Raf激酶抑制蛋白的研究进展

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通过与Raf-1结合,抑制了Ras/Raf-1/MEK/ERK信号转导通路,并在NF-κB及G蛋白偶联受体(GPCR)信号转导通路中也起重要调节作用。RKIP参与细胞凋亡、肿瘤转移、神经发育以及精子发生等病理生理过程,通过研究RKIP能为治疗相关疾病提供新思路新靶点。本文主要介绍RKIP的生物功能,着重于其在神经系统、肿瘤和生殖系统中的研究进展。     

Raf kinase inhibitor protein correlates with sensitivity of nasopharyngeal carcinoma to radiotherapy

Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in nasopharyngeal carcinoma (NPC) tissues and is absent in NPC metastases. To investigate the effect of RKIP on radiosensitivity of NPC, high metastatic 5‐8F with low RKIP expression and non‐metastatic 6‐10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA. Overexpression of RKIP sensitized 5‐8F cells to radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In contrast, downexpression of RKIP in 6‐10B cells protected cells from radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In addition, RKIP expression altered the radiosensitivity of NPC cells through MEK and ERK phosphorylation changes of Raf‐1/MEK/ERK signaling pathway. We further investigated the RKIP expression in NPC patients and its association with patients' survival after radiotherapy. Downexpression of RKIP was significantly correlated with advanced clinical stage, lymph node metastasis and radioresistance. Furthermore, survival curves showed that patients with RKIP downexpression had a poor prognosis and induced relapse. Multivariate analysis confirmed that RKIP expression was an independent prognostic indicator. The data suggested that RKIP was a potential biomarker for the radiosensitivity and prognosis of NPC, and its dysregulation might play an important role in the radioresistance of NPC. J. Cell. Biochem. 110: 975–984, 2010. © 2010 Wiley‐Liss, Inc.     

Raf kinase inhibitor protein mediated signaling inhibits invasion and metastasis of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of liver cancer with high mortality and poor prognosis. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways have been implicated in promoting tumor cell proliferation and invasion of HCC cells.MethodsAs a potential inhibitor of tumor metastasis, the role of Raf kinase inhibitor protein (RKIP) in HCC development and the functional relevance with MAPK and NF-κB signaling pathways were investigated. The levels of RKIP expression were examined in human HCC tissues and correlated with tumor stages and metastatic status. Function of RKIP in cellular proliferation, migration, invasion and apoptosis was investigated in HCC cell lines by either overexpressing or knocking down RKIP expression. Mouse xenograft model was established to assess the effect of RKIP expression on tumor growth.ResultsOur results demonstrated decreased RKIP expression in HCC tissues and a strong correlation with tumor grade and distant metastasis. Manipulation of RKIP expression in HCCLM3 and HepG2 cells indicated that RKIP functioned to inhibit HCC cell motility and invasiveness, and contributed to tumor growth inhibition in vivo. Mechanistic studies showed that the function of RKIP was mediated through MAPK and NF-κB signaling pathways. However, cell type-dependent RKIP regulation on these two pathways was also suggested, indicating the complex nature of signaling network.ConclusionOur study provides a better understanding on the molecular mechanisms of HCC metastasis and sets the foundation for the development of targeted therapeutics for HCC.     

RKIP inhibits the migration and invasion of human prostate cancer PC-3M cells through regulation of extracellular matrix

Raf kinase inhibitor protein (RKIP) plays a pivotal role in several intracellular signaling cascades and has been implicated as a metastasis suppressor in multiple cancer cells including prostate cancer cells, but the mechanism is not very clear. In this study, we investigated the effect of RKIP on cell proliferation, migration and invasion using human prostate cancer PC-3M cells as a model system. Our results indicate that RKIP does not effect cell proliferation in PC-3M cells, but inhibits both cell migration and cell invasion. In association with this inhibitory effect, RKIP down-regulates matrix metalloproteinases (MMP-2 and MMP-9), cathepsin B and urinary plasminogen activator (uPA). Also RKIP has the ability to regulate the expression of E-cadherin. But ectopic expression of RKIP does not affect the level of the Snail protein. As it has been indicated here, RKIP inhibits the migration and invasion ability of human prostate cancer cells through regulation of the extracellular matrix. These findings provide new mechanistic insight how RKIP suppresses metastasis in vitro.     

Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint

Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.     

Effects of raf kinase inhibitor protein expression on metastasis and progression of human epithelial ovarian cancer

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

Modulation of the MAP kinase signaling cascade by Raf kinase inhibitory protein

Proteins like Rafkinase inhibitory protein (RKIP) that serve as modulators of signaling pathways, either by promoting or inhibiting the formation of productive signaling complexes through protein-protein interactions, have been demonstrated to play an increasingly important role in a number of cell types and organisms. These proteins have been implicated in development as well as the progression of cancer. RKIP is a particularly interesting regulator, as it is a highly conserved, ubiquitously expressed protein that has been shown to play a role in growth and differentiation in a number of organisms and can regulate multiple signaling pathways. RKIP is also the first MAP kinase signaling modulator to be identified as playing a role in cancer metastasis, and identification of the mechanism by which it regulates Raf-1 activation provides new targets for theraoeutic intervention.     

Induction of Raf kinase inhibitor protein contributes to macrophage differentiation

Differential gene expression analysis of human blood monocytes has identified the Raf kinase inhibitor protein (RKIP) as a continuously upregulated gene in macrophage and dendritic cell maturation. Using realtime RT-PCR and Western blot analysis we were able to confirm the initial DNA-microarray findings of RKIP induction on mRNA and protein levels. RKIP upregulation in primary cells and overexpression in THP-1 cells did not alter ERK activity but strongly reduced the amount of the NFkappaB subunit p65 in the nucleus. mRNA levels and cell surface expression of maturation markers including the integrin CD11c and the scavenger receptor CD36 were significantly increased in RKIP transfected THP-1 cells. Our data show for the first time that RKIP is upregulated during macrophage and dendritic cell differentiation on mRNA and protein levels and we conclude that RKIP contributes to the monocytic differentiation process via inhibition of the NFkappaB signaling cascade independent from the canonical Ras/Raf/MEK/ERK pathway.     

Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein

Protein kinase C (PKC) regulates activation of the Raf-1 signaling cascade by growth factors, but the mechanism by which this occurs has not been elucidated. Here we report that one mechanism involves dissociation of Raf kinase inhibitory protein (RKIP) from Raf-1. Classic and atypical but not novel PKC isoforms phosphorylate RKIP at serine 153 (Ser-153). RKIP Ser-153 phosphorylation by PKC either in vitro or in response to 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor causes release of RKIP from Raf-1, whereas mutant RKIP (S153V or S153E) remains bound. Increased expression of PKC can rescue inhibition of the mitogen-activated protein (MAP) kinase signaling cascade by wild-type but not mutant S153V RKIP. Taken together, these results constitute the first model showing how phosphorylation by PKC relieves a key inhibitor of the Raf/MAP kinase signaling cascade and may represent a general mechanism for the regulation of MAP kinase pathways.     

Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation

Raf kinase inhibitory protein (RKIP; also known as phosphatidylethanolamine-binding protein or PEBP) is a modulator of the Raf/MAPK signaling cascade and a suppressor of metastatic cancer. Here, we show that RKIP inhibits MAPK by regulating Raf-1 activation; specifically, RKIP acts subsequent to Raf-1 membrane recruitment, prevents association of Raf-1 and p21-activated kinase (PAK), and blocks phosphorylation of the Raf-1 kinase domain by PAK and Src family kinases. Mutation of the PAK and Src phosphorylation sites on Raf-1 to aspartate, a phosphate mimic, prevented RKIP association with or inhibition of Raf-1 signaling. Interestingly, although RKIP can interact with B-Raf, RKIP depletion had no effect on activation of B-Raf. Because c-Raf-1 and B-Raf are both required for maximal MAPK stimulation by epidermal growth factor in neuronal and epithelial cell lines, we determined whether RKIP significantly affects MAPK signaling. In fact, RKIP depletion increased not only the amplitude but also the sensitivity of MAPK and DNA synthesis to epidermal growth factor stimulation by up to an order of magnitude. These results indicate that selective modulation of c-Raf-1 but not B-Raf activation by RKIP can limit the dynamic range of the MAPK signaling response to growth factors and may play a critical role in growth and development.     

A new model for raf kinase inhibitory protein induced chemotherapeutic resistance

Therapeutic resistance remains the most challenging aspect of treating cancer. Raf kinase inhibitory protein (RKIP) emerged as a molecule capable of sensitizing cancerous cells to radio- and chemotherapy. Moreover, this small evolutionary conserved molecule, endows significant resistance to cancer therapy when its expression is reduced or lost. RKIP has been shown to inhibit the Raf-MEK-ERK, NFκB, GRK and activate the GSK3β signaling pathways. Inhibition of Raf-MEK-ERK and NFκB remains the most prominent pathways implicated in the sensitization of cells to therapeutic drugs. Our purpose was to identify a possible link between RKIP-KEAP 1-NRF2 and drug resistance. To that end, RKIP-KEAP 1 association was tested in human colorectal cancer tissues using immunohistochemistry. RKIP miRNA silencing and its inducible overexpression were employed in HEK-293 immortalized cells, HT29 and HCT116 colon cancer cell lines to further investigate our aim. We show that RKIP enhanced Kelch-like ECH-associated protein1 (KEAP 1) stability in colorectal cancer tissues and HT29 CRC cell line. RKIP silencing in immortalized HEK-293 cells (termed HEK-499) correlated significantly with KEAP 1 protein degradation and subsequent NRF2 addiction in these cells. Moreover, RKIP depletion in HEK-499, compared to control cells, bestowed resistance to supra physiological levels of H(2)O(2) and Cisplatin possibly by upregulating NF-E2-related nuclear factor 2 (NRF2) responsive genes. Similarly, we observed a direct correlation between the extent of apoptosis, after treatment with Adriamycin, and the expression levels of RKIP/KEAP 1 in HT29 but not in HCT116 CRC cells. Our data illuminate, for the first time, the NRF2-KEAP 1 pathway as a possible target for personalized therapeutic intervention in RKIP depleted cancers.     

Activation of the RalGEF/Ral pathway promotes prostate cancer metastasis to bone

A hallmark of metastasis is organ specificity; however, little is known about the underlying signaling pathways responsible for the colonization and growth of tumor cells in target organs. Since tyrosine kinase receptor activation is frequently associated with prostate cancer progression, we have investigated the role of a common signaling intermediary, activated Ras, in prostate cancer metastasis. Three effector pathways downstream of Ras, Raf/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase, and Ral guanine nucleotide exchange factors (RalGEFs), were assayed for their ability to promote the metastasis of a tumorigenic, nonmetastatic human prostate cancer cell line, DU145. Oncogenic Ras promoted the metastasis of DU145 to multiple organs, including bone and brain. Activation of the Raf/ERK pathway stimulated metastatic colonization of the brain, while activation of the RalGEF pathway led to bone metastases, the most common organ site for prostate cancer metastasis. In addition, loss of RalA in the metastatic PC3 cell line inhibited bone metastasis but did not affect subcutaneous tumor growth. Loss of Ral appeared to suppress expansive growth of prostate cancer cells in bone, whereas homing and initial colonization were less affected. These data extend our understanding of the functional roles of the Ral pathway and begin to identify signaling pathways relevant for organ-specific metastasis.     

Identification of novel metastasis suppressor signaling pathways for breast cancer

Cancer lethality is mainly caused by metastasis. Therefore, understanding the nature of the genes involved in this process has become a priority. Given the heterogeneity of mutations in cancer cells, considerable focus has been directed toward characterizing metastasis genes in the context of relevant signaling pathways rather than treating genes as independent and equal entities. One signaling cascade implicated in the regulation of cell growth, invasion and metastasis is the MAP kinase pathway. Raf kinase inhibitory protein (RKIP) functions as an inhibitor of the MAP kinase pathway and is a metastasis suppressor in different cancer models. By utilizing statistical analysis of clinical data integrated with experimental validation, we recently identified components of the RKIP signaling pathway relevant to breast cancer metastasis. Using the RKIP pathway as an example, we show how prior biological knowledge can be efficiently combined with genome-wide patient data to identify gene regulatory mechanisms that control metastasis.     

Identification of novel metastasis suppressor signaling pathways for breast cancer

Cancer lethality is mainly caused by metastasis. Therefore, understanding the nature of the genes involved in this process has become a priority. Given the heterogeneity of mutations in cancer cells, considerable focus has been directed toward characterizing metastasis genes in the context of relevant signaling pathways rather than treating genes as independent and equal entities. One signaling cascade implicated in the regulation of cell growth, invasion and metastasis is the MAP kinase pathway. Raf kinase inhibitory protein (RKIP) functions as an inhibitor of the MAP kinase pathway and is a metastasis suppressor in different cancer models. By utilizing statistical analysis of clinical data integrated with experimental validation, we recently identified components of the RKIP signaling pathway relevant to breast cancer metastasis. Using the RKIP pathway as an example, we show how prior biological knowledge can be efficiently combined with genome-wide patient data to identify gene regulatory mechanisms that control metastasis.     

Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis. Some components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf). Mutations also occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. Even in the absence of obvious genetic mutations, this pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia and is also frequently activated in other cancer types (e.g., breast and prostate cancers). Importantly, this increased expression is associated with a poor prognosis. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of activated Akt to phosphorylate and inactivate different Rafs. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell lineage specific effects. For example, Raf/MEK/ERK is usually associated with proliferation and drug resistance of hematopoietic cells, while activation of the Raf/MEK/ERK cascade is suppressed in some prostate cancer cell lines which have mutations at PTEN and express high levels of activated Akt. Furthermore the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways also interact with the p53 pathway. Some of these interactions can result in controlling the activity and subcellular localization of Bim, Bak, Bax, Puma and Noxa. Raf/MEK/ERK may promote cell cycle arrest in prostate cells and this may be regulated by p53 as restoration of wild-type p53 in p53 deficient prostate cancer cells results in their enhanced sensitivity to chemotherapeutic drugs and increased expression of Raf/MEK/ERK pathway. Thus in advanced prostate cancer, it may be advantageous to induce Raf/MEK/ERK expression to promote cell cycle arrest, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK induced proliferation and drug resistance. Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages which may be due to the presence of functional p53 and PTEN and the expression of lineage specific factors.     

A proteomic approach in investigating the hepatoprotective mechanism of Schisandrin B: role of raf kinase inhibitor protein

To identify key proteins involved in the hepatoprotection afforded by schisandrin B (Sch B), we used a proteomic approach to screen proteins that were specifically regulated by Sch B in mouse livers and to investigate the role of the proteins in hepatoprotection. Thirteen proteins were specifically activated or suppressed by Sch B treatment. Among the 13 proteins, Raf kinase inhibitor protein (RKIP) was postulated to be the key regulator involved in the development of hepatotoxin-induced cellular damage. The results indicated that the downregulation of RKIP by antisense RKIP vector transfection led to the activation of the Raf-1/MEK/ERK signaling pathway, as evidenced by increases in the level of MEK/ERK phosphorylation and the level of nuclear factor erythroid 2-related factor 2 in the nucleus. The signaling effect produced by RKIP downregulation resembled that triggered by Sch B, wherein both treatments resulted in a decrease in the extent of carbon tetrachloride-induced apoptotic cell death in AML12 hepatocytes. Overexpression of RKIP by the sense RKIP transfection vector or the inhibition of MEK kinase by PD98059 was able to abrogate the cytoprotective effect of Sch B in the hepatocytes. The results indicate that Sch B triggers the Raf/MEK/ERK signaling pathway, presumably by downregulating RKIP, thereby protecting against carbon tetrachloride-induced cytotoxicity.