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Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5-[(β,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated Cysteinyl amide Inhibitors (PCAIs) constitute a group of potential cancer therapy agents that we designed to specifically disrupt and suppress hyperactive G-protein signaling, such as that caused by mutated RAS proteins. Here we determine the effects of PCAIs on the viability, G-protein levels, downstream mediators, and apoptosis-related proteins on the KRAS-mutated, Black American-derived lung adenocarcinoma cell line, NCI-H23. Of the 17 PCAIs tested, compounds NSL-YHJ-2-27 and NSL-YHJ-2-46 showed the most potency with EC50 values of 2.7 and 3.3 μM, respectively. Western blotting was used to determine the effect of the PCAIs on the phosphorylation levels of MAPK pathway enzymes. After 48 h exposure to 5 μM of the PCAIs, NSL-YHJ-2-46, the MAPK proteins BRAF, MEK1/2, ERK1/2, and p90RSK were activated through phosphorylation by 90, 190, 150 and 120%, respectively. However, CRAF/RAF1 phosphorylation decreased by 40%, suggesting significant changes in the KRAS/MAPK signaling patterns. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 and 76%, respectively. The depletion of these key cytoskeletal proteins may account for the observed inhibition of cell migration and invasion, and spheroid invasion observed on exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46. Treatment with 5 μM of NSL-YHJ-2-27 suppressed full-length inactive caspase 3 and 7 levels by 72 and 91%, respectively. An analysis of cells treated with the fluorescently labeled active caspase 3/7 irreversible inhibitor, CaspaTagTM Caspase-3/7 in situ reagent revealed a 124% increase in active caspase at 3 μM over controls. These findings clearly show the direct effects of the PCAIs on the RAS signaling pathway. Given the profound increases observed in RPS6KA1/p90RSK phosphorylation, future work will involve a determination whether the proapoptotic isoforms of RPS6KA1/p90RSK are phosphorylated due to the PCAIs treatments. These results support the potential use of the PCAIs as targeted therapies against cancers with KRAS mutations. [ABSTRACT FROM AUTHOR]

RAS proteins are necessary for cell survival, and their mutations are closely related to tumor formation and progression. Mutated RAS proteins were undruggable owing to their smooth surface decades ago. Since chemists found that covalently binding to mutated cysteine (G12 C) was a potential therapy for KRASG12C inhibition. Recently, AMG‐510 and MRTX‐849 were approved in 2021 and 2022 respectively for solid tumor treatment. In this paper, started with KRASG12C inhibitor ARS‐1620, we introduced different hydrophilic fragment on it and reached compound K09, which had the best KRASG12C inhibitory activity and antiproliferation activities. Compound K09 is regarded as a lead compound for further exploration. [ABSTRACT FROM AUTHOR]

RAS proteins are peripheral membrane GTPases that activate multiple downstream effectors for cell proliferation and differentiation. The formation of a signaling RAS–RAF complex at the plasma membrane is implicated in a quarter of all human cancers; however, the underlying mechanism remains unclear. In this work, nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses to determine the structure of a hetero‐tetrameric complex comprising KRAS and the RAS‐binding domain (RBD) and cysteine‐rich domain (CRD) of activated RAF1 are employed. The binding of the RBD or RBD–CRD differentially alters the dimerization modes of KRAS on both anionic and neutral membranes, validated by interface‐specific mutagenesis. Notably, the RBD binding allosterically generated two distinct KRAS dimer interfaces in equilibrium, favored by KRAS free and in complex with the RBD–CRD, respectively. Additional interactions of the CRD with both KRAS protomers are mutually cooperative to stabilize a new dimer configuration of KRAS bound to the RBD–CRD. The RAF binding sequentially alters KRAS dimerization, providing new insights into RAF activation, including a configurational transition of the KRAS dimer to provide an interaction site for the CRD and release the autoinhibited RAF complex. These methods are applicable to many other signaling protein complexes on the membrane. [ABSTRACT FROM AUTHOR]

Stress granules (SGs) are non-membranous organelles composed of mRNA and proteins that assemble in the cytosol when the cell is under stress. Although the composition of mammalian SGs is both cell-type and stress-dependent, they consistently contain core components, such as Ras GTPase activating protein SH3 domain binding protein 1 (G3BP1). Upon stress, living cells rapidly assemble micrometric SGs, sometimes within a few minutes, suggesting that SG components may be actively transported by the microtubule and/or actin cytoskeleton. Indeed, SG assembly has been shown to depend on the microtubule cytoskeleton and the associated motor proteins. However, the role of the actin cytoskeleton and associated myosin motor proteins remains controversial. Here, we identified G3BP1 as a novel binding protein of unconventional myosin-5a (Myo5a). G3BP1 uses its C-terminal RNA-binding domain to interact with the middle portion of Myo5a tail domain (Myo5a-MTD). Suppressing Myo5a function in mammalian cells, either by overexpressing Myo5a-MTD, eliminating Myo5a gene expression, or treatment with myosin-5 inhibitor, inhibits the arsenite-induced formation of both small and large SGs. This is different from the effect of microtubule disruption, which abolishes the formation of large SGs but enhances the formation of small SGs under stress conditions. We therefore propose that, under stress conditions, Myo5a facilitates the formation of SGs at an earlier stage than the microtubule-dependent process. [ABSTRACT FROM AUTHOR]

ABSTRACT SGSM proteins are small modulator proteins interacting with proteins in the RAS signaling pathway. Studies with mouse and human tissues indicated that SGSM genes were highly expressed in the brain and could be expressed at different levels at different stages of development in fetal and adult brain tissue. It was first reported by Birnbaum et al. that the SGSM3 gene might be associated with a Mendelian inherited disease in families of Ashkenazi Jews with clinical manifestations of intellectual disability (ID). In this study, a novel homozygous stop‐gain (NM_015705.6: c.1576C>T: p.(Arg526Ter)) variation was detected in the SGSM3 gene in two siblings with short stature and ID findings. The report of two cases with bi‐allelic LOF variants in the SGSM3 gene from different populations with similar clinical manifestations strengthens the potential of this gene as a candidate gene for the nonsyndromic ID phenotype. Functional studies are required to investigate the signaling pathways affected by SGSM3 gene variations to produce the ID phenotype and their effect on the functioning of neurons. [ABSTRACT FROM AUTHOR]

It is widely acknowledged that there is a considerable number of oncogenic mutations within the Ras superfamily of small GTPases which are the driving force behind a multitude of cancers. Ras proteins mediate a plethora of kinase pathways, including the MAPK, PI3K, and Ral pathways. Since Ras was considered undruggable until recently, pharmacological targeting of pathways downstream of Ras has been attempted to varying success, though drug resistance has often proven an issue. Nuances between kinase pathway activation in the presence of various Ras mutants are thought to contribute to the resistance, however, the reasoning behind activation of different pathways in different Ras mutational contexts is yet to be fully elucidated. Indeed, such disparities often depend on cancer type and disease progression. However, we are in a revolutionary age of Ras mutant targeted therapy, with directtargeting KRAS-G12C inhibitors revolutionising the field and achieving FDAapproval in recent years. However, these are only beneficial in a subset of patients. Approximately 90% of Ras-mutant cancers are not KRAS-G12C mutant, and therefore raises the question as to whether other distinct amino acid substitutions within Ras may one day be targetable in a similar manner, and indeed whether better understanding of the downstream pathways these various mutants activate could further improve therapy. Here, we discuss the favouring of kinase pathways across an array of Ras-mutant oncogenic contexts and assess recent advances in pharmacological targeting of various Ras mutants. Ultimately, we will examine the utility of individualised pharmacological approaches to Rasmediated cancer. [ABSTRACT FROM AUTHOR]

Ras-related associated with diabetes (RRAD) is a member of the Ras GTPase superfamily that plays a role in several cellular functions, such as cell proliferation and differentiation. In particular, the superfamily acts as an NF-κB signaling pathway inhibitor and calcium regulator to participate in the immune response pathway. A recent transcriptome study revealed that rrad was expressed in the spleen of disease-resistant Japanese flounder (Paralichthys olivaceus) individuals compared with disease-susceptible individuals, and the results were also verified by qPCR. Thus, the present study aimed to explore how rrad regulates antimicrobial immunity via the NF-κB pathway. First, the coding sequence of P. olivaceus rrad was identified. The sequence was 1092 bp in length, encoding 364 amino acids. Based on phylogenetic and structural relationship analyses, P. olivaceus rrad appeared to be more closely related to teleosts. Next, rrad expression differences between disease-resistant and disease-susceptible individuals in immune-related tissues were evaluated, and the results revealed that rrad was expressed preferentially in the spleen of disease-resistant individuals. In response to Edwardsiella piscicida infection, rrad expression in the spleen changed. In vitro, co-culture was carried out to assess the hypo-methylated levels of the rrad promoter in the disease-resistant spleen, which was consistent with the high mRNA expression. The siRNA-mediated knockdown of rrad performed with the gill cell line of P. olivaceus affected many rrad-network-related genes, i.e., dcp1b, amagt, rus1, rapgef1, ralbp1, plce1, rasal1, nckipsd, prkab2, cytbc-1, sh3, and others, as well as some inflammation-related genes, such as bal2 and Il-1β. In addition, flow cytometry analysis showed that rrad overexpression was more likely to induce cell apoptosis, with establishing a link between rrad's function and its potential roles in regulating the NF-κB pathway. Thus,. the current study provided some clarity in terms of understanding the immune response about rrad gene differences between disease-resistant and disease-susceptible P. olivaceus individuals. This study provides a molecular basis for fish rrad gene functional analysis and may serve as a reference for in-depth of bacterial disease resistance of teleost. [ABSTRACT FROM AUTHOR]

The concomitant activation of both the YAP1 co-transcription factor and RAS GTPases is a hallmark of several aggressive cancers, though the intricacies of their relationship and implications for oncogenesis are still poorly understood. This review has presented a cooperative model where YAP1 and RAS are not independently acting oncogenes but rather interdependently acting ones, with each fulfilling an essential role within the oncogenic process. YAP1 is responsible for initiating the expression of key proteins that contribute to various cancer traits. However, these proteins must often be transported into the cytoplasm to exert their effects. We suggest that oncogenic RAS actually facilitates this transport, enabling the phosphorylation and subsequent activation of the nuclear transporter XPO1 (aka Exportin1). This mechanism is particularly crucial for anti-apoptotic proteins. Instead of being sequestered within the nucleus in an ineffective state, these proteins are rather shuttled into the cytoplasm. Within the cytoplasm, they can effectively inhibit apoptosis, undermining by these means the efficacy of chemotherapeutic agents designed to induce cell death in cancer cells. Therefore, a clearer understanding of the oncogenic partnership between RAS and YAP1 will likely provide new insights into the molecular underpinnings of cancer and highlight as well potential targets for therapeutic interventions designed to disrupt this pernicious interaction. I. YAP1 triggers expression of its target genes, some of which encode cargoes with a nuclear export signal. In response to physiological signals, some cargoes are shuttled into the cytoplasm. Exportin1 serves as the transfer vehicle from the nucleus into the cytoplasm (not shown). For functional nuclear proteins, this shuttling terminates their function, whereas for cytoplasmic proteins, it does initiate their function. In this drawing, the topology within the nucleus is not depicted. II. Oncogenic RAS leads to Exportin1 activation and subsequent unwarranted nuclear-cytoplasmic shuttling of cargoes [17, 35]. The cascade from RAS to Exportin1 is not depicted for the sake of clarity. Some of the cargoes are oncoproteins, cytoplasmic GTPases regulators, or antiapoptotic proteins (example shown: BIRC5 aka Survivin). These latter proteins are now empowered to hinder apoptosis, as expected upon anti-cancer therapies. Of course, the BIRC5 is not the only aberrantly shuttled protein. Another example is Beclin1 (BECN1), which is subjected to the same fate; once in the cytoplasm, it does initiate autophagy in the absence of any autophagic clue [35]. [ABSTRACT FROM AUTHOR]

The KRAS protein, a product of the KRAS gene (V-ki-ras2 Kirsten rat sarcoma viral oncogene homolog), functions as a small GTPase that alternates between an active GTP-bound state (KRAS(ON)) and an inactive GDP-bound state (KRAS (OFF)). The KRASG12C mutation results in the accumulation of KRASG12C(OFF), promoting cell cycle survival and proliferation primarily through the canonical MAPK and PI3K pathways. The KRASG12C mutation is found in 13% of lung adenocarcinomas. Previously considered undruggable, sotorasib and adagrasib are the first available OFF-state KRASG12C inhibitors, but treatment resistance is frequent. In this review, after briefly summarizing the KRAS pathway and the mechanism of action of OFF-state KRASG12C inhibitors, we discuss primary and acquired resistance mechanisms. Acquired resistance is the most frequent, with "on-target" mechanisms such as a new KRAS mutation preventing inhibitor binding; and "off-target" mechanisms leading to bypass of KRAS through gain-of-function mutations in other oncogenes such as NRAS, BRAF, and RET; or loss-of-function mutations in tumor suppressor genes such as PTEN. Other "offtarget" mechanisms described include epithelial-to-mesenchymal transition and histological transformation. Multiple co-existing mechanisms can be found in patients, but few cases have been published. We highlight the lack of data on non-genomic resistance and the need for comprehensive clinical studies exploring histological, genomic, and non-genomic changes at resistance. This knowledge could help foster new treatment initiatives in this challenging context. [ABSTRACT FROM AUTHOR]

Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation. [ABSTRACT FROM AUTHOR]

Linear nevus sebaceous syndrome (LNSS) is a neurocutaneous syndrome associated with systemic complications that involve multiple organs, including the skin, central nervous system, eyes, and skeleton. LNSS is considered to be caused by mosaic RAS gene mutation. In this report, we present an autopsy case of LNSS in a Japanese boy. The affected neonate had hydrops fetalis and was born at 28 weeks and 4 days of gestation, weighing 2104 g. He had bilateral inverted eyelids, verrucous linear nevus separated along Blaschko's line, myocardial hypertrophy, and pharyngeal constriction, and underwent intensive treatment in NICU for arrhythmia, hydrocephalus, and respiratory distress. The hydrocephalus progressed gradually and he died at the age of 181 days, 12 days after a sudden cardiac arrest and recovery. KRAS G12D mutation was found in a skin biopsy specimen but not in blood cells, suggesting a postzygotic mosaicism. Autopsy revealed novel pathological findings related to LNSS, including intracranial lipomatous hamartoma and mesenteric lymphangioma, in addition to previously reported findings such as multicystic dysplastic kidney. There was the limited expression of mutated KRAS protein in kidneys. [ABSTRACT FROM AUTHOR]

Mutation in oncogene KRas plays a crucial role in the occurrence and progression of numerous malignant tumors. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. We hypothesize that oncogene KRas mutations are intrinsic to alterations in cellular mechanics that promote malignant tumor generation and progression. Here, we demonstrate the use of optical tweezers coupled with a confocal fluorescence imaging system and gene interference technique to reveal that the mutant KRas protein can be transported between homogeneous and heterogeneous tumor cells by tunneling nanotubes (TNTs), resulting in a significant reduction of membrane tension and acceleration of membrane phospholipid flow in the recipient cells. Simultaneously, the changes in membrane mechanical properties of the tumor cells also enhance the metastatic and invasive ability of the tumors, which further contribute to the deterioration of the tumors. This finding helps to clarify the association between oncogene mutations and changes in the mechanical properties of tumor cells, which provides a theoretical basis for the development of cancer treatment strategies. Here, we present a laser confocal fluorescence system integrated with optical tweezers to observe the transfer of mutant KRasG12D protein from mutant cells to wild-type cells through TNTs. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. Our results demonstrate a significant decrease in membrane tension and an increase in membrane phospholipid flow in recipient cells. These alterations in mechanical properties augment the migration and invasive capabilities of tumor cells, contributing to tumor malignancy. Our findings propose that cellular mechanical properties could serve as new markers for tumor development, and targeting membrane tension may hold potential as a therapeutic strategy. [Display omitted] [ABSTRACT FROM AUTHOR]

During embryonic development, tissues and organs are gradually shaped into their functional morphologies through a series of spatiotemporally tightly orchestrated cell behaviors. A highly conserved organ shape across metazoans is the epithelial tube. Tube morphogenesis is a complex multistep process of carefully choreographed cell behaviors such as convergent extension, cell elongation, and lumen formation. The identity of the signaling molecules that coordinate these intricate morphogenetic steps remains elusive. The notochord is an essential tubular organ present in the embryonic midline region of all members of the chordate phylum. Here, using genome editing, pharmacology and quantitative imaging in the early chordate Ciona intestinalis we show that Ano10/Tmem16k, a member of the evolutionarily ancient family of transmembrane proteins called Anoctamin/TMEM16 is essential for convergent extension, lumen expansion, and connection during notochord morphogenesis. We find that Ano10/Tmem16k works in concert with the plasma membrane (PM) localized Na+/Ca2+ exchanger (NCX) and the endoplasmic reticulum (ER) residing SERCA, RyR, and IP3R proteins to establish developmental stage specific Ca2+ signaling molecular modules that regulate notochord morphogenesis and Ca2+ dynamics. In addition, we find that the highly conserved Ca2+ sensors calmodulin (CaM) and Ca2+/calmodulin-dependent protein kinase (CaMK) show an Ano10/Tmem16k-dependent subcellular localization. Their pharmacological inhibition leads to convergent extension, tubulogenesis defects, and deranged Ca2+ dynamics, suggesting that Ano10/Tmem16k is involved in both the "encoding" and "decoding" of developmental Ca2+ signals. Furthermore, Ano10/Tmem16k mediates cytoskeletal reorganization during notochord morphogenesis, likely by altering the localization of 2 important cytoskeletal regulators, the small GTPase Ras homolog family member A (RhoA) and the actin binding protein Cofilin. Finally, we use electrophysiological recordings and a scramblase assay in tissue culture to demonstrate that Ano10/Tmem16k likely acts as an ion channel but not as a phospholipid scramblase. Our results establish Ano10/Tmem16k as a novel player in the prevertebrate molecular toolkit that controls organ morphogenesis across scales. The notochord is an essential tubular organ that defines the chordate phylum. This study reveals that an ion channel called Anoctamin 10 orchestrates the robust morphogenesis of the notochord in the urochordate Ciona intestinalis. [ABSTRACT FROM AUTHOR]

RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation. [ABSTRACT FROM AUTHOR]

RAS proteins are conserved guanosine triphosphate (GTP) hydrolases (GTPases) that act as molecular binary switches and play vital roles in numerous cellular processes. Upon GTP binding, RAS GTPases adopt an active conformation and interact with specific proteins termed RAS effectors that contain a conserved ubiquitin-like domain, thereby facilitating downstream signaling. Over 50 effector proteins have been identified in the human proteome, and many have been studied as potential mediators of RAS-dependent signaling pathways. Biochemical and structural analyses have provided mechanistic insights into these effectors, and studies using model organisms have complemented our understanding of their role in physiology and disease. Yet, many critical aspects regarding the dynamics and biological function of RAS-effector complexes remain to be elucidated. In this review, we discuss the mechanisms and functions of known RAS effector proteins, provide structural perspectives on RAS-effector interactions, evaluate their significance in RAS-mediated signaling, and explore their potential as therapeutic targets. RAS proteins act as molecular switches by binding GTP and interacting with effectors to facilitate downstream signaling. This review by Mozzarelli et al. discusses the mechanisms, structures, and functions of RAS effectors, their roles in signaling, and their potential as therapeutic targets, emphasizing the need for deeper insights into RAS-effector interactions. [ABSTRACT FROM AUTHOR]

Rat sarcoma virus (RAS) GTPase is one of the most important drivers of non-small cell lung cancer (NSCLC). RAS has three different isoforms (Harvey rat sarcoma viral oncogene homolog [HRAS], Kirsten rat sarcoma viral oncogene homolog [KRAS] and Neuroblastoma ras viral oncogene homolog [NRAS]), of which KRAS is most commonly mutated in NSCLC. The mutated KRAS protein was historically thought to be "undruggable" until the development of KRASG12C inhibitors. In this review, from the aspect of brain metastasis, we aim to provide an overview of the advances in therapies that target KRASG12C, the limitations of the current treatments, and future prospects in patients with KRAS p.G12C mutant NSCLC. [ABSTRACT FROM AUTHOR]

Aim: Microsatellite instability (MSI) and KRAS mutations change colorectal carcinoma (CRC) treatment protocols. Advanced examinations such as immunohistochemistry and polymerase chain reaction (PCR) are required to determine MSI and KRAS mutations. On the other hand, Crohn-like lymphoid reaction (CLR), tumor-infiltrating lymphocytes (TIL), tumor budding (TB), and desmoplastic response (DR) are histomorphologic features that can be determined only with routine hematoxylin-eosin (H&E) sections. Our study aimed to evaluate relationships between MSI, KRAS mutations, and histomorphologic features. It was thought that the relationships to be determined may be useful in predicting KRAS mutations and MSI by evaluating only H&E sections. Material and Method: One hundred nine metastatic CRC cases were reviewed retrospectively. Polymerase chain reaction results were obtained from the molecular pathology report archive and performed on all cases for KRAS mutation detection upon clinical request during routine pathologic examinations. MLH1, MSH2, MSH6, and PMS2 immunohistochemistry, performed on 70 cases for MSI interpretation upon clinical request during routine pathological examinations, was re-evaluated for standardization. Routine H&E sections with tumors were examined for CLR, TIL, TB, and DR according to study-specific criteria. Results: KRAS mutations were found in 35.77% (39/109), MSI in 24.28% (17/70), CLR in 32.11% (35/109), TIL in 44.95% (49/109), TB in 73.39% (80/109), DR in 84.40% (92/109) of the cases. CLR, TIL, DR, and KRAS mutations were higher in microsatellite stable (MSS) cases, and TB was higher in MSI cases. Crohn-like lymphoid reaction, TIL, DR, and MSI were higher in KRAS wild cases, and TB in KRAS mutant cases. Only the MSS-DR correlation was statistically significant. Conclusion: The MSS-DR correlation was statistically significant in our study. However, desmoplasia was determined in 92.45% of MSS cases, and was also determined in 58.82% of MSI cases. Because DR is an expected feature in tumor stroma, its guidance in terms of MSI was limited. Also, no significant relationship was found between MSI and DR in Turkish or English literature. In our study, histomorphologic features were insufficient to predict MSI and KRAS mutations. It is vital to immediately refer patients with metastases evaluated in centers without immunohistochemistry and PCR facilities to an advanced center for MSI and KRAS mutation determination diagnosing CRC, especially for treatment selection. [ABSTRACT FROM AUTHOR]

The article focuses on the manuscript review of a study by Mohr and colleagues that characterizes the Arabidopsis thaliana small GTPase TITAN5 (TTN5) and its variants. It examines both the biochemical properties of TTN5 and its cellular localization and function, highlighting the strengths in biochemical characterization but noting weaknesses in the cell biology and functional aspects.

The role of KRAS mutation in non‐small cell lung cancer (NSCLC) initiation and progression is well‐established. However, "undruggable" KRAS protein poses the research of small molecule inhibitors a significant challenge. Addressing this, proteolysis‐targeting chimeras (PROTACs) have become a cutting‐edge treatment method, emphasizing protein degradation. A modified ethanol injection method was employed in this study to formulate liposomes encapsulating PROTAC drug LC‐2 (LC‐2 LPs). Precise surface modifications using cell‐penetrating peptide R8 yielded R8‐LC‐2 liposomes (R8‐LC‐2 LPs). Comprehensive cellular uptake and cytotoxicity studies unveiled that R8‐LC‐2 LPs depended on concentration and time, showcasing the superior performance of R8‐LC‐2 LPs compared to normal liposomes. In vivo pharmacokinetic profiles demonstrated the capacity of DSPE‐PEG2000 to prolong the circulation time of LC‐2, leading to higher plasma concentrations compared to free LC‐2. In vivo antitumor efficacy research underscored the remarkable ability of R8‐LC‐2 LPs to effectively suppress tumor growth. This study contributed to the exploration of enhanced therapeutic strategies for NSCLC, specifically focusing on the development of liposomal PROTACs targeting the "undruggable" KRAS protein. The findings provide valuable insights into the potential of this innovative approach, offering prospects for improved drug delivery and heightened antitumor efficacy. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis and high mortality rates. Therefore, it is necessary to identify new targets and therapeutic strategies to improve the prognosis of patients with PDAC. Integrative therapies are increasingly being used to boost the efficacy of the known anticancer therapeutic approaches. Hence, this study aimed to evaluate the effects of a novel combination of different potential anticancer molecules, melatonin (MLT), cannabidiol (CBD), and oxygen–ozone (O2/O3) to treat PDAC using in vitro and in vivo models of human PDAC. The effect of this combination was investigated in combination with gemcitabine (GEM), the most common chemotherapeutic drug used for PDAC treatment. The combination of MLT + CBD + O2/O3 was more effective than the individual treatments in inhibiting PDAC cell viability and proliferation, inducing cell death, and modulating the RAS pathway protein levels. Moreover, different combinations of treatments reduced tumor mass in the PDAC mouse model, thus promoting the effect of GEM. In conclusion, a mixture of MLT + CBD + O2/O3 could serve as a potential adjuvant therapeutic strategy for PDAC. [ABSTRACT FROM AUTHOR]

Ras small GTPases act as molecular switches in various cellular signaling pathways, including cell migration, proliferation, and differentiation. Three Rap proteins are present in Dictyostelium; RapA, RapB, and RapC. RapA and RapC have been reported to have opposing functions in the control of cell adhesion and migration. Here, we investigated the role of RapB, a member of the Ras GTPase subfamily in Dictyostelium, focusing on its involvement in cell adhesion, migration, and developmental processes. This study revealed that RapB, similar to RapA, played a crucial role in regulating cell morphology, adhesion, and migration. rapB null cells, which were generated by CRISPR/Cas9 gene editing, displayed altered cell size, reduced cell-substrate adhesion, and increased migration speed during chemotaxis. These phenotypes of rapB null cells were restored by the expression of RapB and RapA, but not RapC. Consistent with these results, RapB, similar to RapA, failed to rescue the phenotypes of rapC null cells, spread morphology, increased cell adhesion, and decreased migration speed during chemotaxis. Multicellular development of rapB null cells remained unaffected. These results suggest that RapB is involved in controlling cell morphology and cell adhesion. Importantly, RapB appears to play an inhibitory role in regulating the migration speed during chemotaxis, possibly by controlling cell-substrate adhesion, resembling the functions of RapA. These findings contribute to the understanding of the functional relationships among Ras subfamily proteins. [ABSTRACT FROM AUTHOR]

Protein-membrane interactions (PMIs) are ubiquitous in cellular signaling. Initial steps of signal transduction cascades often rely on transient and dynamic interactions with the inner plasma membrane leaflet to populate and regulate signaling hotspots. Methods to target and modulate these interactions could yield attractive tool compounds and drug candidates. Here, we demonstrate that the conjugation of a medium-chain lipid tail to the covalent K-Ras(G12C) binder MRTX849 at a solvent-exposed site enables such direct modulation of PMIs. The conjugated lipid tail interacts with the tethered membrane and changes the relative membrane orientation and conformation of K-Ras(G12C), as shown by molecular dynamics (MD) simulation-supported NMR studies. In cells, this PMI modulation restricts the lateral mobility of K-Ras(G12C) and disrupts nanoclusters. The described strategy could be broadly applicable to selectively modulate transient PMIs.

A T50I substitution in the K-Ras interswitch domain causes Noonan syndrome and emerged as a third-site mutation that restored the in vivo transforming activity and constitutive MAPK pathway activation by an attenuated KrasG12D,E37G oncogene in a mouse leukemia model. Biochemical and crystallographic data suggested that K-RasT50I increases MAPK signal output through a non-GTPase mechanism, potentially by promoting asymmetric Ras:Ras interactions between T50 and E162. We generated a switchable system in which K-Ras mutant proteins expressed at physiologic levels supplant the fms like tyrosine kinase 3 (FLT3) dependency of MOLM-13 leukemia cells lacking endogenous KRAS and used this system to interrogate single or compound G12D, T50I, D154Q, and E162L mutations. These studies support a key role for the asymmetric lateral assembly of K-Ras in a plasma membrane-distal orientation that promotes the formation of active Ras:Raf complexes in a membrane-proximal conformation. Disease-causing mutations such as T50I are a valuable starting point for illuminating normal Ras function, elucidating mechanisms of disease, and identifying potential therapeutic opportunities for Rasopathy disorders and cancer.

Abstract Background Breast cancer (BC) is the most common cancer in women, with triple negative BC (TNBC) accounting for 20% of cases. While early detection and targeted therapies have improved overall life expectancy, TNBC remains resistant to current treatments. Although parity reduces the lifetime risk of developing BC, pregnancy increases the risk of developing TNBC for years after childbirth. Although numerous gene mutations have been associated with BC, no single gene alteration has been identified as a universal driver. RRAS2 is a RAS-related GTPase rarely found mutated in cancer. Methods Conditional knock-in mice were generated to overexpress wild type human RRAS2 in mammary epithelial cells. A human sample cohort was analyzed by RT-qPCR to measure RRAS2 transcriptional expression and to determine the frequency of both a single-nucleotide polymorphism (SNP rs8570) in the 3’UTR region of RRAS2 and of genomic DNA amplification in tumoral and non-tumoral human BC samples. Results Here we show that overexpression of wild-type RRAS2 in mice is sufficient to develop TNBC in 100% of females in a pregnancy-dependent manner. In human BC, wild-type RRAS2 is overexpressed in 68% of tumors across grade, location, and molecular type, surpassing the prevalence of any previously implicated alteration. Still, RRAS2 overexpression is notably higher and more frequent in TNBC and young parous patients. The increased prevalence of the alternate C allele at the SNP position in tumor samples, along with frequent RRAS2 gene amplification in both tumors and blood of BC patients, suggests a cause-and-effect relationship between RRAS2 overexpression and breast cancer. Conclusions Higher than normal expression of RRAS2 not bearing activating mutations is a key driver in the majority of breast cancers, especially those of the triple-negative type and those linked to pregnancy.

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis. Synopsis: RAS family protein ubiquitination at several residues regulates RAS signaling activity and stability. This study shows that monoubiquitination of KRAS and NRAS at lysine 128 (K128) enhances RAS binding to GTPase-activating proteins to restrict RAS activity, a mechanism that is dysregulated in cancer tissues. K128 monoubiquitination of KRAS or NRAS creates an additional interface that facilitates binding to the GTPase activating proteins RASA1 and NF1. The level of KRAS and NRAS ubiquitination at K128 is decreased in cancer samples compared to normal tissue. K128 ubiquitination of wild-type RAS proteins constrains downstream MAPK signalling in a GAP-dependent manner. K128 ubiquitination of the oncogenic KRAS-G12D mutant suppresses the RAL-TBK1 axis and inhibits the autocrine circuit induced by mutant KRAS. Reduction of K128 monoubiquitination activates wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven tumorigenesis. [ABSTRACT FROM AUTHOR]

Background: Breast cancer (BC) is the most common cancer in women, with triple negative BC (TNBC) accounting for 20% of cases. While early detection and targeted therapies have improved overall life expectancy, TNBC remains resistant to current treatments. Although parity reduces the lifetime risk of developing BC, pregnancy increases the risk of developing TNBC for years after childbirth. Although numerous gene mutations have been associated with BC, no single gene alteration has been identified as a universal driver. RRAS2 is a RAS-related GTPase rarely found mutated in cancer. Methods: Conditional knock-in mice were generated to overexpress wild type human RRAS2 in mammary epithelial cells. A human sample cohort was analyzed by RT-qPCR to measure RRAS2 transcriptional expression and to determine the frequency of both a single-nucleotide polymorphism (SNP rs8570) in the 3'UTR region of RRAS2 and of genomic DNA amplification in tumoral and non-tumoral human BC samples. Results: Here we show that overexpression of wild-type RRAS2 in mice is sufficient to develop TNBC in 100% of females in a pregnancy-dependent manner. In human BC, wild-type RRAS2 is overexpressed in 68% of tumors across grade, location, and molecular type, surpassing the prevalence of any previously implicated alteration. Still, RRAS2 overexpression is notably higher and more frequent in TNBC and young parous patients. The increased prevalence of the alternate C allele at the SNP position in tumor samples, along with frequent RRAS2 gene amplification in both tumors and blood of BC patients, suggests a cause-and-effect relationship between RRAS2 overexpression and breast cancer. Conclusions: Higher than normal expression of RRAS2 not bearing activating mutations is a key driver in the majority of breast cancers, especially those of the triple-negative type and those linked to pregnancy. [ABSTRACT FROM AUTHOR]

Hyperactive Ras signalling is found in most cancers. Ras proteins are only active in membrane nanoclusters, which are therefore potential drug targets. We previously showed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering via direct interaction with the Ras binding domain (RBD) of Raf. Here, we establish that the B-Raf preference of Gal1 emerges from the divergence of the Raf RBDs at their proposed Gal1-binding interface. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B- and C-Raf-RBDs. Its 23-mer core fragment is sufficient to interfere with H-Ras nanoclustering, modulate Ras-signalling and moderately reduce cell viability. These latter two phenotypic effects may also emerge from the ability of L5UR to broadly engage with several RBD- and RA-domain containing Ras interactors. The L5UR-peptide core fragment is a starting point for the development of more specific reagents against Ras-nanoclustering and -interactors. The 23mer L5URcore peptide is identified, which binds with low micromolar affinity to Ras-binding domains, notably of Raf proteins, thus disrupting their galectin1-interaction, H-Ras-nanoclustering, -signalling and more broadly cell proliferation. [ABSTRACT FROM AUTHOR]

Background: Oncogenic mutations in the RAS gene are associated with uncontrolled cell growth, a hallmark feature contributing to tumorigenesis. While diverse therapeutic strategies have been diligently applied to treat RAS-mutant cancers, successful targeting of the RAS gene remains a persistent challenge in the field of cancer therapy. In our study, we discover a promising avenue for addressing this challenge. Methods: In this study, we tested the viability of several cell lines carrying oncogenic NRAS, KRAS, and HRAS mutations upon treatment with IkappaBalpha (IκBα) inhibitor BAY 11-7082. We performed both cell culture-based viability assay and in vivo subcutaneous xenograft-based assay to confirm the growth inhibitory effect of BAY 11-7082. We also performed large RNA sequencing analysis to identify differentially regulated genes and pathways in the context of oncogenic NRAS, KRAS, and HRAS mutations upon treatment with BAY 11-7082. Results: We demonstrate that oncogenic NRAS, KRAS, and HRAS activate the expression of IκBα kinase. BAY 11-7082, an inhibitor of IκBα kinase, attenuates the growth of NRAS, KRAS, and HRAS mutant cancer cells in cell culture and in mouse model. Mechanistically, BAY 11-7082 inhibitor treatment leads to suppression of the PI3K-AKT signaling pathway and activation of apoptosis in all RAS mutant cell lines. Additionally, we find that BAY 11-7082 treatment results in the downregulation of different biological pathways depending upon the type of RAS protein that may also contribute to tumor growth inhibition. Conclusion: Our study identifies BAY 11-7082 to be an efficacious inhibitor for treating RAS oncogene (HRAS, KRAS, and NRAS) mutant cancer cells. This finding provides new therapeutic opportunity for effective treatment of RAS-mutant cancers. [ABSTRACT FROM AUTHOR]

Tumor cells reprogram their metabolism to produce specialized metabolites that both fuel their own growth and license tumor immune evasion. However, the relationships between these functions remain poorly understood. Here, we report CRISPR screens in a mouse model of colo-rectal cancer (CRC) that implicates the dual specificity phosphatase 18 (DUSP18) in the establishment of tumor-directed immune evasion. Dusp18 inhibition reduces CRC growth rates, which correlate with high levels of CD8+ T cell activation. Mechanistically, DUSP18 dephosphorylates and stabilizes the USF1 bHLH-ZIP transcription factor. In turn, USF1 induces the SREBF2 gene, which allows cells to accumulate the cholesterol biosynthesis intermediate lanosterol and release it into the tumor microenvironment (TME). There, lanosterol uptake by CD8+ T cells suppresses the mevalonate pathway and reduces KRAS protein prenylation and function, which in turn inhibits their activation and establishes a molecular basis for tumor cell immune escape. Finally, the combination of an anti-PD-1 antibody and Lumacaftor, an FDA-approved small molecule inhibitor of DUSP18, inhibits CRC growth in mice and synergistically enhances anti-tumor immunity. Collectively, our findings support the idea that a combination of immune checkpoint and metabolic blockade represents a rationally-designed, mechanistically-based and potential therapy for CRC. Dual-specificity phosphatases regulate several processes associated with carcinogenesis. Here the authors show that inhibition of the dual-specificity phosphatase DUSP18 improves the activity of tumor-infiltrating CD8 T cells, enhancing response to immune checkpoint blockade in preclinical models of colorectal cancer. [ABSTRACT FROM AUTHOR]

Recently, targeted degradation has emerged as a powerful therapeutic modality. Relying on "event-driven" pharmacology, proteolysis targeting chimeras (PROTACs) can degrade targets and are superior to conventional inhibitors against undruggable proteins. Unfortunately, PROTAC discovery is limited by warhead scarcity and laborious optimization campaigns. To address these shortcomings, analogous protein-based heterobifunctional degraders, known as bioPROTACs, have been developed. Compared to small-molecule PROTACs, bioPROTACs have higher success rates and are subject to fewer design constraints. However, the membrane impermeability of proteins severely restricts bioPROTAC deployment as a generalized therapeutic modality. Here, we present an engineered bioPROTAC template able to complex with cationic and ionizable lipids via electrostatic interactions for cytosolic delivery. When delivered by biocompatible lipid nanoparticles, these modified bioPROTACs can rapidly degrade intracellular proteins, exhibiting near-complete elimination (up to 95% clearance) of targets within hours of treatment. Our bioPROTAC format can degrade proteins localized to various subcellular compartments including the mitochondria, nucleus, cytosol, and membrane. Moreover, substrate specificity can be easily reprogrammed, allowing modular design and targeting of clinically-relevant proteins such as Ras, Jnk, and Erk. In summary, this work introduces an inexpensive, flexible, and scalable platform for efficient intracellular degradation of proteins that may elude chemical inhibition. Targeted degradation has emerged as a powerful therapeutic modality. In this study, the authors develop a lipid-based platform to deliver recombinant bioPROTACs into cells for targeted protein degradation, providing a platform for efficient intracellular degradation of proteins that may elude chemical inhibition. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a highly prevalent and lethal cancer worldwide. Approximately 45% of CRC patients harbor a gain-in-function mutation in KRAS. KRAS is the most frequently mutated oncogene accounting for approximately 25% of all human cancers. Gene mutations in KRAS cause constitutive activation of the KRAS protein and MAPK/AKT signaling, resulting in unregulated proliferation and survival of cancer cells and other aspects of malignant transformation, progression, and metastasis. While KRAS has long been considered undruggable, the FDA recently approved two direct acting KRAS inhibitors, Sotorasib and Adagrasib, that covalently bind and inactivate KRASG12C. Both drugs showed efficacy for patients with non-small cell lung cancer (NSCLC) diagnosed with a KRASG12C mutation, but for reasons not well understood, were considerably less efficacious for CRC patients diagnosed with the same mutation. Thus, it is imperative to understand the basis for resistance to KRASG12C inhibitors, which will likely be the same limitations for other mutant specific KRAS inhibitors in development. This review provides an update on clinical trials involving CRC patients treated with KRASG12C inhibitors as a monotherapy or combined with other drugs. Mechanisms that contribute to resistance to KRASG12C inhibitors and the development of novel RAS inhibitors with potential to escape such mechanisms of resistance are also discussed. [ABSTRACT FROM AUTHOR]

This document is a comprehensive review of the FDA-approved drug selumetinib for the treatment of neurofibromatosis type 1 (NF1) and inoperable plexiform neurofibromas (PN). It discusses the drug's mechanism of action in inhibiting the MAPK/ERK signaling pathway, which is involved in cell growth and differentiation. The review also provides information on the drug's pharmacokinetics and pharmacodynamics, as well as its efficacy and safety in various types of cancer. It highlights the results of clinical trials and studies conducted on selumetinib in pediatric patients with NF1 and plexiform neurofibromas. [Extracted from the article]

The small GTPase Ras plays an important role in intracellular signal transduction and functions as a molecular switch. In this study, we used a photoresponsive protein as the molecular regulatory device to photoregulate Ras GTPase activity. Photo zipper (PZ), a variant of the photoresponsive protein Aureochrome1 developed by Hisatomi et al. was incorporated into the C-terminus of Ras as a fusion protein. The three constructs of the Ras–PZ fusion protein had spacers of different lengths between Ras and PZ. They were designed using an Escherichia coli expression system. The Ras–PZ fusion proteins exhibited photoisomerization upon blue light irradiation and in the dark. Ras–PZ dimerized upon light irradiation. Moreover, Ras GTPase activity, which is accelerated by the Ras regulators guanine nucleotide exchange factors and GTPase-activating proteins, is controlled by photoisomerization. It has been suggested that light-responsive proteins are applicable to the photoswitching of the enzymatic activity of small GTPases as photoregulatory molecular devices. [ABSTRACT FROM AUTHOR]

Leucine zipper-like transcription regulator 1 (LZTR1) acts as a negative factor that suppresses RAS function and MAPK signaling; mutations in this protein may dysregulate RAS ubiquitination and lead to impaired degradation of RAS superfamily proteins. Germline LZTR1 variants are reported in Noonan syndrome, either autosomal dominant or autosomal recessive, and in susceptibility to schwannomatosis. This article explores the genetic and phenotypic diversity of the autosomal dominant LZTR1-related disorders, compiling a cohort of previously published patients (51 with the Noonan phenotype and 123 with schwannomatosis) and presenting two additional adult-onset cases: a male with schwannomatosis and Parkinson's disease and a female with Noonan syndrome, generalized joint hypermobility, and breast cancer. This review confirms that autosomal dominant LZTR1-related disorders exhibit an extreme phenotypic variability, ranging from relatively mild manifestations to severe and multi-systemic involvement, and offers updated frequences of each clinical feature. The aim is to precisely define the clinical spectrum of LZTR1-related diseases, using also two new emblematic clinical cases. Gaining insight into the mechanisms underneath this variability is crucial to achieve precision diagnostics and the development of therapeutic interventions. [ABSTRACT FROM AUTHOR]

The Kirsten rat sarcoma viral oncogene homolog, or KRAS gene is an oncogene that produces the small GTPase transductor protein known as KRAS. Because it may transmit outside signals to the cell nucleus, KRAS plays a role in controlling cell division. Mutations that activate the KRAS gene hinder the KRAS protein's capacity to transition between active and inactive states, which can cause cell transformation and heightened resistance to treatments that target epidermal growth factor receptors, such as chemotherapy and biological therapies. One of the leading causes of cancer-related death worldwide is colon adenocarcinoma. A mutation in KRAS causes your cells to proliferate uncontrollably, leading to the development of cancer. A wild-type gene is an ordinary (unmutated) gene. KRAS, an acronym for Kirsten rat sarcoma, is a gene that is altered in colorectal cancer quite commonly. Additionally connected to pancreatic and lung cancers are mutated forms of this gene. In this research work, we investigated the effect of Lactobacillus plantarum generated SCFA on the expression level of the KRAS gene. The level of KRAS gene expression was significantly lower, according to the data. With a rise in SCFA concentration, the fold change was lessened; at 100 µg/ml, the fold change value was less. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer (GC) is a highly phenotypically heterogeneous disease and is caused by a combination of factors. Retinol binding protein 4 (RBP4) is a member of a family of lipid transport proteins that are involved in the transport of substances between cells and play a crucial role in a variety of cancers. However, the expression and role of RBP4 in GC remain unknown. Methods: In this study, we explored the expression, prognostic significance, immune microenvironment, drug responsiveness and function of associated signaling pathways of RBP4 in GC using web-based bioinformatics tools. Immunohistochemistry and real-time quantitative PCR were utilized to analyze the tissue and cell expression levels of RBP4. CCK-8, colony formation, EDU incorporation, wound healing and transwell assays were applied to demonstrate the effect of RBP4 on GC cell function. Flow cytometric detection of apoptosis after RBP4 knockdown. Nude mice xenograft model elucidates the role of RBP4 for GC in vivo. Related proteins of the RAS signaling pathway were analyzed by employing Western blot assays. Results: RBP4 is highly expressed in GC. RBP4 is closely associated with patient survival and sensitivity to a wide range of antitumor agents. Knockdown of RBP4 promoted apoptosis and inhibited cell proliferation, invasion and migration. RBP4 promotes GC tumorigenesis in vivo. Finally, RBP4 modulates the RAS/RAF/ERK axis. Conclusion: RBP4 may promote gastric carcinogenesis and development through the RAS/RAF/ERK axis and is expected to be a novel target for GC treatment. [ABSTRACT FROM AUTHOR]

In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP‐bound and inactive GDP‐bound states. Various states of GTP‐bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors. Zn2+‐cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H‐Ras(wt).Mg2+.GppNHp crystals soaked with Zn2+‐cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn2+‐cyclen binding is observed at 500 MPa pressure, close to the nucleotide, in Ras protein that is driven by pressure to a state 1 conformer. The unknown binding mode of Zn2+‐cyclen to H‐Ras can thus be fully characterized in atomic details. As a more general conjunction from our study, high pressure x‐ray crystallography turns out to be a powerful method to induce transitions allowing drug binding in proteins that are in low‐populated conformations at ambient conditions, enabling the design of specific inhibitors. [ABSTRACT FROM AUTHOR]

This document explores the concept of self-organizing and excitable signaling networks in cell biology. It discusses how certain signaling networks exhibit spontaneous organization into dynamic spatial patterns and the role of excitability in cellular processes such as cell migration, mitosis, embryonic development, and immune activation. The authors emphasize the importance of understanding these networks in both individual cells and cell populations. The document also references related research papers on topics such as ERK activation, collective cell migration, and actin dynamics. [Extracted from the article]

Rearranged during transfection (RET) rearrangement oncoprotein-mediated Ras/MAPK signaling cascade is constitutively activated in cancers. Here, we demonstrate a unique signal niche. The niche is a ternary complex based on the chimeric RET liquid-liquid phase separation. The complex comprises the rearranged kinase (RET fusion); the adaptor (GRB2), and the effector (SHC1). Together, they orchestrate the Ras/MAPK signal cascade, which is dependent on tyrosine kinase. CCDC6-RET fusion undergoes LLPS requiring its kinase domain and its fusion partner. The CCDC6-RET fusion LLPS promotes the autophosphorylation of RET fusion, with enhanced kinase activity, which is necessary for the formation of the signaling niche. Within the signal niche, the interactions among the constituent components are reinforced, and the signal transduction efficiency is amplified. The specific RET fusion-related signal niche elucidates the mechanism of the constitutive activation of the Ras/MAPK signaling pathway. Beyond just focusing on RET fusion itself, exploration of the ternary complex potentially unveils a promising avenue for devising therapeutic strategies aimed at treating RET fusion-driven diseases. [ABSTRACT FROM AUTHOR]

Background: Carcinomas of the seminal vesicle are exceedingly rare, with a limited number of cases described in the literature. Reported cases span a relatively wide morphological spectrum, and their genomic features remain unexplored. Design: In this study, we interrogated five primary epithelial neoplasms of the seminal vesicle using a targeted DNA sequencing platform (OncoPanel, 447 genes). Results: The tumours included one adenocarcinoma with intestinal phenotype presenting after external beam radiation (for prostatic adenocarcinoma), one carcinoma with Müllerian‐type clear cell phenotype, two mucinous tumours resembling low‐grade mucinous neoplasms of the appendix (LAMN) and one mucinous cystadenoma. The post‐radiation mucinous adenocarcinoma had genomic findings consistent with bi‐allelic inactivation of TP53, as well as multiple copy‐number changes with regional and chromosomal arm‐level copy‐number losses. The Müllerian‐type clear cell carcinoma exhibited a complex copy‐number profile with numerous regional and arm‐level copy‐number changes, as well as focal amplification events, including copy‐number gain of 8q and amplification of a region within 20q13. Both low‐grade mucinous tumours resembling LAMN harboured hot‐spot gain‐of‐function KRAS variants (p.G12V and p.G13D) as the only genomic alteration. No genomic alterations were detected inthe lesion diagnosed as mucinous cystadenoma. Conclusion: Our results suggest that primary low‐grade mucinous neoplasms of the seminal vesicle may represent a distinct entity equivalent to appendiceal counterparts, driven by gain‐of‐function variants of RAS GTPases. The remaining tumours showed genomic features that closely resembled those of neoplasms with comparable phenotypes and/or biological characteristics arising in other sites, suggesting that they could be managed similarly, with special considerations related to their anatomical location. [ABSTRACT FROM AUTHOR]

About 18% of all human cancers carry a mutation in the KRAS gene making it among the most sought-after anticancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. It also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have used the near-diploid hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenous KRAS alleles carries an oncogenic KRAS mutation at glycine 12. Cells with a KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotype, together with WT KRASG12G(WT)/+ cells, reveal that oncogenic KRAS.G12X mutations increase cell proliferation rate and cell motility and reduced focal adhesions in KRASG12V/+ cells. Epidermal growth factor -induced phosphorylation of ERK and AKT was comparable between KRASG12V/+, KRASG12C/+, KRASG12D/+, and KRASG12G(WT)/+ cells. Interestingly, KRASG12X/+ cells showed varying responses to distinct inhibitors with the KRASG12V/+ and KRASG12D/+ cells more sensitive to hydroxyurea and MEK inhibitors, U0126 and trametinib, but more resistant to PI3K inhibitor, PIK-90, than the KRASG12G(WT)/+ cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater in KRASG12V/+ than WT cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signaling and developing effective anti-KRAS reagents with minimum cytotoxicity on WT cells. [ABSTRACT FROM AUTHOR]

Background: Sepsis-associated encephalopathy (SAE) causes acute and long-term cognitive deficits. However, information on the prevention and treatment of cognitive dysfunction after sepsis is limited. The neuropeptide orexin-A (OXA) has been shown to play a protective role against neurological diseases by modulating the inflammatory response through the activation of OXR1 and OXR2 receptors. However, the role of OXA in mediating the neuroprotective effects of SAE has not yet been reported. Methods: A mouse model of SAE was induced using cecal ligation perforation (CLP) and treated via intranasal administration of exogenous OXA after surgery. Mouse survival, in addition to cognitive and anxiety behaviors, were assessed. Changes in neurons, cerebral edema, blood-brain barrier (BBB) permeability, and brain ultrastructure were monitored. Levels of pro-inflammatory factors (IL-1β, TNF-α) and microglial activation were also measured. The underlying molecular mechanisms were investigated by proteomics analysis and western blotting. Results: Intranasal OXA treatment reduced mortality, ameliorated cognitive and emotional deficits, and attenuated cerebral edema, BBB disruption, and ultrastructural brain damage in mice. In addition, OXA significantly reduced the expression of the pro-inflammatory factors IL-1β and TNF-α, and inhibited microglial activation. In addition, OXA downregulated the expression of the Rras and RAS proteins, and reduced the phosphorylation of P-38 and JNK, thus inhibiting activation of the MAPK pathway. JNJ-10,397,049 (an OXR2 blocker) reversed the effect of OXA, whereas SB-334,867 (an OXR1 blocker) did not. Conclusion: This study demonstrated that the intranasal administration of moderate amounts of OXA protects the BBB and inhibits the activation of the OXR2/RAS/MAPK pathway to attenuate the outcome of SAE, suggesting that OXA may be a promising therapeutic approach for the management of SAE. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-β (PDGFR-β)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-β. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-β. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p. [ABSTRACT FROM AUTHOR]

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene that encodes neurofibromin, a RAS GTPase-activating protein. Inactivating NF1 mutations cause hyperactivation of RAS-mediated signaling, resulting in the development of multiple neoplasms, including malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are an aggressive tumor and the main cause of mortality in patients with NF1. MPNSTs are difficult to resect and refractory to chemo- and radiotherapy, and no molecular therapies currently exist. Immune checkpoint blockade (ICB) is an approach to treat inoperable, undruggable cancers like MPNST, but successful outcomes require an immune cell-rich tumor microenvironment. While MPNSTs are noninflamed "cold" tumors, here, we converted MPNSTs into T cell-inflamed "hot" tumors by activating stimulator of IFN genes (STING) signaling. Mouse genetic and human xenograft MPNST models treated with a STING agonist plus ICB exhibited growth delay via increased apoptotic cell death. This strategy offers a potential treatment regimen for MPNSTs. [ABSTRACT FROM AUTHOR]

Background/aim: Alzheimer's disease (AD), one of the most common health issues, is characterized by memory loss, severe behavioral disorders, and eventually death. Despite many studies, there are still no drugs that can treat AD or stop it from progressing. Previous in vitro tests showed that O-demethyl galantamine (ODG) might have therapeutic potential owing to its 10 times higher acetylcholinesterase inhibitory activity than galantamine (GAL). Materials and methods: We aimed to assess the effect of ODG at the molecular level in a 12-month-old 5xFAD Alzheimer's mouse model. To this end, following the administrations of ODG and GAL (used as a positive control), protein alterations were investigated in the cortex, hippocampus, and cerebellum regions of the brain. Surprisingly, GAL altered proteins prominently in the cortex, while ODG exclusively exerted its effect on the cerebellum. Results: GNB1, GNB2, NDUFS6, PAK2, and RhoA proteins were identified as the top 5 hub proteins in the cerebellum of ODG-treated mice. Reregulation of these proteins through Ras signaling and retrograde endocannabinoid signaling pathways, which were found to be enriched, might contribute to reversing AD-induced molecular changes. Conclusion: We suggest that, since it targets specifically the cerebellum, ODG may be further evaluated for combination therapies for AD. [ABSTRACT FROM AUTHOR]

RAS mutations occur in approximately 30% of tumors worldwide and have a poor prognosis due to limited therapies. Covalent targeting of KRAS G12C has achieved significant success in recent years, but there is still a lack of efficient therapeutic approaches for tumors with non-G12C KRAS mutations. A highly promising approach is to target the MAPK pathway downstream of RAS, with a particular focus on RAF kinases. First-generation RAF inhibitors have been authorized to treat BRAF mutant tumors for over a decade. However, their use in RAS-mutated tumors is not recommended due to the paradoxical ERK activation mainly caused by RAF dimerization. To address the issue of RAF dimerization, type II RAF inhibitors have emerged as leading candidates. Recent clinical studies have shown the initial effectiveness of these agents against RAS mutant tumors. Promisingly, type II RAF inhibitors in combination with MEK or ERK inhibitors have demonstrated impressive efficacy in RAS mutant tumors. This review aims to clarify the importance of RAF dimerization in cellular signaling and resistance to treatment in tumors with RAS mutations, as well as recent progress in therapeutic approaches to address the problem of RAF dimerization in RAS mutant tumors. Homo- and heterodimers of RAF kinases play a pivotal role in the signaling transduction and drug resistance in RAS mutant tumors. The red pentagrams represent mutated RAS proteins. [Display omitted] [ABSTRACT FROM AUTHOR]

KRAS regulates several cellular processes, such as cell proliferation, cell cycle regulation, metabolic changes, cell survival, and cell differentiation. Abnormalities in the KRAS gene are found in approximately 30% of patients with non-small cell lung cancer, usually in patients diagnosed with nonsquamous cancer and more often in Caucasian patients, women, and smokers. The p.G12C variant is most frequently found in KRAS-positive patients. Sotorasib is the first drug approved for this population. The superiority of sotorasib over docetaxel after failure of immunochemotherapy was demonstrated in the CodeBreak 200 phase III study for the primary endpoint -- median progression-free survival was 5.6 months [95% confidence interval (CI) 4.3-7.8] vs. 4.5 months (3.0-5.7); hazard ratio = 0.66 (95% CI 0.51-0.86; p = 0.0017), while the 12-month progression-free survival rate was 24.8% for sotorasib and 10.1% for docetaxel. Currently, sotorasib monotherapy, at an initial dose of 960 mg/day, is indicated for use in adults with advanced non-small cell lung cancer with the KRAS p.G12C mutation who have experienced disease progression after at least one previous line of systemic treatment. More randomized trials are needed to determine the optimal place of sotorasib in the systemic treatment sequence in this patient population. [ABSTRACT FROM AUTHOR]

Localized dynamics of RAS, including regions distal to the nucleotide-binding site, is of high interest for elucidating the mechanisms by which RAS proteins interact with effectors and regulators and for designing inhibitors. Among several oncogenic mutants, methyl relaxation dispersion experiments reveal highly synchronized conformational dynamics in the active (GMPPNP-bound) KRASG13D, which suggests an exchange between two conformational states in solution. Methyl and 31P NMR spectra of active KRASG13D in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major Pγ atom peak corresponding to the dominant State 1 conformation and a secondary peak indicating an intermediate state different from the known State 2 conformation recognized by RAS effectors. High-resolution crystal structures of active KRASG13D and KRASG13D-RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively. We use residual dipolar couplings to solve and cross-validate the structure of the intermediate state of active KRASG13D, showing a conformation distinct from those of States 1 and 2 outside the known flexible switch regions. The dynamic coupling between the conformational exchange in the effector lobe and the breathing motion in the allosteric lobe is further validated by a secondary mutation in the allosteric lobe, which affects the conformational population equilibrium.

The article in the Journal of Cell Biology discusses the role of lipid synthesis in driving invasive migration, focusing on the Caenorhabditis elegans anchor cell. The study highlights the importance of fatty acid, phospholipid, and isoprenoid biosynthesis in breaching extracellular matrix barriers. The research sheds light on how lipid biosynthesis aids in the formation of invasive protrusions, providing insights into both developmental processes and potentially cancer invasion. The findings suggest that cells can organize their metabolic machinery to enhance invasive migration efficiency, offering new perspectives on combating metastatic spread. [Extracted from the article]