and in normal liver tissues (P < 0.05), but RKIP-positive rates were lower in tumor tissues than that in paritumoral tissues and normal liver tissues (P < 0.05). RKIP protein expression levels were significantly lower in HCCs with intrahepatic or lymphatic metastasis than that in without. The RKIP positive rates in moderately and well differentiated HCCs were significantly higher than that in poorly differentiated HCCs. There was a relationship between RKIP and pERK expressions (P = 0.04), but RKIP expression was not correlated with p65 expression in HCCs (P = 0.143). Our findings indicate that the down-regulation of RKIP expression may serve as a predictive marker for HCC development, progression and metastasis, which may contribute to the elevated ERK activity. The inhibiting effect of RKIP on invasion and metastasis of liver cancer cells may be due to the down-regulation of pERK expression rather than p65 expression.

The Role Of Raf-1 Kinase Inhibitory Protein (RKIP) In Colorectal Cancer Liver Metastasis

Introduction: Colorectal cancer (CRC) remains the second most frequent cause of cancer-related mortality in the United States. The majority of these deaths are secondary to metastatic disease. Emerging data suggest the idea that RKIP is a metastasis suppressor. The loss of RKIP expression has been reported to be a marker of CRC progression and metastasis. Despite this correlation, the mechanism of RKIP inhibition of CRC metastasis remains undefined. We hypothesize that RKIP expression directly inhibits cell invasion and metastasis by regulating the MAPK/ERK signaling pathway.

Role of Raf Kinase Inhibitor Protein in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) accounts for 80-90% of primary liver tumors and is one of the most common and devastating malignant diseases worldwide. The MAPK signaling pathway is activated in over 90% of HCCs, and RKIP has been identified as an inhibitor of the MAPK pathway. It has been observed that downregulation of RKIP expression in HCC tumors contributes to constitutive activation of the ERK/MAPK pathway and promotes proliferation and migration of HCC cells. More important, activation of IGF-I/ERK/MAPK pathways can be blocked by restoration of RKIP levels. The protein levels of RKIP are significantly reduced in HCC, whereas mRNA levels only decreased in 41% of HCC samples studied, suggesting that the downregulation of RKIP in HCC may be influenced through multiple mechanisms both at the mRNA and protein levels. In this context, mTOR inhibitor, insulin, and proteasome inhibitors were found to modulate RKIP expression in FOCUS HCC cells. A better understating of mechanisms by which RKIP expression is downregulated in HCC may be critical to develop a possible target for therapeutic intervention of HCC.

Clinical Significance of RKIP and Phosphorylated RKIP Expression in Human Cancers

Clinical Significance of RKIP and Phosphorylated RKIP Expression in Human Cancers

Induction of RKIP Expression by Anticancer Therapeutic Antibodies and Its Role in the Reversal of Chemo- and Immunoresistance

Induction of RKIP Expression by Anticancer Therapeutic Antibodies and Its Role in the Reversal of Chemo- and Immunoresistance

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.

Loss of RAF Kinase Inhibitor Protein Is a Frequent Event In Acute Myeloid Leukemia with a Monocytic Phenotype and Cooperates with Mutant RAS In Malignant Transformation

AbstractAbstract 4185RAF kinase inhibitor protein (RKIP) has been described as a negative regulator of the RAS-mitogen activated protein kinase (MAPK) signaling cascade and as a metastasis suppressor gene in solid tumors. As constitutive activation of this pathway occurs frequently in acute myeloid leukemia (AML), we investigated the role of RKIP in the pathogenesis of this disorder. Analysis of RKIP expression by Western blot revealed complete or partial loss in 21/96 (22%) AML patient samples and 5/19 (26%) AML cell lines, but in none of ten purified normal CD34+ hematopoietic stem and precursor cell specimens. To evaluate the biological consequences of RKIP loss in AML, we examined the effects of introducing an RKIP transgene in 32D murine hematopoietic cells and THP-1 AML cells, the latter characterized by low RKIP expression. Overexpression of RKIP induced a significant decrease in the number of viable cells in both cell lines which was due to a decrease in proliferation as measured by cell cycle analysis after bromodeoxyuridine (BrdU) labeling. However, no differences were shown in the percentage of apoptotic cells - subG1 peak – in these assays, a finding that was confirmed by analysis of PARP- and caspase-3 cleavage. Importantly, colony formation of leukemic THP-1 cells in soft agar was significantly decreased following RKIP reconstitution going along with a tumor suppressor function of RKIP in hematopoietic cells. In a next step, we analyzed RKIP expression in a previously described transcriptomic data set of 285 AML patients (Valk et al. NEJM 2004) and sought to identify cooperating alterations. 28/285 (10%) patients harbored mutations in either NRAS or KRAS, 84/285 (29%) in NPM1, 17/285 (6%) in CEBPA, 31/285 (11%) in the FLT3 tyrosine kinase domain and 68/285 (24%) a FLT3 internal tandem duplication. Out of these, only mutations in RAS were significantly associated with decreased expression of RKIP (P = 0.00005) suggesting co-existence of mutant RAS and RKIP loss in AML. To test for a functional synergism, we employed NIH3T3 cells, a system commonly used to study RAS biology. The oncogenic potential of mutant RAS – as assessed by its ability to transform cells and induce focus formation – could be significantly reduced by simultaneous overexpression of RKIP indicating cooperation between RAS mutations and RKIP loss in malignant transformation. We next analyzed if RKIP loss was associated with specific AML features and observed a highly significant correlation with monocytic phenotypes (FAB M4 and M5) in two independent patient cohorts (cohort one with 96 patients, P = 2.13 × 10-7; cohort two with 285 patients, P = 1.6 × 10-8). To test for a causative role of RKIP in monocytic differentiation, we analyzed immature HL-60 AML cells, a cell line which can be converted into a monocytic phenotype by incubation with 1,25-dihydroxyvitamin D3 (1,25 D3). Whereas siRNA knockdown of RKIP alone failed to induce the monocytic phenotype, 1,25 D3 driven effects could be significantly increased by siRNA knockdown of RKIP and inhibited by RKIP overexpression indicating that RKIP loss is involved in the development of a monocytic AML phenotype. In conclusion, our data demonstrate that loss of RKIP is a frequent event in AML, particularly in subgroups with a monocytic phenotype. RKIP acts as a tumor suppressor in hematopoietic cells and cooperates with mutant RAS in malignant transformation. Disclosures:No relevant conflicts of interest to declare.

Functional Roles of Multiple Feedback Loops in Extracellular Signal-Regulated Kinase and Wnt Signaling Pathways That Regulate Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a key event in the generation of invasive tumor cells. A hallmark of EMT is the repression of E-cadherin expression, which is regulated by various signal transduction pathways including extracellular signal-regulated kinase (ERK) and Wnt. These pathways are highly interconnected via multiple coupled feedback loops (CFL). As the function of such coupled feedback regulations is difficult to analyze experimentally, we used a systems biology approach where computational models were designed to predict biological effects that result from the complex interplay of CFLs. Using epidermal growth factor (EGF) and Wnt as input and E-cadherin transcriptional regulation as output, we established an ordinary differential equation model of the ERK and Wnt signaling network containing six feedback links and used extensive computer simulations to analyze the effects of these feedback links in isolation and different combinations. The results show that the feedbacks can generate a rich dynamic behavior leading to various dose-response patterns and have a decisive role in determining network responses to EGF and Wnt. In particular, we made two important findings: first, that coupled positive feedback loops composed of phosphorylation of Raf kinase inhibitor RKIP by ERK and transcriptional repression of RKIP by Snail have an essential role in causing a switch-like behavior of E-cadherin expression; and second, that RKIP expression inhibits EMT progression by preventing E-cadherin suppression. Taken together, our findings provide us with a system-level understanding of how RKIP can regulate EMT progression and may explain why RKIP is downregulated in so many metastatic cancer cells.

T1186 Loss of RKIP Expression is Associated With Metastasis in Esophageal Adenocarcinoma

The PTEN phosphatase acts on phosphatidylinositols resulting from phosphatidylinositol 3-kinase (PI3K) activation. Germinal inactive mutations of PTEN have been identified in Cowden syndrome, a disease characterized by the development of hamartomatous polyps in the digestive tract and associated with an increased risk of cancer. However, little is known about the specific cellular role of PTEN in human intestinal epithelial cells. Objective. To investigate the role of PTEN in human colorectal cancer cells. Methods. PTEN expression was analyzed by Western blot in human colorectal tumours, in normal epithelial crypt cells (HIEC) and in many colorectal cancer cell lines: Caco-2/15, DLD-1, HCT116, HT29, LoVo, Colo205 and SW480. Caco-2/15, HCT116 and CT-26 cells were infected with recombinant lentiviruses expressing a shRNA that specifically knocked-down PTEN. The impact of PTEN downregulation was analyzed on cell migration (wound assay), invasion (matrigel-coated transwells) and on tumour and metastasis formation in mice. Results. 1PTEN expression is decreased in approximately 60% of all colon cancer tumours of all grades. 2PTEN is expressed in all cancer cell lines but at a lower level than normal human intestinal epithelial cells. 3The lentiviral infection of PTEN shRNA significantly increases spreading, migration and invasion capacities of Caco-2/15 and HCT116 cells. 4PTEN downregulation in both Caco-2/15 and HCT116 cells increases tumour size following subcutaneous injection of these cells in nude mice. 5Loss of PTEN expression in CT-26 (mouse colorectal cancer cells) leads to an increase in their metastatic potential following tail-vein and intrasplenic injections in BALB/c mice. Conclusions. Altogether, these results indicate that PTEN is involved in the control of migration/invasion capacity and tumorigenic/metastatic potential of human colon cancer cells. Supported by CIHR Team grant.

The anti-CD20 Mab R603 induces PTEN and RKIP expression in B-NHL cells: role in inhibition of constitutively activated Akt and NF-κB survival anti-apoptotic pathways

The anti-CD20 Mab R603 induces PTEN and RKIP expression in B-NHL cells: role in inhibition of constitutively activated Akt and NF-κB survival anti-apoptotic pathways

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

Raf kinase inhibitor protein (RKIP) has been shown to be a metastasis suppressor in many kinds of malignant tumors. But its function in breast cancer was not yet clarified completely. We detected RKIP expression in clinical samples of primary breast cancer, breast cancer metastases, and in different breast cancer cells. Compared with the normal breast epithelia, benign breast epithelia, or in situ ductal carcinoma, the expression level of RKIP is decreased in invasive carcinoma and significantly reduced or lost in the metastasis lymph node matched to the invasive carcinoma. To explore the potential role of RKIP in breast cancer metastasis, we studied the effect of RKIP on the malignant phenotypes of the breast cancer cells with ectopically overexpression or knockdown of RKIP. Cell proliferation, soft-agar colony formation, in vitro adhesion assay, invasion, and migation assays were done to examine the malignant phenotypes of the transfected cells. Consequently, RKIP has no effect on in vitro proliferation rate or colony-forming ability of MDA-MB-435 cells. In vitro cell invasion and migration assays indicated that the RKIP expression was inversely associated with the invasiveness of MDA-MB-435 cells. Consistent with these results, in the orthotopic murine models, we observed that overexpression of RKIP in breast cancer cells impaired invasiveness and metastasis, whereas down-regulation of RKIP expression promoted invasiveness and metastasis. These results indicate that RKIP is a metastasis suppressor gene of human breast cancer.

BRAF and RKIP are significantly decreased in cutaneous squamous cell carcinoma

Background. Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). However, little is known about the implication of BRAF and RKIP expression, or about the incidence of BRAF mutations in the formation of these cutaneous diseases. The RAS oncogene has been proposed to significantly contribute to skin cancer development. Moreover, numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines.Objectives. This study aimed to measure the mRNA levels of the genes BRAF and RKIP in AK, as well as their possible implication in the progress of AK to SCC. All biopsy specimens were also screened for BRAF mutations within exons 11 and 15.Patients and methods. Expression levels of the genes BRAF and RKIP were examined in 16 AKs and 12 SCCs by RT-qPCR. A novel allele-specific qPCR method, in combination with direct DNA sequencing, was performed in order to inspect the frequency of the V600E mutation in exon 15, as well as to examine the mutation status of the gene within exon 11.Results. Significant down-regulation was noted for both genes in SCC, compared to normal tissue (for BRAF, P=0.002; for RKIP, P

Raf kinase inhibitor protein: mechanism of loss of expression and association with genomic instability

Aims:Raf kinase inhibitory protein (RKIP; also known as PEBP, for phosphatidylethanolamine-binding protein) is an endogenous inhibitor of the Raf– MAPK kinase (MEK)–MAP kinase pathway. It has emerged as a significant...

RKIP and BRAF aberrations in human nasal polyps and the adjacent turbinate mucosae

Background Little is known about the implication of BRAF and RKIP expression, or about the incidence of BRAF mutations in the formation of nasal polyposis. Objectives To determine the expression levels of the genes BRAF and RKIP, and to inspect the frequency of BRAF mutations in exons 11, 14 and 15 in human nasal polyps (NP). Patients and methods We analyzed 24 human NP specimens and their adjacent inferior and middle turbinates (AIT and AMT), as well as 14 control subjects [bearing 14 Control Inferior Turbinates (CIT) and 14 Control Middle Turbinates (CMT), in total]. The expression pattern of BRAF and RKIP was assessed with real-time RT-PCR. A real-time allele-specific PCR method, in combination with direct sequencing, was performed in order to inspect the frequency of the V600E mutation in exon 15, and to examine mutation status within exons 11 and 14. Results The control mucosae presented significantly higher mRNA levels for both genes, compared to the NP and the AIT–AMT. Moreover, in NP, AIT and AMT, RKIP was found to present higher mRNA levels, in relation to the equivalent values of the BRAF gene ( P = 0.003 in NP; P < 0.001 both in AIT and AMT). No mutation was detected in exon 14, whereas a silent mutation (A1380G, G460G) was noted for one NP sample in exon 11. Another NP sample was found to carry two mutations, one T1799A (V600E) and one A1801G (K601E). A significant co-expression of the two genes was noted in NP ( P = 0.012) and AIT ( P = 0.019). Conclusion The results of the expression levels of RKIP and BRAF, reflect the strong connection between the two genes. RKIP could play an important role in the down-regulation of wild-type BRAF, serving thus as an endogenous inhibitor of the MAPK pathway in nasal polyps and their adjacent turbinate mucosa.