Discovering selective antiferroptotic inhibitors of the 15LOX/PEBP1 complex noninterfering with biosynthesis of lipid mediators.

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis in vitro and in vivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators in vivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.

Brain Distribution and Active Efflux of Three panRAF Inhibitors: Considerations in the Treatment of Melanoma Brain Metastases.

Targeted inhibition of RAF and MEK by molecularly targeted agents has been employed as a strategy to block aberrant mitogen-activated protein kinase (MAPK) signaling in melanoma. While the use of BRAF and MEK inhibitors, either as a single agent or in combination, improved efficacy in BRAF-mutant melanoma, initial responses are often followed by relapse due to acquired resistance. Moreover, some BRAF inhibitors are associated with paradoxical activation of the MAPK pathway, causing the development of secondary malignancies. The use of panRAF inhibitors, i.e., those that target all isoforms of RAF, may overcome paradoxical activation and resistance. The purpose of this study was to perform a quantitative assessment and evaluation of the influence of efflux mechanisms at the blood-brain barrier (BBB), in particular, Abcb1/P-glycoprotein (P-gp) and Abcg2/breast cancer resistance protein (Bcrp), on the brain distribution of three panRAF inhibitors: CCT196969 [1-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)phenyl)urea], LY3009120 1-(3,3-Dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido(2,3-d)pyrimidin-6-yl)phenyl)urea, and MLN2480 [4-pyrimidinecarboxamide, 6-amino-5-chloro-N-[(1R)-1-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]amino]carbonyl]-2-thiazolyl]ethyl]-]. In vitro studies using transfected Madin-Darby canine kidney II cells indicate that only LY3009120 and MLN2480 are substrates of Bcrp, and none of the three inhibitors are substrates of P-gp. The three panRAF inhibitors show high nonspecific binding in brain and plasma. In vivo studies in mice show that the brain distribution of CCT196969, LY3009120, and MLN2480 is limited, and is enhanced in transgenic mice lacking P-gp and Bcrp. While MLN2480 has a higher brain distribution, LY3009120 exhibits superior in vitro efficacy in patient-derived melanoma cell lines. The delivery of a drug to the site of action residing behind a functionally intact BBB, along with drug potency against the target, collectively play a critical role in determining in vivo efficacy outcomes.

Inhibition of the Raf-1 kinase inhibitory protein (RKIP) by locostatin induces cell death and reduces the CXCR4-mediated migration of chronic lymphocytic leukemia cells.

The Raf-1 kinase inhibitory protein (RKIP) is an important regulatory element in multiple signaling pathways, including MAPK-ERK1/2. We investigated whether targeted disruption of RKIP is a therapeutic option for chronic lymphocytic leukemia (CLL). The RKIP inhibitor locostatin-induced apoptosis of CLL cells, irrespective of poor prognostic indications or treatment history. Locostatin down-regulated MAPK-ERK1/2 and AKT phosphorylation, decreased expression of the chemokine receptor CXCR4 (p = .04) and reduced the migratory capacity of CLL cells toward stroma-derived factor 1α (SDF-1α, p = .02). Immuno-blotting and immuno-precipitation showed that RKIP is constitutively phosphorylated and highly expressed in CLL cells and that the actions of locostatin may be mediated by binding of G-protein receptor kinase-2 (GRK2) to MEK1 and AKT. Collectively, our data suggest that inhibition of RKIP may be effective against CLL, reducing the survival and migratory capacity of the leukemic cells through down-regulation of MAPK-ERK1/2 and AKT-mediated signaling.

KRAS promotes tumor metastasis and chemoresistance by repressing RKIP via the MAPK-ERK pathway in pancreatic cancer.

Oncogenic KRAS plays a crucial role in pancreatic ductal adenocarcinoma (PDAC) development and progression. However, the mechanism has not been clearly elucidated. RKIP is a tumor repressor, and loss of RKIP has been shown in PDAC. Here, we found that KRAS expression was inversely correlated with RKIP expression in PDAC fresh tissue regardless of the KRAS mutant status. The negative correlation between KRAS and RKIP was further confirmed in our PDAC tissue microarray. KRAS overexpression and RKIP downregulation were associated with poor clinical outcomes. Knockdown or overexpression of KRAS in PDAC cell lines robustly increased or decreased, respectively, RKIP protein and mRNA levels. Furthermore, the MAPK-ERK pathway was involved in the regulation of RKIP. KRAS-regulated RKIP expression, which in turn affected the expression of pivotal epithelial-mesenchymal transition (EMT) and apoptosis factors. The biological function of the KRAS-RKIP axis was demonstrated in human pancreatic cancer cells in vitro and in vivo. KRAS knockdown increased RKIP expression and inhibited metastasis and chemoresistance. Moreover, the feature of metastasis and chemoresistance was rescued in the KRAS-knockdown cells through the inhibition of RKIP by RNA interference. In conclusion, our studies demonstrate how KRAS inhibits the tumor suppressor RKIP, thus offering novel justification for targeting RKIP as a strategy to overcome KRAS-induced tumor metastasis and chemoresistance in PDAC.

Integrated bioinformatics, computational and experimental methods to discover novel Raf/extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells.

Beginning with our previously reported ERK inhibitor BL-EI001, we found Raf1 to be an important regulator in the ERK interactive network, and then we designed and synthesized a novel series of Raf1/ERK dual inhibitors against human breast cancers through integrative computational, synthetic and biological screening methods. Moreover, we found that compound 9d suppressed the proliferation of breast cancer cell lines and induced cellular apoptosis via a mitochondrial pathway with only partial dependence on Raf1 and ERK. Our results suggest that an integrative method including in silico design, chemical synthesis, biological screening and bioinformatics analysis could be an attractive strategy for the discovery of multi-target inhibitors against breast cancer.

Effect of a chemical inhibitor of human phosphatidylethanolamine-binding protein 4 on radiosensitivity of rectal cancer cells.

BACKGROUND: Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a well-established antiapoptosis molecule in recent years. It has also been demonstrated to be involved in the radioresistance of rectal cancer. The objective of this study was to determine whether IOI-42, a chemical inhibitor of hPEBP4, could sensitize rectal cancer cells. METHODS: Rectal cancer cells were treated with IOI-42 alone or in combination with irradiation. Clonogenic survival assays and tumor volume growth analysis were used, respectively, to study the effect of IOI-42 in vitro and in vivo. Western blot was adopted to measure the activation of signal pathway. RESULTS: Clonogenic survival assays showed that IOI-42, combined with irradiation, caused a significant decrease in colony formation compared with radiation alone, which was associated with the downregulation of Akt activation. And we also confirmed the effect of IOI-42 in nude mice transplanted with human rectal cancer subcutaneously. CONCLUSIONS: These data suggest that IOI-42 has a potential to enhance the radiosensitivity of rectal cancer cells, providing a rationale to further investigate the feasibility of combining of IOI-42 with radiation, keeping in mind that this may result in unexpected toxicities.

Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

OBJECTIVE: To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. DESIGN: Laboratory study. SETTING: Human myometrium and leiomyoma. PATIENT(S): Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. INTERVENTION(S): Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 µM) to measure ECM expression by real-time polymerase chain reaction, GSK3ß expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. MAIN OUTCOME MEASURE(S): The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3ß expression, migration, and proliferation in myometrial and leiomyoma cells. RESULT(S): RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3ß expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3ß leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. CONCLUSION(S): Our results indicate that RKIP may be involved in leiomyoma pathophysiology.

Increased chemoresistance via Snail-Raf kinase inhibitor protein signaling in colorectal cancer in response to a nicotine derivative.

A tobacco-specific component, 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK), is a major risk factor for many cancers. Recent reports have demonstrated that NNK exposure may be associated with tumor progression and chemoresistance in certain cancers. However, the underlying NNK-induced mechanism contributing to the aggressiveness of colorectal cancer (CRC) has not been thoroughly studied. In this study, we used HT29 cells treated with NNK to simulate the long-term exposure of cigarette smoke. A comparative analysis was performed to evaluate cell proliferation, migration, and invasion as well as epithelial-mesenchymal transition (EMT) markers and drug-resistance genes expression, cancer stem cell (CSC) properties, and anti-apoptotic activity. Signaling pathways related to chemoresistance were also investigated. As a result, NNK exposure dose-dependently stimulates cell proliferation, enhance abilities of migration and invasion, induce EMT phenomenon, and attenuate apoptosis. Furthermore, NNK exposure also promotes the capabilities of sphere formation, upregulation of Snail, and overexpression of CD133, Nanog, OCT4, and the drug-resistant genes. Knockdown of Snail results in upregulation of Raf kinase inhibitor protein (RKIP), increased apoptosis, reversal of EMT phenomenon, and reducation of expression of CSC markers, all of which contribute to a decrease of chemoresistance. Our study demonstrates a number of related mechanisms that mediate the effect of NNK exposure on increasing CRC therapeutic resistance via the Snail signaling pathway. Targeting Snail may provide a feasible strategy for the treatment of CRC.

Specific regulation of PRMT1 expression by PIAS1 and RKIP in BEAS-2B epithelia cells and HFL-1 fibroblasts in lung inflammation.

Protein arginine methyltransferase 1 (PRMT1) catalyzes methylation of histones and other cellular proteins, and thus regulates gene transcription and protein activity. In antigen-induced pulmonary inflammation (AIPI) PRMT1 was up-regulated in the epithelium, while in chronic AIPI, increased PRMT1 shifted to fibroblasts. In this study we investigated the cell type specific regulatory mechanism of PRMT1. Epithelial cells and fibroblasts were stimulated with IL-4 or IL-1ß. Gene and protein expression were determined by RT-qPCR, immunohistochemistry staining and Western blotting. Signaling pathway inhibitors, siRNAs and shRNA were used to determine the regulatory mechanism of PRMT1. The results showed that IL-4 up-regulated PRMT1 through STAT6 signaling in epithelial cells, while IL-1ß regulated PRMT1 through NF-κB in fibroblasts. The NF-kB inhibitor protein RKIP was highly expressed in epithelial cells and blocked IL-1ß induced PRMT1 up-regulation; while the STAT6 inhibitor protein PIAS1 was expressed in fibroblasts and suppressed IL-4 induced PRMT1 expression. Furthermore, IL-4 stimulated epithelial cells to release IL-1ß which up-regulated PRMT1 expression in fibroblasts. In conclusion, the inhibitor proteins RKIP and PIAS1 regulated the cell type and signaling specific expression of PRMT1. Thus PRMT1 expression in structural lung cells in asthma can be considered as potential target for new therapeutic intervention.

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

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

Polycomb protein EZH2 regulates tumor invasion via the transcriptional repression of the metastasis suppressor RKIP in breast and prostate cancer.

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.

Downregulation of RKIP is associated with poor outcome and malignant progression in gliomas.

Malignant gliomas are highly infiltrative and invasive tumors, which precludes the few treatment options available. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying gliomas aggressive phenotype and poor prognosis. The Raf Kinase Inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. Its role in the prognosis and tumourigenesis of gliomas remains unclear. In the present study, we found that RKIP protein is absent in a low frequency (10%, 20/193) of glioma tumors. Nevertheless, the absence of RKIP expression was an independent prognostic marker in glioma. Additionally, by in vitro downregulation of RKIP, we found that RKIP inhibition induces a higher viability and migration of the cells, having no effect on cellular proliferation and angiogenesis, as assessed by in vivo CAM assay. In conclusion, this is the largest series studied so far evaluating the expression levels of this important cancer suppressor protein in glioma tumors. Our results suggest that in a subset of tumors, the absence of RKIP associates with highly malignant behavior and poor survival of patients, which may be a useful biomarker for tailored treatment of glioma patients.

Didymin induces apoptosis by inhibiting N-Myc and upregulating RKIP in neuroblastoma.

Neuroblastomas arise from the neural crest cells and represent the most common solid tumors outside the nervous system in children. The amplification of N-Myc plays a primary role in the pathogenesis of neuroblastomas, whereas acquired mutations of p53 lead to refractory and relapsed cases of neuroblastomas. In this regard, dietary compounds which can target N-Myc and exert anticancer effects independent of p53 status acquire significance in the management of neuroblastomas. Hence, we investigated the anticancer properties of the flavonoid didymin in neuroblastomas. Didymin effectively inhibited proliferation and induced apoptosis irrespective of p53 status in neuroblastomas. Didymin downregulated phosphoinositide 3-kinase, pAkt, Akt, vimentin, and upregulated RKIP levels. Didymin induced G(2)/M arrest along with decreasing the levels of cyclin D1, CDK4, and cyclin B1. Importantly, didymin inhibited N-Myc as confirmed at protein, mRNA, and transcriptional level by promoter-reporter assays. High-performance liquid chromatography analysis of didymin-treated (2 mg/kg b.w.) mice serum revealed effective oral absorption with free didymin concentration of 2.1 µmol/L. Further in vivo mice xenograft studies revealed that didymin-treated (2 mg/kg b.w.) animals had significant reductions in tumors size compared with controls. Didymin strongly inhibited the proliferation (Ki67) and angiogenesis (CD31) markers, as well as N-Myc expression, as revealed by the histopathologic examination of paraffin-embedded section of resected tumors. Collectively, our in vitro and in vivo studies elucidated the anticancer properties and mechanisms of action of a novel, orally active, and palatable flavonoid didymin, which makes it a potential new approach for neuroblastoma therapy (NANT) to target pediatric neuroblastomas.

ß-N-oxalyl-L-α,ß-diaminopropionic acid regulates mitogen-activated protein kinase signaling by down-regulation of phosphatidylethanolamine-binding protein 1.

ß-N-Oxalyl-L-α,ß-diaminopropionic acid (l-ODAP) an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist activates protein kinase C in white leghorn chick brain. The current study focuses on the protein kinase C downstream signaling targets associated with L-ODAP excitotoxicity in SK-N-MC human neuroblastoma cells and white leghorn male chick (Gallus domesticus) brain extracts. L-ODAP treatment in SK-N-MC cells (1.5 mM) and chicks (0.5 mg/g body weight) results in a decreased expression and increased phosphorylation of phosphatidylehthanolamine-binding protein 1 (PEBP1) up to 4 h which however, returns to normal by 8 h. D-ODAP, the non-toxic enantiomer however, did not affect PEBP1 levels in either chick brain or SK-N-MC cells. Decreased PEBP1 expression correlated with subsequent activation of Raf-1, MEK and ERK signaling components of the mitogen-activated protein kinase cascade and nuclear translocation of hypoxia inducible factor-1α (HIF-1α) in chick brain nuclear extracts and SK-N-MC cells. SK-N-MC cells over-expressing PEBP1 inhibited nuclear translocation of HIF-1α when treated with l-ODAP, indicating that down-regulation of PEBP1 is responsible for HIF-1α stabilization and nuclear localization. Excitotoxicity of L-ODAP may thus be the result of phosphorylation and down-regulation of PEBP1, a crucial signaling protein regulating diverse signaling cascades. L-ODAP induced convulsions and seizures in chicks could be the result of a hypoxic insult to brain.

PEBP1 downregulation is associated to poor prognosis in HCC related to hepatitis B infection.

BACKGROUND & AIMS: Phosphatidylethanolamine-binding protein 1 (PEBP1, also RKIP) plays a pivotal role in cancer by regulating multiple cellular signaling processes and suppressing metastasis in animal models. We examined whether PEBP1 expression in hepatocellular carcinoma (HCC) correlated with the risk of recurrence and survival after resection. METHODS: A randomly selected cohort of 240 Chinese HCC patients, predominantly hepatitis B related, formed the basis of the study. PEBP1 expression levels were evaluated by immunohistochemistry and real-time reverse-transcriptase PCR. Survival analysis was performed by univariate and multivariate analyses. The results were further validated in an independent series of 403 patients. The relevance of PEBP1 to phospho-ERK was determined by Western blot analysis on clinical samples and hepatoma cell lines. RESULTS: PEBP1, prevalently down-regulated in HCC, was significantly associated with tumor invasive characteristics (such as vascular invasion, lack of encapsulation, poor differentiation and large size). Both PEBP1 protein and mRNA levels were independent predictors for tumor recurrence (hazard ratio (HR) = 1.877, p=0.001; HR = 2.633, p = 0.001; respectively), and patient survival (HR = 1.796, p = 0.004; HR = 1.730, p = 0.044; respectively). The prognostic value of PEBP1 was then confirmed in the validation cohort. In addition, Western blot suggested that loss of PEBP1 led to hyperactivity of MAPK signaling. CONCLUSIONS: Down-regulation of PEBP1 in HCC indicated aggressive tumor behaviors and predicted a worse clinical outcome, which may be a useful biomarker to identify the patients at high risk of post-operative recurrence.

Potentiation of tumor necrosis factor-alpha-induced tumor cell apoptosis by a small molecule inhibitor for anti-apoptotic protein hPEBP4.

hPEBP4 (human phosphatidylethanolamine-binding protein 4) has been identified to be able to potentiate the resistance of breast, prostate, and ovarian cancers, with the preferential expression of hPEBP4, to tumor necrosis factor-alpha (TNF-alpha) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, suggesting that inhibitors targeting the anti-apoptotic protein hPEBP4 may be useful to increase the sensitivity of hPEBP4-expressing cancer cells to TNF-alpha or TRAIL-induced apoptosis. By structure-based virtual screening and following surface plasmon resonance-based binding assay, seven small compounds were found to potently bind with hPEBP4. The hit compounds were further functionally screened for their ability to inhibit cancer cell growth, and one small compound, IOI-42, was identified to be able to promote TNF-alpha-mediated growth inhibition of MCF-7 breast cancer cells. IOI-42 could potentiate TNF-alpha-induced apoptosis of MCF-7 cells by inhibiting hPEBP4 and could suppress anchorage-independent cell growth of MCF-7 cells. We further demonstrated that IOI-42 could reduce the endogenous association of hPEBP4 with Raf-1/MEK1 and enhance the activation of ERK1/2 and JNK while inhibiting Akt activation. Furthermore, IOI-42 also promoted TRAIL-induced cell apoptosis of prostate cancer cells. Taken together, our data suggest that IOI-42, as the first chemical inhibitor of anti-apoptotic protein hPEBP4, may serve as a potential anti-tumor drug by sensitizing tumor cells to apoptotic inducers.