Raf Kinase Inhibitor Protein Expression Research Articles

Raf kinase inhibitor protein expression in smooth muscle tumours of the uterus: a diagnostic marker for leiomyosarcoma?

Research questionWhat is the expression pattern of Raf kinase inhibitory protein (RKIP) in different subtypes of leiomyoma (usual type, cellular, apoplectic or haemorrhagic leiomyoma, leiomyoma with bizarre nuclei and lipoleiomyoma) and leiomyosarcoma specimens, and what is its biological role in leiomyosarcoma cells? DesignLeiomyoma and leiomyosarcoma specimens underwent immunohistochemistry staining. Leiomyosarcoma SK-LMS-1 cell line was RKIP knocked down and RKIP overexpressed, and cell viability, wound healing migration and clonogenicity assays were carried out. ResultsA higher immunohistochemical expression of RKIP was observed in bizarre leiomyomas, than in usual-type leiomyomas. Decreased expression was also found in cellular leiomyoma, with generally absent staining in leiomyosarcomas. Upon RKIP expression manipulation in SK-LMS-1 cell line, no major differences were observed in cell viability and migration capacity over time. RKIP knockout, however, resulted in a significant increase in the cell's ability to form colonies (P = 0.011). ConclusionRKIP distinct expression pattern among leiomyoma histotype and leiomyosarcoma, and its effect on leiomyosarcoma cells on colony formation, encourages further studies of RKIP in uterine smooth muscle disorders.

Biological and therapeutic implications of RKIP in Gastrointestinal Stromal Tumor (GIST): an integrated transcriptomic and proteomic analysis

BackgroundGastrointestinal stromal tumors (GIST) represent a significant clinical challenge due to their metastatic potential and limited treatment options. Raf kinase inhibitor protein (RKIP), a suppressor of the MAPK signaling pathway, is downregulated in various cancers and acts as a metastasis suppressor. Our previous studies demonstrated low RKIP expression in GIST and its association with poor outcomes. This study aimed to expand on the previous findings and investigate the biological and therapeutic implications of RKIP loss on GIST.MethodsTo validate the RKIP prognostic significance, its expression was evaluated by immunohistochemistry in 142 bona fide GIST cases. The functional role of RKIP was evaluated in vitro, using the GIST-T1 cell line, which was knocked out for RKIP. The biological and therapeutic implications of RKIP were evaluated by invasion, migration, apoptosis, and 2D / 3D viability assays. Additionally, the transcriptome and proteome of RKIP knockout cells were determined by NanoString and mass spectrometry, respectively.ResultsImmunohistochemical analysis revealed the absence of RKIP in 25.3% of GIST cases, correlating with a tendency toward poor prognosis. Functional assays demonstrated that RKIP knockout increased GIST cells’ invasion and migration potential by nearly 60%. Moreover, we found that RKIP knockout cells exhibited reduced responsiveness to Imatinib treatment and higher cellular viability in 2D and 3D in vitro models, as assessed by apoptosis-related protein expression. Through comprehensive genetic and proteomic profiling of RKIP knockout cells, we identified several putative RKIP-regulated proteins in GIST, such as COL3A1.ConclusionsUsing a multidimensional integrative analysis, we identified, for the first time in GIST, molecules and pathways modulated by RKIP that may potentially drive metastasis and, consequently, poor prognosis in this disease.

Manipulating RKIP reverses the metastatic potential of breast cancer cells.

Breast cancer is a common tumor type among women, with a high fatality due to metastasis. Metastasis suppressors encode proteins that inhibit the metastatic cascade independent of the primary tumor growth. Raf kinase inhibitory protein (RKIP) is one of the promising metastasis suppressor candidates. RKIP is reduced or lost in aggressive variants of different types of cancer. A few pre-clinical or clinical studies have capitalized on this protein as a possible therapeutic target. In this article, we employed two breast cancer cells to highlight the role of RKIP as an antimetastatic gene. One is the low metastatic MCF-7 with high RKIP expression, and the other is MDA-MB-231 highly metastatic cell with low RKIP expression. We used high-throughput data to explore how RKIP is lost in human tissues and its effect on cell mobility. Based on our previous work recapitulating the links between RKIP and SNAI, we experimentally manipulated RKIP in the cell models through its novel upstream NME1 and investigated the subsequent genotypic and phenotypic changes. We also demonstrated that RKIP explained the uneven migration abilities of the two cell types. Furthermore, we identified the regulatory circuit that might carry the effect of an existing drug, Epirubicin, on activating gene transcription. In conclusion, we propose and test a potential strategy to reverse the metastatic capability of breast cancer cells by chemically manipulating RKIP expression.

Cancer resistance via the downregulation of the tumor suppressors RKIP and PTEN expressions: therapeutic implications.

The Raf kinase inhibitor protein (RKIP) has been reported to be underexpressed in many cancers and plays a role in the regulation of tumor cells' survival, proliferation, invasion, and metastasis, hence, a tumor suppressor. RKIP also regulates tumor cell resistance to cytotoxic drugs/cells. Likewise, the tumor suppressor, phosphatase and tensin homolog (PTEN), which inhibits the phosphatidylinositol 3 kinase (PI3K)/AKT pathway, is either mutated, underexpressed, or deleted in many cancers and shares with RKIP its anti-tumor properties and its regulation in resistance. The transcriptional and posttranscriptional regulations of RKIP and PTEN expressions and their roles in resistance were reviewed. The underlying mechanism of the interrelationship between the signaling expressions of RKIP and PTEN in cancer is not clear. Several pathways are regulated by RKIP and PTEN and the transcriptional and post-transcriptional regulations of RKIP and PTEN is significantly altered in cancers. In addition, RKIP and PTEN play a key role in the regulation of tumor cells response to chemotherapy and immunotherapy. In addition, molecular and bioinformatic data revealed crosstalk signaling networks that regulate the expressions of both RKIP and PTEN. These crosstalks involved the mitogen-activated protein kinase (MAPK)/PI3K pathways and the dysregulated nuclear factor-kappaB (NF-κB)/Snail/Yin Yang 1 (YY1)/RKIP/PTEN loop in many cancers. Furthermore, further bioinformatic analyses were performed to investigate the correlations (positive or negative) and the prognostic significance of the expressions of RKIP or PTEN in 31 different human cancers. These analyses were not uniform and only revealed that there was a positive correlation between the expression of RKIP and PTEN only in few cancers. These findings demonstrated the existence of signaling cross-talks between RKIP and PTEN and both regulate resistance. Targeting either RKIP or PTEN (alone or in combination with other therapies) may be sufficient to therapeutically inhibit tumor growth and reverse the tumor resistance to cytotoxic therapies.

NeiyiKangfu tablets control the progression of endometriosis through inhibiting RAF/MEK/ERK signal pathway by targeting RKIP

Background NeiyiKangfu tablets (NYKF) are widely used clinically for the treatment of endometriosis (EMS), whose mechanism of action has been extensively studied. Researchers have found that NYKF may control the development of ectopic lesions by inhibiting angiogenesis and inflammatory cytokine secretion. Nevertheless, NYKF’s mechanism of action remains unclear. Methods In the present study, the function of NYKF in the progression of EMS and the associated underlying mechanism was investigated by in vivo and in vitro experiments. EMS model mice were treated with NYKF and the pro-inflammatory factors and apoptosis of ectopic endometrium as well as RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling activation were assessed. In addition, human endometriosis-derived immortalized entopic stromal (hEM15A) cells transfected with or without RAF kinase inhibitor protein (RKIP)-small-interfering RNA (siRNA) were also treated with NYKF and the proliferation, migration, apoptosis, and RAF/MEK/ERK signaling activation were measured by Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell, and western blot, respectively. Results Results showed that NYKF increased the expression of RKIP, inhibited RAF/MEK/ERK signaling activation, and induced apoptosis while inhibiting proliferation and migration both in EMS mice and hEM15A cells. RKIP knockdown could inhibit the effect of NYKF treatment, leading to the activation of RAF/MEK/ERK signaling and the proliferation and migration of hEM15A cells. Conclusions In conclusion, these results suggest that NYKF treatment promotes apoptosis and inhibits proliferation and migration in EMS by inhibiting the RAF/MEK/ERK signaling pathway by targeting RKIP.

RKIP suppresses the influenza A virus‑induced airway inflammatory response via the ERK/MAPK pathway.

Raf kinase inhibitor protein (RKIP) is an inflammation‑inhibiting mediator that is involved in several diseases; however, the potential mechanism of action of RKIP on the inflammatory response induced by influenza A virus (IAV) remains unclear. The present study aimed to investigate whether RKIP regulated the inflammatory response via the ERK/MAPK pathway. The present study detected the expression levels of RKIP and alterations in the inflammatory response in human normal bronchial epithelialBEAS‑2B cells, diseased human bronchial epithelialcellsand primary human bronchial epithelial cellsinfected with IAV. Cells were treated with locostatin to inhibit the expression of RKIP. RKIP was overexpressed by lentivirus transduction and the small molecule inhibitor SCH772984 was applied to specifically inhibit activation of the ERK/MAPK pathway. In addition, C57BL/6mice were infected with IAV to further confirm the role of RKIP in regulation of the inflammatory response via ERK/MAPK invivo.Western blotting, reverse transcription‑quantitative PCR, ELISA, 5‑ethynyl‑-2'‑deoxyuridine assay, immunofluorescence staining, Cell Counting Kit‑8, cell cycle assay, hematoxylin and eosin staining, and immunohistochemistry were used to detect all of the changes. Notably, RKIP attenuated the inflammatory response that was triggered by IAV infection in airway epithelial cells, which was characterized by augmented inflammatory cytokines and cell cycle arrest. Furthermore, the ERK/MAPK pathway was revealed to be activated by IAV infection and downregulation of RKIP aggravated the airway inflammatory response. By contrast, overexpression of RKIP effectively ameliorated the airway inflammatory response induced by IAV. These findings demonstrated that RKIP may serve a protective role in airway epithelial cells by combating inflammation via the ERK/MAPK pathway. Collectively, the present findings suggested that RKIP may negatively regulate airway inflammation and thus may constitute a promising therapeutic strategy for airway inflammatory‑related diseases that are induced by IAV.

Understanding Mechanisms of RKIP Regulation to Improve the Development of New Diagnostic Tools.

Simple SummaryRaf Kinase Inhibitor protein is a protein that governs multiple intracellular signalling involved primarily in the progression of tumours and the development of metastases. In this review, we discussed the main mechanisms that regulate the expression and activity of RKIP with the aim of identifying the link between the transcriptional, post-transcriptional and post-translational events in different tumour settings. We also tried to analyse the studies that have measured the levels of RKIP in biological fluids in order to highlight the possible advantages and potential of RKIP assessment to obtain an accurate diagnosis and prognosis of various tumours.One of the most dangerous aspects of cancer cell biology is their ability to grow, spread and form metastases in the main vital organs. The identification of dysregulated markers that drive intracellular signalling involved in the malignant transformation of neoplastic cells and the understanding of the mechanisms that regulate these processes is undoubtedly a key objective for the development of new and more targeted therapies. RAF-kinase inhibitor protein (RKIP) is an endogenous tumour suppressor protein that affects tumour cell survival, proliferation, and metastasis. RKIP might serve as an early tumour biomarker since it exhibits significantly different expression levels in various cancer histologies and it is often lost during metastatic progression. In this review, we discuss the specific impact of transcriptional, post-transcriptional and post-translational regulation of expression and activation/inhibition of RKIP and focus on those tumours for which experimental data on all these factors are available. In this way, we could select how these processes cooperate with RKIP expression in (1) Lung cancer; (2) Colon cancer, (3) Breast cancer; (4) myeloid neoplasm and Multiple Myeloma, (5) Melanoma and (6) clear cell Renal Cell Carcinoma. Furthermore, since RKIP seems to be a key marker of the development of several tumours and it may be assessed easily in various biological fluids, here we discuss the potential role of RKIP dosing in more accessible biological matrices other than tissues. Moreover, this objective may intercept the still unmet need to identify new and more accurate markers for the early diagnosis and prognosis of many tumours.

The Role of RKIP in the Regulation of EMT in the Tumor Microenvironment

Simple SummaryRaf kinase inhibitor protein (RKIP) expression in cancer cells is significantly reduced and promoting cancer cells growth and invasiveness. Overexpresssion of RKIP has been reported to mediate pleiotropic anti-cancer activities including the inhibition of survival signaling pathways, sensitization to cell death by cytotoxic drugs, inhibition of invasion, EMT and metastasis. The molecular mechanism by which RKIP inhibits EMT is not clear. In this review, we have examined how RKIP inhibits the selected EMT gene products (Snail, vimentin, N-cadherin, laminin alpha) and found that it involves signaling cross-talks between RKIP and each of the EMT gene products. These findings were validated by bioinformatic analyses demonstrating in various human cancers a negative correlation between the expression of RKIP and the expression of the EMT gene products. These findings suggest that targeting RKIP induction in cancer cells will result in multiple hits by inhibiting tumor growth, metastasis and reversal of chemo-immuno resistance.The Raf Kinase Inhibitor Protein (RKIP) is a unique gene product that directly inhibits the Raf/Mek/Erk and NF-kB pathways in cancer cells and resulting in the inhibition of cell proliferation, viability, EMT, and metastasis. Additionally, RKIP is involved in the regulation of cancer cell resistance to both chemotherapy and immunotherapy. The low expression of RKIP expression in many cancer types is responsible, in part, for the pathogenesis of cancer and its multiple properties. The inhibition of EMT and metastasis by RKIP led to its classification as a tumor suppressor. However, the mechanism by which RKIP mediates its inhibitory effects on EMT and metastases was not clear. We have proposed that one mechanism involves the negative regulation by RKIP of the expression of various gene products that mediate the mesenchymal phenotype as well as the positive regulation of gene products that mediate the epithelial phenotype via signaling cross talks between RKIP and each gene product. We examined several EMT mesenchymal gene products such as Snail, vimentin, N-cadherin, laminin and EPCAM and epithelial gene products such as E-cadherin and laminin. We have found that indeed these negative and positive correlations were detected in the signaling cross-talks. In addition, we have also examined bioinformatic data sets on different human cancers and the findings corroborated, in large part, the findings observed in the signaling cross-talks with few exceptions in some cancer types. The overall findings support the underlying mechanism by which the tumor suppressor RKIP regulates the expression of gene products involved in EMT and metastasis. Hence, the development of agent that can selectively induce RKIP expression in cancers with low expressions should result in the activation of the pleiotropic anti-cancer activities of RKIP and resulting in multiple effects including inhibition of tumor cell proliferation, EMT, metastasis and sensitization of resistant tumor cells to respond to both chemotherapeutics and immunotherapeutics.

Activation of RKIP Binding ASC Attenuates Neuronal Pyroptosis and Brain Injury via Caspase-1/GSDMD Signaling Pathway After Intracerebral Hemorrhage in Mice.

Pyroptosis has been proven to be responsible for secondary brain injury after intracerebral hemorrhage (ICH). A recent study reported that Raf kinase inhibitor protein (RKIP) inhibited assembly and activation of inflammasome in macrophages. Our present study aimed to investigate the effects of RKIP on inflammasome-mediated neuronal pyroptosis and underlying neuroprotective mechanisms in experimental ICH. Here, we showed that RKIP expression was decreased both in cerebrospinal fluid (CSF) samples from patients with ICH and in the peri-hematoma tissues after experimental ICH. In mouse ICH model, activation of RKIP remarkably improved neurological deficits, reduced brain water content and BBB disruption, and promoted hematoma absorption at 24h after ICH, as well as alleviated neuronal degeneration, reduced membrane pore formation, and downregulated pyroptotic molecules NLRP3, caspase-1 P20, GSDMD-N, and mature IL-1β. Besides, RKIP activation decreased the number of caspase-1 P20-positive neurons after ICH. However, RKIP inhibitor reserved the neuroprotective effects of RKIP at 24h following ICH. Moreover, RKIP could bind with ASC, then interrupt the assembly of NLRP3 inflammasome. Mechanistically, inhibiting the caspase-1 by VX-765 attenuated brain injury and suppressed neuronal pyroptosis after RKIP inhibitor-pretreated ICH. In conclusion, our findings indicated that activation of RKIP could attenuate neuronal pyroptosis and brain injury after ICH, to some extent, through ASC/Caspase-1/GSDMD pathway. Thus, RKIP may be a potential target to attenuate brain injury via its anti-pyroptosis effect after ICH.

RKIP Induction Promotes Tumor Differentiation via SOX2 Degradation in NF2-Deficient Conditions.

This study identifies that a selective RKIP inducer inhibits tumor growth and promotes schwannoma cell differentiation under NF2-deficient conditions by reducing SOX2 and increasing SOX10 expression.

Selected Articles from This Issue

Despite previous assumptions that constitutively high Wnt signaling promotes colorectal cancer in each stage of disease, recent studies have demonstrated that reduced Wnt signaling is associated with colorectal cancer invasiveness and poor prognoses. However, little work has been done to elucidate how diminished Wnt signaling underlies aggressive colorectal cancer phenotypes. Chen and colleagues used genetic manipulation strategies and orthotopic mouse models to show that abrogating Wnt signaling increases colorectal cancer invasiveness and decreases immune responsiveness in vivo. Disrupting Wnt signaling by targeting Wnt coreceptor LDL receptor-related protein 6 (LRP6) with CRISPR/Cas9 or transducing a dominant negative version of Wnt transcriptional effector lymphoid enhancer binding factor 1 (LEF1) increases tumor submucosal invasion and dissemination in mice. Using RNA-Seq, the authors revealed that reducing Wnt signaling elevates expression of markers associated with stemness and a mesenchymal phenotype, while repressing expression of immune-stimulatory molecules and collagens. The authors used TCGA data to demonstrate that the low Wnt signaling expression profile corresponds with poor survival in colorectal cancer patients, underscoring the potential clinical relevance of dampened Wnt signaling in colorectal cancer progression.Cholangiocarcinoma (CCA) is an aggressive biliary tract cancer for which effective therapeutics are lacking. Prostaglandin E2 (PGE2)-mediated inflammation is a critical component of CCA carcinogenesis and progression. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) chemically modifies and inactivates PGE2, but its expression is often diminished in CCA. In their work, Zhang and colleagues leveraged public data to show that expression of histone methyltransferase G9a negatively correlates with 15-PGDH expression. The authors validated the G9a-15-PGDH relationship in a mouse model of CCA in which the Notch intracellular domain and constitutively active Akt are expressed via hydrodynamic injection of sleeping beauty transposase-based plasmids. shRNA-mediated G9a silencing increases 15-PGDH expression in CCA cell lines, limiting cell proliferation in vitro and tumor growth in vivo. ChIP assays demonstrated that G9a is recruited to the 15-PGDH promoter by the Myc/Max/E-box complex, which prevents signal transducer and activator of transcription 4 (STAT4) from binding the promoter and inducing 15-PGDH expression. The work presented elucidates a previously unknown mechanism underlying CCA development and progression and reveals a novel potential target for CCA therapeutic strategies.Neurofibromatosis type 2 is characterized by growth of benign nervous system tumors, such as schwannomas, which can lead to deafness, brain stem compression, and death. Neurofibromatosis type 2 is caused by mutations in tumor suppressor gene NF2. Previously, Cho and colleagues demonstrated that NF2 loss promotes elevated TβR1 signaling, resulting in phosphorylation and degradation of tumor suppressor Raf kinase inhibitor protein (RKIP). While TGF-β inhibitors halt schwannoma growth, they are not ideal therapeutics given schwannomas occur around adolescence and TGF-β is required for development. Here, Cho and colleagues developed a novel TβR1 inhibitor, Nf18001, that abrogates TβR1-mediated RKIP degradation while maintaining canonical TGF-β signaling. The authors used human and mouse schwannoma cell lines to demonstrate that Nf18001 disrupts TβR1-RKIP interactions to sustain RKIP expression and lower NF2-mutant cell viability while not influencing normal fibroblast survival. Nf18001 arrests cell cycle progression in schwannoma cells, and augments Schwann cell differentiation marker expression while diminishing expression of markers associated with cell stemness. Nf18001 also displayed efficacy in a schwannoma allograft mouse model, where treatment slowed tumor growth.The pro-tumorigenic role of signal transducer and activator of transcription 3 (STAT3) in multiple myeloma has been established, but therapeutically targeting STAT3 has proven challenging. Similarly, given its canonical role in DNA damage repair, checkpoint kinase 1 (Chk1) inhibition has been tested in anti-cancer therapeutic regimens, but with ambiguous results. In their study, Zhou and colleagues employed a gene expression array to demonstrate that Chk1 inhibition diminishes expression of oncogenic STAT3 target genes in multiple myeloma cells, suggesting Chk1 inhibition may also effectively inhibit STAT3 activity. Subsequent work showed that Chk1 directly binds to STAT3 and phosphorylates it at tyrosine 705, enabling STAT3 dimerization and DNA binding. Chk1 inhibition — pharmacologically and via shRNA silencing — diminishes STAT3 tyrosine 705 phosphorylation and inhibits STAT3 DNA binding, reducing expression of STAT3 transcriptional targets. Chk1 inhibition abrogates STAT3 tyrosine 705 phosphorylation in IL-6–dependent, -independent, and bortezomib-resistant cells, as well as in patient primary myeloma mononuclear cells and multiple myeloma cells implanted into mice, suggesting Chk1 inhibition may provide therapeutic efficacy against multiple myeloma through a STAT3-related mechanism.

Computational Analyses of YY1 and Its Target RKIP Reveal Their Diagnostic and Prognostic Roles in Lung Cancer.

Simple SummaryLung cancer (LC) is the tumor with the highest global mortality rate. Novel personalized therapies are currently being tested (e.g., targeted inhibitors, the immune-checkpoint inhibitors), but they cannot yet prevent the very frequent relapse and generalized metastases observed in a large population of LC patients. Currently, there is an urgent need for novel reliable biomarkers for the prognosis and diagnosis of LC. Through the systematic analysis of multiple deposited expression datasets, this report aims to explore the role of the Yin-Yang 1 (YY1) transcription factor and its target the Raf Kinase Inhibitory Protein (RKIP) in LC. The computational analysis suggested the predictive diagnostic and prognostic roles for both YY1 and RKIP stimulating further studies for proving their implication as novel biomarkers, as well as therapeutically druggable targets in LC.Lung cancer (LC) represents a global threat, being the tumor with the highest mortality rate. Despite the introduction of novel therapies (e.g., targeted inhibitors, immune-checkpoint inhibitors), relapses are still very frequent. Accordingly, there is an urgent need for reliable predictive biomarkers and therapeutically druggable targets. Yin-Yang 1 (YY1) is a transcription factor that may work either as an oncogene or a tumor suppressor, depending on the genotype and the phenotype of the tumor. The Raf Kinase Inhibitory Protein (RKIP), is a tumor suppressor and immune enhancer often found downregulated in the majority of the examined cancers. In the present report, the role of both YY1 and RKIP in LC is thoroughly explored through the analysis of several deposited RNA and protein expression datasets. The computational analyses revealed that YY1 negatively regulates RKIP expression in LC, as corroborated by the deposited YY1-ChIP-Seq experiments and validated by their robust negative correlation. Additionally, YY1 expression is significantly higher in LC samples compared to normal matching ones, whereas RKIP expression is lower in LC and high in normal matching tissues. These observed differences, unlike many current biomarkers, bear a diagnostic significance, as proven by the ROC analyses. Finally, the survival data support the notion that both YY1 and RKIP might represent strong prognostic biomarkers. Overall, the reported findings indicate that YY1 and RKIP expression levels may play a role in LC as potential biomarkers and therapeutic targets. However, further studies will be necessary to validate the in silico results.

Insights of RKIP-Derived Suppression of Prostate Cancer.

Simple SummaryDespite an intensive research effort in the past few decades, prostate cancer (PC) remains a top cause of cancer death in men, particularly in the developed world. The major cause of fatality is the progression of local prostate cancer to metastasis disease. Treatment of patients with metastatic prostate cancer (mPC) is generally ineffective. Based on the discovery of mPC relying on androgen for growth, many patients with mPC show an initial response to the standard of care: androgen deprivation therapy (ADT). However, lethal castration resistant prostate cancers (CRPCs) commonly develop. It is widely accepted that intervention of metastatic progression of PC is a critical point of intervention to reduce PC death. Accumulative evidence reveals a role of RKIP in suppression of PC progression towards mPC. We will review current evidence and discuss the potential utilization of RKIP in preventing mPC progression.Prostate cancer (PC) is a major cause of cancer death in men. The disease has a great disparity in prognosis. Although low grade PCs with Gleason scores ≤ 6 are indolent, high-risk PCs are likely to relapse and metastasize. The standard of care for metastatic PC (mPC) remains androgen deprivation therapy (ADT). Resistance commonly occurs in the form of castration resistant PC (CRPC). Despite decades of research efforts, CRPC remains lethal. Understanding of mechanisms underpinning metastatic progression represents the overarching challenge in PC research. This progression is regulated by complex mechanisms, including those regulating PC cell proliferation, epithelial–mesenchymal transition (EMT), and androgen receptor (AR) signaling. Among this PC metastatic network lies an intriguing suppressor of PC metastasis: the Raf kinase inhibitory protein (RKIP). Clinically, the RKIP protein is downregulated in PC, and showed further reduction in mPC. In xenograft mouse models for PC, RKIP inhibits metastasis. In vitro, RKIP reduces PC cell invasion and sensitizes PC cells to therapeutic treatments. Mechanistically, RKIP suppresses Raf-MEK-ERK activation and EMT, and modulates extracellular matrix. In return, Snail, NFκB, and the polycomb protein EZH2 contribute to inhibition of RKIP expression. In this review, we will thoroughly analyze RKIP’s tumor suppression actions in PC.

Investigation of Marine-Derived Natural Products as Raf Kinase Inhibitory Protein (RKIP)-Binding Ligands.

Raf kinase inhibitory protein (RKIP) is an essential regulator of the Ras/Raf-1/MEK/ERK signaling cascade and functions by directly interacting with the Raf-1 kinase. The abnormal expression of RKIP is linked with numerous diseases including cancers, Alzheimer’s and diabetic nephropathy. Interestingly, RKIP also plays an indispensable role as a tumor suppressor, thus making it an attractive therapeutic target. To date, only a few small molecules have been reported to modulate the activity of RKIP, and there is a need to explore additional scaffolds. In order to achieve this objective, a pharmacophore model was generated that explores the features of locostatin, the most potent RKIP modulator. Correspondingly, the developed model was subjected to screening, and the mapped compounds from Marine Natural Products (MNP) library were retrieved. The mapped MNPs after ensuing drug-likeness filtration were escalated for molecular docking, where locostatin was regarded as a reference. The MNPs exhibiting higher docking scores than locostatin were considered for molecular dynamics simulations, and their binding affinity towards RKIP was computed via MM/PBSA. A total of five molecules revealed significantly better binding free energy scores than compared to locostatin and, therefore, were reckoned as hits. The hits from the present in silico investigation could act as potent RKIP modulators and disrupt interactions of RKIP with its binding proteins. Furthermore, the identification of potent modulators from marine natural habitat can act as a future drug-discovery source.

Prognostic significance of immunohistochemical expression of chemokine receptor (CXCR4) and RAF kinase inhibitor protein (RKIP) in gastric carcinoma and premalignant lesions of the stomach

Background: CXCR4 and RKIP have been implicated in initiation and progression in many cancers, but their role in gastric adenocarcinoma remains vague.Aim: The aim of this work is to assess the possible significance of both markers in gastric adenocarcinoma and premalignant lesions of the stomach.Methods: This retrospective study was carried upon 50 cases of gastric adenocarcinoma and 20 of premalignant lesions. Immunohistochemistry was performed to examine the expression of CXCR4 and RKIP in both gastric adenocarcinoma and premalignant lesions of the stomach.Results: CXCR4 was found to be highly expressed in gastric adenocarcinoma compared to premalignant lesions (P value=0.027). It was significantly correlated with tumor grade (P value=0.027), depth of tumor invasion (P value=0.019), lymph node metastasis (P value=0.025), distant metastasis (P value=0.044) and TNM stage (P value=0.002). No statistically significant correlation between CXCR4 expression and histopathological types (P value=0.8). RKIP expression has no significant correlation with progression from premalignant gastric lesions to adenocarcinoma (P value=0.078). RKIP was negatively associated with advanced tumor grade (P value=0.001), depth of invasion (P value=0.018), distant metastasis (P value=0.029) and TNM stage (P value=0.007). No statistically significant correlation between RKIP expression and histopathological types (P value=0.17) and lymph node status (P value=0.053). No significant correlation between CXCR4 and RKIP expression in studied cases was detected (P value=0.178).Conclusions: These results suggested that CXCR4 might be involved in gastric carcinogenesis.Keyword: Gastric adenocarcinoma, premalignant, CXCR4, RKIP.

P4-13 cMET promotes metastasis and epithelial-mesenchymal transition in colorectal carcinoma via repressing RKIP

Colorectal cancer (CRC) ranks among the most frequent malignancies globally and is associated with high metastasis incidence. Today, it is becoming more prevalent among Asian populations, especially in the Chinese. There is increasing evidence that c-mesenchymal-epithelial transition factor (cMET) plays an important role in malignant progression, and its overexpression is closely linked with poor survival and enhanced resistance to drug therapy, hence abnormal cMET is viewed as a signature event in CRC. As a metastasis suppressor, loss of raf kinase inhibitory protein (RKIP) has been reported in many of cancer types. Both of them may serve to regulate CRC initiation and progression. It is therefore necessary to address the interaction between cMET/ERK signal axis and RKIP. In this study, expression of cMET and RKIP in CRC tissues and cell lines were determined, and their crosstalk and potential biological effects were explored using. Immunofluorescence staining and Chromatin immunoprecipitation. Our results showed cMET was inversely correlated with RKIP. Both cMET upregulation and RKIP downregulation implied poor clinical outcomes. Moreover, the MAPK/ERK signal pathway was involved in the regulation of cMET and RKIP. Overexpression of cMET promoted epithelial mesenchymal transition (EMT), invasion, migration and chemoresistance of tumor cells, whereas the impact could be efficiently impeded by increased RKIP. Notably, shRNA-mediated cMET knockdown dramatically suppressed cell proliferation, although no influence of RKIP on cell growth was observed in CRC. Altogether, cMET overexpression may contribute to tumor progression by inhibiting anti-oncogene RKIP, which provides preclinical justification for targeting RKIP to treat cMET-induced metastasis in CRC.

High Expression of the Resistant Factor YY1 and Low Expression of the RKIP Chemosensitizing Factor in Multiple Myeloma: Delineation of Several Crosstalk Pathways That Regulate the Expression of These Factors

Background: We have reported that multiple myeloma (MM) expresses high levels of the transcription (oncogenic) factor YY1, which regulates cell survival, proliferation, and resistance to both immuno- and chemotherapeutic drugs.1 Inhibition of YY1 sensitizes the tumor cells to cell death/apoptosis to cytotoxic drugs. In contrast, we have also reported that MM expresses very low levels of Raf kinase inhibitor protein (RKIP) and/or high levels of inactive phosphorylated RKIP.2 Overexpression of RKIP or de-phosphorylation of phosphorylated RKIP sensitized drug-resistant MM to drug-induced apoptosisHypothesis: The inverse relationship between YY1 and RKIP expression levels suggested that the two factors may be regulated through crosstalk pathways.Methods: Analysis of the literature revealed the presence of five crosstalk pathways that link RKIP and YY1.Results: The crosstalks consisted of the followings: (1) The NF-κB/YY1/Snail/RKIP loop. In the presence of RKIP, the NF-κB pathway is inhibited and consequently, the NF-κB targets YY1 and Snail are also inhibited. In turn, inhibition of the RKIP repressor Snail resulted in the upregulation of RKIP expression, all of which led to sensitization of drug-resistant tumor cells. (2) The p38 MAPK/RKIP/GSK3β/Snail/YY1 loop. In the presence of RKIP expression, the p38 MAPK-mediated phosphorylation of GSK3β is inhibited and results in the phosphorylation of Snail and its degradation, leading to the de-repression of RKIP expression and inhibition of YY1. (3) The RKIP/Smurf-2/YY1/Snail loop. SMAD ubiquitin regulatory factor 2 (Smurf-2) is an E3 ubiquitin ligase that degrades YY1. In tumor cells, there is low expression of Smurf-2 and, thus, little inhibition of YY1 with high Snail expression in the presence of low expression of RKIP. In the presence of high levels of Smurf-2, YY1 is inhibited, resulting in the inhibition of Snail and de-repression of RKIP. (4) The RKIP/MAPK/Myc/Let-7/HMGA2/Snail/YY1 loop. Let-7/miR98 suppresses Ras and inhibits the MAPK signaling pathway. In the absence of RKIP, the Raf/MEK1-2 pathway is activated and downstream Myc is expressed. In turn, Myc-induced Lin-28 inhibits Let-7, and leads to upregulation of HMGA2. Consequently, HMGA2 activates Snail, in addition to its regulation by YY1. In contrast, in the presence of RKIP, the Raf/MEK pathway is inhibited and there is downstream inhibition of its targets Myc, Lin-28, and HMGA2, leading to an inhibition of Snail and de-repression of RKIP. (5) The RKIP/GPCR/STAT3/miR-34/YY1 loop. In tumor cells expressing low levels of RKIP, there is no efficient inhibition of GPCR and MAKP signaling pathways. Hence, there is a constitutive activation of oncogenic STAT3 that inhibits the tumor suppressor miR-34. Thus, low miR-34 levels de-repress YY1 translation and facilitate its effects on the survival and the resistance of tumor cells to cytotoxic drugs.Conclusion and Implications: The above YY1/RKIP gene signatures might predict the tumor expanding potential and overall aggressiveness and their detriments that contribute to tumor regression. Targeting YY1 inhibition or RKIP induction via interference of the above crosstalk pathways may result in synergistic activity in combination with conventional treatments.References1 Bonavida B, Kaufhold S. Prognostic significance of YY1 protein expression and mRNA levels by bioinformatics analysis in human cancers: a therapeutic target. Pharmacol Ther. 150:149-68, 2015.2 Baritaki, S, Huerta-Yepez, S, Cabrava-Haimandez, M, Sensi, M, Canevari, S, Libra, M, Penichet, M, Chen, H, Berenson, JR, Bonavida, B. Unique Pattern of Overexpression of Raf-1 Kinase Inhibitory Protein in Its Inactivated Phosphorylated Form in Human Multiple Myeloma. Forum on Immunopathological Diseases and Therapeutics, 2: 179-188, 2011. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Drug Resistance Via the Overexpression of Phopho-RKIP (P-RKIP) in Multiple Myeloma: Dephosphorylation of P-RKIP Reverses Drug Resistance

Background: Multiple myeloma (MM) remains an incurable disease. We have reported that MM cell lines and patient-derived MM cells overexpress the gene product Raf-Kinase Inhibitor Protein (RKIP) in its inactivated phosphorylated (p-RKIP) form.1 Active RKIP inhibits both the Raf/MEK/ERK and NF-κB pathways and resulting in the inhibition of proliferation, metastasis, and sensitization to chemo and immune drugs.2Objective: Drug resistance is a common feature of patients with MM. The delineation of factors that regulate resistance and their targeting may reverse resistance and leads to prolongation of survival.Hypothesis: We hypothesized that dephosphorylation of RKIP in MM may restore the activity of RKIP and resulting in the sensitization of drug-resistant MM cells to drug-induced cytotoxicity.Experimental Design: The expression of RKIP and p-RKIP (specific anti-p-RKIP antibody) was analyzed in both MM cell lines and fresh tumor cells derived from bone marrow aspirates of MM patients using Western, flow cytometry and IHC and a specific anti-pRKIP monoclonal antibody. We also examined the effect of a PKC inhibitor, bisindolymalemide-1 (BIM) treatment in MM cell lines and their response to treatment with anti-MM drugs such as bortezomib, melphalan, and bendamustine.Findings: The analyses of MM cell lines and patient-derived MM cells revealed the overexpression of RKIP, the majority of which was in its inactive phosphorylated form, p-RKIP. Treatment of MM cell lines with the PKC inhibitor BIM resulted in the dephosphorylation of p-RKIP and the treated drug resistant MM cells were sensitized to bortezomib, melphalan, and bendamustine-induced cell death. We have also found a correlation between the RKIP/p-RKIP ratios in MM and clinical response; Kaplan-Meyer analysis demonstrated that there was a correlation between the high expression of RKIP and poor survival.Conclusions and Clinical Implication: The findings demonstrated that the overexpression of p-RKIP in MM was involved, in part, on the regulation of drug resistance since dephosphorylation of p-RKIP reversed the drug resistance. The role of p-RKIP in the regulation of drug resistance in MM is corroborated in our prior reported studies in other cancer types that demonstrated that knockout of RKIP rendered the sensitive tumor cells drug resistant, whereas in contrast, overexpression of RKIP sensitized the drug-resistant cells to drug-induced apoptosis. We conclude that the combination treatment of a PKC inhibitor and anti-MM drugs can be clinically relevant in the treatment of patients who are resistant to drug treatment. Also, we propose that the examination of the levels of p-RKIP in MM may reveal a novel prognostic biomarker and the determination of proper treatment regimens.Future studies: We propose to investigate the molecular and biochemical means by which p-RKIP is overexpressed in MM and characterizing the specific PKC that is involved in the phosphorylation of RKIP in MM.References1 Baritaki, S, Huerta-Yepez, S, Cabrava-Haimandez, M, Sensi, M, Canevari, S, Libra, M, Penichet, M, Chen, H, Berenson, JR, Bonavida, B. Unique Pattern of Overexpression of Raf-1 Kinase Inhibitory Protein in Its Inactivated Phosphorylated Form in Human Multiple Myeloma. Forum on Immunopathological Diseases and Therapeutics, 2: 179-188, 2011.2 Bonavida B. RKIP-mediated chemo-immunosensitization of resistant cancer cells via disruption of the NF-κB/Snail/YY1/RKIP resistance-driver loop. Crit Rev Oncog. 19:431-45, 2014. DisclosuresNo relevant conflicts of interest to declare.

Association of low RKIP expression with poor prognosis in non-small cell lung cancer.

3068 Background: Raf1 kinase inhibitor protein (RKIP) is able to bind Raf1 to inhibit Ras-Raf-MEK-ERK signaling, a major oncogenic pathway. It has been reported that reduced RKIP expression associates with poor prognosis in many cancers, including gastric adenocarcinoma, gliomas and bladder cancer. However, there are only several studies on its role in non-small cell lung cancer (NSCLC) and the conclusion is still controversial. Hence, we performed this study to assess the prognostic significance of RKIP in our NSCLC population. Methods: Between June 2017 and June 2020, 156 NSCLC patients treated at our hospital were included for the present study. None of the patients had received chemotherapy, radiotherapy or surgery before. Their tumor tissues and surrounding normal lung tissues were collected for immunostain and western blot analysis of RKIP expression and ERK signaling. We collected information about gender, age, histological differentiation, tumor size, TNM stage, and lymph node status. Survival curves were analyzed using the Kaplan-Meier method. Cox proportional hazards model was used to determine the prognostic value of various variables in a univariate and multivariate setting. Results: Immunostain and western blot results showed a lower RKIP expression and a higher p-ERK level in cancer tissues compared with the surrounding normal tissues. A reduced RKIP expression with high level of p-ERK was also observed in TNM stages III and IV as compared with I and II. Pearson's chi-squared test confirmed low RKIP expression associated with poorer TNM stage ( p< 0.001) and N-stage ( p< 0.05). No significant correlation was observed between RKIP expression level and gender, age, histological type or tumor size. Kaplan-Meier survival analysis revealed that patients with low RKIP expression had significantly worse overall survival than patients with high RKIP expression ( p= 0.019, log-rank). This conclusion was consistent in the stage I&II patients ( p= 0.011, log-rank) but not in the stage III&IV patients ( p= 0.711, log-rank). Univariate Cox proportional hazards regression analysis indicated Tumor size, TNM stage and RKIP expression significantly affected overall survival of the NSCLC patients. Multivariate Cox proportional hazards regression analysis confirmed RKIP expression remained a significant predictor of survival after correcting for the effects of Tumor size and TNM stage (hazard ratio = 1.730, 95% confidence interval = 1.017 – 2.942, p = 0.043). Conclusions: In this study, low RKIP expression was a poor prognostic indicator in NSCLC as it significantly correlated with poorer TNM stage, N-status, and overall survival. Our findings suggest that by inhibiting Ras-Raf-MEK-ERK pathway RKIP may play an anti-tumor role in NSCLC.

Transcriptional Repression of Raf Kinase Inhibitory Protein Gene by Metadherin during Cancer Progression.

Raf kinase inhibitory protein (RKIP), also known as a phosphatidylethanolamine-binding protein 1 (PEBP1), functions as a tumor suppressor and regulates several signaling pathways, including ERK and NF-B. RKIP is severely downregulated in human malignant cancers, indicating a functional association with cancer metastasis and poor prognosis. The transcription regulation of RKIP gene in human cancers is not well understood. In this study, we suggested a possible transcription mechanism for the regulation of RKIP in human cancer cells. We found that Metadherin (MTDH) significantly repressed the transcriptional activity of RKIP gene. An analysis of publicly available datasets showed that the knockdown of MTDH in breast and endometrial cancer cell lines induced the expression RKIP. In addition, the results obtained from qRT-PCR and ChIP analyses showed that MTDH considerably inhibited RKIP expression. In addition, the RKIP transcript levels in MTDH-knockdown or MTDH-overexpressing MCF-7 cells were likely correlated to the protein levels, suggesting that MTDH regulates RKIP expression. In conclusion, we suggest that MTDH is a novel factor that controls the RKIP transcription, which is essential for cancer progression.