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.     

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.     

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.     

The biology of a prostate cancer metastasis suppressor protein: Raf kinase inhibitor protein

Raf kinase inhibitor protein (RKIP) was originally identified as a protein that bound membrane phospholipids and was named phosphatidylethanolamine binding protein-2 (PEBP-2). RKIP was than identified as a protein that bound Raf and blocked its ability to phosphorylate MEK, thus earning its new name of RKIP. Subsequent to identification of its role in the Raf:MEK pathway, RKIP has been demonstrated to regulate several other signaling pathways including G-protein signaling and NF-kappaB signaling. Its involvement in several signaling pathways has engendered RKIP to contribute to several physiological processes including membrane biosynthesis, spermatogenesis, neural development, and apoptosis. RKIP is expressed in many tissues including brain, lung, and liver and thus, dysregulation of RKIP expression or function has potential to contribute to pathophysiology in these tissues. Loss of RKIP expression in prostate cancer cells confers a metastatic phenotype on them. Additionally, restoration of RKIP expression in a 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. In this review, the biology and pathophysiology of RKIP is described.     

MAP kinase meets mitosis: A role for Raf Kinase Inhibitory Protein in spindle checkpoint regulation

Raf Kinase Inhibitory Protein (RKIP) is an evolutionarily conserved protein that functions as a modulator of signaling by the MAP kinase cascade. Implicated as a metastasis suppressor, Raf Kinase Inhibitory Protein depletion correlates with poor prognosis for breast, prostate and melanoma tumors but the mechanism is unknown. Recent evidence indicates that Raf Kinase Inhibitory Protein regulates the mitotic spindle assembly checkpoint by controlling Aurora B Kinase activity, and the mechanism involves Raf/MEK/ERK signaling. In contrast to elevated MAP kinase signaling during the G1, S or G2 phases of the cell cycle that activates checkpoints and induces arrest or senescence, loss of RKIP during M phase leads to bypass of the spindle assembly checkpoint and the generation of chromosomal abnormalities. These results reveal a role for Raf Kinase Inhibitory Protein and the MAP kinase cascade in ensuring the fidelity of chromosome segregation prior to cell division. Furthermore, these data highlight the need for precise titration of the MAP kinase signal to ensure the integrity of the spindle assembly process and provide a mechanism for generating genomic instability in tumors. Finally, these results raise the possibility that RKIP status in tumors could influence the efficacy of treatments such as poisons that stimulate the Aurora B-dependent spindle assembly checkpoint.     

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.     

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.     

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.     

MicroRNA-224 targets RKIP to control cell invasion and expression of metastasis genes in human breast cancer cells

The Raf kinase inhibitor protein (RKIP) is a tumor suppressor that protects against metastasis and genomic instability. RKIP is downregulated in many types of tumors, although the mechanism for this remains unknown. MicroRNAs silence target genes via translational inhibition or target mRNA degradation, and are thus important regulators of gene expression. In the current study, we found that miR-224 expression is significantly upregulated in breast cancer cell lines, and especially in highly invasive MDA-MB-231 cells, compared to human normal breast epithelial cells. In addition, miR-224 inhibits RKIP gene expression by directly targeting its 3'-untranslated region (3'-UTR). Moreover, metastasis, as assayed by Transwell migration, 3D growth in Matrigel, and wound healing, was enhanced by ectopic expression of miR-224 and inhibited by miR-224 downregulation. Promotion of metastasis in response to miR-224 downregulation was associated with derepression of the stroma-associated RKIP target genes, CXCR4, MMP1, and OPN, which are involved in breast tumor metastasis to the bone. Taken together, our data indicate that miR-224 play an important role in metastasis of human breast cancer cells to the bone by directly suppressing the RKIP tumor suppressor.     

Raf Kinase Inhibitory Protein Protects Cells against Locostatin-Mediated Inhibition of Migration

Background

Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function.

Methods/Findings

We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP^−/−) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP^−/− MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle.

Conclusions/Significance

These results suggest that locostatin''s effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.     

Raf kinase inhibitory protein: a signal transduction modulator and metastasis suppressor

Cells have a multitude of controls to maintain their integrity and prevent random switching from one biological state to another. Raf Kinase Inhibitory Protein (RKIP), a member of the phosphatidylethanolamine binding protein (PEBP) family, is representative of a new class of modulators of signaling cascades that function to maintain the "yin yang" or balance of biological systems. RKIP inhibits MAP kinase (Raf-MEK-ERK), G protein-coupled receptor (GPCR) kinase and NFkappaB signaling cascades. Because RKIP targets different kinases dependent upon its state of phosphorylation, RKIP also acts to integrate crosstalk initiated by multiple environmental stimuli. Loss or depletion of RKIP results in disruption of the normal cellular stasis and can lead to chromosomal abnormalities and disease states such as cancer. Since RKIP and the PEBP family have been reviewed previously, the goal of this analysis is to provide an update and highlight some of the unique features of RKIP that make it a critical player in the regulation of cellular signaling processes.     

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。     

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.     

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 regulates MAP kinase signaling in cells with defective B-Raf activity

MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.     

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.