Raf kinase inhibitor protein (RKIP) in cancer

Raf kinase inhibitory protein (RKIP) was initially identified as phosphatidylethanolamine binding protein in bovine brain. It was later identified as a protein that inhibits Raf kinase activation of MEK. Further exploration has revealed that RKIP modulates several other signaling pathways including NF–κB and G-protein signaling. A gene array screen revealed that RKIP expression was low in a metastatic compared with non-metastatic prostate cancer cell line. Further experiments revealed that RKIP fits the criteria for a metastasis suppressor gene. RKIP expression has been shown to be downregulated in metastatic tissues, compared with non-metastatic tissue in multiple cancers, suggesting that loss of RKIP metastasis suppressor activity is a broad mechanism leading to metastasis. Additionally, loss of RKIP has been shown to impact therapy through conferring radioresistance and chemoresistance. Taken together, these data indicate understanding RKIP’s contributions to cancer may lead to important therapeutic strategies to prevent metastasis and promote therapeutic efficacy.     

Raf激酶抑制蛋白与恶性肿瘤

Raf激酶抑制蛋白(RKIP)是磷脂酰乙醇胺结合蛋白家族的成员。RKIP在Raf、核转录因子-κB(NF-κB)及G蛋白偶联受体(GPCR)等蛋白激酶信号传导通路中起重要调节作用。RKIP参与神经发育、心脏功能、精子发生、细胞迁移及巨噬细胞分化,同时还具有丝氨酸蛋白酶活性。近年来众多资料表明,RKIP在多种恶性肿瘤中表达减弱或丢失,其在恶性肿瘤中作用机制已经成为研究的热点,该类研究将为今后肿瘤治疗提供新的靶点。     

Raf激酶抑制蛋白与恶性肿瘤

Raf激酶抑制蛋白（RKIP）是磷脂酰乙醇胺结合蛋白家族的成员。RKIP在Raf、核转录因子-κB（NF-κB）及G蛋白偶联受体（GPCR）等蛋白激酶信号传导通路中起重要调节作用。RKIP参与神经发育、心脏功能、精子发生、细胞迁移及巨噬细胞分化，同时还具有丝氨酸蛋白酶活性。近年来众多资料表明，RKIP在多种恶性肿瘤中表达减弱或丢失，其在恶性肿瘤中作用机制已经成为研究的热点，该类研究将为今后肿瘤治疗提供新的靶点。     

Raf kinase inhibitor protein: a prostate cancer metastasis suppressor gene

Defining the mechanisms that confer metastatic ability on cancer cells is an important goal towards prevention of metastasis. A gene array screen between a non-metastatic prostate cancer cell and its metastatic derivative line revealed decreased expression of Raf kinase inhibitor protein (RKIP) in the metastatic cell line. This finding is consistent with the possibility that loss of RKIP is associated with metastasis. RKIP is expressed in many tissues including brain, lung, and liver. RKIP blocks Raf-induced phosphorylation of MEK. In addition to its modulation of Raf signaling, RKIP modulates both G-protein signaling and NF-kappaB activity. The impact that RKIP has on multiple signaling pathways grants it the ability to play a role in several cellular functions including membrane biosynthesis, spermatogenesis, and neural signaling. Novel cellular functions for RKIP continue to be identified, several of which contribute to cancer biology. For example, RKIP promotes apoptosis of cancer cells, which suggests that loss of RKIP in cancer will protect cancer cells against cell death. Additionally, restoration of RKIP expression ina metastatic prostate cancer cell line does not effect primary tumor growth, but it does inhibit prostate cancer metastasis. These parameters identify RKIP as a metastasis suppressor gene, which suggest that it or proteins it interacts with are putative molecular targets to control metastasis. These findings are supported by the observation that RKIP expression is decreased in metastases of prostate cancer patients, compared to normal prostate or the primary prostate tumor. In this review, RKIP biology and its role in cancer will be described.     

Raf kinase inhibitor protein is downregulated in hepatocellular carcinoma

The Ras/Raf/MEK/ERK signalling cascade is frequently deregulated in tumourigenic diseases and known to be involved in proliferation and transformation of cells. Also in hepatocellular carcinoma (HCC) increased ERK levels are observed and known to correlate with tumour progression, but the underlying molecular mechanism are unknown. We analyzed expression of Raf-1 kinase inhibitory protein (RKIP) in HCC. Expression of RKIP mRNA and protein was downregulated in HCC cell lines and tissue as compared to primary human hepatocytes (PHH) or non-tumorous liver tissue, respectively. Transfection of an HCC cell line with an RKIP expression construct blocked the Raf kinase pathway resulting in decreased activity of ERK1/2 and AP-1. In contrast, downregulation of RKIP by transfection with an antisense RKIP construct led to increased ERK1/2 and AP-1 activity. Since HCC develop in the majority of cases in cirrhotic liver tissue and cirrhosis is the main risk factor for HCC development, we analyzed RKIP expression also in non-cancerous cirrhotic liver tissues by immunohistochemistry. In contrast to normal liver tissue, where the staining was equally distributed within the cytoplasm, hepatocytes in cirrhotic liver revealed an intense RKIP staining of the membrane. It can be speculated that this changed RKIP expression pattern parallels impaired protein function in PHH in cirrhotic livers that may predispose PHH to malignant transformation. In addition, our study demonstrates functional relevance of downregulation of RKIP in HCC that may play an important role in HCC development and progression.     

Raf激酶抑制蛋白启动子甲基化与肺癌患者预后的关系

目的:观察Raf激酶抑制蛋白在肺癌中的表达及其启动子甲基化的情况,研究Raf激酶抑制蛋白启动子甲基化与肺癌患者预后的关系。方法:运用免疫组织化学（SABC）方法检测Raf激酶抑制蛋白在组织中的表达情况,采用甲基化特异性PCR法检测Raf激酶抑制蛋白启动子甲基化的情况,SPSS 15.0统计分析Raf激酶抑制蛋白表达和启动子甲基化的差异及其与肺癌患者预后的相关性。结果:Raf激酶抑制蛋白在肺癌组织的表达明显低于癌旁组织;Raf激酶抑制蛋白启动子甲基化在肺癌组织中发生甲基化或部分甲基化,明显高于癌旁组织;Raf激酶抑制蛋白启动子甲基化与肺癌患者的预后负相关。 结论:肺癌患者预后差的一个重要原因是Raf激酶抑制蛋白表达低,其机制主要是其启动子发生了甲基化。因此Raf激酶抑制蛋白启动子甲基化是预测肺癌患者预后的一个独立因素。     

Reduced expression of Raf kinase inhibitor protein correlates with poor prognosis in pancreatic cancer

Aim

Raf kinase inhibitory protein (RKIP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. But its function in pancreatic cancer was not yet clarified completely. The aim of this study was to investigate the involvement of RKIP in pancreatic cancer.

Methods

RKIP expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumor tissue samples from a series (n?=?99) of consecutive patients with pancreatic cancer. Survival was calculated using Kaplan?CMeier curves. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model.

Results

RKIP expression was high in normal pancreatic epithelium and retained to varying degrees in pancreatic cancer tissues. However, in tumor tissues with lymph node metastasis (P?=?0.008) and high UICC stage (P?=?0.006), RKIP expression was highly significantly reduced or lost. Furthermore, the reduced expression of RKIP significantly correlated with both poor overall and disease-free survival (P?=?0.008 and 0.01, respectively). Multivariate analyses revealed RKIP to be an independent prognosticator.

Conclusion

These findings suggest that RKIP could be a promising marker for predicting a better prognosis in pancreatic cancer.     

肿瘤靶向治疗新探:多靶点Raf激酶抑制剂

随着对肿瘤分子机制的加深理解，对肿瘤分子靶向治疗的研究已获重大进展。蛋白激酶抑制剂是新近研发的靶向治疗药物之一，通过阻碍细胞内分子传导通路，影响肿瘤细胞的存活、增殖以及疾病进展。在Raf／MEK／ERK信号传导通路中，Raf激酶发挥着至关重要的作用。尽管在正常组织中Raf激酶的功能尚未明朗，但现有的基础及临床研究结果均显示，Raf基因的上调及其蛋白的过度表达存在于多种实体肿瘤之中，包括肾细胞癌、肝细胞癌、黑色素瘤以及非小细胞肺癌等。索拉非尼是全球首个口服的Raf激酶抑制剂。此外，作为一个多靶点药物，索拉非尼同时具有针对包括VEGFR与PDGFR的广泛酪氨酸激酶受体抑制功能。目前美国FDA已经批准索拉非尼用于治疗转移性肾癌。另外，该药物在针对黑色素瘤、肝癌、胰腺癌以及非小细胞肺癌的临床研究中也已经显示出一定的疗效。本综述将简要说明Raf激酶在正常与肿瘤细胞中的功能以及在不同肿瘤中的作用机制，并重点介绍索拉非尼的临床应用及研究。     

Immunohistochemical detection of apoptotic markers in gastric cancer

Apoptosis proteins may play a role in prognosis and therapy response; however, they have not been fully investigated in gastric cancer. We aimed to assess the expression of proteins in the Bcl-2 family. Immunohistochemistry was employed to examine the expression of the antiapoptotic proteins Bcl-2 and Bcl-XL and the proapoptotic proteins Bad, Bak, Bax, Bid, Bim, and p53 in 21 cases of gastric cancer. Immunopositivity was observed in 12/21 (57%) cases for p53, 16/21 (76%) cases for Bcl-XL, and 5/21 (23%) cases for Bcl-2. For the proapoptotic members of the Bcl family, loss of protein expression was observed: Bid (14/21 cases; 66%), Bad (13/21 cases; 61%), Bax (12/21 cases; 57%), Bak (9/21 cases; 42%), and Bim (4/21 cases; 19%). This study identified apoptosis proteins that exhibit heterogeneous expression between primary gastric carcinomas.     

Raf激酶抑制蛋白和P-ERK在肝细胞癌中的表达及其临床意义

目的:探讨Raf激酶抑制蛋白(Raf kinase inhibitor protein,RKIP)和磷酸化细胞外信号调节激酶(phos-extracellular signal regulated kinase,P-ERK)在肝细胞癌( hepatocellular carcinoma,HCC )中的表达及其临床意义.方法:应用免疫组织化学方法检测RKIP和P-ERK在HCC、癌旁及正常肝组织中的表达,并分析其与HCC临床病理学特征的关系.结果:HCC组织中RKIP的表达低于癌旁及正常肝组织,而P-ERK的表达高于癌旁及正常肝组织,差异有统计学意义;HCC组织中RKIP与P-ERK 的表达呈显著负相关( r =-0.227, P =0.039);RKIP在HCC组织中的表达与有无肝内或淋巴结转移及肿瘤分化程度相关( P <0.05);P-ERK在HCC组织中的表达与肿瘤分化程度、有无癌栓及有无肝内或淋巴转移相关( P <0.05).结论:RKIP表达的减少或缺失与HCC的发生、发展密切相关,RKIP表达的减少或缺失可能通过上调P-ERK的表达促进HCC的侵袭和转移.     

Loss of Raf kinase inhibitory protein induces radioresistance in prostate cancer

PURPOSE: External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. METHODS AND MATERIALS: The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. RESULTS: RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. CONCLUSION: The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.     

Immunohistochemical detection of lymph node microinvolvement in node-negative gastric cancer

Background. Despite curative resection of the primary tumor with extensive dissection of lymph nodes, some patients with node-negative gastric cancer die of local relapse or metastatic disease. Microinvolvement of regional lymph nodes may play an important role in the prognosis. Methods. To evaluate the incidence and prognostic implications of regional lymph node microinvolvement in node-negative gastric cancer, we retrospectively analyzed tissue samples from 51 patients operated on for primary gastric cancer. They had tumors that invaded beyond the muscularis propria, but without metastasis to the lymph nodes, shown by the conventional H&E staining method. The regional lymph nodes were examined immunohistochemically, using monoclonal antibodies against cytokeratin. Results. Microinvolvement was found in 4.8% of lymph nodes (67/1390) and in 43.2% of the patients (22/51). The clinical outcome of the patients with microinvolvement was not significantly different from those without it. However, no patient died in the no-microinvolvement group, while three patients in the microinvolvement group died of recurrence. Conclusion. The incidence of microinvolvement in conventionally negative lymph nodes cannot be ignored, and detecting microinvolvement may be important for predicting recurrence of gastric cancer. Received for publication on April 26, 1999; accepted on Sept. 14, 1999     

Targeting AKT protein kinase in gastric cancer

Gastric cancer is a highly lethal malignancy with more than 700,000 deaths every year worldwide. Despite significant improvements in our understanding of disease biology, the 5-year survival rates remain low. With the exception of trastuzumab, targeted agents have failed to add any meaningful benefit for this patient population, despite promising pre-clinical data. Protein kinase B (AKT) is essential for cell growth, proliferation, and survival. Aberrant activation of AKT is one of the most common molecular findings in human malignancies including gastric cancer, and it is believed to play an important role in cancer cell survival and chemotherapy resistance. Combining phosphatidylinositol 3-kinase (PI3K)/AKT pathway inhibitors with chemotherapy has successfully attenuated chemotherapeutic resistance in gastric cancer cell lines. Drugs designed to specifically target AKT are now being developed for clinical use. In this article, we will review the current knowledge on AKT signaling in the pathogenesis of gastric cancer and the evolving therapeutic implications of targeting this pathway.     

Reduction in Raf kinase inhibitor protein expression is associated with increased Ras-extracellular signal-regulated kinase signaling in melanoma cell lines

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.     

蛋白激酶C亚型在胃癌中的表达及其意义

目的:研究蛋白激酶C的四种亚型(PKCα、PKCβⅡ、PKCε、PKCζ)在胃癌中的表达及其对胃癌发生发展的影响。方法:采用免疫组织化学方法(S-P法)检测PKCα、PKCβⅡ、PKCε、PKCζ在胃癌中的表达情况,分析其在不同病理类型、不同分化程度和原发灶与转移灶之间的相关性及意义。结果:(1)PKCα在胃癌不同组织类型之间表达依次为管状腺癌92.5%,粘液腺癌57.1%,两者之间比较差异有显著性(P<0.05)。(2)PKCβⅡ在胃癌不同组织类型之间表达依次为管状腺癌82.5%,粘液腺癌64.2%,两者比较差异有显著性(P<0.05)。在有淋巴结转移的胃癌中,淋巴结转移灶中PKCβⅡ的阳性率为77.8%,低于原发灶96.0%的阳性表达率,两者之间比较差异有显著性(P<0.05)。(3)PKCε、PKCζ在胃癌不同组织类型之间及淋巴结转移灶与原发灶之间的表达差异无显著性。结论:PKCβⅡ可能在胃癌的浸润、转移中起重要作用;PKCα、PKCβⅡ表达与肿瘤细胞的类型和组织来源不同有关。     

Immunohistochemical detection of human lung and gastric cancer antigen in human salivary gland tumors

Immunohistochemical identification of human lung and gastric carcinoma antigen detected with monoclonal antibodies (MoAbs) KM-93 and KM-231 respectively, was described in 83 salivary gland tumors, including 67 pleomorphic adenomas, 5 adenolymphomas, 3 mucoepidermoid tumors, 6 sialadenocarcinomas, and 2 adenoid cystic carcinomas as well as in normal salivary glands. The binding patterns of these two MoAbs was compared with that of MoAb recognizing epithelial membrane antigen (EMA). Serous cells of normal salivary glands showed positive KM-93 staining, whereas ductal cells were positive with KM-231, with ductal basal cells being characteristically so. EMA staining was confined to luminal and lateral borders of serous acini and ducts. Pleomorphic adenomas indicated positive depositions for both KM-93 and KM-231 in luminal tumor cells or luminal borders of tubuloductal structures. Adenolymphomas showed positive KM-231 staining in basal tumor cells and a positive KM-93 reaction in luminal tumor cells. Mucoepidermoid tumors revealed positive KM-231 staining in mucous-secreting cells, whereas weak KM-93 staining was found in all tumor cells. Sialoadenocarcinomas exhibited varying degrees of positive staining with KM-93 and KM-231 in their neoplastic cells. Adenoid cystic carcinomas showed luminal staining with KM-93 and KM-231 to their neoplastic cells. Adenoid cystic carcinomas showed luminal staining with KM-93 and MoAb EMA. The histogenesis of these salivary gland tumors is discussed in terms of the immunohistochemical features of staining patterns obtained with MoAbs KM-93 and KM-231.     

Immunohistochemical detection of occult metastases in paraaortic lymph nodes in advanced gastric cancer.

Some Japanese surgeons have examined the utility of super-extended paraaortic lymphadenectomy (PAL) as part of the surgical treatment for advanced gastric cancer. However, therapeutic value of this PAL remains controvertial. The purpose of this study was to evaluate appropriate candidates who might benefit from PAL by the immunostaining with cytokeratin (CK) of the macroscopically intact paraaortic nodes. A total of 525 paraaortic nodes from 35 patients was serially sectioned and stained with hematoxylin-eosin (H&E) and CK staining. A total of 17 nodes (3.2%) from 7 patients (20.0%), among 525 macroscopically intact paraaortic nodes, was determined to be immunopositive for CK. In 4 patients, 8 H&E-positive nodes with metastases were all immunopositive and, in addition, 4 H&E-negative nodes were also immunopositive. Furthermore, 3 patients with H&E-negative nodes had five immunopositive nodes. Immunostaining with CK was useful for detection of occult metastases. Survival was prolonged in 3 of these 7 patients. The incidence of CK-positive nodes was significantly higher in patients with gross type of 3 or 4 gastric cancer and in patients with H&E-detected nodal metastasis within group 3 (N3) nodes. It seems that such patients would benefit from prophylactic PAL.     

Immunohistochemical assay for detection of K-ras protein expression in metastatic colorectal cancer

BackgroundThe monoclonal antibodies (mAbs) that target the epidermal growth factor receptor (EGFR) had expanded the range of treatment options for metastatic colorectal cancer. However, such type of treatment was shown to be ineffective if there is K-ras mutation. In most previous studies K-ras gene mutation was mainly assessed by PCR.AimOur work is designed to detect K-ras protein expression by immunohistochemistry (IHC) aiming to reach a preliminary method that could be confirmed by PCR and considered an alternative way for the detection of K-ras aberration. We are also aiming to find a relation between K-ras protein expression and K-ras gene mutation.Materials and methodsParaffin embedded tissue samples from 26 metastatic colorectal cancer (mCRC) patients were analyzed for K-ras protein expression by IHC using Rap1A polyclonal antibody. Staining patterns were subjectively assessed and correlated with clinicopathological features. The results were statistically evaluated using the Chi-square test.ResultsK-ras cytoplasmic positivity was observed in 42.3% of cases. The positivity was either strong in 26.9% or moderate in 15.4%. With respect to adenocarcinoma variants, 50% of cases were positive for K-ras protein expression while all mucinous and signet ring types were negative. The positivity was noted in 50% of moderately differentiated GII colorectal carcinomas as compared with 38.9% in poorly differentiated GIII. Positive staining was observed in 40% of cases with positive lymph node metastasis while in the absence of nodal metastasis the positivity was 45.5%. No significant correlation was found between clinicopathological parameters and K-ras staining results.ConclusionIHC may compliment PCR in the detection of K-ras mutation.