RKIP localizes to the nucleus through a bipartite nuclear localization signal and interaction with importin α to regulate mitotic progression.

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. 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. 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.

Raf kinase inhibitor protein (RKIP) deficiency decreases latency of tumorigenesis and increases metastasis in a murine genetic model of prostate cancer.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

RKIP: much more than Raf kinase inhibitory protein.

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.

Changes in Expression of Tumor Suppressor Gene RKIP Impact How Cancers Interact with Their Complex Environment.

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.

A Negative Regulatory Role for RKIP in Breast Cancer Immune Response.

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.

The RhoA dependent anti-metastatic function of RKIP in breast cancer.

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 RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK.

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.

Reduced RhoA expression enhances breast cancer metastasis with a concomitant increase in CCR5 and CXCR4 chemokines signaling.

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.

Critical role of miR-10b in B-RafV600E dependent anchorage independent growth and invasion of melanoma cells.

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.

Raf kinase inhibitor protein positively regulates cell-substratum adhesion while negatively regulating cell-cell adhesion.

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.

Raf kinase inhibitory protein knockout mice: expression in the brain and olfaction deficit.

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.

RKIP downregulates B-Raf kinase activity in melanoma cancer cells.

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.

Regulation of RKIP binding to the N-region of the Raf-1 kinase.

The Raf kinase inhibitory protein (RKIP) binds to Raf-1 interfering with binding of the MEK substrate and potentially also Raf-1 activation. In response to mitogen stimulation RKIP dissociates from Raf-1 and later re-associates. Here, using a combination of mutational approaches, biochemical studies, peptide arrays and plasmon surface resonance (BIAcore), we fine map and characterize a minimal 24 amino acid long RKIP binding domain in the Raf-1 N-region, which consists of constitutive elements at both flanks and a center element that is regulated by phosphorylation and enhances the re-binding of RKIP to Raf-1 in the later phase of mitogen stimulation.

Correction: RKIP regulates CCL5 expression to inhibit breast cancer invasion and metastasis by controlling macrophage infiltration.

Present: Due to an error made by the authors while submitting a revision, Dr. Tuan Zea Tan was omitted from the list of authors.Corrected: Correct author list can be found below. Authors sincerely apologize for this oversight. Ila Datar1, Xiaoliang Qiu1, Hong Zhi Ma1, Miranda Yeung1, Shweta Aras1, Ivana de la Serna1, Fahd Al-Mulla2, Tuan Zea Tan3, Jean Paul Thiery3, Robert Trumbly1, Xuan Fan4, Hongjuan Cui4 and Kam C. Yeung1 1 Department of Biochemistry and Cancer Biology, University of Toledo, College of Medicine, Health Science Campus, Toledo, OH, USA 2 Kuwait University, Faculty of Medicine. P.O. Box 24923, Safat, Kuwait 3 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 4 State Key Laboratory Of Silkworm Genome Biology, Chongqing, China Original article: Oncotarget. 2015; 6(36): 39050-61. doi: 10.18632/oncotarget.5176.

RKIP regulates CCL5 expression to inhibit breast cancer invasion and metastasis by controlling macrophage infiltration.

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.

RKIP Inhibits Local Breast Cancer Invasion by Antagonizing the Transcriptional Activation of MMP13.

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.

Engineering the serine/threonine protein kinase Raf-1 to utilise an orthogonal analogue of ATP substituted at the N6 position.

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.

Genetic and epigenetic control of RKIP transcription.

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) blocks signal transducer and activator of transcription 3 (STAT3) activation in breast and prostate cancer.

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