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PDF file, 267KB, Supplementary figure 1. a) Heat maps of RKIP, EZH2, Snail and E-cad expression profiles obtained by interrogating publicly available DNA microarray expression datasets (33). The abbreviations are as follows: NAP: normal adjacent prostate tissue, BPH: benign prostate hyperplasia, PCA: prostate cancer adenocarcinoma, and MPC: metastatic prostate carcinoma. Rows correspond to individual genes and columns represent individual patients. Color density is arranged in order from greatest decrease in expression at left (red) to greatest increase at right (light yellow). EZH2 and Snail mRNA are strongly expressed in MPC, in contrast to RKIP and E-cad. b) DU145 or MDA-MB231 cells were infected with indicated siRNA expressing retroviruses. The siRNA expressing cells were assayed for their ability to proliferate in growth medium containing 0.3% agar and the formation of multi-cellular colonies. Colonies were stained with MTT and quantified after 21 days from plates in triplicate (lower panel). Data shown are representative of three independent experiments and are expressed as mean colony number per plate +/- s.d. c) The effect of the indicated siRNA on DU145 cells proliferation was assayed by direct cell counting. Quiescent cells were counted by coulter counter at the indicated times after stimulation with 20% FBS. Data shown are representative of three independent experiments.

Raf-1 kinase inhibitor protein was first identified as a negative regulator of the Raf signaling pathway. Subsequently, it was shown to have a causal role in containing cancer progression and metastasis. Early studies suggested that RKIP blocks cancer progression by inhibiting the Raf-1 pathway. However, it is not clear if the RKIP tumor and metastasis suppression function involve other targets. In addition to the Raf signaling pathway, RKIP has been found to modulate several other signaling pathways, affecting diverse biological functions including immune response. Recent advances in medicine have identified both positive and negative roles of immune response in cancer initiation, progression and metastasis. It is possible that one way that RKIP exerts its effect on cancer is by targeting an immune response mechanism. Here, we provide evidence supporting the causal role of tumor and metastasis suppressor RKIP in downregulating signaling pathways involved with immune response in breast cancer cells and discuss its potential ramification on cancer therapy.

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells.

Tumor microenvironment (TME) is the immediate environment where cancer cells reside in a tumor. It is composed of multiple cell types and extracellular matrix. Microenvironments can be restrictive or conducive to the progression of cancer cells. Initially, microenvironments are suppressive in nature. Stepwise accumulation of mutations in oncogenes and tumor suppressor genes enables cancer cells to acquire the ability to reshape the microenvironment to advance their growth and metastasis. Among the many genetic events, the loss-of-function mutations in tumor suppressor genes play a pivotal role. In this review, we will discuss the changes in TME and the ramifications on metastasis upon altered expression of tumor metastasis suppressor gene RKIP in breast cancer cells.

The role of RhoA GTPases in breast cancer tumorigenesis and metastasis is unclear. Early studies within which mutations in RhoA were designed based on cancer-associated mutations in Ras supported an oncogene role for RhoA. However, recent whole-genome sequencing studies of cancers raised the possibility that RhoA may have a tumor suppression function. Here, using a syngeneic triple negative breast cancer murine model we investigated the physiological effects of reduced RhoA expression on breast cancer tumorigenesis and metastasis. RhoA knockdown had no effect on primary tumor formation and tumor proliferation, concurring with our in vitro findings where reduced RhoA had no effect on breast cancer cell proliferation and clonogenic growth. In contrast, primary tumors with RhoA knockdown efficiently invaded sentinel lymph nodes and significantly metastasized to lungs compared to control tumors. Mechanistically, the current study demonstrated that this is achieved by promoting a pro-tumor microenvironment, with increased cancer-associated fibroblasts and macrophage infiltration, and by modulating the CCL5-CCR5 and CXCL12-CXCR4 chemokine axes in the primary tumor. To our knowledge, this is the first such mechanistic study in breast cancer showing the ability of RhoA to suppress chemokine receptor expression in breast tumor cells. Our work suggests a physiological lung and lymph node metastasis suppressor role for RhoA GTPase in breast cancer.

One third of newly diagnosed breast cancers in the US are early-stage lesions. The etiological understanding and treatment of these lesions have become major clinical challenges. Because breast cancer risk factors are often linked to aberrant nitric oxide (NO) production, we hypothesized that abnormal NO levels might contribute to the formation of early-stage breast lesions. We recently reported that the basal level of NO in the normal breast epithelia plays crucial roles in tissue homeostasis, whereas its reduction contributes to the malignant phenotype of cancer cells. Here, we show that the basal level of NO in breast cells plummets during cancer progression due to reduction of the NO synthase cofactor, BH4, under oxidative stress. Importantly, pharmacological deprivation of NO in prepubertal to pubertal animals stiffens the extracellular matrix and induces precancerous lesions in the mammary tissues. These lesions overexpress a fibrogenic cytokine, TGFβ, and an oncogene, ERBB2, accompanied by the occurrence of senescence and stem cell-like phenotype. Consistently, normalization of NO levels in precancerous and cancerous breast cells downmodulates TGFβ and ERBB2 and ameliorates their proliferative phenotype. This study sheds new light on the etiological basis of precancerous breast lesions and their potential prevention by manipulating the basal NO level.

Raf-1 kinase inhibitor protein was initially discovered as a physiological kinase inhibitor of the MAPK signaling pathway and was later shown to suppress cancer cell invasion and metastasis. Yet, the molecular mechanism through which RKIP executes its effects is not completely defined. RhoA has both a pro- and anti-metastatic cell-context dependent functions. Given that Rho GTPases primarily function on actin cytoskeleton dynamics and cell movement regulation, it is possible that one way RKIP hinders cancer cell invasion/metastasis is by targeting these proteins. Here we show that RKIP inhibits cancer cell invasion and metastasis by stimulating RhoA anti-tumorigenic functions. Mechanistically, RKIP activates RhoA in an Erk2 and GEF-H1 dependent manner to enhance E-cadherin membrane localization and inhibit CCL5 expression.

The majority of breast cancer-related deaths are due to metastasis. A special class of genes regulates the metastatic process. Metastatic genes that can enhance or suppress metastasis have been identified. Raf kinase inhibitor protein (RKIP) is a novel metastasis suppressor gene in breast cancer and its expression is diminished in triple-negative breast cancer (TNBC). Making up approximately 25% of all breast cancer cases, TNBC carries the worst prognosis because of its high rates of distant recurrence and metastasis and lack of effective targeted therapies. Importantly, the reduced expression of RKIP in TNBC associates with poor relapse free survival in patients. Restoring RKIP expression in TNBC cells is sufficient to inhibit cancer metastasis in experimental mouse models. Emerging evidence suggests that miRNAs might play a pivotal role in regulating RKIP expression in breast cancer. We have identified three miRNAs that might have a causal role in downregulating RKIP expression in TNBC. We hypothesize that targeting miRNAs that regulate RKIP for inhibition is a promising strategy to therapeutically restore RKIP expression to inhibit metastasis.

Recent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the BRaf genetic locus as one of the critical events in melanomagenesis. BRaf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of BRaf is tightly regulated and is required for cell growth and survival. BRaf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of BRaf mutated melanoma cells are stringently dependent on BRaf-MEK-ERK activation, the downstream effector targets that are required for oncogenic BRaf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic BRaf (BRafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of BRaf wild-type melanoma cells, miR-10b silencing decreases BRafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for BRafV600E-mediated anchorage-independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic BRafV600E activity in melanoma.

Recent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the B-Raf genetic locus as one of the critical events in melanomagenesis. B-Raf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of B-Raf is tightly regulated and is required for cell growth and survival. B-Raf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of B-Raf mutated melanoma cells are stringently dependent on B-Raf-MEK-ERK activation, the downstream effector targets that are required for oncogenic B-Raf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic B-Raf (B-RafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of B-Raf wild-type melanoma cells, miR-10b silencing decreases B-RafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for B-RafV600E-mediated anchorage independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic B-RafV600E activity in melanoma.

Accumulating evidence suggests that presence of macrophages in the tumor microenvironment add to the invasive and tumor-promoting hallmarks of cancer cells by secreting angiogenic and growth factors. RKIP is a known metastasis suppressor and interferes with several steps of metastasis. However, the mechanistic underpinnings of its function as a broad metastasis suppressor remain poorly understood. Here, we establish a novel pathway for RKIP regulation of metastasis inhibition through the negative regulation of RANTES/CCL5 thereby limiting tumor macrophage infiltration and inhibition of angiogenesis. Using a combination of loss- and gain-of- function approaches, we show that RKIP hinders breast cancer cell invasion by inhibiting expression of the CC chemokine CCL5 in vitro. We also show that the expression levels of RKIP and CCL5 are inversely correlated among clinical human breast cancer samples. Using a mouse allograft breast cancer transplantation model, we highlight that ectopic expression of RKIP significantly decreases tumor vasculature, macrophage infiltration and lung metastases. Mechanistically, we demonstrate that the inhibition of the CCL5 expression is the cause of the observed effects resulting from RKIP expression. Taken together, our results underscore the significance of RKIP as important negative regulator of tumor microenvironment.

From its discovery as a phosphatidylethanolamine-binding protein in bovine brain to its designation as a physiological inhibitor of Raf kinase protein, RKIP has emerged as a critical molecule for maintaining subdued, well-orchestrated cellular responses to stimuli. The disruption of RKIP in a wide range of pathologies, including cancer, Alzheimer's disease, and pancreatitis, makes it an exciting target for individualized therapy and disease-specific interventions. This review attempts to highlight recent advances in the RKIP field underscoring its potential role as a master modulator of many pivotal intracellular signaling cascades that control cellular growth, motility, apoptosis, genomic integrity, and therapeutic resistance. Specific biological and functional niches are highlighted to focus future research towards an enhanced understanding of the multiple roles of RKIP in health and disease.

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.

Recent high-throughput-sequencing of the cancer genome has identified oncogenic mutations inBRafgenetic locus as one of the critical events in melanomagenesis. In normal cells, the activity ofBRafis tightly regulated. Gain-of-function mutations like those identified in melanoma frequently lead to enhanced cell-survival and unrestrained growth. The activating mutation ofBRafwill also induce the cells to senesce. However, the mechanism by which the oncogenicBRafinduces the senescent barrier remains poorly defined. microRNAs have regulatory functions toward the expression of genes that are important in carcinogenesis. Here we show that expression of several microRNAs is altered when the oncogenic version ofBRafis introduced in cultured primary melanocytes and these cells undergo premature cellular senescence. These include eight microRNAs whose expression rates are significantly stimulated and three that are repressed. While most of the induced microRNAs have documented negative effects on cell cycle progression, one of the repressed microRNAs has proven oncogenic functions. Ectopic expression of some of these induced microRNAs increased the expression of senescence markers and induced growth arrest and senescence in primary melanocytes. Taken together, our results suggest that the change in microRNA expression rates may play a vital role in senescence induced by the oncogenicBRaf.

Background: Tumor metastasis suppressors are proteins that impede secondary tumor formation by inhibiting one or more steps of the metastasis cascade without stimulating primary tumor growth. Raf-1 kinase inhibitor protein (RKIP) is a metastasis suppressor that inhibits metastasis in breast, prostate, melanoma and several other types of cancers. The molecular mechanism through which RKIP executes its anti-metastasis effects is not yet completely defined. The objective of the current study is to understand how RKIP inhibits breast cancer cell invasion and metastasis in molecular level. Given its primary function in regulating actin cytoskeleton and cell movements, Rho GTPases were studied as possible downstream effectors of RKIP. These small GTPases belong to the Ras superfamily and consist of nearly 22 members with RhoA and Rac1 as the major players affecting cell motility. Rho GTPases oscillate between a GTP-bound active form and a GDP-bound inactive form. The GDP-GTP cycle is regulated by GEFs that facilitate the exchange of bound GDP with GTP, and by GAPs that stimulate the Rho GTPase activity. Aberrantly activated GEFs are reported to drive tumorigenesis, while activated GAPs are assumed to inhibit the cancer formation and metastasis. Hence, Rho GTPases are widely accepted as oncogenes. Contrary to this notion, several recent studies suggested a possible metastasis inhibitory function for wild-type RhoA in triple negative breast cancer and several other types of cancers. Experimental procedures and results: In this study, several mouse (4T1, 168 FARN) and human (BT20, MDA-MB 231) breast cancer cell lines were used to eliminate possible cell line specific observations. Using in vitro matrigel invasion assay, we showed that RhoA, not Rac1, acts downstream of RKIP and is needed for RKIP-mediated inhibition of breast cancer cell invasion in vitro. GTPase activity pull-down assay results revealed that RKIP specifically increases RhoA activation, thus inhibiting invasiveness of these cells. Similarly, orthotopic mice tumor implantation experiments showed that ectopic expression of dominant negative RhoA in 4T1 cells confers significantly greater proclivity to metastasize in mice. These observations are consistent with RhoA's emerging role as a metastasis suppressor. In BT20 breast cancer cells, RhoA enhanced E-cadherin expression and negatively affected the cell invasiveness. Interestingly, RKIP phenocopied the RhoA effect on E-cadherin and cell invasion, suggesting that RKIP-RhoA axis inhibits breast cancer cell invasiveness by increasing E-cadherin expression. Conclusions: Our results conclude that RKIP specifically increases RhoA activation in breast cancer cells, and this activated RhoA stabilizes E-cadherin and negatively affects the invasiveness of these cells. Citation Format: Gardiyawasam Kalpana, Vu Bach, Clariza Borile, Miranda Yeung, Rafael Garcia-Mata, Kam C. Yeung. RKIP-RhoA axis inhibits breast cancer invasion and metastasis by increasing E-cadherin expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5153.

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.

The Raf-MEK-ERK pathway regulates many fundamental biological processes, and its activity is finely tuned at multiple levels. The Raf kinase inhibitory protein (RKIP) is a widely expressed negative modulator of the Raf-MEK-ERK signaling pathway. We have previously shown that RKIP inhibits the phosphorylation of MEK by Raf-1 through interfering with the formation of a kinase-substrate complex by direct binding to both Raf-1 and MEK. Here, we show that the evolutionarily conserved ligand-binding pocket of RKIP is required for its inhibitory activity towards the Raf-1 kinase mediated activation of MEK. Single amino acid substitutions of two of the conserved residues form the base and the wall of the pocket confers a loss-of-function phenotype on RKIP. Loss-of-function RKIP mutants still appear to bind to Raf-1. However the stability of the complexes formed between mutants and the N-region Raf-1 phosphopeptide were drastically reduced. Our results therefore suggest that the RKIP conserved pocket may constitute a novel phosphoamino-acid binding motif and is absolutely required for RKIP function.

Diminished expression of the metastasis suppressor protein RKIP was previously reported in a number of cancers. The underlying mechanism remains unknown. Here, we show that the expression of RKIP negatively correlates with that of Snail zinc-transcriptional repressor, a key modulator of normal and neoplastic epithelial-mesenchymal transition (EMT) program. With a combination of loss-of-function and gain-of-function approaches, we showed that Snail repressed the expression of RKIP in metastatic prostate cancer cell lines. The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. Our results therefore suggest that RKIP is a novel component of the Snail transcriptional regulatory network important for the progression and metastasis of cancer.

Raf-1 kinase inhibitor protein (RKIP) has been implicated in the regulation of cell survival pathways and metastases, and is poorly expressed in tumors. We have reported that the NF-κB pathway regulates tumor resistance to apoptosis by the TNF-α family via inactivation of the transcription repressor Yin Yang 1 (YY1). We hypothesized that RKIP overexpression may regulate tumor sensitivity to death ligands via inhibition of YY1 and up-regulation of death receptors (DRs). The TRAIL-resistant prostate carcinoma PC-3 and melanoma M202 cell lines were examined. Transfection with CMV-RKIP, but not with control CMV-EV, sensitized the cells to TRAIL-mediated apoptosis. Treatment with RKIP small interfering RNA (siRNA) inhibited TRAIL-induced apoptosis. RKIP overexpression was paralleled with up-regulation of DR5 transcription and expression; no change in DR4, decoy receptor 1, and decoy receptor 2 expression; and inhibition of YY1 transcription and expression. Inhibition of YY1 by YY1 siRNA sensitized the cells to TRAIL apoptosis concomitantly with DR5 up-regulation. RKIP overexpression inhibited several antiapoptotic gene products such as X-linked inhibitor of apoptosis (XIAP), c-FLIP long, and Bcl-xL that were accompanied with mitochondrial membrane depolarization. RKIP overexpression in combination with TRAIL resulted in the potentiation of these above effects and activation of caspases 8, 9, and 3, resulting in apoptosis. These findings demonstrate that RKIP overexpression regulates tumor cell sensitivity to TRAIL via inhibition of YY1, up-regulation of DR5, and modulation of apoptotic pathways. We suggest that RKIP may serve as an immune surveillance cancer gene, and its low expression or absence in tumors allows the tumor to escape host immune cytotoxic effector cells.

Raf kinase inhibitory protein (RKIP-1) is involved in the regulation of the MAP kinase, NF-kappaB, and GPCR signaling pathways. It is expressed in numerous tissues and cell types and orthologues have been documented throughout the animal and plant kingdoms. RKIP-1 has also been reported as an inhibitor of serine proteases, and a precursor of a neurostimulatory peptide. RKIP-1 has been implicated as a suppressor of metastases in several human cancers. We generated a knockout strain of mice to further assess RKIP-1's function in mammals. RKIP-1 is expressed in many tissues with the highest protein levels detectable in testes and brain. In the brain, expression was ubiquitous in limbic formations, and homozygous mice developed olfaction deficits in the first year of life. We postulate that RKIP-1 may be a modulator of behavioral responses.

Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs) including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP transcription, suggesting a potential mechanism by which RKIP inhibits tumor progression and metastasis.

The Raf-MEK-ERK protein kinase cascade is a highly conserved signaling pathway that is pivotal in relaying environmental cues from the cell surface to the nucleus. Three Raf isoforms, which share great sequence and structure similarities, have been identified in mammalian cells. We have previously identified Raf kinase inhibitor protein (RKIP) as a negative regulator of the Raf-MEK-ERK signaling pathway by specifically binding to the Raf-1 isoform. We show here that RKIP also antagonizes kinase activity of the B-Raf isoform. Yeast two-hybrid and coimmunoprecipitation experiments indicated that RKIP specifically interacted with B-Raf. Ectopic expression of RKIP antagonized the kinase activity of B-Raf. We showed that the effects of RKIP on B-Raf functions were independent of its known inhibitory action on Raf-1. The expression levels of RKIP in melanoma cancer cell lines are low relative to primary melanocytes. Forced expression of RKIP partially reverted the oncogenic B-Raf kinase-transformed melanoma cancer cell line SK-Mel-28. The low expression of RKIP and its antagonistic action on B-Raf suggests that RKIP may play an important role in melanoma turmorgenesis.

Cancer cells are more susceptible to chemotherapeutic agent-induced apoptosis than their normal counterparts. Although it has been demonstrated that the increased sensitivity results from deregulation of oncoproteins during cancer development (Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342-348; Green, D. R., and Evan, G. I. (2002) Cancer Cell 1, 19-30), little is known about the signaling pathways leading to changes in the apoptotic threshold in cancer cells. Here we show that low RKIP expression levels in tumorigenic human prostate and breast cancer cells are rapidly induced upon chemotherapeutic drug treatment, sensitizing the cells to apoptosis. We show that the maximal RKIP expression correlates perfectly with the onset of apoptosis. In cancer cells resistant to DNA-damaging agents, treatment with the drugs does not up-regulate RKIP expression. However, ectopic expression of RKIP resensitizes DNA-damaging agent-resistant cells to undergo apoptosis. This sensitization can be reversed by up-regulation of survival pathways. Down-regulation of endogenous RKIP by expression of antisense and small interfering RNA (siRNA) confers resistance on sensitive cancer cells to anticancer drug-induced apoptosis. Our studies suggest that RKIP may represent a novel effector of signal transduction pathways leading to apoptosis and a prognostic marker of the pathogenesis of human cancer cells and tumors after treatment with clinically relevant chemotherapeutic drugs.

One key area of protein kinase research is the identification of cognate substrates. The search for substrates is hampered by problems in unambiguously assigning substrates to a particular kinase in vitro and in vivo. One solution to this impasse is to engineer the kinase of interest to accept an ATP analogue which is orthogonal (unable to fit into the ATP binding site) for the wild-type enzyme and the majority of other kinases. The acceptance of structurally modified, gamma-(32)P-labelled, nucleotide analogue by active site-modified kinase can provide a unique handle by which the direct substrates of any particular kinase can be displayed in crude mixtures or cell lysates. We have taken this approach with the serine/threonine kinase Raf-1, which plays an essential role in the transduction of stimuli through the Ras-->Raf-->MEK-->ERK/MAP kinase cascade. This cascade plays essential roles in proliferation, differentiation and apoptosis. Here we detail the mutagenesis strategy for the ATP binding pocket of Raf-1, such that it can utilise an N(6)-substituted ATP analogue. We show that these mutations do not alter the substrate specificity and signal transduction through Raf-1. We screen a library of analogues to identify which are orthogonal for Raf-1, and show that mutant Raf-1 can utilise the orthogonal analogue N(6)(2-phenethyl) ATP in vitro to phosphorylate its currently only accepted substrate MEK. Importantly we show that our approach can be used to tag putative direct substrates of Raf-1 kinase with (32)P-N(6)(2-phenethyl) ATP in cell lysates.

Raf kinase inhibitory protein (RKIP) is known to modulate key signaling cascades and regulate normal physiological processes such as cellular proliferation, differentiation, and apoptosis. The expression of RKIP is found to be downregulated in several cancer metastases and the repressed RKIP expression can be reactivated on treatment with chemotherapeutic agents. RKIP is a proven tumor metastasis suppressor gene and investigating the mechanisms of transcriptional regulation of RKIP is therefore of immense clinical importance. In this review, we discuss the basal expression of RKIP in various tissues and the genetic aspects of the RKIP chromosomal locus including the structure of the RKIP promoter as well as gene regulatory elements such as enhancers. We also review the genetic and epigenetic modulation of RKIP transcription through EZH2, a component of the polycomb repressive complex 2 (PRC2) and sequence specific transcription factors (TFs) BACH1 and Snail. Emerging experimental evidence supports a unifying model in which both these TFs repress RKIP transcription in cancers by recruiting the EZH2 containing repressive complex to the proximal RKIP promoter. Finally, we review the known mechanisms employed by different types of chemotherapeutic agents to activate RKIP expression in cancer cells.

Raf kinase inhibitor protein (RKIP) has been shown to act as a metastasis suppressor gene in multiple models of cancer. Loss of RKIP expression promotes invasion and metastasis in cell transplantation animal models. However, it is unknown if RKIP expression can impact the progression of cancer in an autochthonous model of cancer. The goal of this study was to determine if loss of RKIP expression in a genetic mouse model of prostate cancer (PCa) impacts metastasis.Endogenous RKIP expression was measured in the primary tumors and metastases of transgenic adenocarcinoma of the mouse prostate (TRAMP(+) ) mice. RKIP knockout mice (RKIP(-/-) ) were crossbred with (TRAMP(+) ) mice to create RKIP(-/-) TRAMP(+) mice. Mice were euthanized at 10, 20, and 30 weeks for evaluation of primary and metastatic tumor development. To determine if loss of RKIP alone promotes metastasis, RKIP was knocked down in the low metastatic LNCaP prostate cancer cell line.Endogenous RKIP expression decreased in TRAMP(+) mice as tumors progressed. Primary tumors developed earlier in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice. At 30 weeks of age, distant metastases were identified only the RKIP(-/-) TRAMP(+) mice. While prostate epithelial cell proliferation rates were higher at 10 and 20 weeks in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice, by 30 weeks there was no difference. Apoptosis rates in both groups were similar at all timepoints. Decreased RKIP expression did not impact the metastatic rate of LNCaP in an orthotopic PCa model.These results demonstrate that loss of RKIP decreases latency of tumor development and promotes distant metastasis in the TRAMP mouse model in the context of a pro-metastatic background; but loss of RKIP alone is insufficient to promote metastasis. These findings suggest that in addition to its known metastasis suppressor activity, RKIP may promote tumor progression through enhancing tumor initiation. Prostate 75:292-302, 2015. © 2014 Wiley Periodicals, Inc.

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.

The sources and consequences of nongenetic variability in metastatic progression are largely unknown. To address these questions, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we demonstrate that BACH1 acts in a double-negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. Analysis of the BACH1 network reveals the existence of an inverse relationship between BACH1 and RKIP involving both monostable and bistable transitions that can potentially give rise to nongenetic variability. Single-cell analysis confirmed monostable and bistable-like behavior. Treatment with histone deacetylase inhibitors or depletion of the polycomb repressor enhancer of zeste homolog 2 altered relative RKIP and BACH1 levels in a manner consistent with a prometastatic state. Together, our results suggest that the mutually repressive relationship between metastatic regulators such as RKIP and BACH1 can play a key role in determining metastatic progression in cancer.

Raf Kinase inhibitory protein (RKIP) is a well-established metastasis suppressor that is frequently downregulated in aggressive cancers. The impact of RKIP and its phosphorylated form on disease-free survival (DFS) and other clinicopathological parameters in breast cancer is yet to be discovered. To this end, we examined RKIP expression in 3 independent breast cancer cohorts. At the Protein level, loss or reduced total RKIP expression was associated with large-sized tumors characterized by high proliferative index, high-grade and diminished estrogen (ER) and progesterone receptor expression. Loss or diminution of RKIP expression was significantly associated with shorter DFS in all cohorts. Moreover, the complete loss of p-RKIP was an independent prognostic factor using multivariate analysis in operable invasive ductal breast cancer. We show for the first time that ER, partly, drives RKIP expression through MTA3-Snail axis. Consistent with this finding, we found that, at the mRNA level, RKIP expression varied significantly across the different molecular subtypes of breast cancer with the Luminal (ER+) subtype expressing high levels of RKIP and the more aggressive Claudin-low (ER-) subtype, which depicted the highest epithelial to mesenchymal transition (EMT) registered the lowest RKIP expression levels. In conclusion, loss of expression/diminution of RKIP or its phosphorylated form is associated with poor diseases-free survival in breast cancer. Determining the expression of RKIP and p-RKIP adds significant prognostic value to the management and subtyping of this disease.

From its discovery as a phosphatidylethanolamine-binding protein in bovine brain to its designation as a physiological inhibitor of Raf kinase protein, RKIP has emerged as a critical molecule for maintaining subdued, well-orchestrated cellular responses to stimuli. The disruption of RKIP in a wide range of pathologies, including cancer, Alzheimer's disease, and pancreatitis, makes it an exciting target for individualized therapy and disease-specific interventions. This review attempts to highlight recent advances in the RKIP field underscoring its potential role as a master modulator of many pivotal intracellular signaling cascades that control cellular growth, motility, apoptosis, genomic integrity, and therapeutic resistance. Specific biological and functional niches are highlighted to focus future research towards an enhanced understanding of the multiple roles of RKIP in health and disease.

Background/Aims: Raf kinase inhibitory protein (RKIP) is a scaffolding molecule in the PEBP family that sequesters certain signaling molecules away from their pathways, thereby abrogating intracellular growth signals. RKIP has been assigned multiple functions and is associated with an increasing number of diseases through its involvement with signal transduction pathways. We previously demonstrated that RKIP is highly expressed in human normal prostate epithelial cells and plays a pivotal role during prostate cancer (PCa) progression. Whether RKIP is subject to endocrine regulation has not been reported. Methods: The effect of dihydrotestosterone (DHT) on RKIP expression in normal prostate epithelial cells was determined by real-time RTPCR and Western blot. Report assay was performed to determine whether the regulation of RKIP by androgens is at the transcriptional level. The binding of androgen receptor (AR) to the RKIP promoter was determined by EMSA and Chromatin Immunoprecipitation (ChIP) assays. To determine whether RKIP was regulated by androgen in vivo, we examined RKIP expression level in response to castration in 6-8 week old C57BL/6 male mice. Results: Here we report that DHT positively regulates the transcription of RKIP in the normal prostate epithelial cells. The anti-androgen bicalutamide blocked androgen-mediated regulation of RKIP, which indicates that this regulation is mediated through AR. Transfection of the cells with a RKIP promoter-driven luciferase reporter vector showed that DHT increased RKIP promoter activity in parallel with changes in expression. EMSA demonstrates that AR binds to a putative ARE in the RKIP promoter, which was further validated by ChIP assay. Importantly, these data are further supported by our in vivo experiment where castrated mice had less RKIP expression in their prostate glands than sham-operated mice. Conclusions: Collectively, the results establish RKIP as a novel androgen target gene. Androgens induce RKIP expression through AR-mediated transcriptional modulation of the RKIP promoter in the prostate. This is the first demonstration of endocrine regulation of the metastasis suppressor gene RKIP.

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. Cancer Res; 72(12); 3091–104. ©2012 AACR.

Human TFIIA activity is composed of three subunits (alpha, beta, gamma). Here we report the isolation of a human cDNA clone encoding the gamma-subunit and the reconstitution of TFIIA activity from recombinant polypeptides (holo-TFIIA). Protein-protein interaction analysis established that the beta and gamma subunits of TFIIA interact with the TBP component of TFIID. The alpha-subunit is recruited into the complex by association with the gamma-subunit. Functional studies indicate that recombinant TFIIA stimulates basal TFIID-dependent transcription but is without effect on TBP-dependent transcription. Our studies indicate that TFIIA not only functions by physically removing negative components present in TFIID (antirepression), as demonstrated previously, but that it can stimulate basal transcription through components of the TFIID complex. Holo-TFIIA also stimulated activation of transcription in vitro as well as in vivo in transfected HeLa cells.

The human cytomegalovirus (HCMV) immediate-early two (IE2) protein of 579 amino acids significantly activates expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter. Using a proviral HIV-1 genome with a mutated tat gene we demonstrate that the IE2 protein effects an increase in the steady-state level of viral RNA similar to a level as from a wild-type proviral genome. The regions of the HCMV IE2 protein required for transactivation of the HIV-1 LTR promoter were analyzed by mutagenizing the IE2 gene and determining the activity of the mutant protein in human fibroblast cells. The region between amino acids 169 and 194 is required to transactivate the HIV-1 LTR promoter, although we have previously shown that this region is not required to activate a representative HCMV early promoter (C. L. Malone, et al., J. Virol. 64, 1498, (1990)). A region downstream of amino acid 290, which is required to activate a representative HCMV early promoter, is also required to activate the HIV-1 LTR promoter. Three types of mutations within this region were shown to greatly decrease IE2 activity: (1) amino acid substitutions of the cysteine or histidine residues in a putative zinc finger motif between amino acids 428 and 452; (2) substitution of the acidic charged residues between amino acids 558 and 561; (3) substitution of the two prolines at residues 556 and 557 immediately upstream of these acidic residues. Substitution of the other acidic residues near the carboxyl terminus also diminished transactivation by IE2. These data indicate that acidic amino acids and the secondary structure in the carboxyl end of the IE2 protein have an important role in transactivation of the HIV-1 LTR promoter. The other regions of the IE2 protein required for transactivation of the HIV-1 LTR are discussed.

The novel proteasome inhibitor NPI-0052 has been shown to sensitize tumor cells to apoptosis by various chemotherapeutic drugs and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), although the mechanisms involved are not clear. We hypothesized that NPI-0052–mediated sensitization may result from NF-κB inhibition and downstream modulation of the metastasis inducer Snail and the metastasis suppressor/immunosurveillance cancer gene product Raf-1 kinase inhibitory protein (RKIP). Human prostate cancer cell lines were used as models, as they express different levels of these proteins. We show that NPI-0052 inhibits both NF-κB and Snail and induces RKIP expression, thus resulting in cell sensitization to CDDP and TRAIL. The direct role of NF-κB inhibition in sensitization was corroborated with the NF-κB inhibitor DHMEQ, which mimicked NPI-0052 in sensitization and inhibition of Snail and induction of RKIP. The direct role of Snail inhibition by NPI-0052 in sensitization was shown with Snail small interfering RNA, which reversed resistance and induced RKIP. Likewise, the direct role of RKIP induction in sensitization was revealed by both overexpression of RKIP (mimicking NPI-0052) and RKIP small interfering RNA that inhibited NPI-0052–mediated sensitization. These findings show that NPI-0052 modifies the NF-κB-Snail-RKIP circuitry in tumor cells and results in downstream inhibition of antiapoptotic gene products and chemoimmunosensitization. The findings also identified Snail and RKIP as targets for reversal of resistance. [Cancer Res 2009;69(21):8376–85]

The treatment of primary tumors results in an initial response to approved conventional therapeutics. However, recurrences and malignancies develop as a result of tumors' acquisition of anti-apoptotic mechanisms of resistance. Hence, there is an urgent need of novel therapeutics that can reverse resistance. One approach of interest is the inhibition of cell survival and anti-apoptotic pathways by sensitizing agents that can render resistant tumor cells sensitive to respond to various cytotoxic therapies. We have found that nitric oxide donors, similar to DETANONOate, inhibit cell survival anti-apoptotic pathways, such as the constitutively activated NF-kappaB and sensitize drug-resistant tumor cells to apoptosis by both chemotherapy and immunotherapy. Sensitization by DETANONOate was shown to inhibit the transcription repressor Yin Yang1 (YY1) shown to regulate resistance to both Fas ligand and TRAIL. In addition, DETANONOate-induced inhibition of NF-kappaB results downstream in the inhibition of several anti-apoptotic gene products, thus facilitating the activation of the apoptotic pathways with both chemotherapy and immunotherapy. In addition, DETANONOate induces the expression of the metastatic tumor suppressor gene product, Raf-1 Kinase Inhibitor Protein (RKIP), which inhibits the survival pathways induced by NF-kappaB and Raf-1/MEK which also contributes to the sensitizing activity. This indicates a novel finding that RKIP may also play an important role in the prevention of metastasis. Inhibition of NF-kappaB activation by DETANONOate results downstream in the inhibition of the RKIP transcription repressor Snail, resulting in upregulation of RKIP. Inhibition of Snail results in downstream inhibition of the metastatic cascade initiated by the epithelial-mesenchymal transition (EMT). Thus, nitric oxide donors have the dual functions of both sensitizing tumor cells to chemotherapy and immunotherapy and are also involved in the regulation and inhibition of metastasis.

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin-Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E-cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO-1, while expression of the cell-substratum adhesion protein beta1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three-dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP-overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference-based silencing of RKIP expression results in decreased cell-substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell-substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch-wound assay, although not as pronounced as that previously reported for RKIP-knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion while negatively controlling cell-cell adhesion, and underscore the complex functions of RKIP in cell physiology.

The acquisition of resistance to conventional therapies such as radiation and chemotherapeutic drugs remains a major obstacle in the successful treatment of cancer patients [1, 2]. In this regard, one of the major determinants of apoptosis sensitivity in cancer cells is the transcription factor nuclear factor kappa B (NFκB). Constitutive expression of NF-κB has been implicated in decreasing apoptosis in cancer cell lines [3]. NF-κB is an inducible transcription factor required for the upregulation of a large number of genes in response to inflammation, viral and bacterial infection, cell survival, cell adhesion, inflammation, differentiation, growth, and stress stimuli [4, 5]. Genes that are responsive to NF-κB activation include a variety of cytokines, cell adhesion molecules, acute phase response proteins, and apoptotic and anti-apoptotic proteins. It is believed that this reprogramming of gene expression is essential for cell survival during physiologic crisis situations. Active NF-κB is a dimer comprised of various members of the Rel family of proteins, and some form of NF-κB is expressed in most cell types. In unstimulated cells NF-κB is retained in the cytoplasm in an inactive form bound to a family of inhibitory proteins known as IκB (inhibitor of κB). Activation of NF-κB requires the phosphorylation and degradation of I-κB, which allows the NF-κB dimer to translocate to the nucleus [6, 7]. NF-κB can be activated by several signaling cascades and is subject to multiple levels of regulation. Considerable progress has been made in the identification of kinases that phosphorylate IκB and target it for subsequent degradation. In addition to its critical role in re-programming gene expression in response to infection and other stresses, NF-κB also mediates cell survival signals, protecting cells from apoptosis [8–10]. For example, cells derived from NF-κB p65 knockout mice are significantly more sensitive to TNFαinduced cytotoxicity than normal cells. Specificity to NF-κB was demonstrated by transfecting p65 into the knockout cells that reversed the cytotoxicity [11, 12]. Similar effects were also observed when NF-κB activation was ablated with an IκB dominantnegative mutant. Cells with compromised NF-κB activation are also more vulnerable to other proapoptotic signals such as ionizing radiation and cancer chemotherapeutic agents [13]. NF-κB has also been implicated in downregulating apoptosis in cncer cell lines that constitutively express elevated NF-κB activity by the observation that ablation of NF-κB activity by a variety of means induced apoptosis [3]. Many of the target genes that are activated by NF-κB are critical to the

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.

The acquisition of resistance to conventional therapies such as radiation and chemotherapeutic drugs remains the major obstacle in the successful treatment of cancer patients. Tumor cells acquire resistance to apoptotic stimuli and it has been demonstrated that conventional therapies exert their cytotoxic activities primarily by inducing apoptosis in the cells. Resistance to radiation and chemotherapeutic drugs has led to the development of immunotherapy and gene therapy approaches with the intent of overcoming resistance to drugs and radiation as well as enhancing the specificity to eliminate tumor cells. However, cytotoxic lymphocytes primarily kill by apoptosis and, therefore, drug-resistant tumor cells may also be cross-resistant to immunotherapy. To evade apoptosis, tumor cells have adopted various mechanisms that interfere with the apoptotic signaling pathways and promote constitutive activation of cellular proliferation and survival pathways. Thus, modifications of the antiapoptotic genes in cancer cells are warranted for the effectiveness of conventional therapies as well as novel immunotherapeutic approaches. Such modifications will avert the resistant phenotype of the tumor cells and will render them susceptible to apoptosis. Current studies, both in vitro and preclinically in vivo , have been aimed at the modification and regulation of expression of apoptosis-related gene products and their activities. A novel protein designated Raf-1 kinase inhibitor protein (RKIP) has been partially characterized. RKIP is a member of the phosphatidylethanolamine-binding protein family. RKIP has been shown to disrupt the Raf-1–MEK1⧸2 [mitogen-activated protein kinase–ERK (extracellular signal-regulated kinase) kinase-1⧸2]–ERK1⧸2 and NF- κ B signaling pathways, via physical interaction with Raf-1–MEK1⧸2 and NF- κ B-inducing kinase or transforming growth factor β -activated kinase-1, respectively, thereby abrogating the survival and antiapoptotic properties of these signaling pathways. In addition, RKIP has been shown to act as a signal modifier that enhances receptor signaling by inhibiting G protein-coupled receptor kinase-2. By regulating cell signaling, growth, and survival through its expression and activity, RKIP is considered to play a pivotal role in cancer, regulating apoptosis induced by drugs or immune-mediated stimuli. Overexpression of RKIP sensitizes tumor cells to chemotherapeutic drug-induced apoptosis. Also, induction of RKIP by drugs or anti-receptor antibodies sensitizes cancer cells to drug-induced apoptosis. In this review, we discuss the discovery, structure, function, and significance of RKIP in cancer.