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Previous studies have demonstrated that combination therapy involving radiotherapy and aspirin decreases the survival rate of cancer cells. However, the mechanism by which aspirin exerts its radiation sensitization effect at the in vivo level remains largely unclear. In this study, we employed Caenorhabditis elegans (C. elegans) as a model organism to investigate the effect of aspirin combined with radio/chemo-therapy on tumors at the individual level. Here, we illustrate that high-dose aspirin increases the expression of genes involved in core apoptosis pathways (egl-1, ced-9, ced-4 and ced-3) and induces germ cell apoptosis in C. elegans through mitochondrial outer membrane permeabilization (MOMP) and elevation of reactive oxygen species (ROS) levels. Crucially, aspirin-induces ROS upregulates the expression of genes critical for DNA damage response (hus-1, clk-2 and cep-1) and genes involved in MAPK pathways (lin-45, mek-2, mpk-1, sek-1 and pmk-1), thereby mediating the enhanced sensitivity of radio/chemo-therapy by aspirin. Notably, aspirin fails to induce germ cell apoptosis and enhance radio/chemo-therapy in C. elegans lacking the expression of each of those genes. Furthermore, in a C. elegans tumor-like symptom model, aspirin enhances radio/chemo-therapy sensitivity through ROS induction. However, low-dose aspirin can diminish the apoptotic signal of reproductive cells in C. elegans and exert anti-inflammatory effects. Our research results suggest that the tumor-suppressive and radio/chemo-therapy sensitizing effects of aspirin provide robust experimental evidence for improving the clinical efficacy of tumor radio/chemo-therapy and deepening our understanding of aspirin's mechanism of action in cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fish cell lines differ from most mammalian diploid cell lines by the fact that cellular senescence is not readily induced. Previously, we demonstrated that the absence of the p16 gene in the fish genome prevents cells from reaching full senescence even when Ras is activated. Drosophila also lacks p16; however, early senescence triggered by Ras activation progresses to full senescence and is accompanied by a proinflammatory senescence-associated secretory phenotype (SASP), due to mitochondrial deficiency. It is unclear whether mitochondrial deficiency can also induce the maturation of Ras-induced early senescence (RIS) to full senescence along with a proinflammatory SASP in fish cell lines. Here, we investigated whether mitochondrial dysfunction induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in concert with activated Ras results in full senescence and whether this is accompanied by a proinflammatory SASPs in the EPC fish cell line. We found that although EPC cells with mitochondrial dysfunction exhibited a proinflammatory SASP, this did not result in permanent cell proliferation arrest or the upregulation of endogenous Ras expression. These findings suggest that other factors must act in concert with mitochondrial dysfunction to induce full senescence. The proliferation of EPC cells overexpressing a constitutively active mutant of H-Ras (H-Ras V12 ) was markedly reduced, irrespective of CCCP treatment. These findings suggest that there are similarities between the cellular senescence observed in fish and Drosophila cells lacking the p16 gene. However, it should be noted that fish cells differ from Drosophila cells in that mitochondrial dysfunction alone can induce proinflammatory SASP factors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Variants in the RAS family (HRAS, NRAS and KRAS) are among the most common mutations found in cancer. About 19% patients with cancer harbor RAS mutations, which are typically associated with poor clinical outcomes. Over the past four decades, KRAS has long been considered an undruggable target due to the absence of suitable small-molecule binding sites within its mutant isoforms. However, recent advancements in drug design have made RAS-targeting therapies viable, particularly with the approval of direct KRAS G12C inhibitors, such as sotorasib and adagrasib, for treating non-small cell lung cancer (NSCLC) with KRAS G12C mutations. Other KRAS-mutant inhibitors targeting KRAS G12D are currently being developed for use in the clinic, particularly for treating highly refractory malignancies like pancreatic cancer. Herein, we provide an overview of RAS signaling, further detailing the roles of the RAS signaling pathway in carcinogenesis. This includes a summary of RAS mutations in human cancers and an emphasis on therapeutic approaches, as well as de novo, acquired, and adaptive resistance in various malignancies., (© 2024. The Author(s).)

In this issue, four articles highlight the critical role of nongenetic mechanisms and cell plasticity in mediating resistance to different classes of RAS inhibitors in pancreatic ductal adenocarcinoma and non-small cell lung cancer. See related article by Benitz et al., p. 2162 See related article by Dilly et al., p. 2135 See related article by Araujo et al., p. 2183 See related article by Singhal et al., p. 2122., (©2024 American Association for Cancer Research.)

The purpose of this study was to investigate the mechanism of EMP1 action in high glucose (HG)-induced H9c2 cardiac cell pyroptosis and oxidative injury. Rat cardiomyocytes H9c2 were exposed to 33 mM glucose for 24, 48, or 72 h to induce cytotoxicity. EMP1-siRNA, NLRP3 agonist Nigericin, and pcNDA-RAS were used to treat H9c2 cells under HG conditions. Cell Counting Kit (CCK)-8 assay showed that cell proliferation was decreased following HG induction, which was rescued by EMP1 knockdown. Our results also suggested that EMP1 siRNA transfection significantly decreased the apoptosis and pyroptosis of HG-induced cells, as indicated by the reduction of NLRP3 IL-1β, ASC, GSDMD, cleaved-caspase1 and cleaved-caspase3 levels in HG-induced H9c2 cells. In addition, EMP1 knockdown alleviated HG-induced mitochondrial damage and oxidative stress in H9c2 cells. NLRP3 activation reversed the inhibitory effects of EMP1 knockdown on pyroptosis and oxidative stress in HG-induced H9c2 cells. Mechanistically, we found that EMP1 knockdown suppressed the RAS/RAF/MAPK signaling pathway in HG-induced H9c2 cells. RAS overexpression blocked the protective effect of EMP1 knockdown on HG-induced H9c2 cell apoptosis, pyroptosis, and oxidative injury. Our findings suggest that EMP1 knockdown treatment might provide a novel therapy for diabetic cardiomyopathy., (© 2024 Wiley Periodicals LLC.)

A recent report by Yun et al. describes the detection of RAS dimers using intact mass spectrometry and investigates the role that membrane lipids, nucleotide state, and binding partners have in their formation., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 The Author. Published by Elsevier Ltd.. All rights reserved.)

Arteriovenous malformations (AVM) are benign vascular anomalies prone to pain, bleeding, and progressive growth. AVM are mainly caused by mosaic pathogenic variants of the RAS-MAPK pathway. However, a causative variant is not identified in all patients. Using ultra-deep sequencing, we identified novel somatic RIT1 delins variants in lesional tissue of three AVM patients. RIT1 encodes a RAS-like protein that can modulate RAS-MAPK signaling. We expressed RIT1 variants in HEK293T cells, which led to a strong increase in ERK1/2 phosphorylation. Endothelial-specific mosaic overexpression of RIT1 delins in zebrafish embryos induced AVM formation, highlighting their functional importance in vascular development. Both ERK1/2 hyperactivation in vitro and AVM formation in vivo could be suppressed by pharmacological MEK inhibition. Treatment with the MEK inhibitor trametinib led to a significant decrease in bleeding episodes and AVM size in one patient. Our findings implicate RIT1 in AVM formation and provide a rationale for clinical trials with targeted treatments., Competing Interests: Declarations Competing interests Friedrich G. Kapp has received consulting fees from Novartis. Jochen Rössler is currently an employee of Novartis Pharma. All other authors declare no conflicts of interest., (© 2024. The Author(s).)

Deoxynivalenol (DON) is a kind of widespread traditional Fusarium mycotoxins in the environment, and its intestinal toxicity has received considerable attention. Recently, the emerging Fusarium mycotoxin enniatins (ENNs) have also been shown to frequently coexist with DON in animal feed and food with large consumption. However, the mechanism of intestinal damage caused by the two mycotoxins co-exposure remains unclear. In this study, Caco-2 cell line was used to investigate the combined toxicity and potential mechanisms of four representative ENNs (ENA, ENA 1 , ENB, and ENB 1 ) and DON. The results showed that almost all mixed groups showed antagonistic effects, particularly ENB at 1/4 IC 50 (CI = 6.488). Co-incubation of ENNs mitigated the levels of signaling molecule levels disrupted by DON, including reactive oxygen species (ROS), calcium mobilization (Ca 2+ ), adenosine triphosphate (ATP). The differentially expressed genes (DEGs) between the mixed and ENB groups were significantly enriched in the Ras/PI3K/Akt signaling pathway, including 28 up-regulated genes and 40 down-regulated genes. Quantitative real-time PCR further confirmed the lower expression of apoptotic gene in the mixed group, thereby reducing the cytotoxic effects caused by DON exposure. This study emphasizes that co-exposure of ENNs and DON reduces cytotoxicity by regulating the Ras/PI3K/Akt signaling pathway. Our results provide the first comprehensive evidence about the antagonistic toxicity of ENNs and DON on Caco-2 cells, and new insights into mechanisms investigated by transcriptomics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The current treatment for osteosarcoma (OS) is based on surgery combined with systemic chemotherapy, however, gene therapy has been hypothesized to improve patient survival rates. The density-enhanced protein domain 1 protein (DEPDC1) functions as a crucial determinant in the advancement of OS, which is highly expressed in OS cells. The current study was designed to delve into the effect and mechanism of DEPDC1 and phosphotyrosine-picked threonine tyrosine kinase (TTK) in OS. The expression of DEPDC1 and TTK in OS cells was detected by western blotting. Furthermore, the assessment of glycolysis encompassed the quantification of extracellular acidification rate, glucose uptake rate, lactate concentration, and the expression of glucose transporter 1, hexokinase 2, and pyruvate kinase M2. Finally, the functions of DEPDC1 and TTK in autophagy and ras-extracellular signal-regulated kinase signaling were determined by western blotting after interfering with DEPDC1 in SaOS-2 cells. The results revealed that DEPDC1 and TTK were upregulated in OS cell lines and interfering with DEPDC1 inhibited glycolysis and autophagy in OS cells. Furthermore, the STRING database suggested that DEPDC1 and TTK perform targeted binding. Notably, the results of the present study revealed that DEPDC1 upregulated RAS expression through TTK and enhanced ERK activity, thereby affecting glycolysis and autophagy in OS cells. Collectively, the present investigation demonstrated that DEPDC1 affected autophagy-dependent glycolysis levels of OS cells by regulating RAS/ERK signaling through TTK., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The family of Ras-like GTPases consists of over 150 different members, regulated by an even larger number of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs) that comprise cellular switch networks that govern cell motility, growth, polarity, protein trafficking, and gene expression. Efforts to develop selective small molecule probes and drugs for these proteins have been hampered by the high affinity of guanosine triphosphate (GTP) and lack of allosteric regulatory sites. This paradigm was recently challenged by the discovery of a cryptic allosteric pocket in the switch II region of K-Ras. Here, we ask whether similar pockets are present in GTPases beyond K-Ras. We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family., Competing Interests: Declaration of interests K.M.S., J.M., and L.Z. are inventors on patents owned by University of California, San Francisco, covering GTPase-targeting small molecules. K.M.S. has consulting agreements for the following companies, which involve monetary and/or stock compensation: AperTOR, BioTheryX, BridGene Biosciences, Erasca, Exai, G Protein Therapeutics, Genentech, Initial Therapeutics, Kumquat Biosciences, Kura Oncology, Lyterian, Merck, Montara Therapeutics, Nested, Nextech, Revolution Medicines, Rezo, Totus, Type6 Therapeutics, Vevo, Vicinitas, and Wellspring Biosciences (Araxes Pharma). J.E.B. has consulting agreements for the following companies, which involve monetary and/or stock compensation: Reactive Biosciences, Scorpion Therapeutics, and Olema Oncology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epithelial-to-mesenchymal transitions (EMTs) and extracellular matrix (ECM) remodeling are distinct yet important processes during carcinoma invasion and metastasis. Transforming growth factor β (TGF-β) and RAS, signaling through SMAD and RAS-responsive element-binding protein 1 (RREB1), jointly trigger expression of EMT and fibrogenic factors as two discrete arms of a common transcriptional response in carcinoma cells. Here, we demonstrate that both arms come together to form a program for lung adenocarcinoma metastasis and identify chromatin determinants tying the expression of the constituent genes to TGF-β and RAS inputs. RREB1 localizes to H4K16acK20ac marks in histone H2A.Z-loaded nucleosomes at enhancers in the fibrogenic genes interleukin-11 (IL11), platelet-derived growth factor-B (PDGFB), and hyaluronan synthase 2 (HAS2), as well as the EMT transcription factor SNAI1, priming these enhancers for activation by a SMAD4-INO80 nucleosome remodeling complex in response to TGF-β. These regulatory properties segregate the fibrogenic EMT program from RAS-independent TGF-β gene responses and illuminate the operation and vulnerabilities of a bifunctional program that promotes metastatic outgrowth., Competing Interests: Declaration of interests J.M. owns company stock in Scholar Rock. C.M.R. has consulted regarding oncology drug development with Amgen, Astra Zeneca, Chugai, Daiichi Sankyo, Hoffman-La Roche, and Jazz Pharmaceuticals; C.M.R. serves on the scientific advisory boards of Auron, Bridge Medicines, DISCO, Earli, and Harpoon Therapeutics. S.W.L. is a consultant and holds equity in Blueprint Medicines, ORIC Pharmaceuticals, Mirimus, PMV Pharmaceuticals, Faeth Therapeutics, and Senecea Therapeutics, and is a consultant for Fate Therapeutics. T.T. is a consultant and holds equity in Lime Therapeutics; the Tammela Lab receives research support from Ono Pharmaceuticals Co., Ltd. (unrelated to this work). T.T.’s spouse is an employee of Recursion Pharmaceuticals. R.P.K. is a co-founder of and consultant for Econic Biosciences., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Rabex-5 (also called RabGEF1), a protein originally characterized for its Rab5 GEF function, also has an A20-like E3 ubiquitin ligase domain. We and others reported that Rabex-5 E3 activity promotes Ras mono- and di-ubiquitination to inhibit Ras signaling in Drosophila and mammals. Subsequently, we reported that Rabex-5 inhibits Notch signaling in the Drosophila hematopoietic system. Here we report genetic interactions using Rabex-5 transgenes encoding domain-specific mutations that show that Rabex-5 requires an intact E3 domain to inhibit Notch signaling in the epithelial tissue of the developing wing. Surprisingly, we discovered that Rabex-5 with an impaired E3 domain but active Rab5 GEF domain suppresses Notch loss-of-function phenotypes and enhances both Notch duplication phenotypes and activated Ras phenotypes consistent with a model that the Rab5 GEF activity of Rabex-5 might positively regulate Ras and Notch. Positive and negative regulation of developmental signaling by its different catalytic domains could allow Rabex-5 to precisely coordinate developmental signaling to fine-tune patterning. Finally, we report that Rabex-5 also inhibits the overgrowth due to loss of PTEN or activation of PI3K but not activation of AKT. Inhibition of Ras, Notch, and PI3K signaling may explain why Rabex-5 is deleted in some cancers. Paradoxically, Rabex-5 is reported to be an oncogene in other cancers. We propose that Rabex-5 acts as a tumor suppressor via its E3 activity to inhibit Ras, Notch, and PI3K signaling and as an oncogene via its Rab5 GEF activity to enhance Ras and Notch signaling., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Reimels et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is often resistant to therapy. An immune suppressive tumor microenvironment (TME) and oncogenic mutations in KRAS have both been implicated as drivers of resistance to therapy. Mitogen-activated protein kinase (MAPK) inhibition has not yet shown clinical efficacy, likely because of rapid acquisition of tumor-intrinsic resistance. However, the unique PDAC TME may also be a driver of resistance. We found that long-term focal adhesion kinase (FAK) inhibitor treatment led to hyperactivation of the RAS/MAPK pathway in PDAC cells in mouse models and tissues from patients with PDAC. Concomitant inhibition of both FAK (with VS-4718) and rapidly accelerated fibrosarcoma and MAPK kinase (RAF-MEK) (with avutometinib) induced tumor growth inhibition and increased survival across multiple PDAC mouse models. In the TME, cancer-associated fibroblasts (CAFs) impaired the down-regulation of MYC by RAF-MEK inhibition in PDAC cells, resulting in resistance. By contrast, FAK inhibition reprogramed CAFs to suppress the production of FGF1, which can drive resistance to RAF-MEK inhibition. The addition of chemotherapy to combined FAK and RAF-MEK inhibition led to tumor regression, a decrease in liver metastasis, and improved survival in KRAS-driven PDAC mouse models. Combination of FAK and RAF-MEK inhibition alone improved antitumor immunity and priming of T cell responses in response to chemotherapy. These findings provided the rationale for an ongoing clinical trial evaluating the efficacy of avutometinib and defactinib in combination with gemcitabine and nab-paclitaxel in patients with PDAC and may suggest further paths for combined stromal and tumor-targeting therapies.

Ras signaling and glycosylphosphatidylinositol (GPI) biosynthesis are mutually inhibitory in S. cerevisiae (Sc). The inhibition is mediated via an interaction of yeast Ras2 with the Eri1 subunit of its GPI- N -acetylglucosaminyl transferase (GPI-GnT), the enzyme catalyzing the very first GPI biosynthetic step. In contrast, Ras signaling and GPI biosynthesis in C. albicans (Ca) are mutually activated and together control the virulence traits of the human fungal pathogen. What might be the role of Eri1 in this pathogen? The present manuscript addresses this question while simultaneously characterizing the cellular role of CaEri1. It is either nonessential or required at very low levels for cell viability in C. albicans . Severe depletion of CaEri1 results in reduced GPI biosynthesis and cell wall defects. It also produces hyperfilamentation phenotypes in Spider medium as well as in bicarbonate medium containing 5% CO 2 , suggesting that both the Ras-dependent and Ras-independent cAMP-PKA pathways for hyphal morphogenesis are activated in these cells. Pull-down and acceptor-photobleaching FRET experiments suggest that CaEri1 does not directly interact with CaRas1 but does so through CaGpi2, another GPI-GnT subunit. We showed previously that CaGpi2 is downstream of CaEri1 in cross talk with CaRas1 and for Ras-dependent hyphal morphogenesis. Here we show that CaEri1 is downstream of all GPI-GnT subunits in inhibiting Ras-independent filamentation. CaERI1 also participates in intersubunit transcriptional cross talk within the GPI-GnT, a feature unique to C. albicans . Virulence studies using G. mellonella larvae show that a heterozygous strain of CaERI1 is better cleared by the host and is attenuated in virulence.

The abnormal proliferation and differentiation of oral mucosal fibroblasts (FBs) is the key to the progression of oral submucosal fibrosis. To clarify the mechanism of platelet-derived growth factor (PDGF-BB)-induced FBs fibrosis in oral mucosa, real-time quantitative polymerase chain reaction and Western blot were used in this study to detect the expression of miR-503 and the expression of p-MEK, p-ERK, miR-503, RAF, smooth actin and type I collagen under different time and concentration stimulation of PDGF-BB. The effects of overexpression of miR-503 or RAF on the proliferation and migration of FBs were detected by cell counting kit 8 and cell scratch assay, respectively. A dual luciferase reporter gene assay was used to verify the targeting effect of miR-503 on RAF. The results showed that miR-503 was downregulated in a dose- and time-dependent manner in PDGF-BB-induced FBs. In addition, RAF is a direct target of miR-503 and can be negatively regulated. Overexpression of RAF can promote FB proliferation, migration, differentiation, collagen synthesis, and activation of downstream molecules (MEK/ERK), while overexpression of miR-503 can partially reverse the effects of RAF. Therefore, miR-503 regulates the biological behavior of PDGF-BB-induced oral mucosal FBs by influencing the activation of the RAS/RAF/MEK/ERK signaling pathway.

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.

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., (© 2024. The Author(s).)

Glioma represents a primary malignant tumor occurring in the central nervous system. Glutamate decarboxylase (GAD1) plays a significant role in tumor development; however, its function of GAD1 and underlying mechanisms in glioma progression remain unclear. Differentially expressed genes (DEGs) obtained from the GSE12657 and GSE15209 datasets that intersected with cuproptosis-related genes and pivot genes were identified using comprehensive bioinformatics methods. The elesclomol (ES) treatment was used to induce cuproptosis in U251 cells, which was validated by detecting intracellular copper levels and cuproptosis marker expression. Lentivirus-mediated gene overexpression was performed to explore the effects of GAD1 using functional assays in vitro and in a mouse xenograft model. The RAS agonist ML098 was used to verify the effect of GAD1 on the RAS/MAPK pathway in glioma cells. A total of 87 cuproptosis-related DEGs and seven hub genes were obtained, with five genes upregulated and two were downregulated in gliomas. Overexpression of GAD1 inhibited proliferation, invasion, and migration, promoted apoptosis of glioma cells, and suppressed tumorigenesis in vivo. In addition, GAD1 overexpression enhanced the sensitivity of glioma cells to cuproptosis. Additionally, ML098 treatment attenuated the inhibitory effect of GAD1 overexpression on the malignant phenotype of ES-treated cells. GAD1 plays an anti-oncogenic role in glioma by regulating apoptosis via inhibition of the RAS/MAPK pathway., (© 2024 Wiley Periodicals LLC.)

Competing Interests: Declaration of interests The authors declare no competing interests.

The TREX-2 complex of eukaryotes is responsible for the export of a wide range of mRNAs from the nucleus to the cytoplasm. Previously, we showed that a subunit of the D. melanogaster TREX-2 complex, the PCID2 protein, has a domain that specifically interacts with RNA. However, it remains unknown whether other components of the complex are involved in interaction with and recognition of the target mRNA. In the present study, we determined the role of Xmas-2, the core structural subunit of the complex, in the specific recognition of ras2 mRNA fragments. In this work, we showed that Xmas-2 interacts with ras2 mRNA independently of other subunits of the complex. We showed that RNA-binding domains are located in both the N-terminal domain and the C-terminal domain of Xmas-2. However, the interaction of the protein with ras2 mRNA fragments is independent of RNA sequence and structure and is nonspecific. Thus, the Xmas-2 subunit is not involved in the recognition of specific RNA sequences by the complex., (© 2024. Pleiades Publishing, Ltd.)

Pulmonary arterial hypertension (PAH) is a debilitating vascular disorder characterized by abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and collagen synthesis, contributing to vascular remodeling and elevated pulmonary vascular resistance. This study investigated the critical role of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) in cell proliferation and collagen synthesis in PASMCs in PAH. Here we show that ATIC levels are significantly increased in the lungs of monocrotaline (MCT)-induced PAH rat model, hypoxia-induced PAH mouse model, and platelet-derived growth factor (PDGF)-stimulated PASMCs. Inhibition of ATIC attenuated PDGF-induced cell proliferation and collagen I synthesis in PASMCs. Conversely, overexpression or knockdown of ATIC causes a significant promotion or inhibition of Ras and ERK activation, cell proliferation, and collagen synthesis in PASMCs. Moreover, ATIC deficiency attenuated Ras activation in the lungs of hypoxia-induced PAH mice. Furthermore, Ras inhibition attenuates ATIC overexpression- and PDGF-induced collagen synthesis and PASMC proliferation. Notably, we identified that transcription factors MYC, early growth response protein 1 (EGR1), and specificity protein 1 (SP1) directly binds to promoters of Atic gene and regulate ATIC expression. These results provide the first evidence that ATIC promotes PASMC proliferation in pulmonary vascular remodeling through the Ras signaling pathway. NEW & NOTEWORTHY Our findings highlight the important role of ATIC in the PASMC proliferation of pulmonary vascular remodeling through its modulation of the Ras signaling pathway and its regulation by transcription factors MYC, EGR1, and SP1. ATIC's modulation of Ras signal pathway represents a novel mechanism contributing to PAH development.

Background/aims: Potassium channel tetramerization domain containing 17 (KCTD17) protein, an adaptor for the cullin3 (Cul3) ubiquitin ligase complex, has been implicated in various human diseases; however, its role in hepatocellular carcinoma (HCC) remains elusive. Here, we aimed to elucidate the clinical features of KCTD17, and investigate the mechanisms by which KCTD17 affects HCC progression., Methods: We analyzed transcriptomic data from patients with HCC. Hepatocyte-specific KCTD17 deficient mice were treated with diethylnitrosamine (DEN) to assess its effect on HCC progression. Additionally, we tested KCTD17-directed antisense oligonucleotides for their therapeutic potential in vivo., Results: Our investigation revealed the upregulation of KCTD17 expression in both tumors from patients with HCC and mouse models of HCC, in comparison to non-tumor controls. We identified the leucine zipper-like transcriptional regulator 1 (Lztr1) protein, a previously identified Ras destabilizer, as a substrate for KCTD17-Cul3 complex. KCTD17-mediated Lztr1 degradation led to Ras stabilization, resulting in increased proliferation, migration, and wound healing in liver cancer cells. Hepatocyte-specific KCTD17 deficient mice or liver cancer xenograft models were less susceptible to carcinogenesis or tumor growth. Similarly, treatment with KCTD17-directed antisense oligonucleotides (ASO) in a mouse model of HCC markedly lowered tumor volume as well as Ras protein levels, compared to those in control ASO-treated mice., Conclusion: KCTD17 induces the stabilization of Ras and downstream signaling pathways and HCC progression and may represent a novel therapeutic target for HCC.

Lung adenocarcinoma (LUAC) as the most common lung cancer, and its incidence is increasing. Complement factor B (CFB) is an important factor in the alternative complement pathway. CFB has been reported to be involved in the progression of many cancers, including in pancreatic cancer, cutaneous squamous cell carcinoma, and nasopharyngeal carcinoma, but the function and molecular mechanism of CFB in LUAC remains unclear. The present study aimed to explore the role of CFB in LUAC malignant progression. In our previous study, we found that CFB was downregulated expression in LUAC clinical samples. Here, we firstly detected the cell function in vitro. Cell proliferation and migration were increased, while cell apoptosis and cell cycle arrest were suppressed after CFB knockdown. Overexpression of CFB repressed the malignant progression of LUAC in vitro. Besides, in vivo experiments revealed that upregulation of CFB inhibited tumor growth and Ki67 expression. Additionally, our data indicated that CFB negatively regulated Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, upregulation of CFB inhibited the progression of LUAC was reversed by Ras/MAPK pathway activators (ML-098 or C16-PAF). Our study uncovered that CFB acts as a tumor suppressor repressed tumorigenesis of LUAC through inhibiting the Ras/MAPK pathway, suggesting that CFB may be a potential biomarker and therapeutic target for LUAC., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Signaling through the Ras-MAPK pathway can exhibit switch-like activation, which has been attributed to the underlying positive feedback and bimodality in the activation of RasGDP to RasGTP by SOS. SOS contains both catalytic and allosteric Ras binding sites, and a common assumption is that allosteric activation selectively by RasGTP provides the mechanism of positive feedback. However, recent single-molecule studies have revealed that SOS catalytic rates are independent of the nucleotide state of Ras in the allosteric binding site, raising doubt about this as a positive feedback mechanism. Here, we perform detailed kinetic analyses of receptor-mediated recruitment of full-length SOS to the membrane while simultaneously monitoring its catalytic activation of Ras. These results, along with kinetic modeling, expose the autoinhibition release step in SOS, rather than either recruitment or allosteric activation, as the underlying mechanism giving rise to positive feedback in Ras activation., Competing Interests: Declaration of interests All authors declare they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2 , which encodes G αi2 , a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase-mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating G αi2 mutations had clinical presentations that included impaired immunity. Mutant G αi2 impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant G αi2 influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)-activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT S6 signaling to drive cellular growth and proliferation.

The dysregulation of the rat sarcoma (RAS) signaling pathway, particularly the MAPK/ERK cascade, is a hallmark of many cancers, leading to uncontrolled cellular proliferation and resistance to apoptosis-inducing treatments. Dysregulation of the MAPK/ERK pathway is common in various cancers including pancreatic, lung, and colon cancers, making it a critical target for therapeutic intervention. Natural compounds, especially phytochemicals, offer a promising avenue for developing new anticancer therapies due to their potential to interfere with these signaling pathways. This study investigates the potential of anticancer phytochemicals to inhibit the MAPK/ERK pathway through molecular docking and simulation techniques. A total of 26 phytochemicals were screened from an initial set of 340 phytochemicals which were retrieved from Dr. Duke's database using in silico methods for their binding affinity and stability. Molecular docking was performed to identify key interactions with ERK2, followed by molecular dynamics (MD) simulations to evaluate the stability of these interactions. The study identified several phytochemicals, including luteolin, hispidulin, and isorhamnetin with a binding score of -10.1±0 Kcal/mol, -9.86±0.15 Kcal/mol, -9.76±0.025 Kcal/mol, respectively as promising inhibitors of the ERK2 protein. These compounds demonstrated significant binding affinities and stable interactions with ERK2 in MD simulation studies up to 200ns, particularly at the active site. The radius of gyration analysis confirmed the stability of these phytochemical-protein complexes' compactness, indicating their potential to inhibit ERK activity. The stability and binding affinity of these compounds suggest that they can effectively inhibit ERK2 activity, potentially leading to more effective and less toxic cancer treatments. The findings underscore the therapeutic promise of these phytochemicals, which could serve as a basis for developing new cancer therapies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Azmal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Vitamin B12 is an essential nutritional co-factor for the folate and methionine cycles, which together constitute one-carbon metabolism. Here, we show that dietary uptake of vitamin B12 modulates cell fate decisions controlled by the conserved RAS/MAPK signaling pathway in C. elegans. A bacterial diet rich in vitamin B12 increases vulval induction, germ cell apoptosis and oocyte differentiation. These effects are mediated by different one-carbon metabolites in a tissue-specific manner. Vitamin B12 enhances via the choline/phosphatidylcholine metabolism vulval induction by down-regulating fat biosynthesis genes and increasing H3K4 tri-methylation, which results in increased expression of RAS/MAPK target genes. Furthermore, the nucleoside metabolism and H3K4 tri-methylation positively regulate germ cell apoptosis and oocyte production. Using mammalian cells carrying different activated KRAS and BRAF alleles, we show that the effects of methionine on RAS/MAPK-regulated phenotype are conserved in mammals. Our findings suggest that the vitamin B12-dependent one-carbon metabolism is a limiting factor for diverse RAS/MAPK-induced cellular responses., (© 2024. The Author(s).)

Cancer often involves the overactivation of RAS/RAF/MEK/ERK (MAPK) and PI3K-Akt-mTOR pathways due to mutations in genes like RAS, RAF, PTEN, and PIK3CA. Various strategies are employed to address the overactivation of these pathways, among which targeted therapy emerges as a promising approach. Directly targeting specific proteins, leads to encouraging results in cancer treatment. For instance, RTK inhibitors such as imatinib and afatinib selectively target these receptors, hindering ligand binding and reducing signaling initiation. These inhibitors have shown potent efficacy against Non-Small Cell Lung Cancer. Other inhibitors, like lonafarnib targeting Farnesyltransferase and GGTI 2418 targeting geranylgeranyl Transferase, disrupt post-translational modifications of proteins. Additionally, inhibition of proteins like SOS, SH2 domain, and Ras demonstrate promising anti-tumor activity both in vivo and in vitro. Targeting downstream components with RAF inhibitors such as vemurafenib, dabrafenib, and sorafenib, along with MEK inhibitors like trametinib and binimetinib, has shown promising outcomes in treating cancers with BRAF-V600E mutations, including myeloma, colorectal, and thyroid cancers. Furthermore, inhibitors of PI3K (e.g., apitolisib, copanlisib), AKT (e.g., ipatasertib, perifosine), and mTOR (e.g., sirolimus, temsirolimus) exhibit promising efficacy against various cancers such as Invasive Breast Cancer, Lymphoma, Neoplasms, and Hematological malignancies. This review offers an overview of small molecule inhibitors targeting specific proteins within the RAS upstream and downstream signaling pathways in cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Colorectal cancer (CRC) is a common cancer in younger adults. In patients undergoing liver resection with RAS-altered CRCs, there is evidence suggesting younger patients have worse outcomes than older patients. To explain this pattern, differences in associations between RAS status and other cancer-related biomarkers in tumors from younger versus older patients with CRC were evaluated in a cohort of 925 patients with CRC, 277 (30.0%) of whom were ≤50 years old, and 454 (49.1%) who had RAS-altered tumors. For 3 biomarkers, RNF43, APC, and microsatellite instability (MSI), the association with RAS status was significantly modified by age after adjustment for multiple testing. Specifically, younger patients with RAS-altered tumors were more likely to be MSI-high, RNF43 mutated, and APC wild type. These differences might contribute to the observed pattern of diminished survival in younger versus older patients with CRC with RAS-mutated tumors undergoing liver metastasis resection., (© The Author(s) 2024. Published by Oxford University Press.)

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., (© 2024 Chao et al.)

M-RAS plays a crucial role in the RAF-MEK signaling pathway. When activated by GTP, M-RAS forms a complex with SHOC2 and PP1C, initiating downstream RAF-MEK signal transduction. In this study, the crystal structure of the GDP-bound human M-RAS protein is presented with two forms of crystal packing. Both the full-length and truncated human M-RAS structures aligned well with the high-confidence section of the AlphaFold2-predicted structure with low r.m.s.d., except for the Switch regions. Despite high sequence similarity to the available mouse M-RAS structure, the full-length human M-RAS structure exhibits unique crystal packing. This inactive human M-RAS structure could offer novel insights for the design of selective compounds targeting M-RAS.

Cervical cancer poses a significant health burden for women globally, and the rapid proliferation of cervical cancer cells greatly worsens patient prognosis. Long non-coding RNAs (lncRNAs) play a crucial role in regulating tumor cell proliferation. However, the involvement of lncRNAs in cervical cancer cell proliferation remains unclear. In this study, we investigated the lncRNA SIX1-1, which was found to be upregulated in cervical cancer tissues and cell lines. Functional assays revealed that knockdown of SIX1-1 inhibited cell proliferation in vitro and reduced tumor growth in vivo. Mechanistically, SIX1-1 was predominantly localized in the nucleus and could bind with DNMT1 protein. The expression of SIX1-1 enhanced the interaction of DNMT1 with RASD1 promoter, leading to the methylation of the promoter and decreased mRNA transcription. Then RASD1 downregulation activated the cAMP/PKA/CREB signaling pathway, promoting cell proliferation. Rescue experiments showed that knockdown of RASD1 restored the inhibited cell proliferation caused by decreased expression of SIX1-1, indicating that RASD1 acted as the functional mediator of SIX1-1. In conclusion, SIX1-1 promoted cervical cancer cell proliferation by modulating RASD1 expression. This suggests that targeting the SIX1-1/RASD1 axis could be a potential antitumor strategy for cervical cancer., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Exogenous insulin-like growth factor-1 (IGF-1) has been reported to promote wound healing through regulation of vascular endothelial cells (VECs). Despite the existing studies of IGF-1 on VEC and its role in angiogenesis, the mechanisms regarding anti-inflammatory and angiogenetic effects of IGF-1 remain unclear. In this study, we investigated the wound-healing process and the related signaling pathway of IGF-1 using an inflammation model induced by IFN-γ. The results demonstrated that IGF-1 can increase cell proliferation, suppress inflammation in VECs, and promote angiogenesis. In vivo studies further confirmed that IGF-1 can reduce inflammation, enhance vascular regeneration, and improve re-epithelialization and collagen deposition in acute wounds. Importantly, the Ras/PI3K/IKK/NF-κB signaling pathways was identified as the mechanisms through which IGF-1 exerts its anti-inflammatory and pro-angiogenic effects. These findings contribute to the understanding of IGF-1's role in wound healing and may have implications for the development of new wound treatment approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ras guanine nucleotide-releasing protein 1 (RasGRP1), a Ras activator, is upregulated in hepatocellular carcinoma (HCC) and other kinds of cancer and is associated with the poor prognosis of patients. However, little is known about the underlying regulatory mechanisms of RasGRP1 in the context of cancer. Here, we report that RasGRP1 physically interacted with the adaptor protein Src homolog and collagen homolog 3 (Shc3). Moreover, RasGRP1 C-terminus domain (aa 607-797) bound to the central collagen-homology 1 (CH1) domain of Shc3. Subsequently, Shc3 enhanced the RasGRP1 tyrosine phosphorylation rate and stability by inhibiting its ubiquitination. Notably, the phosphorylation-mimicking mutants of RasGRP1, RasGRP1 Y704A, and Y748A, rescued the phosphorylation and ubiquitination levels of RasGRP1 in HCC cells. Further investigation showed that the RasGRP1 and Shc3 interaction induced activation of Ras and c-Jun, resulting in cell proliferation in vitro. Moreover, the regulation of Shc3/RasGRP1/Ras/c-Jun signal transduction was confirmed in vivo using the subcutaneous xenograft mouse model. Thus, we propose that continuous Shc3 overexpression may be a possible mechanism for maintaining RasGRP1 stability and that persistent activation of Ras/c-Jun signaling through the interaction of RasGRP1 and Shc3 is a key event increasing cell proliferation. Our findings suggest that the interaction of RasGRP1 and Shc3 plays an important role in HCC tumorigenesis and suggests the potential clinical usage of novel biomarkers and therapeutic targets in HCC., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Neurofibromatosis type 1 (NF1), an autosomal dominant genetic disorder, is caused by mutations in the NF1 gene, which encodes the GTPase-activating protein neurofibromin. The pathogenesis of the tumor progression of benign plexiform neurofibromas (PNs) and malignant peripheral nerve sheath tumors (MPNSTs) remain unclear. Here, we found that interferon-induced transmembrane protein 1 (IFITM1) was downregulated in MPNST tissues compared to those in PN tissues from patients with NF1. Overexpression of IFITM1 in NF1-associated MPNST cells resulted in a significant decrease in Ras activation (GTP-Ras) and downstream extracellular regulatory kinase 1/2 (ERK1/2) phosphorylation, whereas downregulation of IFITM1 via treatment with small interfering RNA in normal Schwann cells had the opposite result, indicating that expression levels of IFITM1 are closely associated with tumor progression in NF1. Treatment of MPNST cells with interferon-gamma (IFN-γ) significantly augmented the expression of IFITM1, thereby leading to a decrease in Ras and ERK1/2 activation. Despite the small number of patient samples, these findings may potentially provide a new target for chemotherapy in patients with NF1-associated MPNSTs. In xenograft mice injected with MPNST cells, IFN- γ treatment successfully suppressed tumor progression with increased IFITM1 expression and decreased Ras and ERK1/2 activation in tumor tissues. Collectively, these results suggest that IFITM1 is closely involved in MPNST pathogenesis and that IFN-γ is a good candidate for the therapeutic treatment of MPNSTs in NF1.

Src homology 3 (SH3) domains play a critical role in mediating protein-protein interactions (PPIs) involved in cell proliferation, migration, and the cytoskeleton. Despite their abundance in the human proteome, the functions and molecular interactions of many SH3 domains remain unknown, and this is in part due to the lack of SH3-domain-specific reagents available for their study. Affimer proteins have been developed as affinity reagents targeting a diverse range of targets, including those involved in PPIs. In this study, Affimer proteins were isolated against both the N- and C-terminal SH3 domains (NSH3 and CSH3) of growth-factor-receptor-bound protein 2 (Grb2), an adapter protein that provides a critical link between cell surface receptors and Ras signalling pathways. Targeting the CSH3 alone for the inhibition of PPIs appeared sufficient for curtailing Ras signalling in mammalian cell lines stimulated with human epidermal growth factor (EGF), which conflicts with the notion that the predominant interactions with Ras activating Son of sevenless (SOS) occur via the NSH3 domain. This result supports a model in which allosteric mechanisms involved in Grb2-SOS1 interaction modulate Ras activation.

Background: Breast cancer (BC) ranks as the third most fatal malignant tumor worldwide, with a strong reliance on fatty acid metabolism. CLDN6, a candidate BC suppressor gene, was previously identified as a regulator of fatty acid biosynthesis; however, the underlying mechanism remains elusive. In this research, we aim to clarify the specific mechanism through which CLDN6 modulates fatty acid anabolism and its impact on BC growth and metastasis., Methods: Cell function assays, tumor xenograft mouse models, and lung metastasis mouse models were conducted to evaluate BC growth and metastasis. Human palmitic acid assay, triglyceride assay, Nile red staining, and oil red O staining were employed to investigate fatty acid anabolism. Reverse transcription polymerase chain reaction (RT-PCR), western blot, immunohistochemistry (IHC) assay, nuclear fractionation, immunofluorescence (IF), immunoprecipitation and acyl-biotin exchange (IP-ABE), chromatin immunoprecipitation (ChIP), dual luciferase reporter assay, and co-immunoprecipitation (Co-IP) were applied to elucidate the underlying molecular mechanism. Moreover, tissue microarrays of BC were analyzed to explore the clinical implications., Results: We identified that CLDN6 inhibited BC growth and metastasis by impeding RAS palmitoylation both in vitro and in vivo. We proposed a unique theory suggesting that CLDN6 suppressed RAS palmitoylation through SREBP1-modulated de novo palmitic acid synthesis. Mechanistically, CLDN6 interacted with MAGI2 to prevent KLF5 from entering the nucleus, thereby restraining SREBF1 transcription. The downregulation of SREBP1 reduced de novo palmitic acid synthesis, hindering RAS palmitoylation and subsequent endosomal sorting complex required for transport (ESCRT)-mediated plasma membrane localization required for RAS oncogenic activation. Besides, targeting inhibition of RAS palmitoylation synergized with CLDN6 to repress BC progression., Conclusions: Our findings provide compelling evidence that CLDN6 suppresses the palmitic acid-induced RAS palmitoylation through the MAGI2/KLF5/SREBP1 axis, thereby impeding BC malignant progression. These results propose a new insight that monitoring CLDN6 expression alongside targeting inhibition of palmitic acid-mediated palmitoylation could be a viable strategy for treating oncogenic RAS-driven BC., (© 2024. The Author(s).)

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 (SOS cat ) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOS cat . 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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Metastatic prostate cancer is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We recently found that a single clinically relevant specific amino acid change (Proline1597Leucine, (P1597L)), found in metastatic deposits of prostate cancer patients, converts PlexinB1 from a metastasis suppressor to a gene that drives prostate cancer metastasis in vivo. However, the mechanism by which PlexinB1(P1597L) promotes metastasis is not known., Methods: Pull down assays using GST-RalGDS or -GSTRaf1-RBD were used to reveal the effect of mutant or wild-type PlexinB1 expression on Rap and Ras activity respectively. Protein-protein interactions were assessed in GST pulldown assays, Akt/ERK phosphorylation by immunoblotting and protein stability by treatment with cycloheximide. Rho/ROCK activity was monitored by measuring MLC2 phosphorylation and actin stress fiber formation. PlexinB1 function was measured using cell-collapse assays., Results: We show here that the single clinically relevant P1597L amino acid change converts PlexinB1 from a repressor of Ras to a Ras activator. The PlexinB1(P1597L) mutation inhibits the RapGAP activity of PlexinB1, promoting a significant increase in Ras activity. The P1597L mutation also blocks PlexinB1-mediated reduction in Rho/ROCK activity, restraining the decrease in MLC2 phosphorylation and actin stress fiber formation induced by overexpression of wild-type PlexinB1. PlexinB1(P1597L) has little effect on the interaction of PlexinB1 with small GTPases or receptor tyrosine kinases and does not inhibit PlexinB1-stimulated Akt or ERK phosphorylation. These results indicate that the mutation affects Rho signalling via the Rap/Ras pathway. The PlexinB1(P1597L) mutation inhibits morphological cell collapse induced by wild-type PlexinB1 expression, suggesting that the mutation induces a loss of an inhibitory tumour suppressor function., Conclusion: These results suggest that the clinically relevant P1597L mutation in PlexinB1 may transform PlexinB1 from a suppressor to a driver of metastasis in mouse models of prostate cancer by reducing the RapGAP activity of PlexinB1, leading to Ras activation. These findings highlight the PlexinB1-Rap-Ras pathway for therapeutic intervention in prostate cancer., (© 2024. The Author(s).)

Hepatocellular carcinoma (HCC) still presents poor prognosis with low overall survival rates and limited therapeutic options available. Recently, attention has been drawn to peptidomic analysis, an emerging field of proteomics for the exploration of new potential peptide drugs for the treatment of various diseases. However, research on the potential function of HCC peptides is lacking. Here, we analyzed the peptide spectrum in HCC tissues using peptidomic techniques and explored the potentially beneficial peptides involved in HCC. Changes in peptide profiles in HCC were examined using liquid chromatography-mass spectrometry (LC-MS/MS). Analyze the physicochemical properties and function of differently expressed peptides using bioinformatics. The effect of candidate functional peptides on HCC cell growth and migration was evaluated using the CCK-8, colony formation, and transwell assays. Transcriptome sequencing analysis and western blot were employed to delve into the mode of action of potential peptide on HCC. Peptidomic analysis of HCC tissue yielded a total of 8683 peptides, of which 452 exhibited up-regulation and 362 showed down-regulation. The peptides that were differentially expressed, according to bioinformatic analysis, were closely linked to carbon metabolism and the mitochondrial inner membrane. The peptide functional validation identified a novel peptide, PDLC (peptide derived from liver cancer), which was found to dramatically boost HCC cell proliferation through the Ras/Raf/MEK/ERK signaling cascade. Our research defined the peptide's properties and pattern of expression in HCC and identified a novel peptide, PDLC, with a function in encouraging HCC progression, offering an entirely new potential therapeutic target the disease., (© 2024. The Author(s).)

The Ras family genes are proto-oncogenes that are highly conserved from Drosophila to humans. In Drosophila, Ras V12 is a constitutively activated form of the Ras oncoprotein, and its function in cell-cycle progression is context dependent. However, how it influences the cell cycle of female germline stem cells (GSCs) still remains unknown. Using both wild-type GSCs and bam mutant GSC-like cells as model systems, here we determined that Ras V12 overexpression promotes GSC division, not growth, opposite to that in somatic wing disc cells. Ras performs this function through activating the mitogen-activated protein kinase (MAPK) signaling. This signaling is activated specifically in the M phase of mitotic germ cells, including both wild-type GSCs and bam mutant GSC-like cells. Furthermore, Ras V12 overexpression triggers polyploid nurse cells to die through inducing mitotic stress. Given the similarities between Drosophila and mammalian GSCs, we propose that the Ras/MAPK signaling also promotes mammalian GSC division., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

RASopathies are a group of related genetic disorders caused by mutations in genes within the RAS/MAPK signaling pathway. This pathway is crucial for cell division, growth, and differentiation, and its disruption can lead to a variety of developmental and health issues. RASopathies present diverse clinical features and pose significant diagnostic and therapeutic challenges. Studying the landscape of biomarkers in RASopathies has the potential to improve both clinical practices and the understanding of these disorders. This review provides an overview of recent discoveries in RASopathy molecular profiling, which extend beyond traditional gene mutation analysis. mRNAs, non-coding RNAs, protein expression patterns, and post-translational modifications characteristic of RASopathy patients within pivotal signaling pathways such as the RAS/MAPK, PI3K/AKT/mTOR, and Rho/ROCK/LIMK2/cofilin pathways are summarized. Additionally, the field of metabolomics holds potential for uncovering metabolic signatures associated with specific RASopathies, which are crucial for developing precision medicine. Beyond molecular markers, we also examine the role of histological characteristics and non-invasive physiological assessments in identifying potential biomarkers, as they provide evidence of the disease's effects on various systems. Here, we synthesize key findings and illuminate promising avenues for future research in RASopathy biomarker discovery, underscoring rigorous validation and clinical translation.

Cardiac involvement is a major feature of RASopathies, a group of phenotypically overlapping syndromes caused by germline mutations in genes encoding components of the RAS/MAPK (mitogen-activated protein kinase) signaling pathway. In particular, Noonan syndrome (NS) is associated with a wide spectrum of cardiac pathologies ranging from congenital heart disease (CHD), present in approximately 80% of patients, to hypertrophic cardiomyopathy (HCM), observed in approximately 20% of patients. Genotype-cardiac phenotype correlations are frequently described, and they are useful indicators in predicting the prognosis concerning cardiac disease over the lifetime. The aim of this review is to clarify the molecular mechanisms underlying the development of cardiac diseases associated particularly with NS, and to discuss the main morphological and clinical characteristics of the two most frequent cardiac disorders, namely pulmonary valve stenosis (PVS) and HCM. We will also report the genotype-phenotype correlation and its implications for prognosis and treatment. Knowing the molecular mechanisms responsible for the genotype-phenotype correlation is key to developing possible targeted therapies. We will briefly address the first experiences of targeted HCM treatment using RAS/MAPK pathway inhibitors.

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., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Aims: Postoperative cognitive dysfunction (POCD) is prevalent among the elderly, characterized primarily by cognitive decline after surgery. This study aims to explore how extracellular vesicles (EVs) derived from BV2 microglial cells, with and without the C-C chemokine receptor type 5 (CCR5), affect neuroinflammation, neuronal integrity, and cognitive function in a POCD mouse model., Methods: We collected EVs from LPS-stimulated BV2 cells expressing CCR5 (EVs M1 ) and from BV2 cells with CCR5 knockdown (EVs M1-CCR5 ). These were administered to POCD-induced mice. Protein interactions between CCR5, G-protein-coupled receptors (GPCRs), and Ras were analyzed using structure-based docking and co-immunoprecipitation (Co-IP). We assessed the phosphorylation of p38 and Erk, the expression of synaptic proteins PSD95 and MAP2, and conducted Morris Water Maze tests to evaluate cognitive function., Results: Structure-based docking and Co-IP confirmed interactions between CCR5, GPR, and Ras, suggesting a CCR5-GPCRs-Ras-MAPK pathway involvement in neuroinflammation. EVs M1 heightened neuroinflammation, reduced synaptic integrity, and impaired cognitive function in POCD mice. In contrast, EVs M1-CCR5 reduced neuroinflammatory markers, preserved synaptic proteins, enhanced dendritic spine structure, and improved cognitive outcomes., Conclusion: EVs M1 induced neuroinflammation via the CCR5-GPCRs-Ras-MAPK pathway, with EVs M1-CCR5 showing protective effects on POCD progression, suggesting a new therapeutic strategy for POCD management via targeted modification of microglial EVs., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).

Little is known about eukaryotic chemorepulsion. The enzymes phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and CnrN to induce chemorepulsion of cells away from the secreted chemorepellent protein AprA. How D. discoideum cells utilize two proteins with redundant phosphatase activities in response to AprA is unclear. Here, we show that D. discoideum cells require both PTEN and CnrN to locally inhibit Ras activation, decrease basal levels of PI(3,4,5)P3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation and increase filopod sizes. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, and AprA requires PTEN, but not CnrN, to induce cell roundness. Together, our results suggest that CnrN and PTEN play unique roles in AprA-induced chemorepulsion., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric leukemia with few effective treatments and poor outcomes even after stem cell transplantation, the only current curative treatment. We developed a JMML patient-derived xenograft (PDX) mouse model and demonstrated the in vivo therapeutic efficacy and confirmed the target of trametinib, a RAS-RAF-MEK-ERK pathway inhibitor, in this model. A PDX model was created through transplantation of patient JMML cells into mice, up to the second generation, and successful engraftment was confirmed using flow cytometry. JMML PDX mice were treated with trametinib versus vehicle control, with a median survival of 194 days in the treatment group versus 124 days in the control group ( p  = 0.02). Trametinib's target as a RAS pathway inhibitor was verified by showing inhibition of ERK phosphorylation using immunoblot assays. In conclusion, trametinib monotherapy significantly prolongs survival in our JMML PDX model by inhibiting the RAS pathway. Our model can be effectively used for assessment of novel targeted treatments, including potential combination therapies, to improve JMML outcomes.