The Ras/Raf-1/MEK/ERK pathway conveys mitogenic and differentiation signals to the nucleus [13,34]. It is organized in a complex that is nucleated by Ras proteins [35] that are activated by growth factors that bind the Raf-1 kinase with high affinity. These induce Raf-1 recruitment to the cell membrane and its activation via phosphorylation. Raf-1 activation, in turn, leads to the activation of MEK, which results in the phosphorylation and activation of ERK1/2 [13,36,37]. Activated ERKs migrate to the nucleus to regulate gene expression by phosphorylating transcription factors [13,38]. Yeast studies have revealed that the JIP-1 and Ksr proteins are important scaffolding molecules used to assemble components of the MAPK pathway [13,39]. Ksr, a protein kinase, functions by binding to Raf-1, MEK, and ERK, while JIP-1 serves as a scaffolding protein for the stress-activated MAPKs/JNKs [13,40,41,42,43,44,45,46,47]. The above studies helped in the identification of RKIP as an endogenous inhibitor of the MAPK pathway via physical association with different kinases of the cascade [13,48].

RKIP can specifically inhibit the Raf-1/MEK/ERK cascade by two mechanisms: (1) by binding to the N-region of the Raf-1 kinase domain, thus inhibiting its phosphorylation and activation, and (2) by dissociating the Raf-1/MEK complex, hence inhibiting the phosphorylation and activation of MEK [1,13,14,49,50] (Figure 1A). Interestingly, it was further shown that MEK and Raf-1 bind to the same sites on RKIP, even though MEK and RKIP are associated with different domains on Raf-1. Furthermore, it was shown that Raf-1 and RKIP bind to different sites on MEK. In order for the Raf-1/MEK/ERK pathway to be suppressed by RKIP, the MEK binding sites and the Raf-1 binding sites on RKIP must be deleted. This suggests that whether RKIP binds to Raf-1 or MEK, both are ample for their inhibition [13]. Given that the MAPK signaling promotes cell migration and RKIP inhibits MAPK signaling, the restoration of RKIP expression in human hepatoma (HHC) and melanoma cells repressed tumor cell migration and motility, as well as the cellular invasive potential [51,52,53,54].

Active NF-kB acts as a transcriptional regulator of several genes that play a role in immunity, inflammation, cell proliferation, cell migration, and apoptosis [51,55]. RKIP is an inhibitor of NF-κB signaling by interacting with and inhibiting upstream activating kinases, such as the transforming growth factor B-activated kinase-1 (TAK-1), the NF-κB-inducing kinase (NIK), and the IκB kinaseσ (IKKα and IKKβ) [14] (Figure 1B). The translational impact of NF-κB inhibition by RKIP in cancer conveys with cancer cell sensitization to apoptotic signals mediated by chemotherapy, or endogenous immune-related cytotoxicity or exogenous immunotherapy, inhibition of EMT and cell metastatic potential, as well as reduced cell survival [51,56]. Researchers have also proposed that since RKIP interacts with members of the NF-κB signaling pathway, it could also serve as a scaffold protein that plays a role in assembling a multicomponent protein complex [51].

RKIP phosphorylation at Ser153 (pSer153RKIP) increases the survival and invasion of cancerous cells by inhibiting the G-protein-coupled receptor kinase 2 (GRK-2), which negatively regulates G-protein-coupled receptor (GPCR)-mediated signaling [1]. Briefly, under normal conditions, GRK-2 binds to GPCR and blocks its activation and intracellular signaling. When RKIP is unphosphorylated, it associates with Raf-1 and suppresses MAPK signaling. RKIP is phosphorylated at Serine 153 (S153) by GPCR-induced PKC zeta [57]. In turn, this phosphorylation dissociates RKIP from Raf-1, thus de-repressing RKIP-mediated MAPK inhibition. Subsequently, the phosphorylated RKIP binds to GRK2 and desensitizes GPCRs, leading to persistent phosphorylation of Raf-1 and activation of GPCR [18,57,58,59] (Figure 1E).

High levels of pSer153 RKIP are induced by IL-6 and H. pylori infection in colon and gastric cancers, respectively, and are associated with a poor prognosis in stage II colon cancer patients and little to no response to therapy for patients with multiple myeloma [57,60,61]. In gastric cancer models, H. pylori-mediated phosphorylation of RKIP at S153 promotes its nuclear accumulation in cancer cells, while it targets unphosphorylated RKIP for proteasome degradation [62,63]. Unlike the unphosphorylated RKIP form, the pRKIP acts by competitively inhibiting survival signals and promoting apoptosis in cancer cells [64,65,66].

STAT3 is a member of the signal transducer and activator of transcription (STAT) family, which is found in the cytoplasm, and upon being activated by phosphorylation, it translocates in the nucleus acting as a transcription factor for genes involved in the process of apoptosis, cell growth, survival, and differentiation [1,67]. Although the exact mechanism is unknown, studies in colon cancer models have shown that the overexpression of RKIP inhibits IL-6-, Jak-, or Src kinase-mediated phosphorylation of STAT3 known to be necessary for its activation [1,17] (Figure 1C).

RKIP has been reported to regulate glycogen synthase kinase 3 (GSK3β) levels by (1) direct binding to the GSK3β protein that results in better protein stabilization and (2) by preventing GSK3β inhibitory phosphorylation [51] (Figure 1D). Depletion of RKIP induces high levels of oxidative stress response that leads to p38 MAPK activation, which ultimately inhibits GSK3β by phosphorylating its T390 residue, an inhibitory residue [51,68,69]. Furthermore, when RKIP is depleted, downstream GSK3β targets are activated, leading to cyclin D1 stabilization, which is what induces cell cycle progression and expression of β-catenin, SNAIL and SLUG. These three are also responsible for promoting the invasion and EMT [51,69]. In addition, in HEK-293 cells where RKIP was depleted, cell migration was favored by inducing p38-mediated phosphorylation of GSK3β, whereas its degradation stabilized cell migration regulatory molecules, such as β-catenin [51,69].

RKIP also regulates the spindle checkpoint and thus plays a role in the control of the cell cycle and the stability of the genome by associating with the centrosomes and kinetochores in mammalian cells [51,70,71]. RKIP-depleted cells rapidly move to the anaphase displaying a defective spindle checkpoint. Its depletion led to the reduced localization and kinase activity of Aurora B, a kinase that helps with chromosomal alignment as well as spindle checkpoints and cell division [51,70,72]. Furthermore, RKIP deficient cells tend to display decreased localization of Aurora B to kinetochores, which leads to inhibition of the activity of its kinase because of the hyperactivation of the MAPK pathway. This process sometimes leads to chromosomal abnormalities [51,70]. Using comparative genomic hybridization and allelotyping, researchers found that colorectal tumors lacking or weakly expressing RKIP display chromosomal losses and are genomically unstable, unlike cancers expressing it [51,73]. In addition, cells with depleted RKIP exert a shorter transition time from their nuclear envelope breakdown to anaphase, which, together with Aurora B and G2/M downregulation, gives cells a highly proliferative quality due to a faster rate of completion of the cell cycle phases [51,69]. This data further proposes that RKIP does in fact have an influence on cell proliferation and its overexpression reduces cell growth compared to RKIP depleted cells [51]. The depletion of RKIP indirectly influences the acceleration of cellular proliferation and growth by means of modulating the expression of genes that are involved in DNA replication, transition through G1/S phase, G2/M checkpoints, and genomic stability [51,69].

The most well-known transcriptional regulator of RKIP is the epithelial-to-mesenchymal transition (EMT) protein SNAIL. Researchers have demonstrated a negative correlation between SNAIL and RKIP expression in prostate cancer and identified SNAIL as a direct transcriptional repressor. It was proven that SNAIL expression can be induced by NFκB and the transcription factor Yin Yang 1 (YY1), leading to RKIP downregulation [62,84,85]. Furthermore, in melanoma cell lines, a PDZ-domain (the postsynaptic density protein (PSD-95), melanoma differentiation associated gene 9 (MDA-9), discs-large tumor suppressor (Dlg), and tight junction protein-1 (ZO-1)) contains a scaffold protein that can silence RKIP transcription by activating SNAIL expression through ERK1/2 and NFκB signaling due to its role in promoting melanoma progression [54,62].

As for the chemical induction of RKIP, the organic molecule nitric oxide was found to be able to induce RKIP expression [62,86,87]. A nitric oxide donor, DETANONOate, inhibits NFκB signaling and lifts SNAIL-mediated transcriptional suppression on the RKIP promoter [62,86]. In some cases, chemotherapy, immunotherapy, and radiotherapy can also induce RKIP expression.

Snail has been shown to strongly repress E-cadherin transcription. Due to the coexpression between RKIP and E-cadherin, the hypothesis was that Snail may also act similarly towards RKIP [84,88,89]. Suppression of RKIP expression is inversely correlated with the expression levels of Snail; thus, when Snail expression was knocked down by specific siRNA, RKIP expression increased, suggesting that Snail is a repressor of RKIP [84]. Snail represses RKIP by binding to the E-box cis-elements in the RKIP promoter and recruiting mSin3A histone deacetylases and transcriptional repressor complexes [84,90,91]. When Snail is present, the enhancer of Zeste homolog 2 (EZH2) inhibits RKIP expression at the transcriptional level.

BACH1 expression is negatively correlated with the expression of RKIP in breast cancer, suggesting that it may be its key negative regulator [62]. Similar to Snail, BACH1 positively correlates with the expression of EMT-associated genes, implying that it plays a role in EMT [62]. In addition, both molecules negatively regulate their own promoters, reducing their expression in a negative feedback loop manner. In addition, both are downstream targets of RKIP but also its negative regulators [62,92].

The region spanning from −56 to +261 nucleotides in the promoter region of RKIP is needed for its full activity. In this region, cis-acting elements that respond to Sp1, CREB, and histone acetylase p300 are needed for the maintenance of its promoter activity [57,93]. For example, studies have shown that knocking down CREB, p300, or Sp1 or the mutation or deletion of these elements limited RKIP promoter activity, meaning that they positively regulate RKIP transcription [57,93]. Finally, in prostate cells, the binding of the androgen receptor to an androgen responsive element in the RKIP promoter has a positive regulatory effect on the regulation of its transcription [57,94].

The transcription factor C-MET activates β-catenin and acts as a growth factor that promotes cell migration, whereas PAK1 is a protein kinase involved in cell motility. Both molecules were found to be upregulated in RKIP-silenced HEK-293 cells [51,95,96], supporting the idea that RKIP loss influences the cell proteome and transcriptome to favor migration.

It has been hypothesized that RKIP’s suppressive role in metastasis and cell migration is due to its ability to downregulate specific matrix metalloproteinases (MMPs) expression, such as MMP-1 and MMP-2 [51,97]. High expression of MMP-1 and MMP-2 along with a high invasive cellular property as a result of RKIP silencing led to the conclusion that RKIP controls the invasion of cancer cells by negatively regulating NF-kB, which in turn controls the expression of MMP and, therefore, cellular invasion [51,97].

It is known that the downregulation of EZH2 inhibits cancer cell growth, proliferation, and invasion [98,99]. A decreased EZH2 expression can also increase the expression of RKIP in cancer cells [98]. An experiment was done to test whether decreasing cellular proliferation, growth, or invasion via silencing EZH2 could be reversed if RKIP expression was silenced. The inhibition of RKIP had no effect on cellular growth or proliferation, but it did effectively reverse the decrease in invasiveness that was caused by the loss of EZH2 [98], suggesting that the EZH2-mediated inhibition of RKIP is part of the molecular mechanism by which EZH2 promotes cellular invasion and metastasis in prostate and breast malignancies.

The quantification of RKIP transcript levels in different cancer cell lines suggested that its downregulation is due to changes in the stability of its mRNA or the initiation of transcription [84]. Another study was performed to determine whether RKIP is repressed by methylation in metastatic prostate cell lines by examining the effects of trichostatin A (TSA, a histone deacetylase (HDAC) inhibitor) on RKIP expression. The authors reported that TSA significantly increased RKIP expression in the human prostate carcinoma cell line, DU145, but treatment with 3 microM 5-Aza-2dC, a demethylation agent, had no effect on RKIP expression [84,100]. The cellular treatment with 5-Aza-2dC showed that hyper-methylation is not the cause of RKIP downregulation. In addition, the fact that RKIP expression can be induced by TSA in cancer cells implies that it may be actively repressed in them [84]. Several studies on the promoter methylation have shown that the methylation status of the RKIP promoter is correlated with low RKIP expression levels in advanced stages of several tumors, including gastric adenocarcinomas, esophageal squamous cell carcinomas, and colorectal and breast cancers [62,73,101,102,103,104,105,106]. In esophageal and gastric cancers, the RKIP promoter was significantly hypermethylated in poorly differentiated tumors and lymph node metastases and this hypermethylation was associated with worse overall survival [62,102,104]. Furthermore, in the prostate cancer cell line DU145, TSA caused an increase in RKIP levels [62,84], which raises the question of whether histone deacetylation plays a role in RKIP silencing or not. One study showed that treatment of the triple-negative breast cancer (TNBC) cell line SUM159 with histone deacetylase inhibitors induced RKIP expression [107], but this was not verified in a different study [92]. Further research on this noted that BACH1 was induced by treatment with an HDAC inhibitor [62,92]. The authors also confirmed that EZH2 interacts with BACH1 in the TNBC cells to inhibit RKIP transcription, which can also be seen in breast and prostate cancer cells by interaction with Snail [62,98].

As seen in previous studies, RKIP’s expression is inversely correlated with miR-23a in prostate cancer and other malignancies [108,109,110]. To confirm the involvement of miR-23a in the regulation of RKIP in prostate cancer, Du et al., (2017b) computationally predicted that miR-23a binds directly to the 3′UTR of RKIP [108]. This prediction was verified using luciferase reporter assays. Co-transfection with luciferase reporter plasmids and miR-23a mimics showed that the luciferase activity of RKIP was significantly inhibited [108]. The knockdown of miR-23a also led to a significant increase in the expression of RKIP, whereas over-expression of miR-23a caused a significant reduction in RKIP protein expression [108]. Overall, the above results indicate that miR-23a negatively regulates RKIP in prostate cancer cells. Furthermore, RKIP gene expression can be inhibited by certain microRNAs [111], but the overexpression of let-7, miR-1, and miR-16 can enhance RKIP protein translation. On the other hand, the overexpression of miR-155 can destabilize RKIP and reduces its expression [90,91,112,113].

RKIP is reduced in metastasis and has been especially studied in breast cancer cells [114,115]. Several miRNAs, including miR-224, miR-27a, miR-23a, and miR-543 have been shown to target and inhibit the RKIP transcript [102,108,110,114,116,117]. Of these, only miR-224 seems to inhibit RKIP expression in breast cancer [111,114]. After looking at the relationship of RKIP expression and 2238 miRNAs, researchers classified three miRNAs as putative RKIP expression-regulating molecules: miR-224-5p, miR-222-3p and miR-125b-5p [110,111,114]. These were selected based on their correlative expression with RKIP in breast cancer cell lines [84,114]. To establish a regulating effect of the miRNAs on RKIP, three conditions need to be applied. They are: (i) negatively correlated with its expression levels, (ii) physically associated with the RKIP mRNA molecule, and (iii) the existence of miRNA recognition elements (MREs) for the identified miRNA in the 3′UTR and amino acid coding sequence of the RKIP gene is required [110,114]. Experiments showed that miR-224-5p functions as an RKIP repressor in breast cancer cells [111,114,118].

The miR-224 can negatively regulate RKIP, contributing to increased cell proliferation and invasion in gastric and breast tumors and in hepatocellular carcinoma [57,117,119,120]. RKIP can also be targeted by miR-27a, the upregulation of which contributes to chemoresistance in lung adenocarcinoma [57,117].

The protein kinase C (PKC) phosphorylates RKIP, activating its downstream signaling pathways [121]. PKC signal transduction participates in the process of T cell apoptosis and proliferation. It is the key enzyme during the cells inner signal transduction, which can regulate the expression of IL-2, IL-6, GM-CSF, and IL-1B by means of activating the backward position of NF-kB and AP-1 [122]. PKC also plays an important role in the immune inflammation reaction, cell apoptosis and proliferation, and self-immunity disease. PKC also plays a role in the mechanisms by which asthma works [122,123]. PKC phosphorylates RKIP at serine 153, dissociating it from Raf-1 [124].

Du et al., (2017b) investigated whether x-inactive specific transcript (XIST) acts as a competing endogenous RNA (ceRNA) in RKIP regulation, using immunoblotting assays to determine its expression post transfection with XIST (or si-XIST) and miR-23a mimics (or inhibitors, respectively) [108]. The results revealed that XIST overexpression substantially promoted RKIP expression, whereas miR-23a repudiated it [108]. While this was the case for overexpression of XIST, researchers found that the knockdown of XIST led to a reduction in RKIP that could essentially be returned by the inhibition of miR-23a [108]. All in all, these findings suggest that lncRNA XIST regulates RKIP expression in a ceRNA manner and miR-23a plays a main role in XIST-mediated regulatory pathways.

XIST has been known to be responsible for suppressing cellular proliferation and metastasis in prostate cancer cell lines and for negatively regulating miR-23a expression [108]. While investigating the correlation between XIST and miR-23a, miR-23a was identified as a direct target of XIST and its over-expression could overturn the XIST-induced RKIP up-regulation [108]. This suggests that XIST positively regulates RKIP expression through miR-23a binding.

RKIP is widely distributed across different human tissues [12,23,53,115,125]. In patients with adenocarcinoma, the deletion or downregulation of the RKIP gene was predicted to result in a poor prognosis [125]. Scientists found that in gastric cardia adenocarcinoma, the expression of RKIP was substantially lower in precancerous tissues in patients with lymph node metastasis, which further implies that RKIP plays a role in the progression, metastasis, and invasion of gastric cardia adenocarcinoma [125,145].

A further study showed that the overexpression of Snail correlates with high expression of cancer stem cell (CSC) markers and increased chemoresistance in colon cancer cell lines [12]. When looking at the effects of Snail silencing and RKIP upregulation, it was seen that the Snail/RKIP loop is an essential component of CSC existence within the tumor, associated with regulation and tumor chemoresistance [12,146]. The reduction of RKIP led to amplified radio-resistance of non-small-cell lung cancer (NSCLC), which generally accelerates the expression of CSC markers and sustains properties of CSC via the expression of Snail [12,147,148]. This demonstrates that RKIP has a negative effect on radio-resistance regulation and chemoresistance because it affects the number and function of CSCs in the tumor.

Initially, RKIP was found to play a role in cancer through a gene array analysis that was performed to determine the genes that regulate metastasis. Researchers discovered that the prostate cancer (PCa)-associated LNCaP cell line has a low metastatic rate and therefore expresses higher RKIP levels than the cells of the derivative cell line C4-2B, which contained a high metastatic rate [1,23]. When C4-2B PCa cells were transfected with RKIP, its expression was restored and led to a reduction in spontaneous lung metastasis, but not primary tumor growth, proving that RKIP functions as a metastasis suppressor gene [1,56,149]. In addition, the restoration of RKIP expression was shown to inhibit breast cancer metastasis in murine models [91,97]. The downregulation of RKIP induced a high viability and migration of cells but did not have an effect on angiogenesis and cellular proliferation [1,150]. RKIP acts as an inhibitor at the molecular level and its loss promotes metastasis through the inhibition of angiogenesis, local invasion, colonization, and intravasation [1,23,91]. It is possible that the inhibitory protein does this by modulating the extracellular matrix [1,151]. RKIP also acts as a phosphorylation target for pathways that involve MAP kinase (MAPK) and the beta-adrenergic receptor (B-AR) and regulates these pathways by binding as well as inhibiting Raf and G protein-coupled receptor kinase 2 (GRK2) [8,18].

It has been found that RKIP expression is downregulated in several tumors, including those of highly metastatic prostate, colon, and breast cancer, hepatocellular carcinoma, and skin melanomas [12,23,52,53,56,84,115,149,152,153]. Furthermore, the restoration of its expression inhibits prostate cancer metastasis [23,84,154]. In order to study the regulation of RKIP, researchers examined its expression in cancer cell lines with different metastatic capacity. They observed that in highly invasive, metastatic cancers, such as breast and prostate cancers, its expression is repressed, while in noninvasive cell lines RKIP expression is high [84]. Researchers also found that RKIP is correlated with the intracellular adhesion protein E-cadherin (E-cad), which is regulated by the Snail and Slug transcription factors [84,155].

Prostate cancer accounts for 15% of cancers diagnosed in males and for 13% of cancer-related deaths [108,156]. In early state prostate cancer, tumor growth is generally attributed to the presence of androgens in the body. Androgen deprivation therapy (ADT) is the main form of treatment used for androgen-dependent prostate cancer, but a large majority of the tumors tend to continue to grow after remission that lasts about 18–24 months and continue to do so in an androgen-independent manner [108,157,158]. The long non-coding RNAs (lncRNAs) are a relatively new form of non-encoding RNA transcripts (>200 nucleotides) [108,159] that participate in cellular development and differentiation, as well as in tumorigenesis. They can also regulate gene expression in multiple ways, thus affecting several processes, including chromatin structure, nuclear transport, cutting and splicing, transcriptional modification, epigenetic control, and RNA decay [108,160,161,162,163,164,165]. LncRNAs have also been observed in many cancers and studies indicate that they can act as tumor suppressors, oncogenes, or even both [108,166]. For instance, Zeng et al., (2017) reported that lncRNA AF113014 acts as a tumor suppressor in hepatocellular carcinoma cells by promoting Egr2 expression [167], and Li et al., (2017) showed that the lncRNA n340790 enhances cellular proliferation in thyroid cancer by means of targeting miR-1254 [168]. In a study by Du et al. (2017), it was confirmed that the downregulation of XIST in prostate cancer is a common molecular change [116]. The expression of XIST is negatively correlated with metastasis and its low expression is associated with poor prognosis in prostate cancer patients. Furthermore, the overexpression of XIST has the potential to inhibit proliferation, migration, and invasion in prostate cancer cells [108].

Scientists have found evidence that the expression of lncRNAs is strongly associated with the development of cancer. Studies have shown that the lncRNA XIST regulates different cancers but in prostate cancer its underlying mechanism is still unclear [108]. A downregulation of XIST in prostate cancer specimens and cell lines that leads to a low expression of XIST has been correlated with advanced tumor stage in patients with prostate cancer and a poor prognosis in those patients [108].

A study done in 2012 aimed at assessing whether (-)-epigallocatechin 3-gallate (EGCG) regulates the expression of RKIP and invasive metastatic activity in AsPC-1 pancreatic adenocarcinoma cells via epigenetic modifications. Results showed that RKIP expression differed in human pancreatic cancer cell lines. Baseline levels of RKIP were investigated in the cell lines MIA PaCa-2, PANC-1, AsPC-1, and BxPC-3. The AsPC-1 cell line was specifically selected to find out whether RKIP expression was in fact induced by EGCG treatment since it had the lowest level of RKIP of all the chosen cell lines [169]. An MTT assay was used to examine the effect of EGCG on AsPC-1 viability and it was revealed that no toxicity was present up to 10 μM EGCG, which is less than the usual EGCG treatment which is 15 μM [169]. Treatment with 10 μM of EGCG for 24 h led to an increase in RKIP expression in AsPC-1 cells compared to control cells, and in order to confirm if this RKIP regulation was due to histone modification, researchers performed the same experiments along with cellular treatment with TSA [169]. They found that in the presence of TSA, RKIP expression was induced and that the effects were synergistic to the effects of EGCG, allowing them to conclude that in AsPC-1 cells treated with EGCG, RKIP induction is partly due to HDAC modifications [169]. Furthermore, to explain the mechanism in which EGCG inhibits invasion and metastasis, researchers studied the expression of metastasis-related genes, such as MMP-2 and -9, Snail and E-cadherin. Results revealed that compared to the mRNA and protein levels in the control cells, the levels in MMP-2, MMP-9, pERK, and Snail were downregulated in the EGCG-treated AsPC-1 cells [169]. E-cadherin was notably increased by EGCG treatment. Since RKIP is responsible for regulating NF-kB activation via the MEK/ERK signaling pathway, the next step was to investigate whether treatment with EGCG inhibits the ERK phosphorylation [169,170]. The results of this experiment showed that treatment of cells with EGCG suppressed ERK phosphorylation and increased RKIP expression. Essentially, the results proved that when RKIP expression is induced by EGCG, it in turn inhibits the phosphorylation of ERK and NF-κB activation while also decreasing Snail expression, which means that EGCG acts as an HDAC inhibitor and can prevent the invasive metastasis of human PC cells. In conclusion, the results of this study showed that EGCG induced RKIP upregulation through the inhibition of HDAC activity, which in turn increases histone H3 expression and inhibits Snail expression, NF-κB nuclear translocation, MMP-2 and MMP-9 activity, and Matrigel invasion in AsPC-1 cells [169]. The results also infer that EGCG regulates RKIP/ERK/NF-κB and/or RKIP/NF-κB/Snail, as well as inhibiting invasive metastasis in the AsPC-1 human pancreatic adenocarcinoma cell line.

Gliomas are tumors that are aggressive and there are no cures currently. Prior reports have indicated that there was a good correlation between the low expression of RKIP and a higher tumor grade [171,172]. The expression of RKIP in gliomas and its clinical significance in metastasis has been reported [150]. The findings by Martinho et al., were different from the report by Maresch et al., of the correlation between the loss of RKIP and high malignant grade [150,171]. However, the association between the loss of RKIP expression and the poor prognosis of high-grade gliomas reported by Maresch et al., was consistent with the findings of Martinho et al. [150,171]. In gliomas, RKIP expression did not affect cell proliferation, and downregulation of RKIP enhanced cell migration but did not affect tumor angiogenesis [150] in contrast to other murine cancers such as prostate [23] and breast cancers [173]. Overall, these findings suggested that the loss of RKIP expression correlates with a poor clinical outcome in glioma patients.

Clear cell renal cell carcinoma (ccRCC) is a cancer in which there are no reliable biomarkers for either its diagnosis or its prognosis. Papale et al., reported that urine from patients with ccRCC has high levels of RKIP and phospho-RKIP that predicted cell survival and progression-free survival [174]. Down-regulation of RKIP expression has been implicated in the development and progression of renal cell carcinoma (RCC). Recently, a study of 310 RCC cases has suggested that RKIP was a significant prognostic marker because of its close correlation with progression and metastasis of RCC. Furthermore, reduced RKIP expression was related to later disease stage, larger tumor size, sarcomatoid subtype, and poor overall survival. These authors also reported that the genetic polymorphisms in RKIP might be associated with the susceptibility and progression of RCC [175].

RKIP expression in intestinal-type gastric cancer was reported to be significantly lower and the authors proposed that RKIP is an independent prognostic factor for intestinal gastric cancer [17]. Furthermore, immunohistochemical analysis showed that the RKIP expression level was the highest in nonneoplastic gastric tissue, low in primary gastric cancer tissue, and the lowest in metastatic gastric cancer tissue, suggesting that RKIP may play a role in the tumorigenesis and metastasis of gastric cancer [176]. Additional studies revealed that RKIP protein expression was negatively correlated with the depth of invasion, TNM stage, and lymph node metastasis. Further clinical and pathological analyses revealed that RKIP protein expression was negatively correlated with the depth of invasion, TNM stage, and lymph node metastasis [132,176,177].

Lung cancer is the most common and fatal for both male and females. Lung cancer is usually divided into two main categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter is further divided into three major types, squamous cell carcinoma (SCC), adenocarcinoma (AC), and large cell carcinoma [178]. Lung AC is the most common type of lung cancer and has a poor survival rate. Studies on the RKIP expression and its clinical significance in lung cancer are not very conclusive. There is one study that assessed the expression levels of the inactive form of RKIP with phosphorylation at serine 153 (pRKIP) [65]. In agreement with this, studies in melanoma and breast cancer have also shown that low levels of pRKIP could predict poor survival in comparison with relatively higher expression [19,179]. Studies by our group analyzed RKIP mRNA expression across 37 different cancer types and, using data from The Cancer Genome Atlas (TCGA) platform, showed that RKIP is downregulated in lung cancer compared to normal lung tissues, with lung adenocarcinoma being among the eight tumor types with the lowest RKIP expression levels [12].

RKIP plays a major role in physiologic hematopoiesis and myeloid malignancies. In physiologic hematopoiesis, a decrease in RKIP expression in the HSPC pool increases the myelomonocytic lineage commitment of these cells. RKIP loss has been described in acute myeloid leukemia (AML) and a series of other myeloid neoplasia (MNs), and a functional involvement in myeloid leukemogenesis has been proven. These same authors have shown that RKIP inhibits proliferation and transformation of myeloid cells and decreases the transformation that is induced by mutant RAS. Both in vitro and in vivo experiments demonstrated that RKIP is an essential player within the development of these liquid tumors. They postulated that RKIP expression is of prognostic relevance and is a target for enhancing therapeutic strategies in AML [180].

Multiple myeloma (MM) is a clonal plasma-cell neoplastic disorder arising from an indolent premalignant disease known as monoclonal gammopathy of undetermined significance (MGUS). All tumors examined have exhibited low levels of RKIP; in contrast, RKIP is overexpressed primarily in its inactive phosphorylated form in MM cell lines and patient-derived tumor tissues. RKIP and the inactivated p-Ser153 form of RKIP are overexpressed in multiple myeloma cell lines and patients’ tissues compared to other tumors, healthy B cells, and healthy bone marrow. Specifically, about half of the RKIP positive cells in MM are in the phosphorylated form. The high RKIP expression in MM is positively correlated with a more aggressive diagnosis, usually resulting in a worse prognosis [90].

RKIP has been identified as an important protein in various cancer types, several of which have been described above. In the majority of different types of cancers, RKIP exhibits low expression levels and RKIP is often absent in metastasis. RKIP loss has been suggested to result from the hypermethylation of its promoter. RKIP mRNA expression across 37 different cancer types was measured using data from The Cancer Genome Atlas (TCGA) platform, corroborating its downregulation in the majority of them compared to the normal tissues. This analysis showed that RKIP exhibits its highest levels in adrenocortical carcinoma (ACC), liver hepatocellular carcinoma (LIHC), and thyroid carcinoma (THCA), and its lowest expression was detected in acute myeloid leukemia (LAML), esophageal carcinoma (ESCA), and stomach and esophageal carcinomas (STES) [12].