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Background: The effect of DOCK1 gene on the biological behavior of endometrial carcinoma cells and its related pathway has not been reported. Methods: The immunohistochemical method and western blot were utilized to analyze DOCK1 protein expression in endometrial tissues and cells, respectively. CCK-8, BrdU, transwell and flow cytometry were performed to analyze the effect of DOCK1 expression changes on the viability, proliferation, invasion, migration and apoptosis of endometrial cancer cells, respectively. The effects of DOCK1 gene on Bcl-2, MMP9, Ezrin, E-cadherin and c-RAF/ERK1/2 signaling pathway were evaluated by western blot. The xenograft models were constructed to analyze the effect of DOCK1 in vivo. Results: DOCK1 expression was increased in endometrial cancer tissues and cells compared with those in normal adjacent tissues and cells. DOCK1 knockout could inhibit the malignant biological behavior of endometrial cancer cells, while DOCK1 overexpression played the opposite effect. The expression of E-cadherin was upregulated and those of MMP9, Ezrin, Bcl-2, p-c-RAF (S338) and p-ERK1/2 (T202/Y204) were downregulated after DOCK1 knockout, while DOCK1 overexpression played the opposite effect. Additionally, Raf inhibitor LY3009120 reversed the function of DOCK1 on malignant biological behavior. In vivo experiment results showed that the growth and weight of transplanted tumors in nude mice were inhibited after DOCK1 knockout. The changes of E-cadherin, MMP9, Ezrin and Bcl-2 expressions in the transplanted tumors were consistent with those in vitro. Conclusion: DOCK1 could enhance the malignant biological behavior of endometrial cancer cells, which might be through c-RAF/ERK1/2 signaling pathways in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Soluble epoxide hydrolase (sEH) is an enzyme involved in the metabolism of bioactive lipid signaling molecules. sEH converts epoxyeicosatrienoic acids (EET) to virtually inactive dihydroxyeicosatrienoic acids (DHET). The first acids are "medicinal" molecules, the second increase the inflammatory infiltration of cells. Mitogen-activated protein kinases (p38 MAPKs) are key protein kinases involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). p38 MAPK signaling plays an important role in the regulation of cellular processes, especially inflammation. The proto-oncogenic serine/threonine protein kinase Raf (c-Raf) is a major component of the mitogen-activated protein kinase (MAPK) pathway: ERK1/2 signaling. Normal cellular Raf genes can also mutate and become oncogenes, overloading the activity of MEK1/2 and ERK1/2. The development of multitarget inhibitors is a promising strategy for the treatment of socially dangerous diseases. We synthesized 1,3-disubstituted ureas and diureas containing a dichloroadamantyl moiety. The results of computational methods show that soluble epoxide hydrolase inhibitors can act on two more targets in different signaling pathways of mitogen-activated protein kinases p38 MAPK and c-Raf. The two chlorine atoms in the adamantyl moiety may provide additional Cl-π interactions in the active site of human sEH. Molecular dynamics studies have shown that the stability of ligand–protein complexes largely depends on the "spacer effect." The compound containing a bridge between the chloroadamantyl fragment and the ureide group forms more stable ligand–protein complexes with sEH and p38 MAPK, which indicates a better conformational ability of the molecule in the active sites of these targets. In turn, a compound containing two chlorine atoms forms a more stable complex with c-Raf, probably due to the presence of additional halogen bonds of chlorine atoms with amino acid residues. [ABSTRACT FROM AUTHOR]

Paradoxical Raf activation via Raf dimerization is a major drawback of wild/mutant B-Raf inhibitors. Herein, we report that CB-1 a novel, potent B-Raf/c-Raf dual inhibitor, effective against colon cancer cells, irrespective of their genetic status. High-throughput virtual screening of the ChemBridge library against wild B-Raf (B-RafWT), mutant B-Raf (B-RafV600E), and c-Raf was performed using an automated protocol with the AutoDock-VINA. Caco-2 and HT-29 cells were used. Of the 23,365 compounds screened computationally, CB-1 showed the highest binding energy towards B-RafWT with a ΔGbinding score of − 13.0 kcal/mol. The compound was also predicted to be effective against B-RafV600E and c-Raf molecules with ΔGbinding energies of − 10.6 and − 10.1 kcal/mol, respectively. The compound inhibited B-RafWT, B-RafV600E and c-Raf kinases with IC50 values of 27.13, 51.70, and 40.23 nM, respectively. The GI50 value of CB-1 was 247.9 nM in B-RafWT-expressing Caco-2 cells and 352.4 nM in B-RafV600E-expressing HT-29 cells. Dose-dependent increases in total apoptosis and G1 cell cycle phase arrest was observed in CB-1-treated colon cancer cells. The compound decreased B-Raf expression in both wild and mutant colon cancer cells. CB-1, a novel, potent dual B-Raf/c-Raf inhibitor was effective against colon cancer cells bearing wild-type and mutant variants of B-Raf expression. [ABSTRACT FROM AUTHOR]

Over the last three decades, the scaffold proteins prohibitins-1 and -2 (PHB1/2) have emerged as key signaling proteins regulating a myriad of signaling pathways in health and diseases. Small molecules targeting PHBs display promising effects against cancers, osteoporosis, inflammatory, cardiac and neurodegenerative diseases. This review provides an updated overview of the various classes of PHB ligands, with an emphasis on their mechanism of action and therapeutic potential. We also describe how these ligands have been used to explore PHB signaling in different physiological and pathological settings. [ABSTRACT FROM AUTHOR]

C-Raf proto-oncogene serine/threonine kinase is a mitogen-activated protein kinase (MAP) kinase kinase, which can initiate a mitogen-activated protein kinase (MAPK) cascade by phosphorylating the dual-specific MAP kinase kinases (MEK1/2), and in turn activate the extracellular signal-regulated kinases (ERK1/2). To study the function of c-Raf in teleost fish, a c-Raf cDNA sequence from orange-spotted grouper (Epinephelus coioides) was cloned. Ecc-Raf shared 81%–99% amino acid identity with other vertebrate c-Raf molecules, and shared the highest amino acid identity (99%) with Lates calcarifer c-Raf. Genomic structure analysis revealed that grouper c-Raf shared a conserved exon structure with other vertebrates. Tissue distribution showed that Ecc-Raf was mainly transcribed in systemic immune organs. Ecc-Raf was distributed throughout the cytoplasm of transfected GS cells and the overexpression of Ecc-Raf only slightly enhanced the activation of Activator protein 1. The phosphorylation levels of Ecc-Raf can be induced by PMA and H 2 O 2 treatment, in contrast to DMSO or untreated HKLs. Moreover, the phosphorylation level of the Raf-MEK-ERK axis was downregulated after 24 h of SGIV infection. On the other hand, the total level and phosphorylation level of c-Raf significantly increased post C. irritans infection and showed an enhanced level post immunization. The results of this study suggested that the Raf-MEK-ERK cascade was involved in the response to viral or parasitic infections. • Overexpression of Ecc-Raf can only slightly enhance the activation of AP1. • Grouper pc-Raf can by inducing by the stimulation of PMA or H 2 O 2. • Grouper Raf-MEK-ERK axis was activated in SGIV or C. irritans infection. [ABSTRACT FROM AUTHOR]

Unusual lipid modification of K‐Ras makes Ras‐directed cancer therapy a challenging task. Aiming to disrupt electrostatic‐driven protein–protein interactions (PPIs) of K‐Ras with FTase and GGTase I, a series of bivalent dual inhibitors that recognize the active pocket and the common acidic surface of FTase and GGTase I were designed. The structure‐activity‐relationship study resulted in 8 b, in which a biphenyl‐based peptidomimetic FTI‐277 was attached to a guanidyl‐containing gallate moiety through an alkyl linker. Cell‐based evaluation demonstrated that 8 b exhibited substantial inhibition of K‐Ras processing without apparent interference with Rap‐1A processing. Fluorescent imaging showed that 8 b disrupts localization of K‐Ras to the plasma membrane and impairs interaction with c‐Raf, whereas only FTI‐277 was found to be inactive. These results suggest that targeting the PPI interface of K‐Ras may provide an alternative method of inhibiting K‐Ras. [ABSTRACT FROM AUTHOR]

Background: Recent advances in the treatment of melanoma that involve immunotherapy and B-Raf inhibition have revolutionised cancer care for this disease. However, an un-met clinical need remains in B-Raf inhibitor resistant patients where first-generation B-Raf inhibitors provide only short-term disease control. In these cases, B-Raf inhibition leads to paradoxical activation of the C-Raf - MEK - ERK signalling pathway, followed by metastasis. PDE8A has been shown to directly interact with and modulate the cAMP microdomain in the vicinity of C-Raf. This interaction promotes C-Raf activation by attenuating the PKA-mediated inhibitory phosphorylation of the kinase.Methods: We have used a novel cell-penetrating peptide agent (PPL-008) that inhibits the PDE8A - C-Raf complex in a human malignant MM415 melanoma cell line and MM415 melanoma xenograft mouse model to investigate ERK MAP kinase signalling.Results: We have demonstrated that the PDE8A - C-Raf complex disruptor PPL-008 increased inhibitory C-Raf-S259 phosphorylation and significantly reduced phospho-ERK signalling. We have also discovered that the ability of PPL-008 to dampen ERK signalling can be used to counter B-Raf inhibitor-driven paradoxical activation of phospho-ERK in MM415 cells treated with PLX4032 (Vemurafenib). PPL-008 treatment also significantly retarded the growth of these cells. When applied to a MM415 melanoma xenograft mouse model, PPL-008C penetrated tumour tissue and significantly reduced phospho-ERK signalling in that domain.Conclusion: Our data suggests that the PDE8A-C-Raf complex is a promising therapeutic treatment for B-Raf inhibitor resistant melanoma. [ABSTRACT FROM AUTHOR]

Abstract RAF proto-oncogene serine/threonine-protein kinase (c-Raf) is a MAP kinase kinase kinase (MAPKKK) that participates in the Erk1/2 pathway and plays an important role in lymphocyte activation. However, the study on how c-Raf regulates adaptive immunity in non-mammal is still limited. In present study, based on analysis of sequence characteristics of c-Raf from Oreochromis niloticus (On-c-Raf), we investigated its regulation roles on teleost lymphocyte activation. The On-c-Raf was highly conserved during evolution, which was composed of a Raf-like Ras-binding domain (RBD), a protein kinase C conserved region 1 (C1) domain and a serine/threonine protein kinase catalytic (S_TKc) domain. Its mRNA showed a wide distribution in tissues of O. niloticus and with the highest expression in gill. After Aeromonas hydrophila infection, during the adaptive immune stage transcription level of On-c-Raf was significantly upregulated on day 8, but came back to original level on day 16 and 30, suggesting the potential involvement of On-c-Raf in primary response but not memory formation. Furthermore, On-c-Raf mRNA in leukocytes of Nile tilapias was obviously induced by in vitro stimulation of T cell mitogen PHA. More importantly, in vitro stimulation of lymphocytes agonist PMA augmented phosphorylation level of On-c-Raf in leukocytes detected by western-blot and immunofluorescent. Thus, c-Raf regulated lymphocyte activation of Nile tilapia on both mRNA and phosphorylation level. Together, our results revealed that the c-Raf from teleost Nile tilapia engaged in adaptive immune response by regulating lymphocytes activation. Since the regulatory mechanism of lymphocyte-mediated adaptive immunity is largely unknown in teleost, our study provided important evidences to understand teleost adaptive immunity, and also shed a novel perspective for the evolution of adaptive immune system. Highlights • c-Raf from Nile Tilapia O. niloticus is an evolutionary conserved kinase. • On-c-Raf shows a wide distribution in lymphoid-related tissues. • On-c-Raf participates in the primary response of adaptive immunity. • Phosphorylation of c-Raf regulates Nile tilapia lymphocyte activation. [ABSTRACT FROM AUTHOR]

Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell-cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement. [ABSTRACT FROM AUTHOR]

Abstract The Raf/MAPK/ERK kinase (Mek)/extracellular signal-regulated kinases (Erk) pathway is activated in cardiac hypertrophy after a myocardial infarction. Although heat-shock protein 90 (Hsp90) may regulate the Raf/Mek/Erk signal pathway, the role of Hsp90 in pathophysiological cardiac hypertrophy remains unclear. In this study, we examined the role of Hsp90 in this pathway in cardiac hypertrophy under in vivo and in vitro experimental conditions. Cultured rat cardiomyocytes were treated with the Hsp90 inhibitor 17-(allylamino)-17-dimethoxy-geldanamycin (17-AAG) and proteasome inhibitor MG-132, and then incubated with endothelin-1 (ET) to induce hypertrophy of the cells. The ET-induced increase in the cell size was attenuated by 17-AAG pretreatment. Immunoblot analysis revealed that the c-Raf content of ET-treated cardiomyocytes was decreased in the presence of 17-AAG. An increase in phosphorylation levels of Erk1/2 and GATA4 in ET-treated cardiomyocytes was also attenuated by the 17-AAG pretreatment. Myocardial infarction was produced by ligation of the left ventricular coronary artery in rats, and then 17-AAG was intraperitoneally administered to the animals starting from the 2nd week after coronary artery ligation (CAL). CAL-induced increases in the heart weight and cross-sectional area were attenuated by 17-AAG treatment. CAL rats showed signs of chronic heart failure with cardiac hypertrophy, whereas cardiac function in CAL rats treated with 17-AAG was not reduced. Treatment of CAL rats with 17-AAG caused a decrease in the c-Raf content and Erk1/2 and GATA4 phosphorylation levels. These findings suggest that Hsp90 is involved in the activation of the Raf/Mek/Erk pathway via stabilization of c-Raf in cardiomyocytes, resulting in the development of cardiac hypertrophy following myocardial infarction. [ABSTRACT FROM AUTHOR]

C-RAF was the first member of the RAF kinase family to be discovered. Since its discovery, C-RAF has been found to regulate many fundamental cell processes, such as cell proliferation, cell death, and metabolism. However, the majority of these functions are achieved through interactions with different proteins; the genes regulated by C-RAF in its active or inactive state remain unclear. In the work, we used RNA-seq analysis to study the global transcriptomes of C-RAF bearing or C-RAF knockout cells in quiescent or EGF activated states. We identified 3353 genes that are promoted or suppressed by C-RAF. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these genes are involved in drug addiction, cardiomyopathy, autoimmunity, and regulation of cell metabolism. Our results provide a panoramic view of C-RAF function, including known and novel functions, and have revealed potential targets for elucidating the role of C-RAF. [ABSTRACT FROM AUTHOR]

Resistance to chemotherapy is associated with dismal prognosis in patients with gallbladder cancer. Cyclin‑dependent kinase 10 (CDK10) influences the chemosensitivity of gallbladder cancer cells, and cyclin M is the activating factor and binding partner of CDK10. To determine the effect of CDK10 or cyclin M overexpression on chemosensitivity, gemcitabine‑resistant (GR) subclones were established from CDK10 or cyclin M stable transfectants. Stable overexpression of CDK10 increased the sensitivity to gemcitabine in non‑resistant cells and did not further increase the sensitivity to gemcitabine in the GR subclones. GR subclones exhibited a significantly decreased expression of cyclin M while maintaining the expression levels of CDK10, compared with the non‑resistant cells. MicroRNA (miR)‑433 was identified as a candidate factor involved in the mechanism of the downregulation of M cyclin in GR subclones. Luciferase assays confirmed the interaction between miR‑433 and the 3' untranslated region (3'UTR) of cyclin M. Additionally, ectopic expression of miR‑433 significantly decreased the expression of cyclin M. Finally, increased expression of circulating miR‑433 was associated with poor outcome of chemotherapy. The results of the present study suggest that miR‑433 is a potential biomarker for evaluating chemosensitivity in gallbladder cancer. [ABSTRACT FROM AUTHOR]

The activation of Raf kinases by the small GTPase Ras requires two major sets of phosphorylations. One set lies within the activation loop, and the other lies within the N-terminal acidic region (N region). In the most abundant isoform of Raf, C-Raf, N-region phosphorylations occur on serine 338 (S338) and tyrosine 341 (Y341) and are thought to provide allosteric activation of the Raf dimer. We show that the phosphorylations of these N-region sites does not require C-Raf dimerization, but rather, they precede dimerization. One of these phosphorylations (phospho-Y341) is required for C-Raf dimerization, and this action can be replicated by phosphomimetic mutants both in vivo and in vitro. The role of the phosphorylation of Y341 in promoting Raf dimerization is distinct from its well-known function in facilitating S338 phosphorylation. In Ras mutant pancreatic cancer cell lines, the phosphorylation and dimerization of C-Raf are basally elevated. Dimerization is thought to contribute to their elevated growth rate through their activation of the mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase [ERK]) signaling cascade. Blocking the tyrosine phosphorylation of C-Raf with Src family inhibitors blocks growth, basal dimerization, and ERK activation in these cells. We suggest that the kinases mediating C-Raf Y341 phosphorylation are potential candidate drug targets in selected Ras-dependent cancers. [ABSTRACT FROM AUTHOR]

Prolonged exposure to high concentrations of glutamate leads to cell type specific glutathione depletion and resulting oxidative stress, known as oxytosis. As a result of glutathione depletion, accumulation of reactive oxygen species and Ca 2+ influx are increased; however, the specific target of oxytosis has yet to be identified. In the present study, we focused on the effect of glutamate-induced oxidative stress on the extracellular-regulated protein kinase (ERK) pathway using the murine hippocampal HT22 cell line. Although the contribution of the ERK pathway to glutamate-induced oxytosis in HT22 cells is controversial, Western blot analysis revealed that glutamate caused down-regulation of mitogen-activated protein kinase kinase kinase (c-Raf) and a resulting decrease in the phosphorylation of c-Raf, as well as of mitogen-activated protein kinase kinase1/2 (MEK1/2) and ERK1/2, downstream components of the c-Raf/MEK/ERK pathway. Furthermore, neuroprotective (arylthio)cyclopentenone prostaglandins prevented glutamate-induced c-Raf down-regulation and consequently maintained the basal activity of c-Raf and its downstream signaling components. A pull-down assay using biotin-labeled cyclopentenone prostaglandins revealed that they preferentially bound to c-Raf relative to other signaling molecules of the ERK pathway, including Ras, MEK1/2, and ERK. These results suggest that neuroprotective (arylthio)cyclopentenone prostaglandins directly bind to c-Raf protein and protect cells from down-regulation of the c-Raf protein itself, resulting in neuroprotection against oxidative stress. [ABSTRACT FROM AUTHOR]

Regulation of osteoblast activity by mechanical stress is important for bone remodeling. However, the precise mechanotransduction mechanism that triggers the anabolic reaction of osteoblasts is largely unknown. In this study, we performed RNA interference (RNAi) screening to identify the signaling molecules upstream of ERK, which was responsible for osteogenesis. Of twenty-two mitogen-activated protein kinase (MAPK) kinase kinases (MAP3Ks), we identified A-Raf and C-Raf as upstream MAP3Ks of the mechanical stretch-activated ERK pathway. Subsequently we screened the mechanosensitive cation channel, and identified P2X 7 as an upstream molecule of the ERK pathway. Intriguingly, P2X 7 functioned as an upstream activator of A-Raf but not of C-Raf. Furthermore, A-Raf contributed to mechanical stretch-induced osteoblast differentiation. In contrast, C-Raf but not A-Raf protected osteoblasts from mechanical stretch-induced apoptosis. These results suggested that A-Raf and C-Raf were involved in mechanobiological osteogenesis in a distinct way: A-Raf was responsible for osteogenesis while C-Raf for anti-apoptotic protection and promotion. [ABSTRACT FROM AUTHOR]

A new series of benzothiazole amide and urea derivatives tethered with the privileged pyridylamide moiety by ether linkage at the 6-position of benzothiazole (22 final compounds) has been designed and synthesized as potent anticancer sorafenib analogs. A selected group of twelve derivatives was appraised for its antiproliferative activity over a panel of 60 human cancer cell lines at a single dose concentration of 10 μM at National Cancer Institute (NCI, USA). Compounds 4b , 5a , 5b and 5d exhibited promising growth inhibitions and thus were further tested in advanced 5-dose testing assay to determine their GI 50 values. The cellular based assay results revealed that 3,5-bis-trifluoromethylphenyl ( 5b ) urea member is the best derivative with superior potency and efficacy compared to sorafenib as well as notable extended spectrum activity covering 57 human cancer cell lines. Kinase screening of compound 5b showed its kinase inhibitory effect against both B-Raf V600E and C-Raf. Moreover, the most potent derivatives in cells were investigated for their RAF inhibitory activities, and the results were rationalized with the molecular docking study. Profiling of CYP450 and hERG channel inhibitory effects for the active compounds revealed their low possibilities to exhibit undesirable drug–drug interactions and cardiac side effects. [ABSTRACT FROM AUTHOR]

Mutant Kras and chronic pancreatitis are the most common pathological events involved in human pancreatic cancer. It has been demonstrated that c-Raf is responsible for transmitting signals from mutant Ras to its downstream signals including MEK-ERK and for initiating carcinogenesis. The soluble epoxide hydrolase (sEH), a pro-inflammatory enzyme, generally inactivates anti-inflammatory and anti-pain epoxyeicosatrienoic acids (EETs). Herein, we have synthesized a novel compound of trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM) via modifying the central phenyl ring of sorafenib and confirmed its dual inhibition of sEH and c-Raf by recombinant kinase activity assay. Pharmacokinetic analysis revealed that oral dosing of t-CUPM resulted in higher blood levels than that of sorafenib throughout the complete time course (48 h). The effect of t-CUPM on the inhibition of mutant Kras(G12D)-initiated murine pancreatic cancer cell growth was determined using the mouse pancreatic carcinoma cell model obtained from LSL-Kras(G12D)/Pdx1-Cre mice and showed that t-CUPM significantly inhibited this murine pancreatic carcinoma cell growth both in vitro and in mice in vivo. Inhibition of mutant Kras-transmitted phosphorylations of cRAF/MEK/ERK was demonstrated in these pancreatic cancer cells using Western blot assay and immunohistochemical approach. Modulation of oxylipin profile, particularly increased EETs/DHET ratio by sEH inhibition, was observed in mice treated with t-CUPM. These results indicate that t-CUPM is a highly potential agent to treat pancreatic cancer via simultaneously targeting c-Raf and sEH. [ABSTRACT FROM AUTHOR]

Extracellular phosphate plays a key role in growth plate maturation by inducing Erk1/2 (Mapk3/1) phosphorylation, leading to hypertrophic chondrocyte apoptosis. The Raf kinases induce Mek1/2 (Map2k1/2) and Erk1/2 phosphorylation; however, a role for Raf kinases in endochondral bone formation has not been identified. Ablation of both A-Raf (Araf ) and B-Raf (Braf ) in chondrocytes does not alter growth plate maturation. Because c-Raf (Raf1) phosphorylation is increased by extracellular phosphate and c-Raf is the predominant isoform expressed in hypertrophic chondrocytes, chondrocyte-specific c-Raf knockout mice (c-Raff/f;ColII-Cre+) were generated to define a role for c-Raf in growth plate maturation. In vivo studies demonstrated that loss of c-Raf in chondrocytes leads to expansion of the hypertrophic layer of the growth plate, with decreased phospho- Erk1/2 immunoreactivity and impaired hypertrophic chondrocyte apoptosis. However, cultured hypertrophic chondrocytes from these mice did not exhibit impairment of phosphate-induced Erk1/2 phosphorylation. Studies performed to reconcile the discrepancy between the in vitro and in vivo hypertrophic chondrocyte phenotypes revealed normal chondrocyte differentiation in c-Raff/f;ColII-Cre+ mice and lack of compensatory increase in the expression of A-Raf and B-Raf. However, VEGF (Vegfa) immunoreactivity in the hypertrophic chondrocytes of c-Raff/f;ColII-Cre+ mice was significantly reduced, associated with increased ubiquitylation of VEGF protein. Thus, c-Raf plays an important role in growth plate maturation by regulating vascular invasion, which is crucial for replacement of terminally differentiated hypertrophic chondrocytes by bone. [ABSTRACT FROM AUTHOR]

New 2-amido and ureido quinoline derivatives substituted with 2- N -methylamido-pyridin-4-yloxy group at the 5-position of quinoline (18 final compounds) have been designed and synthesized as anticancer sorafenib congeners. Among the synthesized derivatives, fourteen compounds were selected for evaluation of their antiproliferative activity over a panel of 60 cancer cell lines at a single dose concentration of 10 μM at National Cancer Institute (NCI, USA). Four compounds, 9b – d and 9f showed promising mean growth inhibitions and thus were further tested at five-dose testing mode to determine their IC 50 values. The data revealed that 2,4-difluorophenyl ( 9b ) and 4-chloro-3-trifluoromethylphenyl ( 9d ) urea compounds are the most active derivatives with significant efficacies and superior potencies than sorafenib in 36 and 12 cancer cell lines, respectively, belonging particularly to renal carcinoma cell (RCC), ovarian, and non small cell lung cancer (NSCL). Compound 9b and 9d were found to be six and two times more potent than sorafenib against A498 RCC line, with IC 50 values of 0.42 μM and 1.36 μM, respectively. Accordingly, compound 9d was screened over a panel of 41 oncogenic kinases at a single dose concentration of 10 μM to profile its kinase inhibitory activity. Interestingly, the compound showed highly selective inhibitory activities ( 81.8% and 96.3%) against BRAF V600E and C-RAF kinases with IC 50 values of 316 nM and 61 nM, respectively. In addition, molecular docking, cell cycle analysis, compliance to Lipinski's rule of five, and in silico toxicity assessment have been reported. [ABSTRACT FROM AUTHOR]

The multivariate nature of cancer necessitates multi-targeted therapy, and kinase inhibitors account for a vast majority of approved cancer therapeutics. While acute promyelocytic leukemia (APL) patients are highly responsive to retinoic acid (RA) therapy, kinase inhibitors have been gaining momentum as co-treatments with RA for non-APL acute myeloid leukemia (AML) differentiation therapies, especially as a means to treat relapsed or refractory AML patients. In this study GW5074 (a c-Raf inhibitor) and PP2 (a Src-family kinase inhibitor) enhanced RA-induced maturation of t(15;17)-negative myeloblastic leukemia cells and rescued response in RA-resistant cells. PD98059 (a MEK inhibitor) and Akti-1/2 (an Akt inhibitor) were less effective, but did tend to promote maturation-uncoupled G1/G0 arrest, while wortmannin (a PI3K inhibitor) did not enhance differentiation surface marker expression or growth arrest. PD98059 and Akti-1/2 did not enhance differentiation markers and have potential, antagonistic off-targets effects on the aryl hydrocarbon receptor (AhR), but neither could the AhR agonist 6-formylindolo(3,2-b)carbazole (FICZ) rescue differentiation events in the RA-resistant cells. GW5074 rescued early CD38 expression in RA-resistant cells exhibiting an early block in differentiation before CD38 expression, while for RA-resistant cells with differentiation blocked later, PP2 rescued the later differentiation marker CD11b; but surprisingly, the combination of the two was not synergistic. Kinases c-Raf, Src-family kinases Lyn and Fgr, and PI3K display highly correlated signaling changes during RA treatment, while activation of traditional downstream targets (Akt, MEK/ERK), and even the surface marker CD38, were poorly correlated with c-Raf or Lyn during differentiation. This suggests that an interrelated kinase module involving c-Raf, PI3K, Lyn and perhaps Fgr functions in a nontraditional way during RA-induced maturation or during rescue of RA induction therapy using inhibitor co-treatment in RA-resistant leukemia cells. [ABSTRACT FROM AUTHOR]

Design and synthesis of a new series of 5,6-diarylimidazo[2,1- b ]thiazole derivatives possessing terminal aryl sulfonamide moiety are described. Their in vitro antiproliferative activities against a panel of 57 human cancer cell lines of nine different cancer types were tested at the NCI. Compounds 8a , 8b , 8n , 8q , 8t , and 8u showed the highest mean % inhibition values over the 57 cell line panel at 10 μM, and they were further tested in 5-dose testing mode to determine their IC 50 values. Among the six compounds, compound 8u possessing terminal para -hydroxybenzenesulfonamido moiety and ethylene linker showed the highest potency. It demonstrated superior potency than Sorafenib against eight different cell lines, and was equipotent to Sorafenib against COLO 205 colon cancer cell line. Its IC 50 values over NCI-H460 non-small cell lung cancer cell line and MCF7 breast cancer cell line were 0.845 μM and 0.476 μM, respectively. Compounds 8a , 8b , 8q , 8t , and 8u showed high selectivity indices towards cancer cells over L132 normal lung cell line. Compound 8u showed potential inhibitory effects over the components of ERK pathway. Its IC 50 value over V600E-B-RAF and C-RAF kinases were 39.9 nM and 19.0 nM, respectively. [ABSTRACT FROM AUTHOR]

ZLJ33, an oral active multi-kinase inhibitor, was evaluated both in vitro and in vivo against human pancreatic cancer. It could effectively inhibit cell proliferation, induce apoptosis, and cause inhibition of invasion in pancreatic cancer cells. At a dose of 15.0 mg/kg, ZLJ33 induced tumor shrink in Mia-PaCa2, Capan2, and AsPC-1 xenografts models by 60.59%, 74.19%, and 71.54% according to the tumor weight, respectively. The effect of ZLJ33 on pancreatic cancer was mainly mediated by inactivation of p-PDGFRβ, p-c-Raf, and p-RET. Treatment with ZLJ33 did not cause side effect of hematology indexes in the pancreatic cancer xenograft model. ZLJ33 could be a potential therapeutic agent against pancreatic cancer. [ABSTRACT FROM AUTHOR]

Background Left ventricular hypertrophy (LVH) is a potent risk factor for sudden death and congestive heart failure. Methods We tested the effect of sorafenib, a multikinase inhibitor (10 mg/kg, given orally, starting 2 days prior to banding, till sacrifice on day 14), on the development of LVH following aortic banding in rats. Results The latter resulted in significant LVH caused by both an increase in cardiomyocyte volume and interstitial collagen deposition. The observed LVH was entirely blocked by sorafenib downregulating both of these components. LVH was associated with PDGF-BB and TGFβ1 overexpression, as well as phosphorylation of c-raf and ERK1/2. Additionally, the transcription factors c-myc and c-fos leading to proliferation as well as the hypertrophyinducing transcription factor GATA4 and its regulated gene ANP were all upregulated in response to aortic banding. All these overexpressions and upregulations were inhibited upon sorafenib treatment. Conclusion We show that sorafenib exhibits a regulatory role on the occurrence of LVH following AB in rats by blocking the rise in growth factors PDGF-BB and TGFβ1, activation of the corresponding c-Raf-ERK1/2 signaling pathway and effector mechanisms, including GATA4 and ANP. This effect of sorafenib may be of clinical importance in modulating the maladaptive hypertrophic response to pressure overload. [ABSTRACT FROM AUTHOR]

The Raf family of protein kinases are key signaling intermediates, acting as a central link between the membrane-bound Ras GTPases and the downstream kinases MEK and ERK. Raf kinase regulation is well-known for its complexity but only recently has it been realized that many of the mechanisms involved in Raf regulation also modulate Raf dimerization, now acknowledged to be a required step for Raf signaling in multiple cellular contexts. Recent studies have shown that Raf dimerization is necessary for normal Ras-dependent Raf kinase activation and contributes to the pathogenic function of disease-associated mutant Raf proteins with all but high intrinsic kinase activity. Raf dimerization has also been found to alter therapeutic responses and disease progression in patients treated with ATP-competitive Raf inhibitors as well as certain other kinase-targeted drugs. This demonstration of clinical significance has stimulated the recent development of biosensor assays that can monitor inhibitor-induced Raf dimerization as well as studies demonstrating the therapeutic potential of blocking Raf dimerization. [ABSTRACT FROM AUTHOR]

Abstract: Cucurbitacin B is an anti-cancer drug candidate and its efficacy has been demonstrated in hepatocellular carcinoma (HCC). To explore its mechanism against HCC, BEL-7402 cells were treated with cucurbitacin B in vitro. Treatment with cucurbitacin B induced S phase arrest and apoptosis. The growth inhibition effect was associated with cyclin D1 and cdc-2 down regulations. Western blotting analysis of cell signaling molecules indicated that cucurbitacin B inhibited c-Raf activation without affecting STAT3 phosphorylation. Moreover, in vivo study demonstrated that cucurbitacin B is effective against BEL-7402 xenograft when administrated orally. [Copyright &y& Elsevier]

We recently described oncogenic and anti-apoptotic C-RAF germline mutations in patients with therapy-related acute myeloid leukemia (t-AML). Activation of the RAF effector ERK was restricted to transformed cells, suggesting the requirement for cooperating events in leukemogenesis. Western blot analysis of blast cells from patients with C-RAF germline mutations revealed loss of the tumor and metastasis suppressor RAF kinase inhibitor protein (RKIP). Immunohistochemistry of the patients' primary tumors revealed normal RKIP expression levels, indicating that the loss of RKIP is a somatic, t-AML-specific event. In focus formation assays, the oncogenic potential of human mutant C-RAF was strongly influenced by expression levels of RKIP. Although the number of colonies formed by C-RAF(S427G) was significantly increased by RKIP silencing, the opposite was observed after RKIP overexpression. These results show that the loss of RKIP is a functional somatic event in carriers of C-RAF germline mutations, which contributes to the development of t-AML. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor five-year survival rate of less than 10%. Immune suppression along with chemoresistance are obstacles for PDAC therapeutic treatment. Innate immune cells, such as tumor-associated macrophages, are recruited to the inflammatory environment of PDAC and adversely suppress cytotoxic T lymphocytes. KRAS and MYC are important oncogenes associated with immune suppression and pose a challenge to successful therapies. Here, we targeted KRAS, through inhibition of downstream c-RAF with GW5074, and MYC expression via difluoromethylornithine (DFMO). DFMO alone and with GW5074 reduced in vitro PDAC cell viability. Both DFMO and GW5074 showed efficacy in reducing in vivo PDAC growth in an immunocompromised model. Results in immunocompetent syngeneic tumor-bearing mice showed that DFMO and combination treatment markedly decreased tumor size, but only DFMO increased survival in mice. To further investigate, immunohistochemical staining showed DFMO diminished MYC expression and increased tumor infiltration of macrophages, CD86+ cells, CD4+ and CD8+ T lymphocytes. GW5074 was not as effective in modulating the tumor infiltration of total CD3+ lymphocytes or tumor progression and maintained MYC expression. Collectively, this study highlights that in contrast to GW5074, the inhibition of MYC through DFMO may be an effective treatment modality to modulate PDAC immunosuppression. [ABSTRACT FROM AUTHOR]

Abstract: BAD, a member of the BCL2 family, exhibits an original mode of regulation by phosphorylation. In the present report, we examine the role of the kinase C-RAF in this process. We show that the inducible activation of C-RAF promotes the rapid phosphorylation of BAD on Serine-112 (Ser-75 in the human protein), through a cascade involving the kinases MEK and RSK. Our findings reveal a new aspect of the regulation of BAD protein and its control by the RAF pathway: we find that C-RAF activation promotes BAD poly-ubiquitylation in a phosphorylation-dependent fashion, and increases the turn-over of this protein through proteasomal degradation. [Copyright &y& Elsevier]

Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit cancer cell growth, induce apoptosis and decrease tumor metastasis. We have previously reported that a NSAID NS398 repressed the expression of matrix metalloproteinase-2 (MMP-2) via inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we investigate the underlying mechanism of this inhibition. In vitro kinase assay indicated that NS398 could not directly inhibit c-Raf, MEK1 and ERK enzymatic activity. We found that NS398 increased the inhibitory phosphorylation of Ser259 in c-Raf, attenuated membrane recruitment of c-Raf and inhibited Ras/c-Raf interaction to attenuate activation of this kinase. This is a general effect for NSAIDs because sulindac sulfide, aspirin and indomethacin also inhibited the binding of c-Raf to Ras. Immunofluorescent staining verified that NS398 reduced the serum-induced membrane recruitment of c-Raf in cells. However, overexpression of constitutively active c-Raf only partly reversed NS398-induced inhibition of MMP-2 expression. Interestingly, we found that NS398 up-regulated the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and MKP-3. Block of MKP activity by sodium orthovanadate also partly counteracted the inhibitory effect of NS398. Overexpression of constitutively active c-Raf and treatment of sodium orthovanadate together completely reversed the inhibition of MMP-2 by NS398. Taken together, we conclude that NS398 and other NSAIDs act via inhibition of Ras/c-Raf interaction and up-regulation of MKPs to suppress the ERK-mediated signaling. [Copyright &y& Elsevier]

GW5074 a brain-permeable 3′ substituted indolone, protects neurons from death in culture and in an in vivo paradigm of neurodegeneration. Using low potassium (LK) induced apoptosis of cerebellar granule neurons, we report here that the protective action of GW5074 is mediated through the activation of B-Raf. Over-expression of a kinase-dead form of B-Raf blocks the ability of GW5074 to neuroprotect, whereas over-expression of active forms of B-Raf protect even in the absence of GW5074. Although mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK) are activated by GW5074, pharmacological inhibition of MEK-ERK signaling by U0126 or PD98059 does not reduce neuroprotection suggesting that B-Raf signals through a non-canonical signaling pathway. GeneChip microarray analyses identified activating transcription factor-3 (ATF-3) as a gene whose expression is induced by LK but that is negatively regulated by GW5074. Forced inhibition of ATF-3 expression using siRNA protects neurons against LK-induced apoptosis, whereas the over-expression of ATF-3 blocks GW5074-mediated neuroprotection. Not unexpectedly, expression of active B-Raf inhibits the apoptosis-associated increase in ATF-3 expression. We extended our work to include three other 3′ substituted indolones – a commercially available inhibitor of RNA-dependent protein kinase and two novel compounds designated as SK4 and SK6. Like GW5074, RNA-dependent protein kinase inhibitor, SK4, and SK6 all inhibited c-Raf in vitro but activated B-Raf in neuronal cultures. All four compounds also inhibited ATF-3 expression. Taken together our results indicate that all four indolones mediate neuroprotection by a common mechanism which involves B-Raf activation, and that a downstream target of B-Raf is ATF-3. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis of several novel aza-stilbene derivatives was carried out. The compounds were tested for their c-RAF enzyme inhibition. Compound 27 possesses significant potency against c-RAF and demonstrates selectivity over other protein kinases. A hypothesis for the binding mode, activity, and selectivity is proposed. [Copyright &y& Elsevier]

During mitosis, a select pool of MEK1 and p42/p44 MAPK becomes activated at the kinetochores and spindle poles, without substantial activation of the bulk of the cytoplasmic p42/p44 MAPK. Recently, we set out to identify the MAP kinase kinase kinase (MAPKKK) responsible for this mitotic activation, using cyclin-treated Xenopus egg extracts as a model system, and presented evidence that Mos was the relevant MAPKKK . However, a second MAPKKK distinct from Mos was readily detectable as well. Here, we partially purify this second MAPKKK and identify it as B-Raf. No changes in the activity of B-Raf were detectable during progesterone-induced oocyte maturation, after egg fertilization, or during the early embryonic cell cycle, arguing against a role for B-Raf in the mitotic activation of MEK1 and p42 MAPK. Ras proteins can bring about activation of MEK1 and p42 MAPK in extracts, and Ras may contribute to signaling from the classical progesterone receptor during oocyte maturation and from receptor tyrosine kinases during early embryogenesis. We found that both B-Raf and C-Raf, but not Mos, are required for Ras-induced MEK1 and p42 MAPK activation. These data indicate that two upstream stimuli, active Ras and active Cdc2, utilize different MAPKKKs to activate MEK1 and p42 MAPK.Oncogene (2006) 25, 3307–3315. doi:10.1038/sj.onc.1209354; published online 23 January 2006 [ABSTRACT FROM AUTHOR]

One of the hallmarks of cAMP is its ability to inhibit proliferation in many cell types, but stimulate proliferation in others. Clearly cAMP has cell type specific effects and the outcome on proliferation is largely attributed to crosstalk from cAMP to the RAS/RAF/mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway. We review the crosstalk between these two ancient and conserved pathways, describing the molecular mechanisms underlying the interactions between these pathways and discussing their possible biological importance. [ABSTRACT FROM AUTHOR]

Alterations in signalling via the Raf/MEK/ERK pathway interfere with influenza A virus replication in cell culture. While virus yields are reduced in cells expressing dominant-negative Raf or ERK, virus propagation is enhanced upon expression of constitutively active Raf or MEK. To study the impact of active Raf on influenza virus propagation in vivo, we investigated transgenic mice expressing an activated mutant of c-Raf (Raf-BxB) in the main target tissue of influenza virus, the lung. Raf-BxB expression results in multicentric alveolar adenomas. Influenza virus A infection of Raf-BxB mice results in increased disease symptoms and higher mortality rates. The immune response against viral pathogens in transgenic animals did not differ from wild-type mice as determined by the use of a Pseudorabies virus (PRV) as a model for a viral infection not affecting the lung. No significant differences of influenza virus titers in the lung of Raf-BxB and wild-type mice were observed. However, immunohistology revealed increased numbers of influenza NP-positive cells in the alveolar linings of Raf-BxB mice, demonstrating the strong tropism of influenza virus for cells expressing active Raf. These findings disclose the possibility to use modified influenza virus for the therapy of tumors with an activated Ras/Raf signalling pathway.Oncogene (2004) 23, 6639-6646. doi:10.1038/sj.onc.1207883 Published online 5 July 2004 [ABSTRACT FROM AUTHOR]

We have performed an exhaustive unbiased yeast two-hybrid analysis to identify interaction partners of two human Raf kinase isoforms, A-Raf and C-Raf, using their N-terminal regulatory domain as “bait.” A total of 20 different human proteins were found to interact with Raf isoforms. Several of these interactions were novel and an extensive bioinformatics evaluation was performed for each. The novel putative interactions include a signalosome component, TOPK/PBK kinase, and two new putative protein phosphatases. The cysteine-rich zinc-binding domain (CRD) of Raf was found to interact with all 20 proteins and to achieve isoform-specific interactions. Since similar putative CRDs are present in a variety of protein serine–threonine kinases, the data suggest that the CRD may function as a major protein–protein interaction domain of these kinases. We propose possible functional consequences of these novel Raf interactions. [Copyright &y& Elsevier]

Activating and inhibitory phosphorylation mechanisms play an essential role in regulating Raf kinase activity. Here we demonstrate that phosphorylation of C‐Raf in the kinase activation loop (residues T491 and S494) is necessary, but not sufficient, for activation. C‐Raf has additional activating phosphorylation sites at S338 and Y341. Mutating all four of these residues to acidic residues, S338D/Y341D/T491E/S494D (DDED), in C‐Raf results in constitutive activity. However, acidic residue substitutions at the corresponding activation loop sites in B‐Raf are sufficient to confer constitutive activity. B‐Raf and C‐Raf also utilize similar inhibitory phosphorylation mechanisms to regulate kinase activity. B‐Raf has multiple inhibitory phosphorylation sites necessary for full kinase inhibition where C‐Raf requires only one. We examined the functional significance of these inhibitory and activating phosphorylations in Caenorhabditis elegans lin‐45 Raf. Eliminating the inhibitory phosphorylation or mimicking activating phosphorylation sites is sufficient to confer constitutive activity upon lin‐45 Raf and induce multi‐vulva phenotypes in C.elegans. Our results demonstrate that different members of the Raf family kinases have both common and distinct phosphorylation mechanisms to regulate kinase activity and biological function. [ABSTRACT FROM AUTHOR]

Cover Feature: A Guanidyl-Based Bivalent Peptidomimetic Inhibits K-Ras Prenylation and Association with c-Raf (Chem. Eur. J. 59/2019) Keywords: bivalent inhibitors; c-Raf; K-Ras; prenylation; protein-protein interactions Bivalent inhibitors, c-Raf, K-Ras, prenylation, protein-protein interactions. [Extracted from the article]

Five-year survival for pancreatic ductal adenocarcinoma (PDAC) patients remains below 7% due to the lack of effective treatments. Here, we report that combined ablation of EGFR and c-RAF expression results in complete regression of a significant percentage of PDAC tumors driven by Kras/Trp53 mutations in genetically engineered mice. Moreover, systemic elimination of these targets induces toxicities that are well tolerated. Response to this targeted therapy correlates with transcriptional profiles that resemble those observed in human PDACs. Finally, inhibition of EGFR and c-RAF expression effectively blocked tumor progression in nine independent patient-derived xenografts carrying KRAS and TP53 mutations. These results open the door to the development of targeted therapies for PDAC patients. • Combined Egfr / Raf1 ablation results in complete regression of a subset of PDACs • Mouse mutant Kras / Trp53- induced PDACs display distinct transcriptional profiles • PDAC transcriptional profiles determine their response to Egfr / Raf1 ablation • EGFR/c-RAF inhibition also prevents proliferation of PDX-derived tumor cells Blasco et al. show that ablation of Egfr and Raf1 results in complete regression of a subset of mutant Kras / Trp53 -induced pancreatic ductal adenocarcinomas (PDAC) without overt toxicity. Inhibition of EGFR and c-RAF expression also blocks the progression of patient-derived PDAC xenografts with mutant KRAS / TP53. [ABSTRACT FROM AUTHOR]

c-Raf is a central component of the extracellular signal-regulated kinase (ERK) pathway which is implicated in the development of many cancer types. RanBPM (Ran-Binding Protein M) was previously shown to inhibit c-Raf expression, but how this is achieved remains unclear. RanBPM is part of a recently identified E3 ubiquitin ligase complex, the CTLH (C-terminal to LisH) complex. Here, we show that the CTLH complex regulates c-Raf expression through a control of its degradation. Several domains of RanBPM were found necessary to regulate c-Raf levels, but only the C-terminal CRA (CT11-RanBPM) domain showed direct interaction with c-Raf. c-Raf ubiquitination and degradation is promoted by the CTLH complex. Furthermore, A-Raf and B-Raf protein levels are also regulated by the CTLH complex, indicating a common regulation of Raf family members. Finally, depletion of CTLH subunits RMND5A (required for meiotic nuclear division 5A) and RanBPM resulted in enhanced proliferation and loss of RanBPM promoted tumour growth in a mouse model. This study uncovers a new mode of control of c-Raf expression through regulation of its degradation by the CTLH complex. These findings also uncover a novel target of the CTLH complex, and suggest that the CTLH complex has activities that suppress cell transformation and tumour formation. [ABSTRACT FROM AUTHOR]

Summary K-Ras4B preferentially activates Raf-1. The high-affinity interaction of Ras-binding domain (RBD) of Raf with Ras was solved, but the relative position of Raf's cysteine-rich domain (CRD) in the Ras/Raf complex at the membrane and key question of exactly how it affects Raf signaling are daunting. We show that CRD stably binds anionic membranes inserting a positively charged loop into the amphipathic interface. Importantly, when in complex with Ras/RBD, covalently connected CRD presents the same membrane interaction mechanism, with CRD locating at the space between the RBD and membrane. To date, CRD's role was viewed in terms of stabilizing Raf-membrane interaction. Our observations argue for a key role in reducing Ras/RBD fluctuations at the membrane, thereby increasing Ras/RBD affinity. Even without K-Ras, via CRD, Raf-1 can recruit to the membrane; however, by reducing the Ras/RBD fluctuations and enhancing Ras/RBD affinity at the membrane, CRD promotes Raf's activation and MAPK signaling over other pathways. [ABSTRACT FROM AUTHOR]

Summary A quarter of all solid tumors harbor KRAS oncogenes. Yet, no selective drugs have been approved to treat these malignancies. Genetic interrogation of the MAPK pathway revealed that systemic ablation of MEK or ERK kinases in adult mice prevent tumor development but are unacceptably toxic. Here, we demonstrate that ablation of c-RAF expression in advanced tumors driven by Kras G12V / Trp53 mutations leads to significant tumor regression with no detectable appearance of resistance mechanisms. Tumor regression results from massive apoptosis. Importantly, systemic abrogation of c-RAF expression does not inhibit canonical MAPK signaling, hence, resulting in limited toxicities. These results are of significant relevance for the design of therapeutic strategies to treat K-RAS mutant cancers. [ABSTRACT FROM AUTHOR]