Raf-1 Protein Research Articles

Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering.

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling.

RAF1 is increased in labouring myometrium and modulates inflammation-induced pro-labour mediators.

Inflammation plays a central role in the terminal process of human labour and delivery, including myometrial contractions. RAF1 proto-oncogene serine/threonine-protein kinase (RAF1) can activate ERK (official gene symbol MAPK1) and/or nuclear factor-kappa B (NF-κB) to regulate genes involved in inflammation. There are, however, no studies on the role of RAF1 in the processes of human labour and delivery. Thus, the aims of this study were to determine the effect of i) human labour and pro-inflammatory cytokines interleukin 1 beta (IL1B) and tumour necrosis factor (TNF) alpha on RAF1 protein expression in myometrium and ii) siRNA knockdown of RAF1 on pro-inflammatory and pro-labour mediators in human myometrial primary cells. Term labour was associated with an increase in RAF1 protein expression. Furthermore, RAF1 protein expression was increased in myometrial cells treated with IL1B and TNF, two likely factors contributing to preterm birth. Knockdown of RAF1 by siRNA in primary myometrial cells significantly decreased IL1B- and TNF-induced IL1A, IL1B, IL6, (C-X-C motif) ligand 8 (CXCL8)and chemokine (C-C motif) ligand 2 (CCL2) mRNA abundance and IL6, IL8 and CCL2; prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels and prostaglandin PGF2 α release; and NF-κB activation. Furthermore, RAF1 knockdown was associated with decreased activation of ERK in the presence of IL1B but not TNF. Concordantly, the ERK inhibitor U0126 significantly decreased IL1B-induced IL6, CXCL8, CCL2 and PTGS2 mRNA abundance; IL6, CXCL8, CCL2 and PGF2 α release; and NF-κB activation. In conclusion, IL1B induces the expression and secretion of pro-labour mediators through the RAF1-MAPK1-NF-κB signalling pathway. TNF, on the other hand, regulates pro-labour mediators through the RAF1-NF-κB signalling pathway via an MAPK1-independent mechanism.

Optogenetically controlled RAF to characterize BRAF and CRAF protein kinase inhibitors.

Here, we applied optoRAF, an optogenetic tool for light-controlled clustering and activation of RAF proteins that mimics the natural occurring RAS-mediated dimerization. This versatile tool allows studying the effect on BRAF and CRAF homodimer- as well as heterodimer-induced RAF signaling. Vemurafenib and dabrafenib are two clinically approved inhibitors for BRAF that efficiently suppress the kinase activity of oncogenic BRAF (V600E). However in wild-type BRAF expressing cells, BRAF inhibitors can exert paradoxical activation of wild-type CRAF. Using optoRAF, vemurafenib was identified as paradoxical activator of BRAF and CRAF homo- and heterodimers. Dabrafenib enhanced activity of light-stimulated CRAF at low dose and inhibited CRAF signaling at high dose. Moreover, dabrafenib increased the protein level of CRAF proteins but not of BRAF proteins. Increased CRAF levels correlate with elevated RAF signaling in a dabrafenib-dependent manner, independent of light activation.

MicroRNAs mediated regulation of MAPK signaling pathways in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a severe problem throughout the world and requires identification of novel targets for its treatment. This multifactorial disease accounts for about 15% of the all diagnosed leukemia cases. Mitogen-activated protein kinase (MAPK) signaling pathway is crucial for the cell survival and its dysregulation is being implicated in various types of cancers. In here, we have discussed the potential role of various miRNAs that are found involved in regulating the proteins cascades of MAPK signaling pathway associated with CML. An emphasis has been paid to summarize the influence of various miRNAs in elevating or suppressing the expression level of significant proteins such as miR-203, miR-196a, miR-196b, miR-30a, miR-29b, miR-138 in BCR-ABL tyrosine kinase; miR-126, miR-221, miR-128, miR-15a, miR-188-5p, miR-17 in CRK family proteins; miR-155, miR-181a with SOS proteins; miR-155, miR-19a, with KRAS proteins; miR-19a with RAF1 protein; and miR-17, miR-19a, miR-17-92 cluster with MAPK/ERK proteins. In light of ever-increasing importance and ever-widening regulatory roles of miRNAs in cells, we have reviewed the recent progress in the field of miRNAs and have tried to suggest them as controlling targets for various protein cascades of MAPK signaling pathway. An understanding of the supervisory mechanism of MAPK by miRNAs might provide novel targets for treating CML.

Advanced Glycation End Products (AGE) Potently Induce Autophagy through Activation of RAF Protein Kinase and Nuclear Factor κB (NF-κB)

Advanced glycation end products (AGE) accumulate in diabetic patients and aging people because of high amounts of three- or four-carbon sugars derived from glucose, thereby causing multiple consequences, including inflammation, apoptosis, obesity, and age-related disorders. It is important to understand the mechanism of AGE-mediated signaling leading to the activation of autophagy (self-eating) that might result in obesity. We detected AGE as one of the potent inducers of autophagy compared with doxorubicin and TNF. AGE-mediated autophagy is inhibited by suppression of PI3K and potentiated by the autophagosome maturation blocker bafilomycin. It increases autophagy in different cell types, and that correlates with the expression of its receptor, receptor for AGE. LC3B, the marker for autophagosomes, is shown to increase upon AGE stimulation. AGE-mediated autophagy is partially suppressed by inhibitor of NF-κB, PKC, or ERK alone and significantly in combination. AGE increases sterol regulatory element binding protein activity, which leads to an increase in lipogenesis. Although AGE-mediated lipogenesis is affected by autophagy inhibitors, AGE-mediated autophagy is not influenced by lipogenesis inhibitors, suggesting that the turnover of lipid droplets overcomes the autophagic clearance. For the first time, we provide data showing that AGE induces several cell signaling cascades, like NF-κB, PKC, ERK, and MAPK, that are involved in autophagy and simultaneously help with the accumulation of lipid droplets that are not cleared effectively by autophagy, therefore causing obesity.

Griffipavixanthone, a dimeric xanthone extracted from edible plants, inhibits tumor metastasis and proliferation via downregulation of the RAF pathway in esophageal cancer

Metastasis causes a large number of deaths among esophageal cancer patients. The activation of RAF family proteins elevates tumor metastasis and proliferation. In screen targeting the RAF protein, we identified that Griffipavixanthone (GPX), a dimeric xanthone isolated from Garcinia esculenta, is a B-RAF and C-RAF inhibitor against esophageal cancer cells. Using wound healing, transwell migration and matrigel invasion assays, we confirmed that GPX significantly inhibited cell migration and invasion. Furthermore, exposure to GPX rendered cell proliferation and induced G2/M cell cycle arrest. Our mechanistic study showed that GPX suppressed cancer metastasis and proliferation through downregulation of RAF-MEK-ERK cascades proteins as well as RAF mRNA levels. In a pulmonary metastasis model, the intraperitoneal injection of GPX significantly suppressed esophageal tumor metastasis and ERK protein level in vivo. In conclusion, our present study suggested that GPX could inhibit tumor migration, invasion and proliferation in vitro and in vivo, which indicated the potential of GPX for preventing and treating esophageal cancer.

Cooperative Activity of BRAF F595L and Mutant HRAS in Histiocytic Sarcoma Provides New Insights into Oncogenic BRAF Signaling

Activating BRAF mutations, in particular V600E/K, drive many cancers, including a substantial proportion of systemic histiocytic disorders, and mutant BRAF-selective inhibitors are promising therapeutics for these diseases. Activating BRAF alleles are considered mutually exclusive with mutations in RAS family members, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif can coexist with oncogenic RAS and cooperate via transactivation of wildtype RAF proteins and paradoxical MEK/ERK activation. Due to the increasing use of global approaches to tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We used several in vitro experimental systems, including Braf-deficient murine embryonic fibroblasts expressing a regulatable HRAS oncogene, to determine the biochemical properties and cellular effects of a largely uncharacterized mutation, F595L, in the DFG motif of the BRAF activation segment that was identified by clinical exome sequencing in a patient with histiocytic sarcoma and multiorgan involvement and also occurs as somatic alteration in colorectal adenoma or carcinoma, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, melanoma, and neuroblastoma and as germline mutation in cardio-facio-cutaneous syndrome. In addition, we investigated the interaction between BRAF F595L and a concomitant HRAS Q61R allele, which was present in the same tumor cell clone and occurs as acquired alteration in multiple tumor types and as inherited variant in Costello syndrome. Unlike previously described DFG motif mutants, BRAF F595L is a gain-of-function variant with intermediate activity towards MEK that, in sharp contrast to BRAF V600E, requires an intact dimer interface for downstream signaling. Furthermore, BRAF F595L does not act paradoxically, but nevertheless cooperates with mutant HRAS to induce maximal activity of the MEK-ERK signaling pathway. Of immediate clinical relevance, BRAF F595L shows divergent responses to the mutant BRAF-selective inhibitors vemurafenib and dabrafenib, whereas signaling driven by BRAF F595L with and without mutant HRAS is efficiently blocked by the pan-RAF inhibitors sorafenib and AZ628 and the MEK inhibitor trametinib. Consistent with this, sorafenib treatment led to abrogation of aberrant MEK/ERK signaling in the index case with histiocytic sarcoma driven by BRAF F595L and HRAS Q61R. Mutation data from patients and cell lines, representing 18 different tumor entities, show that BRAF F595L as well as other BRAF mutants with intermediate signaling activity coincide with mutant RAS in at least 40% and 23% of cases, respectively. These data define a distinct class of activating BRAF mutations that cooperate with oncogenic RAS in a non-paradoxical fashion, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway, and underscore the value of comprehensive genomic profiling for uncovering the vulnerabilities of individual tumors. DisclosuresOff Label Use: Administration of sorafenib in a patient with histiocytic sarcoma.

Differentiation and apoptosis induction by lovastatin and γ-tocotrienol in HL-60 cells via Ras/ERK/NF-κB and Ras/Akt/NF-κB signaling dependent down-regulation of glyoxalase 1 and HMG-CoA reductase

Glyoxalase 1 (GLO1) and HMG-CoA reductase (HMGCR) are highly expressed in most tumor cells and little in normal cells. In this study, treatment of HL-60 cells with lovastatin induced characteristic apoptosis in a dose-dependent manner. We demonstrated that lovastatin treatment inhibited Ras and Raf protein translocation to cell membrane and eliminated the phosphorylation of the downstream effectors Akt and ERK, and the subsequent NF-κB translocation into nucleus. Specific inhibitors and γ-tocotrienol confirmed the Ras/Raf/ERK/NF-κB/GLO1 and Ras/Akt/NF-κB/GLO1 pathways. Data revealed that lovastatin induced HL-60 cell death was attenuated by mevalonate treatment. We demonstrated also that γ-tocotrienol showed its apoptotic effect on the HL-60 cell through the same pathway. γ-Tocotrienol enhanced the apoptotic effect of lovastatin through the down-regulation of GLO1 and HMGCR resulting in an increase of methylglyoxal and a decrease of cholesterol and led to the apoptosis of HL-60 cells.Data also revealed that both lovastatin and gamma-tocotrienol induced significant HL-60 cell differentiation. These results suggest that both lovastatin and gamma-tocotrienol could induce differentiation and followed by apoptosis.

Repurposing FDA-approved drugs as therapeutics to treat Rift Valley fever virus infection.

There are currently no FDA-approved therapeutics available to treat Rift Valley fever virus (RVFV) infection. In an effort to repurpose drugs for RVFV treatment, a library of FDA-approved drugs was screened to determine their ability to inhibit RVFV. Several drugs from varying compound classes, including inhibitors of growth factor receptors, microtubule assembly/disassembly, and DNA synthesis, were found to reduce RVFV replication. The hepatocellular and renal cell carcinoma drug, sorafenib, was the most effective inhibitor, being non-toxic and demonstrating inhibition of RVFV in a cell-type and virus strain independent manner. Mechanism of action studies indicated that sorafenib targets at least two stages in the virus infectious cycle, RNA synthesis and viral egress. Computational modeling studies also support this conclusion. siRNA knockdown of Raf proteins indicated that non-classical targets of sorafenib are likely important for the replication of RVFV.

Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding.

RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.

Regulation of RAF protein kinases in ERK signalling

RAF family kinases were among the first oncoproteins to be described more than 30 years ago. They primarily act as signalling relays downstream of RAS, and their close ties to cancer have fuelled a large number of studies. However, we still lack a systems-level understanding of their regulation and mode of action. The recent discovery that the catalytic activity of RAF depends on an allosteric mechanism driven by kinase domain dimerization is providing a vital new piece of information towards a comprehensive model of RAF function. The fact that current RAF inhibitors unexpectedly induce ERK signalling by stimulating RAF dimerization also calls for a deeper structural characterization of this family of kinases.

MP55-01 CYTOPLASMIC FOXO1 NEGATIVELY REGULATES ERK TO OVERCOME TAXOL RESISTANCE IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2015MP55-01 CYTOPLASMIC FOXO1 NEGATIVELY REGULATES ERK TO OVERCOME TAXOL RESISTANCE IN PROSTATE CANCER Chunwu Pan, Jian An, Yunqian Pan, Liguo Wang, Jun Zhang, Jun Qi, and Haojie Huang Chunwu PanChunwu Pan More articles by this author , Jian AnJian An More articles by this author , Yunqian PanYunqian Pan More articles by this author , Liguo WangLiguo Wang More articles by this author , Jun ZhangJun Zhang More articles by this author , Jun QiJun Qi More articles by this author , and Haojie HuangHaojie Huang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2044AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES O-class forkhead factor 1 (FoxO1) acts as a tumor suppressor in the nucleus through its transcriptional function. FOXO1 can be phosphorylated by activated protein kinase B (AKT) and exported to cytoplasm, thereby losing its tumor suppressor function. The PI3K/AKT signaling is frequently hyperactivated in human cancers, and inhibitors of PI3K/AKT are currently tested in various preclinical and clinical settings. Intriguingly, inhibition of PI3K/AKT invariably triggers activation of extracellular signal-regulated kinase (ERK) signaling, which is an important mechanism of chemoresistance. Here we identify a transcription-independent tumor suppressor function of FOXO1 in the cytoplasm, finding of which has significant applications in overcoming taxol chemoresistance through inhibiting ERK signaling in prostate cancer. METHODS We employed unbiased tandem affinity purification (TAP) and mass spectrometry to identify interacting proteins of FOXO1. We used GST-Pulldown and co-immuniprecipitation (co-IP) to define the interaction domains in both IQGAP1 and FOXO1, Western-blot to detect the protein expression level, immunocytochemistry (ICC) to detect FOXO1 cellular localization, immunohistochemistry (IHC) to analysize FOXO1 and phosphor-ERK level in human prostate cancer using tissue microarray (TMA), and MTS assay to determine cells proliferation. RESULTS We identified the scaffold protein IQGAP1 as a binding partner of FOXO1 (Fig A). We demonstrated that serine-319 phosphorylation on FOXO1 is important for FOXO1 binding to IQGAP1 (Fig B). Cytoplasmic FOXO1 inhibits IQGAP1-augmented phosphorylation of ERK1/2 by blocking IQGAP1 interaction with RAF, MEK and ERK proteins (Fig C-E). We also found that there is an inverse correlation between FOXO1 and phosphorylated ERK1/2 in human prostate cancer tissues (Fig F, G). We further demonstrated that low expression of FOXO1 and increased expression of ERK1/2 phosphorylation significantly associates with prostate cancer progression (Fig H). Finaly, we demonstrated that cytoplasmic FOXO1 inhibits PI3K/AKT inhibitor or taxol-induced ERK activation and thereby overcoming taxol-resistance in prostate cancer (Fig I-L). CONCLUSIONS Cytoplasmic FOXO1 overcomes taxol resistance in prostate cancer by negatively regulating ERK activation. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e672-e673 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chunwu Pan More articles by this author Jian An More articles by this author Yunqian Pan More articles by this author Liguo Wang More articles by this author Jun Zhang More articles by this author Jun Qi More articles by this author Haojie Huang More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

MP55-02 PROSTATE ADENOCARCINOMAS COMPARED WITH NON-TUMOR PROSTATE TISSUE. EXPRESSION OF COLLAGEN, ELASTIC SYSTEM FIBERS, SMOOTH MUSCLE CELLS AND VESSELS, ACCORDING TO THE GLEASON SCORE

METHODS: We employed unbiased tandem affinity purification (TAP) and mass spectrometry to identify interacting proteins of FOXO1. We used GST-Pulldown and co-immuniprecipitation (co-IP) to define the interaction domains in both IQGAP1 and FOXO1, Western-blot to detect the protein expression level, immunocytochemistry (ICC) to detect FOXO1 cellular localization, immunohistochemistry (IHC) to analysize FOXO1 and phosphor-ERK level in human prostate cancer using tissue microarray (TMA), and MTS assay to determine cells proliferation. RESULTS: We identified the scaffold protein IQGAP1 as a binding partner of FOXO1 (Fig A). We demonstrated that serine-319 phosphorylation on FOXO1 is important for FOXO1 binding to IQGAP1 (Fig B). Cytoplasmic FOXO1 inhibits IQGAP1-augmented phosphorylation of ERK1/2 by blocking IQGAP1 interaction with RAF, MEK and ERK proteins (Fig C-E). We also found that there is an inverse correlation between FOXO1 and phosphorylated ERK1/2 in human prostate cancer tissues (Fig F, G). We further demonstrated that low expression of FOXO1 and increased expression of ERK1/2 phosphorylation significantly associates with prostate cancer progression (Fig H). Finaly, we demonstrated that cytoplasmic FOXO1 inhibits PI3K/AKT inhibitor or taxol-induced ERK activation and thereby overcoming taxol-resistance in prostate cancer (Fig I-L). CONCLUSIONS: Cytoplasmic FOXO1 overcomes taxol resistance in prostate cancer by negatively regulating ERK activation.

Methionine Adenosyltransferase 2B–GIT1 Complex Serves as a Scaffold to Regulate Ras/Raf/MEK1/2 Activity in Human Liver and Colon Cancer Cells

Methionine adenosyltransferase 2B (MAT2B) encodes for variant proteins V1 and V2 that interact with GIT1 to increase ERK activity and growth in human liver and colon cancer cells. MAT2B or GIT1 overexpression activates MEK. This study explores the mechanism for MEK activation. We examined protein-protein interactions by co-immunoprecipitation and verified by confocal microscopy and pull-down assay using recombinant or in vitro translated proteins. Results were confirmed in an orthotopic liver cancer model. We found that MAT2B and GIT1-mediated MEK1/2 activation was not mediated by PAK1 or Src in HepG2 or RKO cells. Instead, MAT2B and GIT1 interact with B-Raf and c-Raf and enhance recruitment of Raf proteins to MEK1/2. MAT2B-GIT1 activates c-Raf, which is the key mediator for MEK/12 activation, because this still occurred in RKO cells that express constitutively active B-Raf mutant. The mechanism lies with the ability of MAT2B-GIT1 to activate Ras and promote B-Raf/c-Raf heterodimerization. Interestingly, MAT2B but not GIT1 can directly interact with Ras, which increases protein stability. Finally, increased Ras-Raf-MEK signaling occurred in phenotypically more aggressive liver cancers overexpressing MAT2B variants and GIT1. In conclusion, interaction between MAT2B and GIT1 serves as a scaffold and facilitates signaling in multiple steps of the Ras/Raf/MEK/ERK pathway, further emphasizing the importance of MAT2B/GIT1 interaction in cancer growth.

Current Understanding of BRAF Alterations in Diagnosis, Prognosis, and Therapeutic Targeting in Pediatric Low-Grade Gliomas.

The mitogen-activated protein kinase (MAPK) pathway is known to play a key role in the initiation and maintenance of many tumors as well as normal development. This often occurs through mutation of the genes encoding RAS and RAF proteins which are involved in signal transduction in this pathway. BRAF is one of three RAF kinases which act as downstream effectors of growth factor signaling leading to cell cycle progression, proliferation, and survival. Initially reported as a point mutation (V600E) in the majority of metastatic melanomas, other alterations in the BRAF gene have now been reported in a variety of human cancers including papillary thyroid cancer, colon carcinomas, hairy cell leukemia, and more recently in gliomas. The identification of oncogenic mutations in the BRAF gene have led to a revolution in the treatment of metastatic melanoma using targeted molecular therapies that affect the MAPK pathway either directly through BRAF inhibition or downstream through inhibition of MEK. This review describes the molecular biology of BRAF in the context of pediatric low-grade gliomas, the role of BRAF as a diagnostic marker, the prognostic implications of BRAF, and evidence for therapeutic targeting of BRAF.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1.

Downregulation of SNAIL sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis by regulating the NF-κB pathway.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the second most lethal cancer worldwide. Evidence has shown HCC cell resistance to TRAIL‑mediated apoptosis. In a previous study, we verified that silencing SNAIL downregulated the growth of HCC cells. In addition, the mechanism of resistance to TRAIL in HCC cells was connected with the activation of nuclear factor-κB (NF-κB). Thus, it was hypothesized that the downregultaion of SNAIL sensitizes HCC cells to TRAIL-induced apoptosis by regulating the NF-κB pathway. In the present study, the most effective lentiviral vectors carrying shRNA against SNAIL were selected and adenoviral vectors harboring TRAIL were constructed. The expression of SNAIL and TRAIL was detected by quantitative PCR and western blotting. HCC cell viability and apoptosis were assessed using an MTT assay and the Hoechst test. To determine how to sensitize HCC cells to TRAIL-induced apoptosis after silencing SNAIL, p53 was assessed by western blot analysis. We also investigated the expression of Bcl-xL, cIAP2, survivin and Raf-1 protein using western blot analysis and the apoptotic degree of HuH-7 cells was detected using the Hoechst test following the suppression of each gene, which was a possible molecular mechanism to sensitive TRAIL-induced apoptosis through the downregulation of SNAIL in HCC cells. Silencing SNAIL resulted in increased apoptosis by enhancing sensitization to TRAIL in all the HCC cells. Additionally, p53 protein was upregulated in HuH-7 cells. Expression of Bcl-xL, cIAP2, survivin and Raf-1 was downregulated following silencing of SNAIL, while down-regulation of any of the proteins contributed to SNAIL suppression enhancing HCC cell sensitivity to TRAIL‑induced apoptosis, with the exception of cIAP2. The results demonstrated that silencing SNAIL can sensitize TRAIL-induced apoptosis in HCC cells by upregulating p53 protein and by regulating related genes of the NF-κB pathway such as Bcl-xL, survivin and Raf-1.

Black Rice Anthocyanins Suppress Metastasis of Breast Cancer Cells by Targeting RAS/RAF/MAPK Pathway.

Overexpression of human epidermal growth factor receptor 2 (HER2) drives the biology of 30% of breast cancer cases. As a transducer of HER2 signaling, RAS/RAF/MAPK pathway plays a pivotal role in the development of breast cancer. In this study, we examined the molecular mechanisms underlying the chemopreventive effects of black rice anthocyanins (BRACs) extract and identified their molecular targets in HER2+ breast cancer cells. Treatment of MDA-MB-453 cells (HER2+) with BRACs inhibited cell migration and invasion, suppressed the activation of mitogen-activated protein kinase kinase kinase (RAF), mitogen-activated protein kinase kinase (MEK), and c-Jun N-terminal kinase (JNK), and downregulated the secretion of matrix metalloproteinase 2 (MMP2) and MMP9. BRACs also weakened the interactions of HER2 with RAF, MEK, and JNK proteins, respectively, and decreased the mRNA expression of raf, mek, and jnk. Further, we found combined treatment with BRACs and RAF, MEK, or JNK inhibitors could enhance the antimetastatic activity, compared with that of each treatment. Transient transfection with small interfering RNAs (siRNAs) specific for raf, mek, and jnk inhibited their mRNA expression in MDA-MB-453 cells. Moreover, cotreatment with BRACs and siRNA induces a more remarkable inhibitory effect than that by either substance alone. In summary, our study suggested that BRACs suppress metastasis in breast cancer cells by targeting the RAS/RAF/MAPK pathway.

Rigosertib Blocks RAS Signaling By Acting As a Small Molecule RAS Mimetic That Binds to the RAS-Binding Domains of RAS Effector Proteins

Oncogenic activation of RAS via point mutations occurs in more than 30% of all human cancers, including hematopoietic malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Investigations to understand the critical biochemical and biological mechanisms of RAS function are at the forefront of cancer research. Studies have shown that RAS interacts with a large number of effector proteins by a highly conserved mechanism that involves the switch region of RAS and the RAS-binding domains (RBDs) of its effector proteins. Because these interactions play an essential role in oncogenic RAS function, inhibiting them constitutes an attractive and important therapeutic approach for myeloid neoplasias and other cancers. Rigosertib is a novel styryl benzyl sulfone, which is in a Phase III clinical trial (ONTIME) for MDS. Here, we delineate the way rigosertib interacts with the RBDs of several RAS effector proteins: RAF, the PI3K family of proteins and RalGDS. To identify residues in the B-RAF RBD that interact with rigosertib, we recorded a series of 15N-1H HSQC spectra of 15N-labeled B-RAF RBD with increasing concentration of rigosertib. Strikingly, the chemical shift perturbations caused by addition of rigosertib are localized to the very region of the B-RAF-RBD implicated in RAS binding, namely the beta1 and beta2 strands and alpha3 helix (Fig 1). Additionally, this cluster of residues with largest chemical shift perturbation contains many of the same residues involved in RAS binding, namely Ile156, Lys164, Arg166, Thr167, Val168, Ala184 and Met187. These key residues are conserved within RAF RBDs, suggesting that rigosertib would bind to similar regions of the A- and c-RAF RBDs. Next, we examined the binding of rigosertib and GTP-RAS to wild type and mutant forms of c-RAF RBD that harbor mutations in residues that mediate binding to rigosertib. Our studies show that all mutations that cause dissociation of GTP-RAS binding also inhibit rigosertib binding to these mutant proteins. Taken together, the chemical shift data and mutagenesis data provide powerful evidence that rigosertib binds the B-RAF RBD at the same location as the RAS switch I region.A consequence of inhibiting RAS binding to RAF appears to be a block in growth factor-induced activation of RAF kinase activity. We also show that a result of this block in RAS/RAF interactions is an inability of RAF proteins to form dimers and activate MEK and ERK. This block in the activation of MEK/ERK pathways can be seen in cells that express wild-type RAS and RAF proteins (HeLa), in cells that express a constitutively active form of oncogenic RAS (HeLa-N-RAS-G12D), and in cells that exhibit amplification of EGF receptors (A431). Rigosertib also inhibits the phosphorylation of c-RAF serine 338, which has been shown to be essential for the activation of its kinase activity and for its association with and activation of PLK-1. Our results showing rigosertib-mediated inhibition of the PLK-1/RAF interaction might help explain the ability of this compound to induce mitotic arrest of human tumor cells and the ability of rigosertib to reduce blast counts in MDS patients (Seetharam et al, Leuk Res 2012).We have also demonstrated the binding of rigosertib to the RBDs of the PI3K family of kinases and RalGDS, both of which constitute important effectors of RAS. A consequence of the interaction of rigosertib with the RBD domains of PI3Ks appears to be a block in growth factor-induced AKT activation. These studies suggest that the disruption of multiple RAS-driven signaling pathways by rigosertib is mediated via rigosertib’s binding to RBDs of RAS effector proteins, leading to their inactivation. [Display omitted] DisclosuresReddy:Onconova Therapeutics Inc: Research Funding. Divakar:Onconova Therapeutics Inc: Research Funding. Vasquez-Del Carpio:Onconova Therapeutics Inc: Research Funding. Dutta:Onconova Therapeutics Inc: Research Funding. Baker:Onconova Therapeautics Inc: Consultancy. Reddy:Onconova Therapeutics Inc: Consultancy. Aggarwal:Onconova Therapeutics Inc: Research Funding.

A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.