The tobacco phosphatidylethanolamine-binding protein NtFT4 simultaneously improves vitality, growth, and protein yield in human cells.

The production of biopharmaceutical proteins in mammalian cells by transient expression or stable transformation requires robust and viable cells. Cell line engineering must therefore balance improved cell growth and viability with high productivity. We tested the ability of nonmammalian phosphatidylethanolamine-binding proteins to enhance cell proliferation in monolayers and suspension cultures. The tobacco protein NtFT4 improved the proliferation of multiple human cell lines. Viable cell density is usually impaired by efficient transfection, but we found that the number of HEK-293TNtFT4 cells at the peak of protein expression was twice that of standard HEK-293T cells, and the antibody yield increased by approximately one-third. Improved growth and viability were observed in different cell lines, in different culture media, and also after transient transfection, suggesting the beneficial trait is consistent and transferable. Additional modifications could boost the productivity of high-density HEK-293TNtFT4 cells even further as we showed for a fluorescent marker protein and recombinant antibody expressed in monolayer cultures. The HEK-293TNtFT4 cell line provides a new human model platform that increases cell proliferation, also achieving a fundamental improvement in recombinant protein expression.

Downregulation of Raf-1 kinase inhibitory protein as a sorafenib resistance mechanism in hepatocellular carcinoma cell lines.

PURPOSE: Although sorafenib enhances overall survival, sorafenib resistance has been reported to be a significant limiting factor for improved prognosis in patients with hepatocellular carcinoma (HCC). Therefore, it is important to identify the mechanism of sorafenib resistance. This study aimed to identify the causative factor of sorafenib resistance and suggest methods for overcoming it. METHODS: The sensitivity to sorafenib was compared in human HCC cell lines and patient-derived HCC primary cells. Based on its cytotoxicity, signaling pathways altered by sorafenib and the causative factors were examined through assays. The mechanism by which sorafenib modified the sorafenib-resistance inducer through gene or protein expression or stability was also investigated. We also designed a treatment option to overcome sorafenib resistance. RESULTS: Sorafenib activated the Raf/MEK/ERK pathway and caused sorafenib resistance in HCC cell lines and patient-derived HCC primary cells. Sorafenib reactivated the MAPK pathway by down-regulating RKIP at the post-translational level. Knockdown of RKIP increased phosphorylated ERK and thus suppressed sorafenib-mediated cell death. We also found that sorafenib-reactivated ERK maybe an attractive target for second-line therapy for patients with sorafenib resistance. Sequential combination treatment with sorafenib and PD98059 significantly reduced the viability and proliferation of sorafenib-resistant cells, while their increasing apoptosis efficacy. CONCLUSION: Reactivation of the Raf/MEK/ERK pathway through aberrant expression of RKIP is one of the mechanisms behind sorafenib resistance in HCC. Sequential combination treatment with sorafenib and PD98059 could provide a new strategy to overcome sorafenib resistance in future clinical studies.

Raf Kinase Inhibitor Protein (RKIP) Inhibits Tumor Necrosis Factor-α (TNF-α) Induced Adhesion Molecules Expression in Vascular Smooth Muscle Bells by Suppressing (Nuclear Transcription Factor-κB (NF-kappaB) Pathway.

BACKGROUND Raf kinase inhibitor protein (RKIP) regulates growth and differentiation and plays a role in key signal transduction cascades in mammalian cells. Nevertheless, the underlying mechanism for which RKIP regulates cell-cell adhesion remains unknown. Our study investigated the function of the RKIP overexpression on adhesion molecules expression induced by tumor necrosis factor (TNF)-α in cultured mouse vascular smooth muscle cells (MOVACs). MATERIAL AND METHODS The expression levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were detected by ELISA kit, reverse transcription-PCR, and western blot assays. The protein expression of RKIP, p65, and inhibitor of nuclear factor (NF)-κBα (IκBα) were detected by western blot analysis. The activity of NF-kappaB was determined using a Dual-Luciferase Reporter assay. RESULTS The results showed that MOVACs transfected with pCMV5-HA-RKIP significantly inhibited TNF-α induced mRNA and protein expression of ICAM-1 and VCAM-1. The adhesion of THP-1 cells was also detected and inhibited by pCMV5-HA-RKIP in TNF-α-treated MOVACs. RKIP also suppressed the TNF-α-induced activation of NF-kappaB and the protein expression of phosphorylated IκB-α, and promoted the protein expression of IkB-α and nuclear translocation of p65 NF-kappaB. Furthermore, RKIP and the inhibitor of NF-kappaB (BAY11-7082) reduced the upregulation of ICAM-1 and VACM-1 induced by TNF-α. CONCLUSIONS Taken together, these results suggested that RKIP may inhibit the TNF-α-induced expression of adhesion molecules in MOVACs through inactivation of the NF-kappaB pathway.

Inverse correlation between the metastasis suppressor RKIP and the metastasis inducer YY1: Contrasting roles in the regulation of chemo/immuno-resistance in cancer.

Several gene products have been postulated to mediate inherent and/or acquired anticancer drug resistance and tumor metastasis. Among these, the metastasis suppressor and chemo-immuno-sensitizing gene product, Raf Kinase Inhibitor Protein (RKIP), is poorly expressed in many cancers. In contrast, the metastasis inducer and chemo-immuno-resistant factor Yin Yang 1 (YY1) is overexpressed in many cancers. This inverse relationship between RKIP and YY1 expression suggests that these two gene products may be regulated via cross-talks of molecular signaling pathways, culminating in the expression of different phenotypes based on their targets. Analyses of the molecular regulation of the expression patterns of RKIP and YY1 as well as epigenetic, post-transcriptional, and post-translational regulation revealed the existence of several effector mechanisms and crosstalk pathways, of which five pathways of relevance have been identified and analyzed. The five examined cross-talk pathways include the following loops: RKIP/NF-κB/Snail/YY1, p38/MAPK/RKIP/GSK3ß/Snail/YY1, RKIP/Smurf2/YY1/Snail, RKIP/MAPK/Myc/Let-7/HMGA2/Snail/YY1, as well as RKIP/GPCR/STAT3/miR-34/YY1. Each loop is comprised of multiple interactions and cascades that provide evidence for YY1's negative regulation of RKIP expression and vice versa. These loops elucidate potential prognostic motifs and targets for therapeutic intervention. Chiefly, these findings suggest that targeted inhibition of YY1 by specific small molecule inhibitors and/or the specific induction of RKIP expression and activity are potential therapeutic strategies to block tumor growth and metastasis in many cancers, as well as to overcome anticancer drug resistance. These strategies present potential alternatives for their synergistic uses in combination with low doses of conventional chemo-immunotherapeutics and hence, increasing survival, reducing toxicity, and improving quality of life.

Reduced RKIP Expression is Associated With Breast Neoplastic Progression and is Correlated With Poor Outcomes and Aberrant Methylation in Breast Carcinoma.

Raf kinase inhibitor protein's (RKIP) downregulation can predict poor outcome in patients with various types of malignancy. In this study, we aimed to assess the potential involvement of RKIP in breast carcinogenesis and to evaluate its association with outcome variables and aberrant promoter methylation in breast carcinoma (BC). Tissue microarray sections were immunostained for RKIP in 26 normal breasts, 25 usual ductal hyperplasia, 76 ductal carcinoma in situ, and 198 BC specimens. The methylation status of RKIP was also determined in BC. In addition, the mRNA and protein level of RKIP was analyzed in 8 pairs of BC tissues and surrounding normal tissues by quantitative real-time polymerase chain reaction and Western blot analysis, respectively. RKIP mRNA and protein expression was significantly downregulated in BC tissues compared with the surrounding normal tissues (P<0.05 and P<0.01, respectively). Reduced RKIP expression seemed to increase progressively from normal breast to BC (P<0.001). Reduced RKIP expression was significantly associated with metastatic relapse (P<0.001) and was identified as an independent adverse prognostic indicator for disease-free survival (P=0.003). Reduced RKIP expression in BC was significantly correlated with its aberrant promoter methylation (P<0.05). In conclusion, downregulation of RKIP plays an important role in the breast neoplastic progression and correlates with poor prognosis in patients with BC. Aberrant RKIP methylation is one of the mechanisms that lead to downregulation of RKIP in BC.

Effects of Raf kinase inhibitor protein expression on pancreatic cancer cell growth and motility: an in vivo and in vitro study.

PURPOSE: Raf kinase inhibitor protein (RKIP) is a tumor suppressor that inhibits cell growth and metastasis of malignant tumors. Pancreatic cancer is a leading cause of cancer death with a low survival rate. RKIP expression and its role in tumorigenesis and metastasis in pancreatic cancer are poorly understood. The aims of our study were to assess the effects of RKIP on pancreatic carcinoma cells in vitro and in tumor tissues in vivo. METHODS: This study included 84 patients with histologically confirmed pancreatic adenocarcinoma. The expression levels of RKIP were measured in pancreatic cancer tissues and adjacent normal tissues using real-time PCR and immunohistochemistry. Overexpression plasmid of RKIP was transfected into SW1990 and AsPC-1 cell lines, and the effects on cell proliferation were studied using a Cell Counting Kit-8 assay. MEK1/2 and ERK1/2 were detected by Western blot and immunofluorescence assay. RESULTS: Results showed a reduced expression of RKIP in pancreatic carcinoma tissues compared with adjacent normal tissues, which closely correlated with patient outcomes. Overexpression of RKIP suppressed cell proliferation and promoted apoptosis in cultured SW1990 and AsPC-1 cell lines. Transwell assay showed RKIP can inhibit cell migration and invasion, and in vivo RKIP can suppress tumorigenesis by diminishing the volume of the tumors. CONCLUSIONS: In conclusion, expression of RKIP is closely correlated with the survival of pancreatic cancer patients. RKIP can inhibit pancreatic adenocarcinoma cells proliferation, activities of migration and invasion, through downregulating Raf-1-MEK1/2-ERK1/2 signaling pathway.

Role of Raf-kinase inhibitor protein in colorectal cancer and its regulation by hydroxycamptothecine.

BACKGROUND: Recently accumulated evidence suggests that Raf kinase inhibitor protein (RKIP) participates in regulation of many signaling pathways and plays an important role in tumorigenesis and tumor metastasis. However, studies investigating the role of RKIP in colorectal cancer have not been reported. The aim of this study was to investigate the role of RKIP on colorectal cancer cell differentiation, progression and its correlation with chemosensitivity. RESULTS: Immunohistochemical analysis revealed that RKIP expression was higher in non-neoplastic colorectal tissue (NCRCT) and colorectal cancer tissue (CRCT) than that in metastatic lymph node tissue (MLNT) (P <0.05). P-ERK protein expression was higher in MLNT and CRCT than that in NCRCT (P = 0.02). Immunocytochemical analysis further revealed that RKIP expression was higher in the well differentiated cell line SW1116 as compared to that in the poorly differentiated cell line LoVo. Matrigel invasive assay demonstrated that the inhibition of RKIP by short hairpin RNA (shRNA) 271 transfection significantly increased the number of migrated cells (90.67 ± 4.04 vs. 37.33 ± 2.51, P <0.05), whereas over-expression of RKIP by PEBP-1 plasmid transfection significantly suppressed the number of migrated cells (79.24 ± 5.18 vs. 154.33 ± 7.25, P <0.05). Meanwhile, down-regulation of RKIP induced an increase in the cell survival rate by inhibiting apoptosis induced by hydroxycamptothecine. CONCLUSIONS: RKIP was also found to be associated with cell differentiation, with a higher activity in well differentiated colorectal cancer cells than in poorly differentiated ones. The upregulated expression of RKIP in colorectal cancer cells inhibited cell invasion and metastasis, while downregulation of RKIP reduced chemosensitivity by inhibiting apoptosis induced by HCPT.

Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling.

The role and underlying mechanism of Raf kinase inhibitory protein (RKIP) in nasopharyngeal carcinoma (NPC) metastasis remain unclear. Here, we showed that RKIP was downregulated in the NPC with high metastatic potentials, and its decrement correlated with NPC metastasis and poor patient survival, and was an independent predictor for reduced overall survival. With a combination of loss-of-function and gain-of-function approaches, we observed that high expression of RKIP reduced invasion, metastasis and epithelial to mesenchymal transition (EMT) marker alternations of NPC cells. We further showed that RKIP overexpression attenuated while RKIP knockdown enhanced Stat3 phosphorylation and activation in NPC cells; RKIP reduced Stat3 phosphorylation through interacting with Stat3; Stattic attenuated NPC cell migration, invasion and EMT marker alternations induced by RKIP knockdown, whereas Stat3 overexpression restored NPC cell migration, invasion and EMT marker alternations reduced by RKIP overexpression. In addition, there was an inverse correlation between RKIP and phospho-Stat3 expression in the NPC tissues and xenograft metastases. Our data demonstrate that RKIP is a metastatic suppressor and predictor for metastasis and prognosis in NPC, and RKIP downregulation promotes NPC invasion, metastasis and EMT by activating Stat3 signaling, suggesting that RKIP/Stat3 signaling could be used as a therapeutic target for NPC metastasis.

Oncolytic adenovirus-mediated short hairpin RNA targeting MYCN gene induces apoptosis by upregulating RKIP in neuroblastoma.

The amplification of MYCN is a typical characteristic of aggressive neuroblastomas, whereas acquired mutations of p53 lead to refractory and relapsed cases. We had previously examined the applicability of the replication-competent oncolytic adenovirus, ZD55-shMYCN, to deliver a short hairpin RNA targeting MYCN gene for p53-null and MYCN-amplified neuroblastoma cell line LA1-55N. Our data have shown that ZD55-shMYCN has an additive tumor growth inhibitory response through shRNA-mediated MYCN knockdown and ZD55-mediated cancer cell lysis. In this regard, ZD55-shMYCN can downregulate MYCN and perform anticancer effects, thereby acquiring significance in the administration of MYCN-amplified and p53-null neuroblastomas. Hence, we further investigated the anticancer properties of ZD55-shMYCN in neuroblastomas. Our data showed that ZD55-shMYCN induced G2/M arrest via decreasing the levels of cyclin D1 and cyclin B1 irrespective of p53 status. ZD55-shMYCN effectively induced apoptosis in neuroblastomas through activation of caspase-3 and enhancing PARP cleavage. Furthermore, ZD55-shMYCN could downregulate phosphoinositide 3-kinase and pAkt and upregulate RKIP levels. Similarly, pro-apoptosis was revealed by the histopathologic examination of paraffin-embedded section of resected tumors of mice xenograft. In vitro and in vivo studies, we elucidate the apoptosis properties and mechanisms of action of ZD55-shMYCN, which provide a promising approach for further clinical development.

Common reduction of the Raf kinase inhibitory protein in clear cell renal cell carcinoma.

Despite the recent progress in our understanding of clear cell renal cell carcinomas (ccRCCs), the etiology of ccRCC remains unclear. We reported here a prevailing reduction of the raf kinase inhibitory protein (RKIP) in ccRCC. In our examination of more than 600 ccRCC patients by western blot and immunohistochemistry, RKIP was significantly reduced in 80% of tumors. Inhibition of RKIP transcription in ccRCC occurs to greater levels than VHL transcription based on the quantification analysis of their transcripts in six large datasets of DNA microarray available in Oncomine™ with the median rank of suppression being 582 and 2343 for RKIP and VHL, respectively. Collectively, the magnitude of RKIP reduction and the levels of its downregulation match those of VHL. Furthermore, RKIP displays tumor suppressing activity in ccRCC. While modulation of RKIP expression did not affect the proliferation of A498 and 786-0 ccRCC cells and neither their ability to form xenograft tumors in NOD/SCID mice, ectopic expression or knockdown of RKIP inhibited or enhanced A498 and 786-0 ccRCC cell invasion, respectively. This was associated with robust changes in vimentin expression, a marker of EMT. Taken together, we demonstrate here that downregulation of RKIP occurs frequently at a rate that reaches that of VHL, suggesting RKIP being a critical tumor suppressor for ccRCC. This is consistent with RKIP being a tumor suppressor for other cancers.

Expression of Raf Kinase Inhibitor Protein (RKIP) is a predictor of uveal melanoma metastasis.

Melanoma arising from melanocytes within the choroid is the most frequent primary intraocular neoplasm in adults. It is biologically distinct from cutaneous melanoma by a very strong propensity to metastasize the liver. Raf kinase inhibitor protein is a member of an evolutionarily conserved group of proteins called phosphatidylethanolamine-binding proteins. It is an interacting partner of Raf-1 and a negative regulator of the mitogen-activated protein kinase cascade initiated by Raf-1. Raf kinase inhibitor protein expression is low in many human cancers and represents an indicator of poor prognosis and/or induction of metastasis. In the present study, we examined the immunohistochemical expression levels of Raf kinase inhibitor protein and phosphorylated Raf kinase inhibitor protein in primary uveal melanoma with and without metastasis, and evaluated their association with other high risk characteristics for metastasis in order to assess whether Raf kinase inhibitor protein and phosphorylated Raf kinase inhibitor protein can be used to predict metastasis. A significant low expression of Raf kinase inhibitor protein was seen in patients with metastasis but not in patients without metastasis. The latter more frequently had a high expression of Raf kinase inhibitor protein. No significant difference was seen in phosphorylated Raf kinase inhibitor protein expression between patients with and without metastasis. Raf kinase inhibitor protein expression is a suitable and easily determinable marker in the primary tumour that could predict the risk of uveal melanoma to metastasize, and hence guide strategies for monitoring and therapy.

Raf kinase inhibitor protein (RKIP) blocks signal transducer and activator of transcription 3 (STAT3) activation in breast and prostate cancer.

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding-protein (PEBP) family that modulates the action of many kinases involved in cellular growth, apoptosis, epithelial to mesenchymal transition, motility, invasion and metastasis. Previously, we described an inverse association between RKIP and signal transducers and activators of transcription 3 (STAT3) expression in gastric adenocarcinoma patients. In this study, we elucidated the mechanism by which RKIP regulates STAT3 activity in breast and prostate cancer cell lines. RKIP over expression inhibited c-Src auto-phosphorylation and activation, as well as IL-6-, JAK1 and 2-, and activated Raf-mediated STAT3 tyrosine and serine phosphorylation and subsequent activation. In MDA-231 breast cancer cells that stably over express RKIP, IL-6 treatment blocked STAT3 phosphorylation and transcriptional activation. Conversely, in RKIP knockdown MDA-231 cells: STAT3 phosphorylation and activation increased in comparison to parental MDA-231 cells. RKIP over expression resulted in constitutive physical interaction with STAT3 and blocked c-Src and STAT3 association. The treatment of DU145 prostate, but not PC3 prostate or MDA-231 breast, cancer cell lines with ENMD-1198 or MKC-1 dramatically increased expression of RKIP. Overexpression of RKIP sensitized PC3 and MDA-231 cells to MTI-induced apoptosis. Moreover, MTI treatment resulted in a decrease in Src-mediated STAT3 tyrosine phosphorylation and activation, an effect that was significantly enhanced by RKIP over expression. In stable RKIP over expressing MDA-231 cells, tumor xenograft growth induced by activated STAT3 is inhibited. RKIP synergizes with MTIs to induce apoptosis and inhibit STAT3 activation of breast and prostate cancer cells. RKIP plays a critical role in opposing the effects of pro-oncogenic STAT3 activation.

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.

Raf kinase inhibitor protein expression combined with peritoneal involvement and lymphovascular invasion predicts prognosis in Dukes' B colorectal cancer patients.

AIMS: There is controversy regarding the use of adjuvant therapy in patients with Dukes' B colorectal cancer (CRC). New markers, identifying high-risk Dukes' B patients, are needed. Here, we examine the utility of Raf kinase inhibitor protein (RKIP) as such a marker and promoter methylation as a mechanism of RKIP down-regulation. METHODS AND RESULTS: We used a tissue microarray of 220 patients with Dukes' B CRC to examine the effect of RKIP expression on survival. Pyrosequencing was used to assess RKIP promoter methylation status.RKIP expression correlated inversely with disease-specific survival in this cohort. In multivariate analysis, RKIP was found to be an independent prognostic indicator, along with peritoneal invasion and lymphovascular invasion (LVI). RKIP promoter hypermethylation was seen in only one of 29 tumours analysed by pyrosequencing. CONCLUSIONS: Raf kinase inhibitor protein, peritoneal invasion and LVI provide independent prognostic information in this cohort of Dukes' B CRC patients.This demonstrates the potential utility of RKIP in identifying 'high-risk' Dukes' B patients. It is this high-risk group which is most likely to benefit from close postoperative monitoring and may derive the most benefit from adjuvant therapy.

Positive effect of high RKIP expression on reduced distant metastasis by chemotherapy when combined with radiotherapy in locoregionally advanced nasopharyngeal carcinoma: a prospective study.

The purpose of this prospective study is to investigate the predictive and prognostic significance of the Raf kinase inhibitory protein (RKIP) in locoregionally advanced nasopharyngeal carcinoma (NPC). Immunohistochemical assays were performed to detect the RKIP protein expression of samples from 212 patients with locoregionally advanced NPC. All patients were assigned randomly into the inductive chemotherapy plus radiation therapy (IC + RT) group, the concurrent chemoradiotherapy (CCRT) group, the inductive chemotherapy plus concurrent chemoradiotherapy (IC + CCRT) group, and the radiation therapy alone (RT) group. The patients in the IC + RT group were treated with IC using 2-3 cycles of cisplatin (80 mg/m(2)) and fluorouracil (500 mg/m(2)), repeated every 3 weeks, followed by radiotherapy. Those in the CCRT group were treated with weekly cisplatin (40 mg/m(2)) for 6-7 cycles during radiotherapy. In the IC + CCRT group, the chemotherapy prior to radiation was similar to the cisplatin-fluorouracil regimen in the IC + RT group, whereas it cisplatin regimen was identical to that in the CCRT group. The results show that RKIP is an independent prognostic factor for 5-year distant metastasis-free survival (DMFS), overall survival (OS), and progression-free survival (PFS). Patients with high RKIP expression benefited more from reduced metastasis in the IC + RT and the IC + CCRT group, with improved OS and PFS in each treatment group compared with that among patients with low RKIP expression. In the high RKIP expression subgroup, chemotherapy combined with radiotherapy improved the DMFS when compared with the RT group, but this effect was not observed in the low RKIP expression subgroup. RKIP was predictive of distant metastasis with good sensitivity and specificity. Clinically, high RKIP expression inhibited distant metastasis in advanced NPC, and its detection might be used to predict distant metastasis with good sensitivity and specificity. The effect of chemotherapy on distant metastasis in combined chemoradiotherapy might be related to the RKIP expression level. Patients with high RKIP expression showed more improved OS and PFS than their low RKIP expression counterparts. Higher RKIP expression improves the DMFS of patients who receive inductive high-dose cisplatin-based chemoradiotherapy, with or without concurrent cisplatin. Low RKIP expression is also a predictive marker for cancer progression and metastasis, which could be used to stratify patients with high risk of metastasis and death.

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.

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.

Decreased expression and aberrant methylation of Raf kinase inhibitory protein gene in esophageal squamous cell carcinoma.

Raf kinase inhibitory protein (RKIP) gene is considered to be a suppressor of metastasis involved in various carcinomas. In the present study, we observed that promoter methylation repressed the expression of RKIP in TE-13 cell line. 5-Aza treatment and stable transfection of RKIP resulted in a significant inhibition of TE-13 cell proliferation. The promoter hypermethylation of RKIP was found to occur in dysplastic tissues and a close correlation was noted between RKIP methylation and the loss of mRNA and protein expression of the gene in ESCC specimens. In summary, RKIP may act as a tumor suppressor gene in esophageal cancer.

Suppression of astrocyte lineage in adult hippocampal progenitor cells expressing hippocampal cholinergic neurostimulating Peptide precursor in an in vivo ischemic model.

Hippocampal cholinergic neurostimulating peptide (HCNP) is known to promote differentiation of septohippocampal cholinergic neurons. The HCNP precursor protein (HCNP-pp) may play several roles, for example, as an ATP-binding protein, a Raf kinase inhibitor protein, and a phosphatidylethanolamine-binding protein, as well as a precursor for HCNP. This study therefore aimed to elucidate the involvement of HCNP-pp in specific neural lineages after stroke using a hypoxic-ischemic (HI) rat model of brain ischemia. The specific neural lineages in the hippocampus were investigated 14 days after ischemia. Some bromodeoxyuridine (BrdU)(+) neural progenitor cells in the hippocampus of hypoxic, HI, or sham-operated rats expressed HCNP-pp. Almost half of the BrdU(+)/HCNP-pp(+) cells also expressed the oligodendrocyte lineage marker 2',3'-cyclic nucleotide 3'-phosphodiesterase, whereas only a few BrdU(+)/HCNP-pp(+) cells in the hippocampus in HI brains expressed the neuronal lineage marker, doublecortin (DCX). Interestingly, no BrdU(+)/HCNP-pp(+) progenitor cells in hypoxic, HI, or sham-operated brains expressed the astrocyte lineage marker, glial fibrillary acidic protein. Together with previous in vitro data, the results of this study suggest that the expression level of HCNP-pp regulates the differentiation of neural progenitor cells into specific neural lineages in the HI hippocampus, indicating that neural stem cell fate can be controlled via the HCNP-pp mediating pathway.

Polycomb protein EZH2 regulates tumor invasion via the transcriptional repression of the metastasis suppressor RKIP in breast and prostate cancer.

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.