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Background Recurrent hepatocellular carcinoma (HCC) shows strong resistance to sorafenib, and the tumor-repopulating cells (TRCs) with cancer stem cell-like properties are considered a driver for its high recurrent rate and drug resistance. Methods Suppression of TRCs may thus be an effective therapeutic strategy for treating this fatal disease. We evaluated the pharmacology and mechanism of sulfarotene, a new type of synthetic retinoid, on the cancer stem cell-like properties of HCC TRCs, and assessed its preclinical efficacy in models of HCC patient-derived xenografts (PDXs). Results Sulfarotene selectively inhibited the growth of HCC TRCs in vitro and significantly deterred TRC-mediated tumor formation and lung metastasis in vivo without apparent toxicity, with an IC50 superior to that of acyclic retinoid and sorafenib, to which the recurrent HCC exhibits significant resistance at advanced stage. Sulfarotene promoted the expression and activation of RARα, which down-regulated SOS2, a key signal mediator associated with RAS activation and signal transduction involved in multiple downstream pathways. Moreover, sulfarotene selectively inhibited tumorigenesis of HCC PDXs with high expression for SOS2. Conclusions Our study identified sulfarotene as a selective inhibitor for the TRCs of HCC, which targets a novel RARα-SOS2-RAS signal nexus, shedding light on a new, promising strategy of target therapy for advanced liver cancer.

Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains, we performed NMR studies of Drk-CSH3. The resulting solution structure and the

Development of a method for detecting protein–protein interactions (PPIs) in living cells is important for therapeutic drug screening against various diseases including infectious diseases. We have recently developed a method named SOS localization-based interaction screening (SOLIS), in which we designed membrane-anchored and SOS-fused chimeric proteins, whose PPI-dependent association triggers membrane localization of the SOS-fused chimeric protein, activates the Ras/MAPK pathway, and induces cell growth. While SOLIS was able to detect relatively strong PPIs, further sensitivity was required for detecting intracellular endogenous PPIs typically having a micromolar order of dissociation constant (Kd). Here we develop high-sensitive SOLIS (H-SOLIS) that could universally detect PPIs with lower affinities. In order to improve the sensitivity, H-SOLIS introduces a heterodimeric helper interaction, in which addition of a small-molecule helper ligand could accommodate association of the two chimeric proteins and regulate the sensitivity. Four types of domain–peptide interactions having known Kd values are employed to examine the versatility and detection limit of H-SOLIS. Consequently, the heterodimer-inducible helper ligand dramatically enhances detection sensitivity, lowering the detection limit to a ten-micromolar order of Kd. Thus, H-SOLIS could be a platform to detect disease-related domain–peptide interactions for drug discovery screening.

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.

Ras family proteins play an essential role in several cellular functions, including growth, differentiation, and survival. The mechanism of action of Ras mutants in Costello syndrome and cancers has been identified, but the contribution of Ras mutants to Noonan syndrome, a genetic disorder that prevents normal development in various parts of the body, is unknown. Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor. In response to Ras-activating cell signaling, SOS autoinhibition is released and is followed by accelerative allosteric feedback autoactivation. Here, using mutagenesis-based kinetic and pulldown analyses, we show that Noonan syndrome Ras mutants I24N, T50I, V152G, and D153V deregulate the autoactivation of SOS to populate their active form. This previously unknown process has been linked so far only to the development of Noonan syndrome. In contrast, other Noonan syndrome Ras mutants—V14I, T58I, and G60E—populate their active form by deregulation of the previously documented Ras GTPase activities. We propose a novel mechanism responsible for the deregulation of SOS autoactivation, where I24N, T50I, V152G, and D153V Ras mutants evade SOS autoinhibition. Consequently, they are capable of forming a complex with the SOS allosteric site, thus aberrantly promoting SOS autoactivation, resulting in the population of active Ras mutants in cells. The results of this study elucidate the molecular mechanism of the Ras mutant–mediated development of Noonan syndrome.

RASopathies are caused by variants in genes encoding components or modulators of the RAS/MAPK signaling pathway. Noonan syndrome is the most common entity among this group of disorders and is characterized by heart defects, short stature, variable developmental delay, and typical facial features. Heterozygous variants in SOS2, encoding a guanine nucleotide exchange factor for RAS, have recently been identified in patients with Noonan syndrome. The number of published cases with SOS2-related Noonan syndrome is still limited and little is known about genotype-phenotype correlations. We collected previously unpublished clinical and genotype data from 17 individuals carrying a disease-causing SOS2 variant. Most individuals had one of the previously reported dominant pathogenic variants; only four had novel changes at the established hotspots for variants that affect protein function. The overall phenotype of the 17 patients fits well into the spectrum of Noonan syndrome and is most similar to the phenotype observed in patients with SOS1-related Noonan syndrome, with ectodermal anomalies as common features and short stature and learning disabilities as relatively infrequent findings compared to the average Noonan syndrome phenotype. The spectrum of heart defects in SOS2-related Noonan syndrome was consistent with the known spectrum of cardiac anomalies in RASopathies, but no specific heart defect was particularly predominating. Notably, lymphatic anomalies were extraordinarily frequent, affecting more than half of the patients. We therefore conclude that SOS2-related Noonan syndrome is associated with a particularly high risk of lymphatic complications that may have a significant impact on morbidity and quality of life.

Across the Bilateria, FGF/FGFR signaling is critical for normal development, and in bothDrosophilaand vertebrates, docking proteins are required to connect activated FGFRs with downstream pathways. While vertebrates use Frs2 to dock FGFR to the RAS/MAPK or PI3K pathways, the unrelated protein, downstream of FGFR (Dof/stumps/heartbroken), fulfills the corresponding function inDrosophila. To better understand the evolution of the signaling pathway downstream of FGFR, the available sequence databases were screened to identify Frs2, Dof, and other key pathway components in phyla that diverged early in animal evolution. While Frs2 homologues were detected only in members of the Bilateria, canonical Dof sequences (containing Dof, ankyrin, and SH2/SH3 domains) were present in cnidarians as well as bilaterians (but not in other animals or holozoans), correlating with the appearance of FGFR. Although these data suggested that Dof coupling might be ancestral, gene expression analysis in the cnidarianHydrarevealed thatDofis not upregulated in the zone of strongFGFRaandFGFRbexpression at the bud base, where FGFR signaling controls detachment. In contrast, transcripts encoding other, known elements of FGFR signaling in Bilateria, namely the FGFR adaptors Grb2 and Crkl, which are acting downstream of Dof (and Frs2), as well as the guanyl nucleotide exchange factor Sos, and the tyrosine phosphatase Csw/Shp2, were strongly upregulated at the bud base. Our expression analysis, thus, identified transcriptional upregulation of known elements of FGFR signaling at theHydrabud base indicating a highly conserved toolkit. Lack of transcriptionalDofupregulation raises the interesting question, whetherHydraFGFR signaling requires either of the docking proteins known from Bilateria.

Son of Sevenless (SOS), one of guanine nucleotide exchange factors (GEFs), activates Ras. We discovered that the allosteric domain of SOS yields SOS to proceed a previously unrecognized autoactivation kinetics. Its essential feature is a time-dependent acceleration of SOS feedback activation with a reaction initiator or with the priming of active Ras. Thus, this mechanistic autoactivation feature explains the notion, previously only conjectured, of accelerative SOS activation followed by the priming of active Ras, an action produced by another GEF Ras guanyl nucleotide-releasing protein (RasGRP). Intriguingly, the kinetic transition from gradual RasGRP activation to accelerative SOS activation has been interpreted as an analog to digital conversion; however, from the perspective of autoactivation kinetics, it is a process of straightforward RasGRP-mediated SOS autoactivation. From the viewpoint of allosteric protein cooperativity, SOS autoactivation is a unique time-dependent cooperative SOS activation because it enables an active SOS to accelerate activation of other SOS as a function of time. This time-dependent SOS cooperativity does not belong to the classic steady-state protein cooperativity, which depends on ligand concentration. Although its hysteretic or sigmoid-like saturation curvature is a classic hallmark of steady-state protein cooperativity, its hyperbolic saturation figure typically represents protein noncooperativity. We also discovered that SOS autoactivation perturbs the previously predicted hysteresis of SOS activation in a steady state to produce a hyperbolic saturation curve. We interpret this as showing that SOS allostery elicits, through SOS autoactivation, cooperativity uniquely time-dependent but not ligand concentration dependent.

Intervention in protein-protein interactions (PPIs) has tremendous effects in the molecular therapy of many diseases. To fulfill the requirements for targeting intracellular proteins, here we develop SOS-localization-based interaction screening (SOLIS), which elaborately mimics signaling via the Ras-mitogen-activated protein kinase pathway. SOLIS employs two chimeric proteins in which a membrane localization motif (CaaX) is fused at the C-terminus of a protein of interest and the catalytic domain of SOS is fused at the C-terminus of another protein of interest. Interaction between the two proteins of interest induces membrane localization of the SOS chimera and cell proliferation. Thus, the SOLIS system enables enrichment of superior binders based on cell proliferation in an intracellular PPI-dependent manner. This was verified by three major modalities against intracellular PPIs (small molecules, peptide aptamers, and intrabodies). The system worked over a broad range of affinities (KD = 0.32-140 nM). In a screening of a site-directed randomized library, novel intrabody clones were selected on the basis of the potency of cell proliferation. Three other PPI detection methods (NanoBiT, SPR, and pull-down assays) were employed to characterize the SOLIS system, and several intrabody clones were judged as false negatives in these assays. SOLIS signals would be less sensitive to the orientation/conformation of the chimeric proteins, and this feature emerges as the advantage of SOLIS as a mammalian cytosolic PPI detection system with few false negatives.

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras–GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility–mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOS(cat)) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOS(cat) engaging KRas. We also find KRas(G13D) exhibits high affinity for SOS(cat) and is a potent allosteric modulator of its activity. A structure of the KRas(G13D)•SOS(cat) complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRas(G13D)–GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas–GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRas(G13D)•SOS(cat) complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

© 2021 by the authors., Recent breakthroughs have reignited interest in RAS GEFs as direct therapeutic targets. To search for new inhibitors of SOS GEF activity, a repository of known/approved compounds (NIH-NACTS) and a library of new marine compounds (Biomar Microbial Technologies) were screened by means of in vitro RAS-GEF assays using purified, bacterially expressed SOS and RAS constructs. Interestingly, all inhibitors identified in our screenings (two per library) shared related chemical structures belonging to the anthraquinone family of compounds. All our anthraquinone SOS inhibitors were active against the three canonical RAS isoforms when tested in our SOS GEF assays, inhibited RAS activation in mouse embryonic fibroblasts, and were also able to inhibit the growth of different cancer cell lines harboring WT or mutant RAS genes. In contrast to the commercially available anthraquinone inhibitors, our new marine anthraquinone inhibitors did not show in vivo cardiotoxicity, thus providing a lead for future discovery of stronger, clinically useful anthraquinone SOS GEF blockers., Work supported by grants from ISCIII-MCIU (PI19/00934; CIBERONC-CB16/12/00352), JCyL (SA264P18-UIC 076) and Foundations Ramón Areces (CIVP19A5942, Madrid) and Samuel Solórzano (FS/22-2019, USAL). R.F.M. was supported by a predoctoral fellowship from Consejería de Educación, JCyL. Research co-financed by FEDER funds.

© 2020 The Author(s)., SOS1 and SOS2 are the most universal and widely expressed family of guanine exchange factors (GEFs) capable or activating RAS or RAC1 proteins in metazoan cells. SOS proteins contain a sequence of modular domains that are responsible for different intramolecular and intermolecular interactions modulating mechanisms of self-inhibition, allosteric activation and intracellular homeostasis. Despite their homology, analyses of SOS1/2-KO mice demonstrate functional prevalence of SOS1 over SOS2 in cellular processes including proliferation, migration, inflammation or maintenance of intracellular redox homeostasis, although some functional redundancy cannot be excluded, particularly at the organismal level. Specific SOS1 gain-of-function mutations have been identified in inherited RASopathies and various sporadic human cancers. SOS1 depletion reduces tumorigenesis mediated by RAS or RAC1 in mouse models and is associated with increased intracellular oxidative stress and mitochondrial dysfunction. Since WT RAS is essential for development of RAS-mutant tumors, the SOS GEFs may be considered as relevant biomarkers or therapy targets in RAS-dependent cancers. Inhibitors blocking SOS expression, intrinsic GEF activity, or productive SOS protein-protein interactions with cellular regulators and/or RAS/RAC targets have been recently developed and shown preclinical and clinical effectiveness blocking aberrant RAS signaling in RAS-driven and RTK-driven tumors., ES and FCB were supported by grants from ISCIII-MCUI (FIS PI19/00934), JCyL (SA264P18-UIC 076) and Areces Foundation (CIVP19A5942). JMRC and NZ received grant support from MINECO-FEDER (SAF2016-78852-R and Spanish Association against Cancer (AECC/CGB14142035THOM). ES and JMRC were also supported by ISCIII-CIBERONC (groups CB16/12/00352 and CB16/12/00273, respectively). Research co-financed by FEDER funds.

In all living organisms, genome replication and cell division must be coordinated to produce viable offspring. In the event of DNA damage, bacterial cells employ the SOS response to simultaneously express damage repair systems and halt cell division. Extensive characterization of SOS-controlled cell division inhibition in Escherichia coli has laid the ground for a long-standing paradigm where the cytosolic SulA protein inhibits polymerization of the central division protein, FtsZ, and thereby prevents recruitment of the division machinery at the future division site. Within the last decade, it has become clear that another, likely more general, paradigm exists, at least within the broad group of Gram-positive bacterial species, namely membrane-localized, SOS-induced cell division inhibition. We recently identified such an inhibitor in Staphylococci, SosA, and established a model for SosA-mediated cell division inhibition in Staphylococcus aureus in response to DNA damage. SosA arrests cell division subsequent to the septal localization of FtsZ and later membrane-bound division proteins, while preventing progression to septum closure, leading to synchronization of cells at this particular stage. A membrane-associated protease, CtpA negatively regulates SosA activity and likely allows growth to resume once conditions are favorable. Here, we provide a brief summary of our findings in the context of what already is known for other membrane cell division inhibitors and we emphasize how poorly characterized these intriguing processes are mechanistically. Furthermore, we put some perspective on the relevance of our findings and future developments within the field.

Background/Aims: Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. Methods: In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. Results: SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. Conclusion: SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1.

The guanine-nucleotide exchange factor (GEF) Son-of-Sevenless (SOS) plays a critical role in metazoan signaling by converting Ras•GDP (guanosine diphosphate) to Ras•GTP (guano-sine triphosphate) in response to tyrosine kinase activation. Structural studies have shown that SOS differs from other Ras-specific GEFs in that SOS is itself activated by Ras•GTP binding to an allosteric site, distal to the site of nucleotide exchange. The activation of SOS involves membrane recruitment and conformational changes, triggered by lipid binding, that open the allosteric binding site for Ras•GTP. This is in contrast to other Ras-specific GEFs, which are activated by second messengers that more directly affect the active site. Allosteric Ras•GTP binding stabilizes SOS at the membrane, where it can turn over other Ras molecules processively, leading to an ultrasensitive response that is distinct from that of other Ras-specific GEFs.

Ras is a molecular switch cycling between an active, GTP-bound and an inactive, GDP-bound state. Mutations in Ras, mostly affecting the off-switch, are found in many human tumours. Recently, it has been shown that K-Ras 4B is targeted by lysine acetylation at K104. Based on results obtained for an acetylation mimetic Ras mutant (K104Q), it was hypothesised that K104-acetylation might interfere with its oncogenicity by impairing SOS-catalysed guanine-nucleotide exchange. We prepared site-specifically K104-acetylated K-Ras 4B and the corresponding oncogenic mutant protein G12V using the genetic-code expansion concept. We found that SOS-catalysed nucleotide exchange, also of allosterically activated SOS, was neither affected by acetylation of K104 in wildtype K-Ras 4B nor in the G12V mutant, suggesting that glutamine is a poor mimetic for acetylation at this site. In vitro, the lysine-acetyltransferases CBP and p300 were able to acetylate both, wildtype and G12V K-Ras 4B. In addition to K104 we identified further acetylation sites in K-Ras 4B, including K147, within the important G5/SAK-motif. However, the intrinsic and the SOS-catalysed nucleotide exchange was not affected by K147-acetylation of K-Ras 4B. Finally, we show that Sirt2 and HDAC6 do neither deacetylate K-Ras 4B if acetylated at K104 nor if acetylated at K147 in vitro.

Activation of small GTPases of the Ras superfamily by guanine nucleotide exchange factors (GEFs) is a key step in numerous cell signaling processes. Unveiling the detailed molecular mechanisms of GEF-GTPase signaling interactions is of great importance due to their central roles in cell biology, including critical disease states, and their potential as therapeutic targets. Here we present an assay to monitor individual Ras activation events catalyzed by single molecules of the GEF Son of Sevenless (SOS) in the natural membrane environment. The assay employs zero-mode waveguide (ZMW) nanostructures containing a single Ras-functionalized liposome. The ZMWs facilitate highly localized excitation of fluorophores in the vicinity of the liposome membrane, allowing direct observation of individual Ras activation events as single SOS enzymes catalyze exchange of unlabeled nucleotides bound to Ras with fluorescently labeled nucleotides from solution. The system is compatible with continuous recording of long sequences of individual enzymatic turnover events over hour-long time scales. The single turnover waiting time sequence is a molecular footprint that details the temporal characteristics of the system. Data reported here reveal long-lived activity states that correspond to well-defined conformers of SOS at the membrane. Liposome functionalized ZMWs allow for studies of nucleotide exchange reactions at single GTPase resolution, providing a platform to gauge the mechanisms of these processes.

K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers.

© 2019 The Authors., Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen‐killing responses. In neutrophils G protein‐coupled receptor (GPCR)‐driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)‐trisphosphate (PIP3), are highly dependent on PI3K‐γ, a Ras‐GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR‐triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR–PIP3 signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM‐CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP3 accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation., This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/J004456/1 and BB/P013384/1).

About a third of tumors harbor activating mutations in HRAS, NRAS, or KRAS, genes encoding guanine triphosphatases (GTPases) of the RAS family. In these tumors, wild-type RAS signaling cooperates with mutant RAS to promote downstream effector activation. This cooperation between wild-type and mutant RAS drives proliferation and transformation, suggesting that upstream activators of wild-type RAS are important modulators of mutant RAS-driven oncogenesis. Previous studies investigated the role of the guanine nucleotide exchange factor (GEF) SOS1 in KRAS-driven proliferation, but little is understood about the role of SOS2. Here, we found that RAS family members have a hierarchical requirement for SOS2 expression and activity to drive transformation. In Sos2(−/−) mouse embryonic fibroblasts, SOS2 critically mediated mutant KRAS-driven transformation, but was dispensable for transformation driven by HRAS. Sos2 deletion reduced EGF-dependent wild-type HRAS activation and phosphorylation of the kinase AKT in cells expressing mutant RAS, and assays using pharmacological inhibition revealed a hierarchical requirement for signaling by the kinase PI3K in promoting RAS-driven transformation that mirrored the requirement for SOS2. KRAS-driven transformation required the GEF activity of SOS2 and was restored in Sos2(−/−) MEFs by expression of constitutively activated PI3K. Finally, CRISPR/Cas9-mediated deletion of SOS2 reduced EGF-stimulated AKT phosphorylation and synergized with MEK inhibition to block transformation of KRAS-mutant tumor cells. These results indicate that SOS2-dependent PI3K signaling plays an important role in mutant KRAS-driven transformation; hence, SOS2 may be a therapeutic target in KRAS-driven cancers. Additionally, our data reveal the importance of 3D-culture systems in investigating important mediators of mutant KRAS.

Using Sos1 knockout (Sos1-KO), Sos2-KO, and Sos1/2 double-knockout (Sos1/2-DKO) mice, we assessed the functional role of Sos1 and Sos2 in skin homeostasis under physiological and/or pathological conditions. Sos1 depletion resulted in significant alterations of skin homeostasis, including reduced keratinocyte proliferation, altered hair follicle and blood vessel integrity in dermis, and reduced adipose tissue in hypodermis. These defects worsened significantly when both Sos1 and Sos2 were absent. Simultaneous Sos1/2 disruption led to severe impairment of the ability to repair skin wounds, as well as to almost complete ablation of the neutrophil-mediated inflammatory response in the injury site. Furthermore, Sos1 disruption delayed the onset of tumor initiation, decreased tumor growth, and prevented malignant progression of papillomas in a DMBA (7,12-dimethylbenz[α]anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate)-induced skin carcinogenesis model. Finally, Sos1 depletion in preexisting chemically induced papillomas resulted also in decreased tumor growth, probably linked to significantly reduced underlying keratinocyte proliferation. Our data unveil novel, distinctive mechanistic roles of Sos 1 and Sos2 in physiological control of skin homeostasis and wound repair, as well as in pathological development of chemically induced skin tumors. These observations underscore the essential role of Sos proteins in cellular proliferation and migration and support the consideration of these RasGEFs as potential biomarkers/therapy targets in Ras-driven epidermal tumors., This study was supported by grants FIS PI16/02137 from ISCIII (MINECO), SA043U16 (UIC 076) from JCyL, and AECC Spain (to E.S.); by MINECO grant SAF2015-66015-R; and by MSyC grants ISCIII-RETIC RD12/0036/0009, PIE 15/00076, and CB/16/00228 (to J.M.P.). This research was cofinanced by FEDER funds

Inhibition of Ras signalling has been a goal almost since its central role in cell signalling and its deregulation in disease were discovered. Early attempts at inhibiting its post-translational modification using peptidomimetics were successful in cell culture but failed spectacularly in clinical trials, making industry wary of targeting this critical oncoprotein. Small molecule inhibition of the protein-protein interactions involving Ras has also been difficult due to the nature of the interaction interface. Recent improvements in design, synthesis and selection of stabilised peptides, peptidomimetics and macrocycles have suggested that these biologics may represent a new hope in Ras inhibition. Here we review the various ways in which Ras has been targeted with these molecules. We also describe work on related small G proteins of the Ras superfamily, since many of the principles may be applicable to Ras, and these also provide inhibition of pathways downstream of Ras.

International audience; Rationale: The life-threatening drug rash with eosinophilia and systemic symptoms (DRESS) syndrome occurs most commonly after exposure to drugs, clinical features mimic those found with other serious systemic disorders. It is rarely associated with thrombotic microangiopathy.Patient concerns: We describe the unique case of a 44-year-old man who simultaneously experienced DRESS syndrome with thrombotic microangiopathy (TMA) after a 5 days treatment with fluindione.Diagnoses: Clinical evaluation leads to the discovery of an underlying lymphangiomatosis, due to a Noonan syndrome. Intervetions: The anticoagulant was withdrawn, and corticosteroids (1 mg/kg/day) and acenocoumarol were started.Outcomes: Clinical improvement ensued. At follow-up the patient is well.Lessons: The association of DRESS with TMA is a rare condition; we believe that the presence of the underlying Noonan syndrome could have been the trigger. Moreover, we speculate about the potential interrelations between these entities.

Cell signaling is the process by which extracellular information is transmitted into the cell to perform useful biological functions. The ERK (extracellular-signal-regulated kinase) signaling controls several cellular processes such as cell growth, proliferation, differentiation and apoptosis. The ERK signaling pathway considered in this work starts with an extracellular stimulus and ends with activated (double phosphorylated) ERK which gets translocated into the nucleus. We model and analyze this complex pathway by decomposing it into three functional subsystems. The first subsystem spans the initial part of the pathway from the extracellular growth factor to the formation of the SOS complex, ShC-Grb2-SOS. The second subsystem includes the activation of Ras which is mediated by the SOS complex. This is followed by the MAPK subsystem (or the Raf-MEK-ERK pathway) which produces the double phosphorylated ERK upon being activated by Ras. Although separate models exist in the literature at the subsystems level, a comprehensive model for the complete system including the important regulatory feedback loops is missing. Our dynamic model combines the existing subsystem models and studies their steady-state and dynamic interactions under feedback. We establish conditions under which bistability and oscillations exist for this important pathway. In particular, we show how the negative and positive feedback loops affect the dynamic characteristics that determine the cellular outcome., Scientific and Technological Research Council of Turkey (TÜBİTAK); Koç University

Son of sevenless (SOS) is one of the guanine nucleotide exchange factors that can regulate the mitogen-activated protein kinase/extracellular signal regulated kinase signal pathway via controlling the activation of Ras. microRNAs are key regulon of gene expression and would be treated as tumor biomarkers or therapeutic targets. In this study, we find that miR-148a-3p acts as a tumor-suppressor in the development and progression of non-small-cell lung cancer (NSCLC). miR-148a-3p inhibits NSCLC cells proliferation and epithelial-mesenchymal transition by reducing the expression of SOS2, which refers Ras activating. Our findings demonstrate that the miR-148a-3p may play a significant role in NSCLC including the kind of lung cancer with K-Ras gene mutation, and it exerted the tumor inhibitor function by targeting SOS2. Because of that, miR-148a-3p and SOS2 may be an efficient target in developing more useful therapies against NSCLC.

Salt stress is one of the major abiotic stresses that severely impact plant growth and development. In this study, we investigated the physiological and transcriptomic responses of Chinese cabbage "Qingmaye" to salt stress, a main variety in North China. Our results showed that the growth and photosynthesis of Chinese cabbage were significantly inhibited by salt treatment. However, as a glycophyte, Chinese cabbage could cope with high salinity; it could complete an entire life cycle at 100 mM NaCl. The high salt tolerance of Chinese cabbage was achieved by accumulating osmoprotectants and by maintaining higher activity of antioxidant enzymes. Transcriptomic responses were analyzed using the digital gene expression profiling (DGE) technique after 12 h of treatment by 200 mM NaCl. A total of 1235 differentially expressed genes (DEGs) including 740 up- and 495 down-regulated genes were identified. Functional annotation analyses showed that the DEGs were related to signal transduction, osmolyte synthesis, transcription factors, and antioxidant proteins. Taken together, this study contributes to our understanding of the mechanism of salt tolerance in Chinese cabbage and provides valuable information for further improvement of salt tolerance in Chinese cabbage breeding programs.

T cell receptor (TCR) stimulation activates diverse kinase pathways, which include the mitogen-activated protein kinases (MAPKs) ERK and p38, the phosphoinositide 3-kinases (PI3Ks), and the kinase mTOR. Although TCR stimulation activates the p38 pathway through a "classical" MAPK cascade that is mediated by the adaptor protein LAT, it also stimulates an "alternative" pathway in which p38 is activated by the kinase ZAP70. Here, we used dual-parameter, phosphoflow cytometry and in silico computation to investigate how both classical and alternative p38 pathways contribute to T cell activation. We found that basal ZAP70 activation in resting T cell lines reduced the threshold ("primed") TCR-stimulated activation of the classical p38 pathway. Classical p38 signals were reduced after T cell- specific deletion of the guanine nucleotide exchange factors Sos1 and Sos2, which are essential LAT signalosome components. As a consequence of Sos1/2 deficiency, production of the cytokine IL-2 was impaired, differentiation into regulatory T cells was reduced, and the autoimmune disease EAE was exacerbated in mice. These data suggest that the classical and alternative p38 activation pathways exist to generate immune balance.

Genome-wide association studies have identified >50 common variants associated with kidney function, but these variants do not fully explain the variation in eGFR. We performed a two-stage meta-analysis of associations between genotypes from the Illumina exome array and eGFR on the basis of serum creatinine (eGFRcrea) among participants of European ancestry from the CKDGen Consortium (nStage1: 111,666; nStage2: 48,343). In single-variant analyses, we identified single nucleotide polymorphisms at seven new loci associated with eGFRcrea (PPM1J, EDEM3, ACP1, SPEG, EYA4, CYP1A1, and ATXN2L; PStage1

Hereditary hyperplastic gingivitis (HHG) is an autosomal recessive condition found predominantly in farmed silver foxes, first documented in Europe in the 1940s. Hereditary gingival fibromatosis (HGF) is an analogous condition occurring in humans. HGF has a heterogeneous aetiology with emphasis placed on the autosomal dominant forms of inheritance for which there are three known loci: HGF1, HGF2, and HGF3. Among these, only one causative mutation has been determined, in the Son of sevenless homolog 1 (SOS1) gene. The goal of this study was to explore potential molecular or cellular mechanisms underlying HHG by analysis of global gene expression patterns from Affymetrix Canine 2.0 microarrays cross-referenced against candidate genes within the human loci. We conclude that the SOS1 gene involved in HGF1 is not significantly up-regulated in HHG. However, the structurally and functionally similar SOS2 gene is up-regulated in affected foxes, and we propose this as a candidate gene for HHG. At HGF2 we identify RASA1 (rat sarcoma viral p21 protein activator 1) as a candidate gene for HHG, as it is up-regulated in affected foxes and is involved in MAPK signalling. From comparison to the genes within the HGF3 locus, we find evidence for a role of androgens in HHG phenotype severity by differential up-regulation of SRD5A2 in HHG-affected foxes. We hypothesize that the putative mutation occurs upstream of RAS in the extracellular signal-regulated kinase component of MAPK signalling.

The etiology of chronic periodontitis clearly includes a heritable component. Our purpose was to perform a small exploratory genome-wide association study in adults ages 18–49 years to nominate genes associated with periodontal disease−related phenotypes for future consideration. Full-mouth periodontal pocket depth probing was performed on participants (N = 673), with affected status defined as two or more sextants with probing depths of 5.5 mm or greater. Two variations of this phenotype that differed in how missing teeth were treated were used in analysis. More than 1.2 million genetic markers across the genome were genotyped or imputed and tested for genetic association. We identified ten suggestive loci (p-value ≤ 1E-5), including genes/loci that have been previously implicated in chronic periodontitis: LAMA2, HAS2, CDH2, ESR1, and the genomic region on chromosome 14q21-22 between SOS2 and NIN. Moreover, we nominated novel loci not previously implicated in chronic periodontitis or related pathways, including the regions 3p22 near OSBPL10 (a lipid receptor implicated in hyperlipidemia), 4p15 near HSP90AB2P (a heat shock pseudogene), 11p15 near GVINP1 (a GTPase pseudogene), 14q31 near SEL1L (an intracellular transporter), and 18q12 in FHOD3 (an actin cytoskeleton regulator). Replication of these results in additional samples is needed. This is one of the first research efforts to identify genetic polymorphisms associated with chronic periodontitis-related phenotypes by the genome-wide association study approach. Though small, efforts such this are needed in order to nominate novel genes and generate new hypotheses for exploration and testing in future studies.

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

Mutated RAS is present in 30% of human tumors, appearing in 90% of pancreatic, 45% of colon and 35% of lung cancers. These high occurrences make RAS one of the most important drug targets in oncology. Three decades of effort to target RAS have been unsuccessful in generating drug therapies suggesting that it might represent an 'undruggable' target. However, recent reports highlighting new approaches for targeting RAS have uncovered more information on protein structure and identified new binding pockets. Efforts to target the KRAS G12C mutation specifically have shown promising results whereas other approaches have targeted various protein complexes. These advances could lead to development of new effective cancer drugs targeting RAS.

Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2-KO counterparts. The Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation, in comparison with WT and Sos2-KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Cellular proliferation and migration were impaired in Sos1-KO and Sos1/2-DKO MEFs in comparison with WT and Sos2-KO MEFs, whereas cell adhesion was only impaired upon depletion of both Sos isoforms. RasGTP formation was practically absent in Sos1/2-DKO MEFs as compared with the other genotypes and extracellular signal-regulated kinase phosphorylation showed only significant reduction after combined Sos1/2 depletion. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1-KO and the Sos1/2-DKO cells as compared with Sos2-KO and WT MEFs. Interestingly, treatment of the MEF cultures with antioxidants corrected the altered phenotypes of Sos1-KO and Sos1/2-DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2-KO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability., This work was supported by grants FIS PI13/02846, RTICC RD12/0036/0001 and RD12/0036/0048 from ISCIII (MINECO) and grants SA181U13 and BIO/SA03/14 from JCyL, Spain. PL-B, RG-N and CG were supported by FEDER-JCyL, Fondo Social Europeo and AECC, respectively.

K-Ras is a small GTPase that functions as a molecular switch cycling between inactive (GDP-bound) and active (GTP-bound) states. The conversion of K-Ras-GDP to K-Ras-GTP is the rate-limiting step in the activation of K-Ras and is catalyzed by guanine nucleotide exchange factors such as the son of sevenless (Sos). Mutations in K-Ras fix the protein in the active state and endow cells with capabilities that represent the hallmarks of cancer.[1] These include the ability to proliferate, evade apoptosis, reprogram cell metabolism, induce angiogenesis, activate invasion and metastasis, and escape immune destruction.[2] Indeed, aberrant K-Ras signaling plays a role in 30% of all human cancers, with the highest incidence of activating mutations found in pancreatic (70-90%), colon (30-50%), and lung (20-30%) carcinomas.[3] Downregulation of activated Ras reverses the transformed phenotype of cells and results in the dramatic regression of tumors in murine xenograft models.[4] Thus, K-Ras inhibition represents an attractive therapeutic strategy for many cancers. However, Ras activation and signaling is accomplished primarily through protein-protein interactions. Such protein interfaces typically lack well-defined binding pockets and have been difficult to target with small molecules.[5]

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

we previously showed that epidermal growth factor receptor tyrosine kinase (EGFRtk) is essential in the development of myogenic tone. GRB2-SOS, protein kinase B (Akt), Janus kinase (JAK), and Signal Transducer and Activator of Transcription 3 (STAT3) are activated by stretch. Thus, we hypothesized that GRB2-SOS, Akt, JAK and STAT3 are downstream signaling of the EGFR and play role in myogenic tone.myogenic tone was determined in freshly isolated coronary arterioles from C57/BL6 mice with and without inhibitors. Pressurized coronary arterioles under 25 and 75mm Hg were subjected to Western blot analysis to determine signaling phosphorylation. Smooth muscle cells (SMC) stimulated with EGF were used to determine the interaction between signaling.coronary arteriole myogenic tone was significantly reduced under EGFRtk, GRB2-SOS, JAK, and STAT3 inhibition (53.6 ± 2 vs. 83.4 ± 1.3; 82.8 ± 1; 83.6 ± 1; 86.1 ± 1% of passive diameter at 75mm Hg, p0.05, respectively). However, Akt inhibition had no effect on coronary arteriole myogenic tone. Western blot analysis showed increased EGFRtk, STAT3, JAK, and Akt phosphorylation at 75mm Hg, which was significantly inhibited under EGFRtk inhibition. Interestingly, immunoprecipitation/Western blot analysis showed two intracellular complexes (ERK1/2-JAK-STAT3) involved in myogenic tone and (Akt-JAK-STAT3) not involved in myogenic tone.these findings demonstrate that ERK1/2-JAK-STAT3 complex and GRB2-SOS, down stream signaling of the EGFRtk, are critical in the development of myogenic tone, thereby highlighting these signaling events as potential therapeutic targets in cardiovascular disease states associated with altered myogenic tone.

Many G protein-coupled receptors (GPCRs) are known to modulate cell growth and differentiation by stimulating the extracellular signal-regulated protein kinases (ERKs). In growth factor signaling, ERKs are typically stimulated through an elaborate network of modules consisting of adaptors, protein kinases, and the small GTPase Ras. The mechanism by which G protein signals tap into the ERK signaling pathway has thus far remain elusive. Members of the Gq family of G proteins, in particular Galpha16, have been shown to associate with tetratricopeptide repeat 1 (TPR1), an adaptor protein which preferentially binds to Ras. Here, we examined if TPR1 is indeed the missing link between Galpha16 signaling and Ras activation. Expression of Galpha16QL, a constitutively active mutant of Galpha16, in HEK 293 cells led to the formation of GTP-bound Ras and the subsequent phosphorylation of ERK. Likewise, stimulation of endogenou G16-coupled CCR1 chemokine receptors produced the same responses in human erythroleukemia cells. siRNA-mediated knockdown of TPR1 or expression of a dominant negative mutant of TPR1 effectively abolished the ability of Galpha16QL to induce Ras activation in HEK 293 cells. In contrast, these manipulations had no inhibitory effect on Galpha16QL induced activation of phospholipase Cbeta. Galpha16QL-induced phosphorylations of downstream targets including ERK, signal transducer and activator of transcription 3, and IkappaB kinase were significantly suppressed upon expression of the dominant negative mutant of TPR1. Furthermore, SOS2, a Ras guanine nucleotide exchange factor, was found to form a complex with TPR1 and Galpha16QL. Expression of SOS2 enhanced Galpha16QL-induced Ras activation and its subsequent signaling. Collectively, our results suggest that Galpha16 regulates multiple signaling pathways by activating Ras through its association with TPR1, but TPR1 is not required for Galpha16 to stimulate phospholipase Cbeta.

Cell viability depends on the stable transmission of genetic information to each successive generation. Therefore, in the event of intrinsic or extrinsic DNA damage, it is important that cell division be delayed until DNA repair has been completed. In Bacillus subtilis , this is accomplished in part by YneA, an inhibitor of division that is induced as part of the SOS response. We sought to gain insight into the mechanism by which YneA blocks cell division and the processes involved in shutting off YneA activity. Our data suggest that YneA is able to inhibit daughter cell separation as well as septum formation. YneA contains a LysM peptidoglycan binding domain and is predicted to be exported. We established that the YneA signal peptide is rapidly cleaved, resulting in secretion of YneA into the medium. Mutations within YneA affect both the rate of signal sequence cleavage and the activity of YneA. YneA does not stably associate with the cell wall and is rapidly degraded by extracellular proteases. Based on these results, we hypothesize that exported YneA is active prior to signal peptide cleavage and that proteolysis contributes to the inactivation of YneA. Finally, we identified mutations in the transmembrane segment of YneA that abolish the ability of YneA to inhibit cell division, while having little or no effect on YneA export or stability. These data suggest that protein-protein interactions mediated by the transmembrane region may be required for YneA activity.

Signal transduction originates at the membrane, where the clustering of signaling proteins is a key step in transmitting a message. Membranes are difficult to study, and their influence on signaling is still only understood at the most rudimentary level. Recent advances in the biophysics of membranes, surveyed in this review, have highlighted a variety of phenomena that are likely to influence signaling activity, such as local composition heterogeneities and long-range mechanical effects. We discuss recent mechanistic insights into three signaling systems-Ras activation, Ephrin signaling and the control of actin nucleation-where the active role of membrane components is now appreciated and for which experimentation on the membrane is required for further understanding.

Pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuronal differentiation, in part via a Rit GTPase signaling cascade. Here we show that PACAP-mediated Rit activation involves Src kinase-dependent TrkA receptor transactivation and identify TrkA-Rit signaling as a key contributor to PACAP-dependent neuronal differentiation., Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent neuropeptide that possesses both neurotrophic and neurodevelopmental effects. Recently, the Rit GTPase was found to be activated by a novel Gα/cAMP/exchange protein activated by cyclic AMP (Epac)-dependent signaling pathway and required for PACAP-dependent cAMP response element-binding protein activation and neuronal differentiation. However, Epac did not function as a Rit guanine nucleotide exchange factor (GEF), and the nature of the PACAP regulatory cascade remained unclear. Here, we show that PACAP-mediated Rit activation involves Src family kinase-dependent TrkA receptor transactivation. PACAP receptor (PACR1) stimulation triggered both Giα and Gsα/cAMP/Epac regulatory cascades resulting in Src kinase activity, which in turn induced TrkA kinase tyrosine phosphorylation. Importantly, Src inhibition, or the lack of functional Trk receptors, was found to inhibit PACAP-mediated Rit activation, whereas constitutively active Src alone was sufficient to stimulate Rit-guanosine triphosphate levels. A single tyrosine (Y499) phosphorylation event was identified as critical to both PACAP-mediated transactivation and TrkA-dependent Rit activation. Accordingly, PACAP stimulation resulted in TrkA-dependent phosphorylation of both the Shc adaptor and son of sevenless (SOS)1/2 GEFs, and Rit activation was inhibited by RNA interference silencing of SOS1/2, implicating a TrkA/Shc/SOS signaling complex in Rit regulation. Together, these observations expand upon the nature of PACR1-mediated transactivation and identify TrkA-Rit signaling as a key contributor to PACAP-dependent neuronal differentiation.

Ras activation is common to many human cancers and promotes cell proliferation and survival by initiating multiple signaling cascades. Accordingly, Ras-transformed cells are generally considered too resourceful to become addicted to a single effector. In contrast to this tenet, we now demonstrate an absolute, cell autonomous requirement for Raf-1 in the development and maintenance of Ras-induced skin epidermis tumors. Mechanistically, Raf-1 functions as an endogenous inhibitor dimming the activity of the Rho-dependent kinase Rok-alpha in the context of a Ras-induced Raf-1:Rok-alpha complex. Raf-1-induced Rok-alpha inhibition allows the phosphorylation of STAT3 and Myc expression and promotes dedifferentiation in Ras-induced tumors. These data link the Raf-1:Rok-alpha complex to STAT3/Myc activation and delineate a pathway crucial for cell fate decision in Ras-induced tumorigenesis.

Progression of cancer invasion is believed to be dependent on the remodeling of extracellular matrix induced by tumor cells. Rhein has been shown to inhibit the growth and proliferation of human nasopharyngeal carcinoma (NPC) cells. However, the molecular mechanism underlying rhein-induced inhibition of cancer invasion has not been explored. Herein, we show that rhein could inhibit the invasion and migration of NPC cells in vitro. Rhein inhibits invasion by reducing the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). Moreover, we demonstrate that the pathway involved in rhein-inhibited invasion is presumably through the growth factor receptor bound protein 2/son of sevenless-Ras-mitogen-activated protein kinase (GRB2/SOS-Ras-MAPK) pathway, as shown by an decrease in the expression levels of GRB2, SOS-1 and Ras as well as led to suppression of the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK. Further study has shown that rhein also inhibited activation of transcription factor nuclear factor kappaB (NF-kappaB), which is known to implicate the regulation of MMP-9 and VEGF gene expression in cancer invasion. Our findings suggest that rhein inhibits the invasion of NPC cells may be mediated in part through the suppression of MMP-9 and VEGF expression via the modulation of NF-kappaB signaling pathway.