RAF and MEK Inhibitors in Non-Small Cell Lung Cancer.

Lung cancer, despite recent advancements in survival rates, represents a significant global health burden. Non-small cell lung cancer (NSCLC), the most prevalent type, is driven largely by activating mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and receptor tyrosine kinases (RTKs), and less in v-RAF murine sarcoma viral oncogene homolog B (BRAF) and mitogen-activated protein-kinase kinase (MEK), all key components of the RTK-RAS-mitogen-activated protein kinase (MAPK) pathway. Learning from melanoma, the identification of BRAFV600E substitution in NSCLC provided the rationale for the investigation of RAF and MEK inhibition as a therapeutic strategy. The regulatory approval of two RAF-MEK inhibitor combinations, dabrafenib-trametinib, in 2017, and encorafenib-binimetinib, in 2023, signifies a breakthrough for the management of BRAFV600E-mutant NSCLC patients. However, the almost universal emergence of acquired resistance limits their clinical benefit. New RAF and MEK inhibitors, with distinct biochemical characteristics, are in preclinical and clinical development. In this review, we aim to provide valuable insights into the current state of RAF and MEK inhibition in the management of NSCLC, fostering a deeper understanding of the potential impact on patient outcomes.

SIRT4 as a novel interactor and candidate suppressor of C-RAF kinase in MAPK signaling.

Cellular responses leading to development, proliferation, and differentiation depend on RAF/MEK/ERK signaling, which integrates and amplifies signals from various stimuli for downstream cellular responses. C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, necessitating the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain, whereas SIRT4 predominantly requires the C-terminus for full interaction with C-RAF. Interestingly, SIRT4 specifically interacts with C-RAF in a pre-signaling inactive (serine 259-phosphorylated) state. Consistent with this finding, the expression of SIRT4 in HEK293 cells results in an up-regulation of pS259-C-RAF levels and a concomitant reduction in MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, we propose an additional extra-mitochondrial function of SIRT4 as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its metabolic tumor suppressor role of glutamate dehydrogenase and glutamate levels in mitochondria.

Conditional Cooperativity in RAS Assembly Pathways on Nanodiscs and Altered GTPase Cycling.

Self-assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size-tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of the cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition of the membrane. Notably, the oncogenic mutants assemble in nonsequential pathways with two mutually cooperative 'α/α' and 'α/ß' interfaces, while α/α dimerization of wild-type KRAS promotes the secondary α/ß interaction sequentially. Mutation-based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and probe their favorable interface interactions. Transiently exposed interfaces were available for the assembly. Real-time NMR demonstrated that higher-order oligomers retain higher numbers of active GTP-bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster-enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and lipid nanoparticle-based formulations of stable protein oligomers with enhanced cooperativity.

Targeting the RAS/RAF/MAPK pathway for cancer therapy: from mechanism to clinical studies.

Metastatic dissemination of solid tumors, a leading cause of cancer-related mortality, underscores the urgent need for enhanced insights into the molecular and cellular mechanisms underlying metastasis, chemoresistance, and the mechanistic backgrounds of individuals whose cancers are prone to migration. The most prevalent signaling cascade governed by multi-kinase inhibitors is the mitogen-activated protein kinase (MAPK) pathway, encompassing the RAS-RAF-MAPK kinase (MEK)-extracellular signal-related kinase (ERK) pathway. RAF kinase is a primary mediator of the MAPK pathway, responsible for the sequential activation of downstream targets, such as MEK and the transcription factor ERK, which control numerous cellular and physiological processes, including organism development, cell cycle control, cell proliferation and differentiation, cell survival, and death. Defects in this signaling cascade are associated with diseases such as cancer. RAF inhibitors (RAFi) combined with MEK blockers represent an FDA-approved therapeutic strategy for numerous RAF-mutant cancers, including melanoma, non-small cell lung carcinoma, and thyroid cancer. However, the development of therapy resistance by cancer cells remains an important barrier. Autophagy, an intracellular lysosome-dependent catabolic recycling process, plays a critical role in the development of RAFi resistance in cancer. Thus, targeting RAF and autophagy could be novel treatment strategies for RAF-mutant cancers. In this review, we delve deeper into the mechanistic insights surrounding RAF kinase signaling in tumorigenesis and RAFi-resistance. Furthermore, we explore and discuss the ongoing development of next-generation RAF inhibitors with enhanced therapeutic profiles. Additionally, this review sheds light on the functional interplay between RAF-targeted therapies and autophagy in cancer.

Effect of electroacupuncture on urodynamics and Raf/MEK/ERK signaling pathway in spinal cord tissue of rats with detrusor hyperreflexia after suprasacral spinal cord injury. / ç”µé’ˆå¯¹éª¶ä¸Šè„Šé«“æŸä¼¤åŽé€¼å°¿è‚Œåå°„äº¢è¿›åž‹å¤§é¼ å°¿æµåŠ¨åŠ›å­¦åŠè„Šé«“Raf/MEK/ERK信号通路的影响.

OBJECTIVES: To observe the effect of electroacupuncture (EA) on urodynamics and Raf/MEK/ERK signaling pathway in spine cord tissue of rats after suprasacral spinal cord injury (SSCI), so as to explore its possible mechanism in improving bladder function in rats with detrusor hyperreflexia after SSCI. METHODS: Female SD rats were randomly divided into blank, sham operation, model, EA and EA+PD98059 groups, with 12 rats in each group. Thorax (T) 10 spinal cord transection was performed by surgery. Rats in the EA group were given EA (10 Hz/50 Hz, 20 min) at "Ciliao" (BL32), "Zhongji" (CV3), "Sanyinjiao" (SP6) and "Dazhui" (GV14) once daily for 7 d. Rats of the EA+PD98059 group received intraperitoneal injection of PD98059 (5 mg/kg) 2 h before EA intervention. The urodyna-mics was used to measure the base pressure, leak point pressure, maximum pressure, maximum capacity and comp-liance of bladder, and the morphology of bladder detrusor tissue was observed with HE staining. The TUNEL staining was used to detect the cell apoptosis of the spinal cord tissue. The expression levels of exchange protein directly activated by cAMP 2 (Epac2), Rap, phosphorylated rapidly accelerated fibrosarcoma (p-Raf), phosphorylated mitogen-activated extracellular signal-regulated kinase (p-MEK), phosphorylated extracellular signal regulated kinase 1 and 2 (p-ERK1/2), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) were determined by Western blot. RESULTS: Compared with the sham operation group, the base pressure, leak point pressure and maximum pressure of bladder were significantly increased (P<0.01), the maximum bladder capacity and bladder compliance were decreased (P<0.01), the cell apoptosis rate of spinal cord tissue was increased (P<0.01), and the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were decreased (P<0.01), while the expression level of Bax protein was increased (P<0.01) in the model group. After the treatment and compared with the model group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate of spinal cord tissue, the expression level of Bax protein were decreased (P<0.05) in the EA group, while the maximum bladder capacity and bladder compliance, the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were all increased (P<0.05, P<0.01). In comparison with the EA group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate, the expression level of Bax protein were significantly increased (P<0.05), whereas the maximum bladder capacity, bladder compliance, and the expression levels of p-MEK, p-ERK1/2, and Bcl-2 protein were decreased (P<0.05) in the EA+PD98059 group. Results of HE staining showed disordered transitional epithelial cells and destroyed lamina propria in bladder detrusor tissue, with the infiltration of monocytes in the model group, which was obviously milder in both EA and EA+PD98059 groups, especially in the EA group. CONCLUSIONS: EA can improve the bladder function in detrusor hyperreflexia rats after SSCI, which may be related to its effect in up-regulating Epac2 and Rap, activating the Raf-MEK-ERK pathway, and reducing the cell apoptosis of spinal cord tissue.

Theoretical analysis reveals a role for RAF conformational autoinhibition in paradoxical activation.

RAF kinase inhibitors can, under certain conditions, increase RAF kinase signaling. This process, which is commonly referred to as 'paradoxical activation' (PA), is incompletely understood. We use mathematical and computational modeling to investigate PA and derive rigorous analytical expressions that illuminate the underlying mechanism of this complex phenomenon. We find that conformational autoinhibition modulation by a RAF inhibitor could be sufficient to create PA. We find that experimental RAF inhibitor drug dose-response data that characterize PA across different types of RAF inhibitors are best explained by a model that includes RAF inhibitor modulation of three properties: conformational autoinhibition, dimer affinity, and drug binding within the dimer (i.e., negative cooperativity). Overall, this work establishes conformational autoinhibition as a robust mechanism for RAF inhibitor-driven PA based solely on equilibrium dynamics of canonical interactions that comprise RAF signaling and inhibition.

Unraveling the interplay between RAS/RAF/MEK/ERK signaling pathway and autophagy in cancer: From molecular mechanisms to targeted therapy.

RAS/RAF/MEK/ERK signaling pathway is one of the most important pathways of Mitogen-activated protein kinases (MAPK), which widely participate in regulating cell proliferation, differentiation, apoptosis and signaling transduction. Autophagy is an essential mechanism that maintains cellular homeostasis by degrading aged and damaged organelles. Recently, some studies revealed RAS/RAF/MEK/ERK signaling pathway is closely related to autophagy regulation and has a dual effect in tumor cells. However, the specific mechanism by which RAS/RAF/MEK/ERK signaling pathway participates in autophagy regulation is not fully understood. This article provides a comprehensive review of the research progress with regard to the RAS/RAF/MEK/ERK signaling pathway and autophagy, as well as their interplay in cancer therapy. The impact of small molecule inhibitors that target the RAS/RAF/MEK/ERK signaling pathway on autophagy is discussed in this study. The advantages and limitations of the clinical combination of these small molecule inhibitors with autophagy inhibitors are also explored. The findings from this study may provide additional perspectives for future cancer treatment strategies.

Allosteric site variants affect GTP hydrolysis on Ras.

RAS GTPases are proto-oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP-bound and GTP-bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF-RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.

Analysis of the ERK Pathway Cysteinome for Targeted Covalent Inhibition of RAF and MEK Kinases.

The ERK pathway is one of the most important signaling cascades involved in tumorigenesis. So far, eight noncovalent inhibitors of RAF and MEK kinases in the ERK pathway have been approved by the FDA for the treatment of cancers; however, their efficacies are limited due to various resistance mechanisms. There is an urgent need to develop novel targeted covalent inhibitors. Here we report a systematic study of the covalent ligandabilities of the ERK pathway kinases (ARAF, BRAF, CRAF, KSR1, KSR2, MEK1, MEK2, ERK1, and ERK2) using constant pH molecular dynamics titration and pocket analysis. Our data revealed that the hinge GK (gate keeper)+3 cysteine in RAF family kinases (ARAF, BRAF, CRAF, KSR1, and KSR2) and the back loop cysteine in MEK1 and MEK2 are reactive and ligandable. Structure analysis suggests that the type II inhibitors belvarafenib and GW5074 may be used as scaffolds for designing pan-RAF or CRAF-selective covalent inhibitors directed at the GK+3 cysteine, while the type III inhibitor cobimetinib may be modified to label the back loop cysteine in MEK1/2. The reactivities and ligandabilities of the remote cysteine in MEK1/2 and the DFG-1 cysteine in MEK1/2 and ERK1/2 are also discussed. Our work provides a starting point for medicinal chemists to design novel covalent inhibitors of the ERK pathway kinases. The computational protocol is general and can be applied to the systematic evaluation of covalent ligandabilities of the human cysteinome.

Consensus on the RAS dimerization hypothesis: Strong evidence for lipid-mediated clustering but not for G-domain-mediated interactions.

One of the open questions in RAS biology is the existence of RAS dimers and their role in RAF dimerization and activation. The idea of RAS dimers arose from the discovery that RAF kinases function as obligate dimers, which generated the hypothesis that RAF dimer formation might be nucleated by G-domain-mediated RAS dimerization. Here, we review the evidence for RAS dimerization and describe a recent discussion among RAS researchers that led to a consensus that the clustering of two or more RAS proteins is not due to the stable association of G-domains but, instead, is a consequence of RAS C-terminal membrane anchors and the membrane phospholipids with which they interact.

Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS.

Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.

FDA-Approved Inhibitors of RTK/Raf Signaling Potently Impair Multiple Steps of In Vitro and Ex Vivo Influenza A Virus Infections.

Influenza virus (IV) infections pose a burden on global public health with significant morbidity and mortality. The limited range of currently licensed IV antiviral drugs is susceptible to the rapid rise of resistant viruses. In contrast, FDA-approved kinase inhibitors can be repurposed as fast-tracked host-targeted antivirals with a higher barrier of resistance. Extending our recent studies, we screened 21 FDA-approved small-molecule kinase inhibitors (SMKIs) and identified seven candidates as potent inhibitors of pandemic and seasonal IV infections. These SMKIs were further validated in a biologically and clinically relevant ex vivo model of human precision-cut lung slices. We identified steps of the virus infection cycle affected by these inhibitors (entry, replication, egress) and found that most SMKIs affected both entry and egress. Based on defined and overlapping targets of these inhibitors, the candidate SMKIs target receptor tyrosine kinase (RTK)-mediated activation of Raf/MEK/ERK pathways to limit influenza A virus infection. Our data and the established safety profiles of these SMKIs support further clinical investigations and repurposing of these SMKIs as host-targeted influenza therapeutics.

Structure of the SHOC2-MRAS-PP1C complex provides insights into RAF activation and Noonan syndrome.

SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions.

ALDH6A1 weakens the progression of colon cancer via modulating the RAS/RAF/MEK/ERK pathway in cancer cell lines.

BACKGROUND: Aldehyde dehydrogenase 6 family member A1 (ALDH6A1) is associated with multiple diseases, but its pathogenesis in colon cancer (CC) is ambiguous and needs further study so that this research explores the function of ALDH6A1 in CC. METHODS: The level of ALDH6A1 in colon adenocarcinoma (COAD), CC tissues, and cells was measured by starBase v2.0, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot. Post transfection with overexpressed (oe)-ALDH6A1, cell biological behaviors, as well as apoptosis-, matrix metalloproteinase (MMP)-, and rat sarcoma virus (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway-related markers were measured by cell function experiments, qRT-PCR, and western blot. Next, the effects of small interfering RNA targeting ALDH6A1 (si-ALDH6A1) and RAS/RAF inhibitor (MCP110) on cell biological behaviors, as well as apoptosis-, MMP-, and RAS/RAF/MEK/ERK pathway-related markers were detected again. RESULTS: ALDH6A1 was low-expressed in COAD, CC tissues, and cells. Oe-ALDH6A1 weakened cell vitality, migration and invasionbut facilitated apoptosis; while it reduced expression levels of Bcl-2, MMP-2, MMP-9 and the RAS/RAF/MEK/ERK pathway-related markers but promoted Bax level. However, the regulation of si-ALDH6A1 on cell biological behaviors and related genes was opposite to that of oe-ALDH6A1. Moreover, MCP110 rescued the regulation of si-ALDH6A1 on cell biological behaviors, expressions of apoptosis- MMP- as well as RAS/RAF/MEK/ERK pathway-related markers. To sum up, ALDH6A1 attenuated CC progression by down-regulating the expressions of RAS/RAF/MEK/ERK pathway-related markers.

Syringin exerts anti-breast cancer effects through PI3K-AKT and EGFR-RAS-RAF pathways.

BACKGROUND: Breast cancer (BC) is one of the most common malignant tumors with the highest mortality in the world. Modern pharmacological studies have shown that Syringin has an inhibitory effect on many tumors, but its anti-BC efficacy and mechanism are still unclear. METHODS: First, Syringin was isolated from Acanthopanax senticosus (Rupr. & Maxim.) Harms (ASH) by systematic solvent extraction and silica gel chromatography column. The plant name is composed of genus epithet, species additive words and the persons' name who give its name. Then, the hub targets of Syringin against BC were revealed by bioinformatics. To provide a more experimental basis for later research, the hub genes which could be candidate biomarkers of BC and a ceRNA network related to them were obtained. And the potential mechanism of Syringin against BC was proved in vitro experiments. RESULTS: Syringin was obtained by liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), and high-performance liquid chromatography (HPLC). Bioinformatics results showed that MAP2K1, PIK3CA, HRAS, EGFR, Caspase3, and PTGS2 were the hub targets of Syringin against BC. And PIK3CA and HRAS were related to the survival and prognosis of BC patients, the PIK3CA-hsa-mir-139-5p-LINC01278 and PIK3CA-hsa-mir-375 pathways might be closely related to the mechanism of Syringin against BC. In vitro experiments confirmed that Syringin inhibited the proliferation and migration and promoted apoptosis of BC cells through the above hub targets. CONCLUSIONS: Syringin against BC via PI3K-AKT-PTGS2 and EGFR-RAS-RAF-MEK-ERK pathways, and PIK3CA and HRAS are hub genes for adjuvant treatment of BC.

Mechanisms of isoform-specific residue influence on GTP-bound HRas, KRas, and NRas.

HRas, KRas, and NRas are GTPases with a common set of effectors that control many cell-signaling pathways, including proliferation through Raf kinase. Their G-domains are nearly identical in sequence, with a few isoform-specific residues that have an effect on dynamics and biochemical properties. Here, we use accelerated molecular dynamics (aMD) simulations consistent with solution x-ray scattering experiments to elucidate mechanisms through which isoform-specific residues associated with each Ras isoform affects functionally important regions connected to the active site. HRas-specific residues cluster in loop 8 to stabilize the nucleotide-binding pocket, while NRas-specific residues on helix 3 directly affect the conformations of switch I and switch II. KRas, the most globally flexible of the isoforms, shows greatest fluctuations in the switch regions enhanced by a KRas-specific residue in loop 7 and a highly dynamic loop 8 region. The analysis of isoform-specific residue effects on Ras proteins is supported by NMR experiments and is consistent with previously published biochemical data.

Structural basis for SHOC2 modulation of RAS signalling.

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers1-3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5-8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11-13, but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

Millisecond molecular dynamics simulations of KRas-dimer formation and interfaces.

Ras dimers have been proposed as building blocks for initiating the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cellular signaling pathway. To better examine the structure of possible dimer interfaces, the dynamics of Ras dimerization, and its potential signaling consequences, we performed molecular dynamics simulations totaling 1 ms of sampling, using an all-atom model of two full-length, farnesylated, guanosine triphosphate (GTP)-bound, wild-type KRas4b proteins diffusing on 29%POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine)-mixed POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membranes. Our simulations unveil an ensemble of thermodynamically weak KRas dimers spanning multiple conformations. The most stable conformations, having the largest interface areas, involve helix α2 and a hypervariable region (HVR). Among the dimer conformations, we found that the HVR of each KRas has frequent interactions with various parts of the dimer, thus potentially mediating the dimerization. Some dimer configurations have one KRas G-domain elevated above the lipid bilayer surface by residing on top of the other G-domain, thus likely contributing to the recruitment of cytosolic Raf kinases in the context of a stably formed multi-protein complex. We identified a variant of the α4-α5 KRas-dimer interface that is similar to the interfaces obtained with fluorescence resonance energy transfer (FRET) data of HRas on lipid bilayers. Interestingly, we found two arginine fingers, R68 and R149, that directly interact with the beta-phosphate of the GTP bound in KRas, in a manner similar to what is observed in a crystal structure of GAP-HRas complex, which can facilitate the GTP hydrolysis via the arginine finger of GTPase-activating protein (GAP).

Correlation of LAGE3 with unfavorable prognosis and promoting tumor development in HCC via PI3K/AKT/mTOR and Ras/RAF/MAPK pathways.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common clinical malignancies quite susceptible to recurrence and metastasis. Despite several improvements in therapeutic approaches, the prognosis remains poor due to the limited treatment options. A bioinformatics analysis based on TCGA databases revealed that the recombinant human L antigen family member 3 (LAGE3) might function as an effective prognostic and diagnostic biomarker for HCC, as LAGE3, a protein-coding gene, maintains several important biological functions and has a physiological significance in the CTAG family while simultaneously being involved in regulating the occurrence and invasion of numerous types of tumors. However, the LAGE3 gene's functional and regulatory mechanism in the progression of HCC remains unclear. METHODS: The LAGE3 level was investigated in 79 HCC tissues cases, ten HCC adjacent tissue cases, and six cases of normal liver tissues by IHC, while the LAGE3 level was evaluated in BEL-7404, SMCC-7721, Huh-7, HepG2, and MIHA cell lines by qRT-PCR and Western blot tests. Although the proliferation, migration, invasion, and apoptotic abilities of HCC cells were measured in vitro after silencing assay to probe the role of LAGE3 in HCC cells, the tumor xenograft growth experiment was used to verify the in vivo effect of LAGE3 gene knockdown on the growth of HCC tumors combined with bioinformatics analysis to study the LAGE3 mechanisms regulating HCC proliferation. RESULTS: Our results implied that LAGE3 was extensively expressed in HCC cell lines like BEL-7404, SMCC-7721, and Huh-7 cells as well as HCC tissues, but a lower expression was observed in HepG2 cells. Additionally, LAGE3 restrains cellular proliferation, promotes apoptotic pathways in HCC cells, and inhibits the growth of HCC tumors in vivo. Lastly, it was stated that LAGE3 might promote tumor development in HCC via PI3K/AKT/mTOR and Ras/RAF/MAPK pathways. CONCLUSION: This study shows that the development of specific LAGE3 target drugs might become new effective treatment modalities for HCC patients.