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Hyperactive Ras in developmental disorders and cancer
Abstract
Ras genes are the most common targets for somatic gain-of-function mutations in human cancer. Recently, germline mutations that affect components of the Ras-Raf-mitogen-activated and extracellular-signal regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway were shown to cause several developmental disorders, including Noonan, Costello and cardio-facio-cutaneous syndromes. Many of these mutant alleles encode proteins with aberrant biochemical and functional properties. Here we will discuss the implications of germline mutations in the Ras-Raf-MEK-ERK pathway for understanding normal developmental processes and cancer pathogenesis.
... Although most cancers in humans originate from epithelial cells and fibroblasts are part of the tumor microenvironment, studying fibroblasts can provide insights into whether a species tends to have anticancer properties by assessing their resistance to malignant transformation. For example, previous studies have shown that the expression of oncogenic HRAS(G12V) and SV40 large antigen (SV40 LT) effectively induces malignant transformation in mouse fibroblasts 10,11 . However, this transformation is not observed in fibroblasts derived from naked mole-rats and blind mole-rats, which are well-known for their anticancer characteristics 12,13 . ...
... In this study, we offered experimental evidence, both in vitro and in vivo, for the malignant transformation resistance of MPI cells, strongly implying that the big-footed bat (Myotis pilosus) is a mammalian species resistant to cancers. It is important to note that while the oncogenic HRAS(G12V) is the most common cancer-associated substitution and is also commonly used to induce malignant transformation of cells 11 , particularly in non-model organisms like naked mole-rats and blind mole-rats 12,13 , neoplastic transformation of cells may require species-or cell type-specific mutations of HRAS or other RAS members 10,11 . Therefore, our results cannot completely rule out the possibility that the other six bat species examined in this study may have evolved different anticancer mechanisms compared to MPI. ...
... In this study, we offered experimental evidence, both in vitro and in vivo, for the malignant transformation resistance of MPI cells, strongly implying that the big-footed bat (Myotis pilosus) is a mammalian species resistant to cancers. It is important to note that while the oncogenic HRAS(G12V) is the most common cancer-associated substitution and is also commonly used to induce malignant transformation of cells 11 , particularly in non-model organisms like naked mole-rats and blind mole-rats 12,13 , neoplastic transformation of cells may require species-or cell type-specific mutations of HRAS or other RAS members 10,11 . Therefore, our results cannot completely rule out the possibility that the other six bat species examined in this study may have evolved different anticancer mechanisms compared to MPI. ...
Mammals exhibit different rates of cancer, with long-lived species generally showing greater resistance. Although bats have been suggested to be resistant to cancer due to their longevity, this has yet to be systematically examined. Here, we investigate cancer resistance across seven bat species by activating oncogenic genes in their primary cells. Both in vitro and in vivo experiments suggest that Myotis pilosus (MPI) is particularly resistant to cancer. The transcriptomic and functional analyses reveal that the downregulation of three genes (HIF1A, COPS5, and RPS3) largely contributes to cancer resistance in MPI. Further, we identify the loss of a potential enhancer containing the HIF1A binding site upstream of COPS5 in MPI, resulting in the downregulation of COPS5. These findings not only provide direct experimental evidence for cancer resistance in a bat species but also offer insights into the natural mechanisms of cancer resistance in mammals.
... We presented a unique case of a younger patient who was presented with multiple immune dysregulations with a single underlying root cause of NRAS gene positive. The scientific evidence suggests that varying RAS genes are mutated in distinct malignancies [12,13]. Hematological malignancies are mostly unusual where both NRAS and KRAS, responsible for encoding a GAP (GTPase activating protein) called neurofibromin, are mutated with varying frequencies [14]. ...
... Hematological malignancies are mostly unusual where both NRAS and KRAS, responsible for encoding a GAP (GTPase activating protein) called neurofibromin, are mutated with varying frequencies [14]. However, NRAS than KRAS mutations are predominant in hematological malignancies [12]. In addition, the studies demonstrated that somatic mutations of NRAS are found in about 20 to 40% of the myelodysplastic syndrome (MDs), heterogenous clustering of hematopoietic disorders featured by abnormal hematopoiesis, cytopenia, and higher risk of acute myeloid leukemia [15,16]. ...
Hematological malignancies in particular are recognized to be linked to immunological dysregulated disorders such hematolytic anemia, immunodeficiency, and autoimmune illnesses. We describe a rare case that had multiple immune dysregulation symptoms and was subsequently found to be positive for the NRAS gene. A male, 29, complained of pleuritic chest pain. On the ECG, he showed broad PR depression and elevated PR in aVR, along with elevated troponin. He received colchicine and NSAIDs as treatment with a good response and resolution of his symptoms. After four weeks, he developed a fever which thought to be secondary to perianal abscess, and a surgical incision and drainage along with broad spectrum antibiotics was used to treat a perianal abscess. Despite receiving extensive medical and surgical care, his fever persisted. His blood film started to show circulating blast cells. Following an immediate bone marrow aspiration and biopsy, there were no notable abnormalities seen, and a 1% blast was present, which was assumed to be the result of stress or infection. Later, a weak positive +1 Coombs (DAT) test is developed and caused active hemolytic anemia required blood transfusion. Along with the anemia, a wrist expanding wound was discovered in the previous IV cannula site. Clinically wound appears as a pyoderma gangrenosum. Histopathology showed purulent dermal inflammation including many neutrophils and macrophages. This feature of micro abscess formation was consisting with the diagnosis of pyoderma gangrenosum. An intravenous steroid (1 mg/kg) was administered in response to a positive Coombs test and pyoderma gangrenosum. The patient's condition greatly improved overall. His wound began to heal as his white blood cell count dropped. After being given oral steroids with a titration-down plan, the patient was sent home. A whole genome sequence was performed on him because he had peri myocarditis, pyoderma gangrenosum, and hematolytic anemia. The results show that he is NRAS gene positive. He was attentively observed clinically with regular out-patient follow-ups as this gene is strongly associated with hematological cancers. He developed symptomatic normocytic anemia a few months later, and a bone marrow aspiration and biopsy were performed to confirm the presence of Myelodysplastic syndromes (MDS). He was transferred to another facility and underwent chemotherapy and bone marrow transplant. In otherwise healthy individuals, the idea of offering a single explanation—Occam's razor—must be considered and investigated. Finding the underlying reason could improve patient care, enable precision therapy, and alter the course of events.
... Approximatively 19% of all patients with cancer harbor KRAS, NRAS or HRAS activating mutations (7). Whereas KRAS mutations are frequent in NSCLC, NRAS mutations are mostly found in melanoma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, thyroid carcinoma (7)(8)(9). These alterations disrupt the guanine exchange cycle of the RAS protein and favor an active GTP bound state, which leads to the activation of downstream signaling pathways involved in proliferation and cell survival such as the MAPK or the PIK3/AKT/mTOR pathways (8,(10)(11)(12). ...
... Whereas KRAS mutations are frequent in NSCLC, NRAS mutations are mostly found in melanoma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, thyroid carcinoma (7)(8)(9). These alterations disrupt the guanine exchange cycle of the RAS protein and favor an active GTP bound state, which leads to the activation of downstream signaling pathways involved in proliferation and cell survival such as the MAPK or the PIK3/AKT/mTOR pathways (8,(10)(11)(12). ...
... This signaling pathway is tightly regulated under physiological conditions, and its dysfunction causes many human diseases, particularly cancers [2][3][4] . Hyperactive ERK signaling that arises from genetic alterations of pathway components or upstream regulators exists in almost half of cancers, and plays a critical role in the transformation, overgrowth, and organ infiltration of cancer cells [5][6][7] . Targeting this signaling pathway with small molecular inhibitors as well as other advanced approaches has achieved promising outcomes in clinic cancer treatment though remarkable challenges (i.e. ...
... Hyperactive ERK signaling that arises from genetic mutations/alterations is responsible for many human diseases. It is well known that germ-line mutations of components or regulators of ERK signaling induce developmental disorders termed as RASopathies 3,4 , whereas somatic dysregulations of this signaling cause cancers [5][6][7] . Here we focus on cancer-related mutations/alterations. ...
... Aberrant Ras function drives cancer cell proliferation, survival, and metastasis, which result from the well-established genomic mutations and posttranslational modifications of Ras proteins (2). Among which, KRAS is the most frequently mutated, while HRAS is the less in all cancer-associated RAS mutations (3,4). However, whether RAS genes are regulated in posttranscriptional level remains unexplored. ...
... MAPK pathway is a major downstream of Ras, which plays an essential role in cancer (4). As expected, dm 6 ACRISPR targeting HRAS reduced expression of p-ERK and p-MEK (Fig. 5K). ...
Overexpression of Ras, in addition to the oncogenic mutations, occurs in various human cancers. However, the mechanisms for epitranscriptic regulation of RAS in tumorigenesis remain unclear. Here, we report that the widespread N6-methyladenosine (m6A) modification of HRAS, but not KRAS and NRAS, is higher in cancer tissues compared with the adjacent tissues, which results in the increased expression of H-Ras protein, thus promoting cancer cell proliferation and metastasis. Mechanistically, three m6A modification sites of HRAS 3' UTR, which is regulated by FTO and bound by YTHDF1, but not YTHDF2 nor YTHDF3, promote its protein expression by the enhanced translational elongation. In addition, targeting HRAS m6A modification decreases cancer proliferation and metastasis. Clinically, up-regulated H-Ras expression correlates with down-regulated FTO and up-regulated YTHDF1 expression in various cancers. Collectively, our study reveals a linking between specific m6A modification sites of HRAS and tumor progression, which provides a new strategy to target oncogenic Ras signaling.
... Guanine nucleotide exchange factors (GEFs), such as Son of Sevenless (SOS), promote the exchange of GDP for GTP to activate Ras (6,7), while hydrolysis of GTP to GDP is promoted by GTPase-activating proteins (GAPs) (8). Under physiological conditions, Ras activation and deactivation reactions are in constant competition and must be tightly regulated; misregulation of Ras is a major cause of cancer (9). Decades of focused studies on the Ras signaling mechanism have revealed many detailed biochemical insights (10), such as its interaction with various effectors (3,7,8), conformational dynamics (11,12), and its potential to dimerize and form clusters (13)(14)(15)(16)(17). ...
Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays. The results reveal a bimodal Ras activation response, which is not a result of deterministic bistability but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts deterministic bistability and may be more resistant to pharmacological inhibition.
... Several landmark studies and research papers have established the importance of oncogenes in cancer. Many studies unveiled the identification of RAS as an oncogene, highlighting its significance in cancer development [34][35][36][37][38]. Our results identify proteins that act as oncogenes, including PDGFRA, NRAS, HRAS, CSF1R, KIT, MET, ABL1, FGFR2, FGFR3, and KRAS, which are essential critical proteins in various cellular processes and are often associated with different types of cancer and other diseases. ...
The RAS signaling pathway is a crucial cell transduction pathway central to transmitting signals from outside the cell to the cell nucleus and influencing fundamental biological mechanisms like cell growth, division, and specialization. This signaling pathway has recently received much attention in scientific research because of its involvement in various diseases, especially carcinogenesis. Our study identified the significance of crucial proteins in the RAS signaling cascade in cancer development and progression. We found that proteins such as PDGFRB, PDGFB, IGF1, HRAS, HGF, FGF10, and ABL1 are involved in various types of cancer and could serve as potential therapeutic targets. Misregulation of these proteins may result in unregulated cell proliferation and contribute to cancer development and maintenance. The study also emphasizes the importance of oncogenes in cancer development, with RAS being identified as a pivotal oncogene. In addition, the findings indicate several proteins, including PDGFRA, NRAS, HRAS, CSF1R, KIT, MET, ABL1, FGFR2, FGFR3, and KRAS, function as oncogenes and are related to different forms of cancer and diseases. Targeted therapies for these proteins are being investigated in various cancer types, including gastrointestinal stromal tumors, chronic myelogenous leukemia, and bladder cancer. Moreover, we identified NF1 as a critical tumor suppressor gene essential in regulating cellular proliferation. Mutations in the NF1 gene lead to neurofibromatosis category 1. This paper emphasizes the significance of crucial proteins implicated in the RAS signaling pathway in cancer growth and advancement. Understanding the complexity of these proteins and their dysregulation could offer essential insights into the progression of practical treatment approaches that enhance and refine cancer therapies. These findings provide promising avenues for further research and advances in cancer treatment and give us hope for better outcomes in the fight against this challenging disease.
... In addition, one of the neoplasmic sources in older children and adults is a tissue anomaly during development. Also, latent oncogenes in anomalous tissues may activate and transform into malignant tumors under certain environmental conditions for example mouse dams' exposure to chemicals as BPA even with low doses leading to mammary gland morphogenesis anomalous which induce carcinoma [6][7][8] ...
... Ostrander et al (42) reported that the TET2 deletion mutation increases dendritic cell production, increases hematopoietic stem cell self-renewal and polarizes toward the myeloid lineage. NRAS is a member of the RAS family, and mutations in NRAS facilitate GTP binding of RAS, thereby promoting constitutive activation of the RAS-RAF-MEK-ERK/MAPK signaling pathway and causing abnormal proliferation of myeloid cells (41,43). This may be involved in the occurrence of MF. ...
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an extremely rare and aggressive tumor with an unknown pathogenesis. Myelofibrosis (MF) is a type of myeloproliferative neoplasm. MF can be secondary to several hematological malignancies, including chronic myeloid leukemia, myelodysplastic syndrome and hairy cell leukemia. In the present report, a rare case of BPDCN secondary to MF is described. A 70-year-old male patient developed a large purplish-red rash with recurrent symptoms. BPDCN was confirmed by immunohistochemistry of a biopsy specimen and flow cytometry of bone marrow cells. Bone marrow histopathology revealed MF. Next-generation sequencing of peripheral blood revealed mutations in the Tet methylcytosine dioxygenase 2 and NRAS proto-oncogene GTPase genes. The patient underwent one cycle of chemoimmunotherapy, but the condition progressed, an infection developed and the patient eventually died. The present case suggests that BPDCN can occur in conjunction with MF and that the prognosis of such patients is poor. Pathological examination and genetic testing aided in the diagnosis and treatment. This case emphasizes the need to raise awareness of BPDCN among clinicians and to be alert to the potential for fatal infection in patients with BPDCN combined with MF following myelosuppression triggered during chemotherapy.
... We found that the lack of Ku70 does not affect the phosphorylation of EGFR (Fig. 7A). In addition to the plasma membrane, Ras-ERK signaling has been observed at endosomes, the endoplasmic reticulum, the Golgi apparatus, and the mitochondria (60,61). Coimmunoprecipitation revealed that endogenous Ku70 from the colon tissue lysate of WT mice interacted with the early endosome marker Rab5 and the late endosome marker Rab7 (Fig. 7B). ...
The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5 ⁺ Rab7 ⁺ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.
... The FGFRs share a similar structure containing an extracellular ligand binding domain composed of three immunoglobulin-like domains, a single transmembrane helix segment, and an intracellular tyrosine kinase domain (Dionne et al. 1990;Houssaint et al. 1990;Gilbert et al. 1993;Pellegrini et al. 2000). These receptors are expressed on numerous cell types in various tissues and regulate crucial biological processes, including cell proliferation, migration, survival, and differentiation by activation of downstream signaling pathways, such as the PI3K-AKT-mTOR, PLCγ-PKC, or the RAS-MAPK signaling cascades (Kouhara et al. 1997;Thisse and Thisse 2005;Schubbert et al. 2007;Turner and Grose 2010). Pathogenic FGFR variants affecting ligand binding and specificity or tyrosine kinase activity can lead to aberrant receptor signaling causing diverse inherited conditions, e.g., skeletal disorders such as Pfeiffer syndrome (MIM 101600), Muenke syndrome (MIM 602849), or achondroplasia (MIM 100800). ...
The fibroblast growth factor receptors comprise a family of related but individually distinct tyrosine kinase receptors. Within this family, FGFR2 is a key regulator in many biological processes, e.g., cell proliferation, tumorigenesis, metastasis, and angiogenesis. Heterozygous activating non-mosaic germline variants in FGFR2 have been linked to numerous autosomal dominantly inherited disorders including several craniosynostoses and skeletal dysplasia syndromes. We report on a girl with cutaneous nevi, ocular malformations, macrocephaly, mild developmental delay, and the initial clinical diagnosis of Schimmelpenning–Feuerstein–Mims syndrome, a very rare mosaic neurocutaneous disorder caused by postzygotic missense variants in HRAS, KRAS, and NRAS. Exome sequencing of blood and affected skin tissue identified the mosaic variant c.1647=/T > G p.(Asn549=/Lys) in FGFR2, upstream of the RAS signaling pathway. The variant is located in the tyrosine kinase domain of FGFR2 in a region that regulates the activity of the receptor and structural mapping and functional characterization revealed that it results in constitutive receptor activation. Overall, our findings indicate FGFR2-associated neurocutaneous syndrome as the accurate clinical-molecular diagnosis for the reported individual, and thereby expand the complex genotypic and phenotypic spectrum of FGFR-associated disorders. We conclude that molecular analysis of FGFR2 should be considered in the genetic workup of individuals with the clinical suspicion of a mosaic neurocutaneous condition, as the knowledge of the molecular cause might have relevant implications for genetic counseling, prognosis, tumor surveillance and potential treatment options.
... AKT/mTOR [30][31][32] and Ras/MAPK 33 Overexpression of AKT and Ras triggered strong activation of the mTORC1/2 pathway. Here, we showed Fat-1 can strongly inhibit AKT/mTOR pathway in vivo. ...
Background and Aim:
n-3 PUFAs have been shown to have multiple biological effects on cancers. The fat-1 gene encodes an n-3 desaturase that introduces a double bond at the n-3 position of the hydrocarbon chain in n-6 fatty acids to form an n-3 fatty acid. Here we investigated the anti-tumorigenesis effect of Fat-1 gene.
Methods: Mounting evidence indicates that activation of AKT and Ras pathways is a key oncogenic event in human hepatocarcinogenesis. Our previous results demonstrate that co-expression of AKT and N-Ras in the mouse liver promotes rapid hepatocarcinogenesis in vivo. Here we utilized hydrodynamic tail vein injection of Fat-1 gene to AKT/Ras hepatocarcinogenesis' model to testify its anti-tumorigenesis effect.
Results: Hydrodynamic injection of Fat-1 gene could inhibit lipogenesis and affects liver tumor development in co-expressing AKT and Ras oncogenes' mice. Molecular analysis showed that Fat-1 gene strongly inhibited the Ras/MAPK and Akt/ mTORC cell signal pathway, and significantly inhibited de novo lipogenesis by inhibition of fatty acid biosynthesis gene (FASN, ACLY) and transcription factors (LXR-β, SREBP1), which were highly elevated in AKT/Ras mice. The AKT/Ras cells lines in vitro also showed the similar results. Noticeably, although the hydrodynamic injection of fat-1 gene showed significant inhibition effect of hepatocarcinogenesis, it didn't change fatty acid profile in various tissues of mice, which is different from previous studies by using Fat-1 transgenic mice.
Conclusions Local expression of Fat-1 gene can achieve sufficient suppression of AKT/Ras-induced hepatocarcinogenesis. Thus, with multiple biological effects on various signal pathways, Fat-1 gene can prevent of hepatocarcinogenesis.
... Mutations in the KRAS isoform, in particular codon-12 missense mutations, promote its constitutive activation and are common oncogenic drivers for human cancer (1-4). These mutations inhibit transition of active, GTP-bound KRAS to its inactive GDP-bound state by rendering it insensitive to the action of GTPase activating proteins that promote GTP hydrolysis (5)(6)(7). A common oncogenic KRAS mutation that bears a cysteine-for-glycine substitution at codon 12 (KRAS G12C ) is associated with residual GTP hydrolysis rates that are amenable to targeting the KRAS G12C -nucleoside complex in its inactive (GDPbound) form (8,9). Covalent modification of the C12 thiol irreversibly traps KRAS G12C in the inactive state by preventing GTP loading by nucleotide exchange factors such as Son of Sevenless (SOS), and several such covalent KRAS G12C inhibitors (i)-with target specificity conferred by a dynamic binding pocket under the KRAS Switch II loop adjacent to the nucleoside binding site-have demonstrated clinical efficacy to date against KRAS G12C -dependent solid tumors (10)(11)(12). ...
Although KRASG12C inhibitors show clinical activity in patients with KRAS G12C mutated non–small cell lung cancer (NSCLC) and other solid tumor malignancies, response is limited by multiple mechanisms of resistance. The KRASG12C inhibitor JDQ443 shows enhanced preclinical antitumor activity combined with the SHP2 inhibitor TNO155, and the combination is currently under clinical evaluation. To identify rational combination strategies that could help overcome or prevent some types of resistance, we evaluated the duration of tumor responses to JDQ443 ± TNO155, alone or combined with the PI3Kα inhibitor alpelisib and/or the cyclin-dependent kinase 4/6 inhibitor ribociclib, in xenograft models derived from a KRASG12C-mutant NSCLC line and investigated the genetic mechanisms associated with loss of response to combined KRASG12C/SHP2 inhibition. Tumor regression by single-agent JDQ443 at clinically relevant doses lasted on average 2 weeks and was increasingly extended by the double, triple, or quadruple combinations. Growth resumption was accompanied by progressively increased KRAS G12C amplification. Functional genome-wide CRISPR screening in KRASG12C-dependent NSCLC lines with distinct mutational profiles to identify adaptive mechanisms of resistance revealed sensitizing and rescuing genetic interactions with KRASG12C/SHP2 coinhibition; FGFR1 loss was the strongest sensitizer, and PTEN loss the strongest rescuer. Consistently, the antiproliferative activity of KRASG12C/SHP2 inhibition was strongly enhanced by PI3K inhibitors. Overall, KRAS G12C amplification and alterations of the MAPK/PI3K pathway were predominant mechanisms of resistance to combined KRASG12C/SHP2 inhibitors in preclinical settings. The biological nodes identified by CRISPR screening might provide additional starting points for effective combination treatments.
Significance
Identification of resistance mechanisms to KRASG12C/SHP2 coinhibition highlights the need for additional combination therapies for lung cancer beyond on-pathway combinations and offers the basis for development of more effective combination approaches.
See related commentary by Johnson and Haigis, p. 4005
... The biochemical function of these proteins involves cycling of the catalytic domain between an active GTPbound and inactive GDP-bound states. While regulated cycling between these states is required for a wide variety of physiological processes that control cell growth, motility and trafficking [35][36][37] , dysregulation of the cycle causes many intractable diseases including cancer [38][39][40][41][42][43][44][45] . In addition to the GTPase cycle, membrane binding is required for the cellular activity of Ras and Rh. ...
We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. Here we show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we find that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.
... H-ras (19,20,21). Ras oncogenes play a fundamental role in regulation of cell growth and survival and they are frequently activated in cancer (35). Previous studies showed that active oncogenic Ras induced autophagy to promote and facilitate oncogenic transformation by maintaining and improving cell metabolism (16,17,36). ...
GABARAP gene has an essential role in the autophagic process through its involvement in the maturation of the autophagosome. The role of GABARAP in tumorigenesis is not yet clarified. It is ubiquitously expressed in all tested normal tissues, while its expression in tumors is divers. Autophagy could induce by Oncogenic Ras to handle the metabolic stress and support cell survival. In this study, we found that GABARAP knockout mice exhibited significantly less tumor formation than wild-type mice after 7,12-dimethylbenz(a)anthracene treatment. Different types of tumor developed in the mice (skin, mammary, lymphoma and liver tumors). Furthermore, the tumor occurrence started earlier in wild-type mice compared to GABARAP knockout animals, and the tumor sizes in wild-type mice were obviously larger in most of induced tumors compared to the tumors formed in GABARAP KO mice. No H-ras mutation detected in the tumors of GABARAP knockout mice compared to 5 mutations in 14 tumors of the wild-type mice which revealed by mutation analysis of tumors induced by DMBA. In conclusion, the absence of H-ras mutation in DMBA-induced tumors of GABARAP KO mice indicates the significance of GABARAP gene in tumor progression that need further studies to clarify the exact role.
... Genome sequencing studies of AML patients show it to be an intricate and diverse disease characterized by the existence of numerous leukemic genes, some repeatedly and other uncommonly mutated [13]. Different subtypes of mutations have been recognized which have broadly been classified into two groups; Class I mutations include stimulated signaltransduction pathways, enhancing the expansion or existence of hematopoietic precursor cells which include the mutations in FLT3 and RAS gene family, while class II mutations affect transcription factors and allow distinction to be impeded which include mutations in CEBPA, MLL, and NPM1 [15][16][17][18].Clinical characteristics and genetic analysis of about 45% of all AML cases among a wide subgroup of AML patients having a normal karyotype has been carried out. In the case of AML, translocation t(8;21) leads to the generation of AML1-ETO fusion oncogene which has been shown to influence differentiation, proliferation and apoptosis in both in-vitro and in-vivo models having association with a favorable prognosis [19]. ...
ackground: The onset and progression of leukemia is associated with many genetic abnormalities including gene mutations and production of fusion oncogenes. Molecular studies on fusion oncogenes and mutations in different populations have been done. However, not much research on correlation of the fusion oncogenes with acute myeloid leukemia have been done in Pakistan. Methods: Genetic analysis of 105 AML patients was done to investigate AML1-ETOand CBFB-MYH11 fusion oncogenes and mutations in NPM1 and NRAS genes. The genomic DNA and cDNA were subjected to amplification, electrophoresis, and Sanger sequencing. Results: The frequency of AML1-ETO was 26% in AML patients and 34.2% in AML-M2 patients. CBFB-MYH11 was present in 11.4% AML patients. A total of six mutations in 4 regions of NPM1 gene and 2 regions of NRAS gene were detected. 3'UTR of NPMI gene had three variants; g.1128C>T (57.1%), g.1185-/T insertion (80.95%), and g.1163A>T (57.14%) while c.867_871subGTGGA >CAAGTTTGC (2.86%) was present in exon 12. NRAS gene had two mutations c.12C>T (51.4%) and c.33A>T (11.43%) in exon 2. c.867_871subGTGGA >CAAGTTTGC , and g.1163A>T in NPM1 gene and c.33A>T in NRAS gene were the novel findings in this ethnic population. Conclusion: This genetic analysis may help to modulate the treatment strategies and improve survival of patients. B Abstract www.als-journal.com/
... Ras oncogenes have been discovered in human tumors' genomes and are demonstrated to be the mutated and constitutively active form of the Ras p21 protein. More than one-third of human tumors harbor activating RAS mutations [5,6]. Like other small GTPases, Ras p21 proteins are active when bound to GTP and inactive in the GDP-bound form. ...
Quinine, a bitter compound, can act as an agonist to activate the family of bitter taste G protein-coupled receptor family of proteins. Previous work from our laboratory has demonstrated that quinine causes activation of RalA, a Ras p21-related small G protein. Ral proteins can be activated directly or indirectly through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using normal mammary epithelial (MCF-10A) and non-invasive mammary epithelial (MCF-7) cell lines, we investigated the effect of quinine in regulating Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF-10A and MCF-7 cells; however, RalA was inhibited in MCF-10A cells, and no effect was observed in the case of MCF-7 cells. MAP kinase, a downstream effector for Ras p21, was activated in both MCF-10A and MCF-7 cells. Western blot analysis confirmed the expression of RalGDS in MCF-10A cells and MCF-7 cells. The expression of RalGDS was higher in MCF-10A cells in comparison to the MCF-7 cells. Although RalGDS was detected in MCF-10A and MCF-7 cells, it did not result in RalA activation upon Ras p21 activation with quinine suggesting that the Ras p21-RalGDS-RalA pathway is not active in the MCF-10A cells. The inhibition of RalA activity in MCF-10A cells due to quinine could be as a result of a direct effect of this bitter compound on RalA. Protein modeling and ligand docking analysis demonstrated that quinine can interact with RalA through the R79 amino acid, which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change that results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that regulate Ral activity in mammary epithelial cells.
... RAS mutations cause tumor initiation and drive uncontrollable tumor cell proliferation (13,14); they are also associated with poor prognosis (15)(16)(17). RAS proteins are guanosine triphosphatases (GTPases) that function as binary switches cycling between inactive (guanosine diphosphate-bound) and active [guanosine-5'-triphosphate (GTP)-bound] states (18,19). Activated RAS proteins can bind to numerous downstream effectors, such as RAF and PI3K, which regulate critical cellular processes, including metabolism, proliferation and survival (20). ...
The aim of the present study was to examine the effects of alisertib (ALS) on RAS signaling pathways against a panel of colorectal cancer (CRC) cell lines and engineered Flp-In stable cell lines expressing different Kirsten rat sarcoma virus (KRAS) mutants. The viability of Caco-2KRAS wild-type, Colo-678KRAS G12D, SK-CO-1KRAS G12V, HCT116KRAS G13D, CCCL-18KRAS A146T and HT29BRAF V600E cells was examined by Cell Titer-Glo assay, and that of stable cell lines was monitored by IncuCyte. The expression levels of phosphorylated (p-)Akt and p-Erk as RAS signal outputs were measured by western blotting. The results suggested that ALS exhibited different inhibitory effects on cell viability and different regulatory effects on guanosine triphosphate (GTP)-bound RAS in CRC cell lines. ALS also exhibited various regulatory effects on the PI3K/Akt and mitogen-activated protein kinase (MAPK) pathways, the two dominant RAS signaling pathways, and induced apoptosis and autophagy in a RAS allele-specific manner. Combined treatment with ALS and selumetinib enhanced the regulatory effects of ALS on apoptosis and autophagy in CRC cell lines in a RAS allele-specific manner. Notably, combined treatment exhibited a synergistic inhibitory effect on cell proliferation in Flp-In stable cell lines. The results of the present study suggested that ALS differentially regulates RAS signaling pathways. The combined approach of ALS and a MEK inhibitor may represent a new therapeutic strategy for precision therapy for CRC in a KRAS allele-specific manner; however, this effect requires further study in vivo.
... As small GTPases, Ras proteins play various roles of molecular switches, which can transduce signals from their activated receptors to the downstream effectors for regulating cell growth, self-renewal, cell differentiation, and cell survival. Over one-third of cancers in humans carry active Ras mutations [9]. The BCR-ABL1 oncoprotein can constitutively activate the Ras-associated pathways in CML. ...
BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalARosa26-Tg/+) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.
... bone development in addition to increased FGF23 production through the MAPK pathway. 11,12 This is an interesting observation as altered FGF signaling also affects pinna development in lacrimoauriculo-dento-digital (LADD) and labyrinthine aplasia, microtia, and microdontia (LAMM) syndrome due to pathogenic variants in FGFR2, FGFR3, FGF10, and FGF3. 13,14 In addition, Fgf8 and À10 mutant mice as well as hypomorphic Fgfr1 À/À mice present with variable microtia. ...
Epidermal nevus syndrome (ENS) comprises a heterogeneous group of neurocutaneous syndromes associated with the presence of epidermal nevi and variable extracutaneous manifestations. Postzygotic activating HRAS pathogenic variants were previously identified in nevus sebaceous (NS), keratinocytic epidermal nevus (KEN), and different ENS, including Schimmelpenning–Feuerstein–Mims and cutaneous‐skeletal‐hypophosphatasia syndrome (CSHS). Skeletal involvement in HRAS‐related ENS ranges from localized bone dysplasia in association with KEN to fractures and limb deformities in CSHS. We describe the first association of HRAS‐related ENS and auricular atresia, thereby expanding the disease spectrum with first branchial arch defects if affected by the mosaic variant. In addition, this report illustrates the first concurrent presence of verrucous EN, NS, and nevus comedonicus (NC), indicating the possibility of mosaic HRAS variation as an underlying cause of NC. Overall, this report extends the pleiotropy of conditions associated with mosaic pathogenic variants in HRAS affecting ectodermal and mesodermal progenitor cells.
... Glutamine 61 is essential for GTP hydrolysis, and substitution of any amino acid at this position blocks hydrolysis. Accordingly, the active GTP-bound conformation (Ras-GTP) accumulates in cells, thereby causing abnormal cell proliferation and differentiation (Schubbert et al., 2007). ...
Introduction:
Bladder tumors of cattle are very uncommon accounting from 0.1% to 0.01% of all bovine malignancies. Bladder tumors are common in cattle grazing on bracken fern-infested pasturelands. Bovine papillomaviruses have a crucial role in tumors of bovine urinary bladder.
Aim of the study:
To investigate the potential association of ovine papillomavirus (OaPV) infection with bladder carcinogenesis of cattle.
Methods:
Droplet digital PCR was used to detect and quantify the nucleic acids of OaPVs in bladder tumors of cattle that were collected at public and private slaughterhouses.
Results:
OaPV DNA and RNA were detected and quantified in 10 bladder tumors of cattle that were tested negative for bovine papillomaviruses. The most prevalent genotypes were OaPV1 and OaPV2. OaPV4 was rarely observed. Furthermore, we detected a significant overexpression and hyperphosphorylation of pRb and a significant overexpression and activation of the calpain-1 as well as a significant overexpression of E2F3 and of phosphorylated (activated) PDGFβR in neoplastic bladders in comparison with healthy bladders, which suggests that E2F3 and PDGFβR may play an important role in OaPV-mediated molecular pathways that lead to bladder carcinogenesis.
Conclusion:
In all tumors, OaPV RNA could explain the causality of the disease of the urinary bladder. Therefore, persistent infections by OaPVs could be involved in bladder carcinogenesis. Our data showed that there is a possible etiologic association of OaPVs with bladder tumors of cattle.
... These common mutations interfere with the intrinsic GTPase activity of RAS and confer resistance to GAPs, which stabilize the transition state of the RAS-GTP hydrolysis reaction, resulting in the accumulation of active, GTP-bound RAS proteins. The glutamine at codon 61 is required for GTP hydrolysis, and an amino acid substitution other than glutamic acid at this position blocks this reaction [61]. The mutation found in our study was in codon 61 with a histidinesubstituted for the glutamine. ...
... Activation of EGFR will cause KRAS to bind to GTP, resulting in activation of downstream signaling pathways such as RAF/ MEK/ERK pathways. Mutations in KRAS codons 12 or 13 lead to the accumulation of an active GTP-bound conformation, resulting in sustained activation of downstream signaling pathways, which may predict cetuximab resistance [94,95]. Lievre et al. found that KRAS mutations were present in 43% of tumors in metastatic CRC patients treated with cetuximab and were significantly associated with cetuximab resistance [96], which was consistent with Di Fiore et al.'s findings in mCRC patients treated with cetuximab in combination with chemotherapy [97]. ...
... GGPP is a major substrate for anchoring Ras to the membrane. Decrease of GGPP is associated with dissociation of Ras from the membrane and consequently inhibiting the Rasmediated growth signaling (McTaggart 2006;Schubbert et al. 2007). These authors demonstrated that suppression of the membrane localization of Ras and up-regulation of Bim contributed to inhibition of the ERK and mTOR signaling pathways. ...
Inflammation plays a critical role in several diseases such as cancer, gastric, heart and nervous system diseases. Data suggest that the activation of mammalian target of rapamycin (mTOR) pathway in epithelial cells leads to inflammation. Statins, the inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), seem to be able to inhibit the mTOR. Statins are considered to have favorable effects on inflammatory diseases by reducing the complications caused by inflammation and by regulating the inflammatory process and cytokines secretion. This critical review collected data on this topic from clinical, in vivo and in vitro studies published between 1998 and June 2022 in English from databases including PubMed, Google Scholar, Scopus, and Cochrane libraries.
... KRAS protein acts as a binary switch, the active GTP-bound form is ON and inactive GDP-bound form is OFF, to activate several important cellular signaling pathways. When GDP exchange to GTP by GEFs, KRAS will be active and able to stimulate downstream pathways which are important in cell growth, differentiation and cell survival (16,17,18). Mutations that convert KRAS protooncogenes to oncogenes are typically point mutations causing amino acid changes, particularly at positions 12, 13, and 61. ...
Helicobacter pylori (H. pylori) is the most important etiologic factor for gastric cancer ,it is one of the most common human pathogens, which colonizes in the mucus layer of the gastric epithelium in more than 50% of the population. It is the only bacterium that classified as a class I carcinogen by the WHO .The clinical outcomes of H. pylori infection is determine by host genetic predisposition, bacterial virulence factors, and environmental factors. H. pylori can induce chronic inflammation and oxidative stress that provides a permissive environment to DNA damage, this damage can lead to genetic instability and eventually, neoplastic transformation. KRAS is one of the most frequently mutated oncogenes in human cancers, in particular in pancreatic, colorectal and lung cancers. However, oncogenic mutations of KRAS are infrequent in gastric cancer. Point mutations of the KRAS are found predominantly in adenocarcinomas. Codons 12 and 13 are the most frequently detected mutation "hot spots", they make the protein resistant to GTP hydrolysis by GTPases, thereby leading to constitutive KRAS activity. The results revealed that no mutations in codons 12 or13 detected in all patient groups, but the sequencing analysis detected other mutations in exon 2. These mutations were found in 7 /20 (35%) patients of HIP-GC group, 5/16(31.25%) patients of HIN-GC group and 4/15(26.7%) patients of the ulcer group.
... BRAF mutants are present in >50% of melanoma patients as well as in colorectal (5−10%), thyroid carcinomas (25−45%), hairy cell leukemia (∼100%) and less commonly in ovarian and lung malignancies. In colorectal cancer (CRC), the missense mutation V600E in BRAF genes occurs in a mutually exclusive fashion with RAS genes alterations, and often indicates a poor outcome in the metastatic setting [13][14][15][16][17]. ...
BRAF is a serine/threonine kinase frequently mutated in human cancers. BRAFV600E mutated protein is targeted through the use of kinase inhibitors which are approved for the treatment of melanoma; however, their long-term efficacy is hampered by resistance mechanisms. The PROTAC-induced degradation of BRAFV600E has been proposed as an alternative strategy to avoid the onset of resistance. In this study, we designed a series of compounds where the BRAF kinase inhibitor encorafenib was conjugated to pomalidomide through different linkers. The synthesized compounds maintained their ability to inhibit the kinase activity of mutated BRAF with IC50 values in the 40–88 nM range. Selected compounds inhibited BRAFV600E signaling and cellular proliferation of A375 and Colo205 tumor cell lines. Compounds 10 and 11, the most active of the series, were not able to induce degradation of mutated BRAF. Docking and molecular dynamic studies, conducted in comparison with the efficient BRAF degrader P5B, suggest that a different orientation of the linker bearing the pomalidomide substructure, together with a decreased mobility of the solvent-exposed part of the conjugates, could explain this behavior.
... The Ras signaling pathway (hsa04014) controls many cellular responses, such as proliferation, survival, and differentiation. Developmental disorders associated with mutations in the Ras pathway share common phenotypic features, including facial abnormalities, cardiac defects, and impaired growth and development [48]. Ubiquitin-mediated proteolysis pathway (hsa04120) regulate nearly every aspect of cellular events in eukaryotes. ...
Background
Chromosomal aneuploidy is the most common birth defect. However, the developmental mechanism and gene expression profile of fetuses with chromosomal aneuploidy are relatively unknown, and the maternal immune changes induced by fetal aneuploidy remain unclear. The inability to obtain the placenta multiple times in real-time is a bottleneck in research on aneuploid pregnancies. Plasma cell-free DNA (cfDNA) carries the gene expression profile information of its source cells and may be used to evaluate the development of fetuses with aneuploidy and the immune changes induced in the mother owing to fetal aneuploidy.
Methods
Here, we carried out whole-genome sequencing of the plasma cfDNA of 101 pregnant women carrying a fetus with trisomy (trisomy 21, n = 42; trisomy 18, n = 28; trisomy 13, n = 31) based on non-invasive prenatal testing (NIPT) screening and 140 normal pregnant women to identify differential genes according to the cfDNA nucleosome profile in the region around the transcription start sites (TSSs).
Results
The plasma cfDNA promoter profiles were found to differ between aneuploid and euploid pregnancies. A total of 158 genes with significant differences were identified, of which 43 genes were upregulated and 98 genes were downregulated. Functional enrichment and signaling pathway analysis were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases found that these signal pathways were mainly related to the coordination of developmental signals during embryonic development, the control of cell growth and development, regulation of neuronal survival, and immune regulation, such as the MAPK, Hippo, TGF-β, and Rap1 signaling pathways, which play important roles in the development of embryonic tissues and organs. Furthermore, based on the results of differential gene analysis, a total of 14 immune-related genes with significant differences from the ImmPort database were collected and analyzed. These significantly different immune genes were mainly associated with the maintenance of embryonic homeostasis and normal development.
Conclusions
These results suggest that the distribution characteristics of cfDNA nucleosomes in maternal plasma can be used to reflect the status of fetal development and changes of the immune responses in trisomic pregnancies. Overall, our findings may provide research ideas for non-invasive detection of the physiological and pathological states of other diseases.
... When K-ras gene is mutated, its GTPase activity is affected due to the lack of hydrolysis of active GTP and GAP-mediated guanine nucleotide exchange factors. In consequence, it is activated via the transduction pathway of growth signals and cellular differentiation, giving rise to the initiation, promotion, and/or progression of CRC [6,7]. Some studies showed that the K-ras oncogene has the necessary characteristics to be used as a biomarker capable of preventing, diagnosing, and even predicting CRC [8][9][10][11]. ...
In the scientific literature, it has been documented that electrochemical genosensors are novel analytical tools with proven clinical diagnostic potential for the identification of carcinogenic processes due to genetic and epigenetic alterations, as well as infectious diseases due to viruses or bacteria. In the present work, we describe the construction of an electrochemical genosensor for the identification of the k12p.1 mutation; it was based on use of Screen-Printed Gold Electrode (SPGE), Cyclic Voltammetry (CV), and Atomic Force Microscopy (AFM), for the monitoring the electron transfer trough the functionalized nanostructured surface and corresponding morphological changes. The sensitivity of the genosensor showed a linear response for the identification of the k12p.1 mutation of the K-ras gene in the concentration range of 10 fM to 1 μM with a detection limit of 7.96 fM in the presence of doxorubicin (Dox) as DNA intercalating agent and indicator of the hybridization reaction. Thus, the electrochemical genosensor developed could be useful for the identification of diseases related with the K-ras oncogene.
... Strong drivers, such as G12D, G12V, and G12C, as well as the Q61L substitution, abolish GAP's action (Fig. 1B). The consequent GTP-bound active K-Ras4B molecules stimulate oncogenic signaling (98)(99)(100)(101)(102). Intrinsic hydrolysis and especially exchange of GDP by GTP to maintain Ras activity are stimulated by weaker drivers such as K-Ras4B A146T (103). ...
A vast effort has been invested in the identification of driver mutations of cancer. However, recent studies and observations call into question whether the activating mutations or the signal strength are the major determinant of tumor development. The data argue that signal strength determines cell fate, not the mutation that initiated it. In addition to activating mutations, factors that can impact signaling strength include (i) homeostatic mechanisms that can block or enhance the signal, (ii) the types and locations of additional mutations, and (iii) the expression levels of specific isoforms of genes and regulators of proteins in the pathway. Since signal levels are largely decided by chromatin structure, they vary across cell types, states, and time windows. A strong activating mutation can be restricted by low expression, whereas a weaker mutation can be strengthened by high expression. Strong signals can be associated with cell proliferation, but too strong a signal may result in oncogene-induced senescence. Beyond cancer, moderate signal strength in embryonic neural cells may be associated with neurodevelopmental disorders, and moderate signals in aging may be associated with neurodegenerative diseases, like Alzheimer's disease. The challenge for improving patient outcomes therefore lies in determining signaling thresholds and predicting signal strength.
... Therefore, whether p53 equips a pro-or antiautophagic signal in any described situation is context dependent. Ras, a small family of GTPases, has a regulatory role in cellular growth and survival [53]. Autophagy, with the assistance of H Ras and K Ras oncoproteins, plays a prosurvival role in tumor cells to help them cope with stress. ...
Autophagy is an evolutionary conserved catabolic process that plays physiological and pathological roles in a cell. Its effect on cellular metabolism, the proteome, and the number and quality of organelles, diversely holds the potential to alter cellular functions. It acts paradoxically in cancer as a tumor inhibitor as well as a tumor promoter. In the early stage of tumorigenesis, it prevents tumor initiation by the so-called “quality control mechanism” and suppresses cancer progression. For late-staged tumors that are exposed to stress, it acts as a vibrant process of degradation and recycling that promotes cancer by facilitating metastasis. Despite this dichotomy, the crucial role of autophagy is evident in cancer, and associated with mammalian targets of rapamycin (mTOR), p53, and Ras-derived major cancer networks. Irrespective of the controversy regarding autophagic manipulation, promotion and suppression of autophagy act as potential therapeutic targets in cancer treatment and may provide various anticancer therapies.
... The RAS-RAF-MEK-ERK protein kinase cascade is a major signaling pathway that stimulates cell proliferation in response to extracellular mitogenic signals [1,2]. Not surprisingly, this pathway is frequently activated in human cancers [3,4]. The complexity of this pathway is compounded by the presence of multiple components [3]: three RAS proteins (H-, N-, and K-RAS), three RAF proteins (A-, B-, and C-RAF), two MEK versions (MEK1 and -2), and two ERK proteins (ERK1 and 2) [5,6]. ...
The RAS–BRAF signaling is a major pathway of cell proliferation and their mutations are frequently found in human cancers. Adenylate kinase 2 (AK2), which modulates balance of adenine nucleotide pool, has been implicated in cell death and cell proliferation independently of its enzyme activity. Recently, the role of AK2 in tumorigenesis was in part elucidated in some cancer types including lung adenocarcinoma and breast cancer, but the underlying mechanism is not clear. Here, we show that AK2 is a BRAF-suppressor. In in vitro assays and cell model, AK2 interacted with BRAF and inhibited BRAF activity and downstream ERK phosphorylation. Energy-deprived conditions in cell model and the addition of AMP to cell lysates strengthened the AK2-BRAF interaction, suggesting that AK2 is involved in the regulation of BRAF activity in response to cell metabolic state. AMP facilitated the AK2–BRAF complex formation through binding to AK2. In a panel of HCC cell lines, AK2 expression was inversely correlated with ERK/MAPK activation, and AK2-knockdown or -knockout increased BRAF activity and promoted cell proliferation. Tumors from HCC patients showed low-AK2 protein expression and increased ERK activation compared to non-tumor tissues and the downregulation of AK2 was also verified by two microarray datasets (TCGA-LIHC and GSE14520). Moreover, AK2/BRAF interaction was abrogated by RAS activation in in vitro assay and cell model and in a mouse model of HRASG12V-driven HCC, and AK2 ablation promoted tumor growth and BRAF activity. AK2 also bound to BRAF inhibitor-insensitive BRAF mutants and attenuated their activities. These findings indicate that AK2 monitoring cellular AMP levels is indeed a negative regulator of BRAF, linking the metabolic status to tumor growth.
... Activating somatic mutations of this proto-oncogene disrupt this complex interplay between GEFs and GAPs [18,19] . GAPs can no longer increase the GTP hydrolyzation of oncogenic K-RAS, leading to a constitutive active mutant (mut) form driving neoplastic transformations in many cancer entities [20][21][22] . With a frequency of 21.6% somatic point mutations in K-RAS are the most common mutations in the RAS gene of all human cancers, followed by an 8.0% incidence of N-RAS mutations and a 3.3% rate of H-RAS mutations [23] . ...
K-RAS is the most frequently mutated oncogene in solid tumors, such as pancreatic, colon or lung cancer. The GTPase K-RAS can either be in an active (GTP-loaded) or inactive (GDP-loaded) form. In its active form K-RAS forwards signals from growth factors, cytokines or hormones to the nucleus, regulating essential pathways, such as cell proliferation and differentiation. In turn, activating somatic mutations of this proto-oncogene deregulate the complex interplay between GAP (GTPase-activating) - and GEF (Guanine nucleotide exchange factor) - proteins, driving neoplastic transformation. Due to a rather shallow surface, K-RAS lacks proper binding pockets for small molecules, hindering drug development over the past thirty years. This review summarizes recent progress in the development of low molecular antagonists and further shows insights of a newly described interaction between mutant K-RAS signaling and PD-L1 induced immunosuppression, giving new hope for future treatments of K-RAS mutated cancer.
... In addition to the high aggressiveness of PC, the poor survival rate might be due to chemotherapeutic resistance and a lack of effective therapeutic targets [3]. Several genetic variations are distinctly related to pancreatic carcinogenesis, such as KRAS, BRCA1, and BRCA2 mutations and Wnt and Notch mutations [4][5][6]. Targeted therapy has not yielded impressive outcomes for PC patients. Thus, exploration of the molecular mechanism of PC and development of new therapeutic targets and agents are urgent. ...
Fbxo45, a conserved F-box protein, comprises of an atypical SKP1, CUL1, F-box protein (SCF) ubiquitin ligase complex that promotes tumorigenesis and development. However, the biological function and molecular mechanisms of Fbxo45 involved in pancreatic carcinogenesis are ambiguous. We conducted several approaches, including transfection, coIP, real-time polymerase chain reaction (RT-PCR), Western blotting, ubiquitin assays, and animal studies, to explore the role of Fbxo45 in pancreatic cancer. Here, we report that USP49 stability is governed by Fbxo45-mediated ubiquitination and is enhanced by the absence of Fbxo45. Moreover, Fbxo45 binds to a short consensus sequence of USP49 through its SPRY domain. Furthermore, Fbxo45-mediated USP49 ubiquitination and degradation are enhanced by NEK6 kinase. Functionally, Fbxo45 increases cell viability and motility capacity by targeting USP49 in pancreatic cancer cells. Xenograft mouse experiments demonstrated that ectopic expression of Fbxo45 enhanced tumor growth in mice and that USP49 overexpression inhibited tumor growth in vivo. Notably, Fbxo45 expression was negatively associated with USP49 expression in pancreatic cancer tissues. Fbxo45 serves as an oncoprotein to facilitate pancreatic oncogenesis by regulating the stability of the tumor suppressor USP49 in pancreatic cancer.
... Approximately 55% of colorectal cancers (CRCs) are driven by mutational activation of KRAS, BRAF, NRAS, and thus are hard-wired to oncogenic RAS/RAF/MEK/ ERK pathway signaling [1][2][3]. CRC tumors with activated RAS/RAF pathway appear to be associated with poor outcomes [4]. Although RAS/RAF-mutated CRC has been targeted for the development of therapeutic inhibitors, they have been largely ineffective, likely due to complex resistance mechanisms such as intrinsic and adaptive resistance [5][6][7][8][9][10][11][12]. ...
Abstract Background Over half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s). Methods We classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi “bypass” resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154). Results Here we report a central role of SRC in mediating “bypass”-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P
... proteins being locked in the active GTP-bound state with consequent permanent activation of downstream RAS/MEK/ERK signaling, which controls cancer cell proliferation and survival [9][10][11] . Following decades of failures, inhibition of KRAS specific subtypes has had recent success [12][13][14][15][16] . ...
The lack of effective RAS inhibition represents a major unmet medical need in the treatment of pancreatic ductal adenocarcinoma (PDAC). Here, we investigate the anticancer activity of RRSP-DTB, an engineered biologic that cleaves the Switch I of all RAS isoforms, in KRAS-mutant PDAC cell lines and patient-derived xenografts (PDXs). We first demonstrate that RRSP-DTB effectively engages RAS and impacts downstream ERK signaling in multiple KRAS-mutant PDAC cell lines inhibiting cell proliferation at picomolar concentrations. We next tested RRSP-DTB in immunodeficient mice bearing KRAS-mutant PDAC PDXs. Treatment with RRSP-DTB led to 95% tumor regression after 29 days. Residual tumors exhibited disrupted tissue architecture, increased fibrosis and fewer proliferating cells compared to controls. Intratumoral levels of phospho-ERK were also significantly lower, indicating in vivo target engagement. Importantly, tumors that started to regrow without RRSP-DTB shrank when treatment resumed, demonstrating resistance to RRSP-DTB had not developed. Tracking persistence of the toxin activity following intraperitoneal injection showed that RRSP-DTB is active in sera from immunocompetent mice for at least one hour, but absent after 16 hours, justifying use of daily dosing. Overall, we report that RRSP-DTB strongly regresses hard-to-treat KRAS-mutant PDX models of pancreatic cancer, warranting further development of this pan-RAS biologic for the management of RAS-addicted tumors.
The RREB1 is a zinc finger transcription factor that plays a role in regulating gene expression and inactivating MAPK signalling components. To date, no pathogenic variant in the RREB1 gene has been associated with any disease, but several cases of 6p terminal deletions affecting the RREB1 gene have been reported. In this study, we report the first case of RREB1‐associated Noonan‐like RASopathy caused by a pathogenic variant within this gene. Genetic testing included whole‐genome sequencing (WGS) of the proband and Sanger sequencing of the proband, his parents, and his sibling. The proband had a de novo c.2677del, p.(Ala893Argfs*20) variant, likely resulting in RREB1 haploinsufficiency. Comparative analysis of patients with microdeletions, including in the RREB1 gene, confirmed shared clinical traits while highlighting unique features, such as blue sclerae and absence of cardiac anomalies. This study reinforces previous data on RREB1 haploinsufficiency as the driver of a new Noonan‐like RASopathy variant, which includes intellectual disability, delayed motor skills, short stature, short neck, and distinctive facial dysmorphisms as key clinical indicators. These findings shed light on this RREB1‐related syndrome and underscore the necessity for further investigation into the functional consequences of RREB1 mutations.
We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. We show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we found that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.
Endometrial cancer (EC) ranks as the second most common gynaecological cancer worldwide. EC patients are diagnosed at an early clinical stage and generally have a good prognosis. Therefore, there is a dire need for development of a specific marker for early detection of endometrial adenocarcinoma. The development of EC is conditioned by a multistep process of oncogenic upregulation and tumor suppressor downregulation as shown by molecular genetic evidence. In this setting, microRNAs appear as significant regulators of gene expression and several variations in the ex-pression of microRNAs have been implicated in normal endometrium, endometrial tissue, metrorrhagia, and endometrial cancer. Furthermore, microRNAs act as highly precise, sensitive, and robust molecules, making them potential markers for diagnosing specific cancers and their progression. With the rising incidence of EC, its management remains a vexing challenge and diagnostic methods for the disease are limited to invasive, expensive, and inaccurate tools. Therefore, the prospect of exploiting the utility of microRNAs as potential candidates for diagnosis and therapeutic use in EC seems promising.
Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal, or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays. The results reveal a bimodal Ras activation response, which is not a result of classic kinetic bistability, but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts classic kinetic bistability and is distinctly more resistant to pharmacological inhibition.
Antecedentes: El síndrome de Noonan es un trastorno genético relacionado principalmente con la mutación del gen PTPN11. Reporte del caso: Recién nacido varón de 34 semanas de edad gestacional con ultrasonidos obstétricos que muestran higroma quístico, hidronefrosis renal bilateral, y polihidramnios. Al nacimiento, presentó edema nucal, puente nasal ancho, pabellón auricular de implantación baja, y criptorquidia derecha. Además, defecto del tabique auricular, ausencia de vena cava inferior, hipertensión pulmonar, conducto arterioso persistente y dificultad respiratoria. El resultado del análisis del panel de 14 genes mostró una mutación del gen MAP2K1 y una variante de significado incierto en el gen CBL, confirmando el diagnóstico del síndrome de Noonan negativo para PTPN11. Durante el seguimiento, también se le diagnosticó blefaroptosis izquierda y reflujo gastroesofágico. Conclusión: El presente caso destaca la amplia variedad de características fenotípicas en un paciente con síndrome de Noonan, con sospecha al nacimiento y confirmado durante el seguimiento.
The purpose of this study is intended to detect and evaluate serum lipid concentrations and establish the interrelationship between the lipid profile variables for both oral and non oral cancer. This case control study included 100 clinically diagnosed and histopathologically confirmed OSCC cases and 100 age and gender matched healthy control subjects who had neither any history of cancer nor suffered from any major illness. Serum lipid concentrations such as total cholesterol, HDL cholesterol and triglycerides were determined by enzymatic colorimetric assays. LDL and VLDL cholesterol concentrations were calculated from the above findings. ANOVA was performed to compare mean, standard deviation and P- values of the parameters using SPSS 15.0 version for windows. The results have shown significantly decreased levels of serum lipid concentrations in OSCC cases when compared to normal control subjects, showing an inverse relation between serum lipid levels and Oral Squamous cell carcinomas. The detected lower concentrations of serum lipid components in the head and neck cancer cases might be due to the utilization of lipids by the cancer cells to maintain cell integrity. Plasma lipid status may be a useful bio-marker indicator for initial changes occurring in neoplastic cells
En dépit de l’émergence des thérapies ciblées et de l’immunothérapie, la chimiothérapie reste un gold-standard pour le traitement de nombreux cancers. Parmi les agents chimiothérapeutiques conventionnels, les poisons du fuseau interférant avec la dynamique des microtubules (taxanes, vinca-alcaloïdes) sont très largement utilisés. Cependant, leurs nombreux effets indésirables et l’émergence de chimiorésistance limitent leur efficacité et soulignent la nécessité d’identifier de nouveaux inhibiteurs de la tubuline.Notre équipe a réalisé un criblage cellulaire à haut-débit sur plus de 7500 molécules chimiques et identifié une famille de composés pyrrolo-pyrimidine, active sur les formes de cancer pulmonaire résistantes à l’apoptose et aux thérapies ciblées. Notre objectif était de caractériser in vitro et in vivo 15 molécules de cette famille afin d’identifier à terme un potentiel candidat-médicament pour le traitement des cancers résistants.Des essais préliminaires de cytotoxicité et d’apoptose sur différentes lignées cancéreuses pulmonaires ont permis de sélectionner, parmi les 15 composés pyrrolo-pyrimidine, 2 molécules prometteuses : PP-2 et PP-13. PP-2 et PP-13 ont des effets cytotoxiques sur de nombreuses lignées cancéreuses humaines, incluant les lignées résistantes aux thérapies ciblées. En perturbant l’organisation et la dynamique des microtubules, PP-2 et PP-13 induisent le blocage transitoire des cellules en prométaphase puis aboutissent aux phénomènes de catastrophe mitotique, de glissement mitotique ou de division asymétrique. Des études mécanistiques avancées montrent que PP-2 et PP-13 sont des agents poisons du fuseau et entrent en compétition avec la colchicine pour la liaison sur la tubuline. Contrairement aux antimitotiques conventionnels, PP-2 et PP-13 ne sont pas sensibles aux mécanismes de chimiorésistance par surexpression de pompes d’efflux. De plus, à l’IC50, ces 2 composés n’affectent pas le réseau microtubulaire des cellules à l’interphase suggérant un effet toxique (et principalement neurotoxique) moindre. La molécule PP-13 semble être la molécule anticancéreuse la plus prometteuse en raison de son IC50 10 fois inférieure à celle de PP-2 dans les différentes lignées cancéreuses étudiées et d’une affinité pour la tubuline 2 fois plus élevée. In vivo, PP-13 réduit significativement la croissance tumorale et étastatique. L’ensemble de ces résultats suggère que PP-13 pourrait être une alternative intéressante pour le traitement de nombreux cancers y compris des cancers chimiorésistants.
Background & objective:
Emerging evidence suggests that KRAS could play an important role in squamous cell carcinoma; however, its role in oral squamous cell carcinoma (OSCC) is largely unknown. The aim of the current study was to investigate the expression of KRAS, Ki-67, Cyclin D1, and Bcl2 in OSCC and their association with clinicopathological features.
Methods:
Forty paraffin blocks of retrospective histologically diagnosed cases of OSCC and 20 blocks of oral leukoplakia with epithelial dysplasia were obtained from two hospitals between 2018 and 2021. The paraffin-embedded tissue was analyzed for the expression of KRAS for oral epithelial dysplasia and OSCC, and ki-67, Cyclin D1, and bcl2 were analyzed only for OSCC. The results were correlated with each other and with different clinicopathological features and were statistically analyzed.
Results:
KRAS expression was significantly associated with histological tumor grade, tumor extent, presence of nodal and distant metastasis, pathological stage, and the presence of lymphovascular invasion (P=<0.001, 0.001, 0.001, 0.009, <0.001, and <0.001, respectively). The KRAS expression was positively correlated with the histological grade, tumor extent, nodal status, and the pathological stage (r=0.712, 0.649, 0.646, and 0.865, respectively). A positive correlation was also found with the expression of Bcl2, Cyclin D1, and Ki-67 (r=0.81, 0.723, and 0.698, respectively). The KRAS expression in oral epithelial dysplasia was significantly lower than that in OSCC (P=0.003).
Conclusion:
KRAS may be a potential prognostic marker for OSCC and may play a role in its progression.
Introduction. The ferns of the genus Pteridium are characterized by their wide habitat and adaptive power that allows their establishment in various types of soils and altitudes. They have the ability to produce a toxin called ptaquiloside. Objective. To gather a current body of updated knowledge on the ptaquiloside toxin present in ferns of the genus Pteridium. Development. The ferns of the genus Pteridium produce ptaquiloside biotoxin, especially in young phenological stages and in the fronds. The hydrolysis of this toxin at the biological level generates compounds with cytotoxic and genotoxic properties. Ptaquiloside can produce enzootic bovine hematuria, be deposited in the milk of the animal that consumes it and affects the health of dairy consumers. The biotoxin has an immunosuppressive effect in cattle, which can favor the papilloma virus, and also causes blindness in sheep. It is associated with esophageal and gastric cancer in humans living in areas populated by ferns of the genus Pteridium. The carcinogenic effect of ptaquiloside is generated because it causes DNA alkylation at N3 adenine or N7 guanine, 24 h after ingestion. For the eradication of ferns in pastures, mechanical control, biological control, and chemical control, are the most important. Conclusion. The biotoxin ptaquiloside represents a risk to human and animal health, which can spread to soil, water bodies, and food. More research is needed to take measures to reduce its effects.
NRas is a key mediator of the mitogenic pathway in normal cells and in cancer. Its dynamics and nanoscale organization at the plasma-membrane (PM) facilitate its signalling. Here, we employed two-color photoactivated localization microscopy to resolve the organization of individual NRas and associated signalling proteins in live melanoma cells, with resolution down to ∼20nm. Upon EGF activation, a fraction of NRas and BRAF (dis)assembled synchronously at the PM in co-clusters. NRas and BRAF clusters associated with GPI-enriched domains, serving as possible nucleation sites for these clusters. NRas and BRAF association in mutual clusters was reduced by the NRas farnesylation inhibitor lonafarnib, yet enhanced by the BRAF inhibitor vemurafenib. Surprisingly, dispersed NRas molecules associated with the periphery of self-clusters of either Grb2 or NF1. Thus, NRas-mediated signalling, which is critical in health and disease, is regulated by dynamic interactions with functional clusters of BRAF or other related proteins at the PM.
Medullary thyroid cancer (MTC) is a rare neuroendocrine tumor originating from parafollicular C cells that produce calcitonin (Ct). It constitutes 2–5% of all thyroid malignancies. It can be seen in sporadic form or hereditary form as a component of multiple neuroendocrine neoplasia (MEN) type 2. Its prognosis is poor compared to differentiated thyroid cancers. At the time of diagnosis, 35–50% of MTC patients are diagnosed with regional metastasis; 13–15% are diagnosed with distant metastasis to organs such as lungs, bones, and liver [1, 2]. According to SEER data, 10-year survival in gland-limited MTCs is approximately 95.6%, and overall survival in patients with regional disease is 75.5% [1]. The 10-year survival rate of those with distant metastasis at the time of diagnosis is approximately 20% [1, 2].
Background
Noonan syndrome (NS) is a genetic disorder characterized by developmental delays, typical facial gestalt and cardiovascular defects. LZTR1 variants have been recently described in patients with NS and schwannomatosis, but the association, inheritance pattern and management strategy has not been fully elucidated. Here, we review the contribution of LZTR1 in NS and describe a patient with a novel, likely pathogenic variant in LZTR1 .
Case presentation
A female patient was diagnosed with clinical NS at 8 months of age. She presented in adulthood when a brain and spine MRI identified plexiform neurofibromas; however, she did not meet the clinical criteria for Neurofibromatosis type 1. No pathogenic variants were identified through molecular genetic analysis of NF1 , SPRED1 and a multigene NS panel. Whole exome sequencing at age 23 identified a novel de novo likely pathogenic heterozygous variant in the LZTR1 gene denoted as c.743G>A (p.Gly248Glu). Serial MRIs have shown stable imaging findings and the patient is being followed clinically by cardiology, neurology and medical genetics.
Conclusions
We identified a novel mutation in the LZTR1 gene, not previously reported in association with NS. This report provides additional evidence to support for the assessment of schwannomatosis in patients with LZTR1 -NS and may have overlap with Neurofibromatosis type 1.
We recently characterized the association of the 44-kDa mitogen-activated protein kinase, also known as extracellular-regulated kinase 1 (ERK1), with the 90-kDa ribosomal S6 kinase (pp90rsk), one of its putative substrates in intact PC12 cells. Using antibodies to ERK1 that precipitate a functional ERK1.pp90rsk phosphoprotein complex, we demonstrate here the regulation of both kinases by various stimuli. In mouse fibroblasts expressing human insulin receptors, insulin and vanadate swiftly stimulated ERK1 activity within 5 min. While the hormonal effect was short-lived, vanadate led to a first peak followed by a progressively increasing second phase. In PC12 cells, epidermal growth factor, which is a growth promoting factor, provokes a rapid but evanescent activation of ERK1. In contrast, nerve growth factor (NGF), which acts as a neuronal differentiation factor for PC12 cells, induced a swift monophasic response followed by a sustained second phase. This strikingly different pattern of ERK1 stimulation by NGF and epidermal growth factor was associated to a contrasting effect on ERK1 cellular translocation. Thus, NGF induced a nuclear translocation of ERK1, while epidermal growth factor was without noticeable effect on ERK1 localization. In both cell systems all effectors tested stimulated ERK1 phosphorylation on both threonine and tyrosine residues in an 1:1 ratio. During ERK1 inactivation, phosphothreonine and phosphotyrosine were dephosphorylated in a similar fashion. Concurrent with ERK1 activation was the de novo appearance of phosphothreonine and an increase in phosphoserine on pp90rsk. The pp90rsk phosphothreonine content paralleled the ERK1 activity more closely than the phosphoserine level. These results provide compelling evidence that in fibroblasts and PC12 cells ERK1 plays a direct role in the phosphorylation of pp90rsk and that pp90rsk represents a physiologically relevant substrate of extracellular-regulated kinases. Finally, we would like to suggest that the differentiating action of NGF in PC12 cells might be due, at least in part, to the conjunction of its sustained and robust stimulation of ERK1 and pp90rsk, and of its induction of ERK1 nuclear translocation.
The discovery of mutations in cancer genes has advanced our understanding of cancer. These results are dispersed across the scientific literature and with the availability of the human genome sequence will continue to accrue. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website have been developed to store somatic mutation data in a single location and display the data and other information related to human cancer. To populate this resource, data has currently been extracted from reports in the scientific literature for somatic mutations in four genes, BRAF, HRAS, KRAS2 and NRAS. At present, the database holds information on 66 634 samples and reports a total of 10 647 mutations. Through the web pages, these data can be queried, displayed as figures or tables and exported in a number of formats. COSMIC is an ongoing project that will continue to curate somatic mutation data and release it through the website.Keywords: somatic, mutation, database, website
Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras+/− mice produced H-ras−/− offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-ras did not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras−/− mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras−/− mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras−/−/N-ras−/−) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-than-expected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development. This work was supported by FEDER grant 1FD1997-1735 from Ministerio de Ciencia y Tecnología, Spain. Peer reviewed
Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's “two hit” model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1−/− murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W41 mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras–mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W41) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.
Expression of the mouse beta-PDGF receptor by gene transfer confers PDGF-dependent and reversible neuronal differentiation of PC12 pheochromocytoma cells similar to that observed in response to NGF and basic FGF. A common property of the PDGF, NGF, and basic FGF-induced differentiation response is the requirement for constant exposure of cells to the growth factor. To test the hypothesis that a persistent level of growth factor receptor signaling is required for the maintenance of the neuronal phenotype, we examined the regulation of the serine/threonine-specific MAP kinases after either short- (10 min) or long-term (24 h) stimulation with growth factors. Mono Q FPLC resolved two peaks of growth factor-stimulated MAP kinase activity that coeluted with tyrosine phosphorylated 41- and 43-kDa polypeptides. MAP kinase activity was markedly stimulated (approximately 30-fold) within 5 min of exposure to several growth factors (PDGF, NGF, basic FGF, EGF, and IGF-I), but was persistently maintained at 10-fold above basal activity after 24 h only by the growth factors that also induce PC12 cell differentiation (PDGF, NGF, and basic FGF). Thus the beta-PDGF receptor is in a subset of tyrosine kinase-encoded growth factor receptors that are capable of maintaining continuous signals required for differentiation of PC12 cells. These signals include the constitutive activation of cytoplasmic serine/threonine protein kinases.
The rate constants have been determined for elementary steps in the basal GTPase mechanism of normal p21N-ras (Gly-12) and an oncogenic mutant (Asp-12): namely GTP binding, hydrolysis, phosphate release, and GDP release. By extrapolation from data at lower temperatures, the GTP association rate constant at 37 degrees C is 1.4 x 10(8) M-1 s-1 for the normal protein and 4.8 x 10(8) M-1 s-1 for the mutant. Other rate constants were measured directly at 37 degrees C, and three processes have similar slow values. GTP dissociation is at 1.0 x 10(-4) s-1 (normal) and 5.0 x 10(-4) s-1 (mutant). The hydrolysis step is at 3.4 x 10(-4) s-1 (normal) and 1.5 x 10(-4) s-1 (mutant). GDP dissociates at 4.2 x 10(-4) s-1 (normal) and 2.0 x 10(-4) s-1 (mutant). GDP association rate constants are similar to those for GTP, 0.5 x 10(8) M-1 s-1 for normal and 0.7 x 10(8) M-1 s-1 for mutant. Both hydrolysis and GDP release therefore contribute to rate limitation of the basal GTPase activity. There are distinct differences (up to 5-fold) between rate constants for the normal and mutant proteins at a number of steps. The values are consistent with the reduced GTPase activity for this mutant and suggest little difference between normal and mutant proteins in the relative steady-state concentrations of GTP and GDP complexes that may represent active and inactive states. The results are discussed in terms of the likely role of p21ras in transmembrane signalling.
Kinetic studies performed on p21H guanine nucleotide complexes with and without Mg2+ show that point mutations at positions 12, 59, and 61 each have a different effect on the rate of nucleotide dissociation. Double mutants with a combination of these amino acid substitutions reveal that the effects of each mutation on these kinetics are interactive (nonadditive) for positions 12 and 59 and approximately additive for the positions 12 and 61. The magnitude and direction of the effects seen are dependent on the nature of the nucleotide and whether or not the complexes contain Mg2+. All the mutants have reduced GTPase activity. It is also shown that the autophosphorylation reaction velocity is of first order with respect to the protein concentration and that this reaction is an intramolecular one, which takes place as a side reaction of the GTPase reaction. The autophosphorylation is not reversible under the experimental conditions. The covalently bound phosphate does not decrease the nucleotide-binding ability of the protein nor does it change the relative affinity of the protein for GTP versus GDP. The results are discussed in terms of the structural model and function of p21H.
We expressed six forms of p21-ras polypeptides in Escherichia coli with differing transformation potentials resulting from
amino acid substitutions at position 12. The ability of the encoded p21's to autophosphorylate, bind guanine nucleotides,
and hydrolyze GTP was assessed. All versions of p21 bound GTP equivalently; the kinase activity, while dependent upon residue
12, did not correlate with the transforming potential of the polypeptide. All transforming versions exhibited an impaired
GTPase activity, while a novel nontransforming derivative [p21(pro-12)] possessed an enhanced GTPase activity. These results
provide strong support for the proposal that an impairment of the cellular p21 GTPase activity can unmask its transforming
potential.
Because the protein-tyrosine phosphatase (PTP) Syp associates with the tyrosine-phosphorylated platelet-derived growth factor beta receptor (beta PDGFR), the beta PDGFR is a likely Syp substrate. We tested this hypothesis by determining whether recombinant Syp (rSyp) and a control PTP, recombinant PTP1B (rPTP1B), were able to dephosphorylate the beta PDGFR. The beta PDGFR was phosphorylated at multiple tyrosine residues in an in vitro kinase assay and then incubated with increasing concentrations of rSyp or rPTP1B. While the receptor was nearly completely dephosphorylated by high concentrations of rPTP1B, receptor dephosphorylation by rSyp plateaued at approximately 50%. Two-dimensional phosphopeptide maps of the beta PDGFR demonstrated that rSyp displayed a clear preference for certain receptor phosphorylation sites; the most efficiently dephosphorylated sites were phosphotyrosines (Tyr(P)-771 and -751, followed by Tyr(P)740, while Tyr(P)-1021 and Tyr(P)-1009 were very poor substrates. In contrast, rPTP1B displayed no selectivity for the various rPTP1B displayed no selectivity for the various beta PDGFR tyrosine phosphorylation sites and dephosphorylated all of them with comparable efficiency. A Syp construct that lacked the SH2 domains was still able to discriminate between the various receptor phosphorylation sites, although less effectively than full-length Syp. These in vitro studies predicted that Syp can dephosphorylate the receptor in vivo. Indeed, we found that a beta PDGFR mutant (F1009) that associates poorly with Syp, had a much slower in vivo rate of receptor dephosphorylation than the wild type receptor. In addition, the GTPase-activating protein of Ras (GAP) and phosphatidylinositol 3-kinase were less stably associated with the wild type beta PDGFR than with the F1009 receptor. These findings are consistent with the in vitro experiments showign that Syp prefers to dephosphorylate sites on the beta PDGFR, that are important for binding phosphatidylinositol 3-kinase (Tyr(P)-740 and Tyr(P)-751) and GAP (Tyr(P)-771). These studies reveal that Syp is a substrate-selective PTP and that both the catalytic domain and the SH2 domains contribute to Syp's ability to choose substrates. Furthermore, it appears that Syp plays a role in PDGF-dependent intracellular signal relay by selectively dephosphorylating the beta PDGFR and thereby regulating the binding of a distinct group of receptor-associated signal relay enzymes.
The mammalian ras gene family encodes key cell-signaling, cell growth-related proteins that have been highly conserved in species from yeast to man. Specific point mutations in the ras genes are associated with various mammalian tumors. To understand the developmental role of the N-ras protooncogene in the mouse, we have disrupted its gene function by homologous recombination in embryonic stem cells. Mice derived from these cells that are homozygous for the N-ras mutation do not produce any detectable N-Ras protein and are morphologically and histologically indistinguishable from their heterozygous and wild-type siblings. Since N-ras is expressed at high levels in hematopoietic cells, we examined different populations of cells in peripheral blood and found no differences between mutant and normal animals. Our results show that N-ras gene function is dispensable for normal mouse development, growth, and fertility.
Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.
Ras (p21ras) interacts directly with the catalytic subunit of phosphatidylinositol-3-OH kinase in a GTP-dependent manner through the Ras effector site. In vivo, dominant negative Ras mutant N17 inhibits growth factor induced production of 3' phosphorylated phosphoinositides in PC12 cells, and transfection of Ras, but not Raf, into COS cells results in a large elevation in the level of these lipids. Therefore Ras can probably regulate phosphatidylinositol-3-OH kinase, providing a point of divergence in signalling pathways downstream of Ras.
GTPases of the Ras superfamily regulate many aspects of cell growth, differentiation and action. Their functions depend on their ability to alternate between inactive and active forms, and on their cellular localization. Numerous proteins affecting the GTPase activity, nucleotide exchange rates and membrane localization of Ras superfamily members have now been identified. Many of these proteins are much larger and more complex than their targets, containing multiple domains capable of interacting with an intricate network of cellular enzymes and structures.
Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.
The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase
(GTPase)–activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly
hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of
the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active
site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334,
thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural
arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation
for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van
der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb
the arrangements of residues in the transition state.
ras genes encode members of the small GTP-binding proteins. Ras protein in highly conserved in various species from yeast to humans and plays a key role in signal transduction. Ras is related to cell proliferation and differentiation. While, in addition, mutations in the ras genes are implicated in a variety of tumors. However, the physiological functions and specific roles of each ras gene, H-ras, K-ras and N-ras, are still not fully understood. To clarify the role of the K-Ras in vivo, we generated K-ras mutant mice by gene targeting. In contrast to the findings that H-Ras-deficient mice and N-Ras-deficient mice are born and grow normally, the K-Ras-deficient embryos die progressively between embryonic day 12.5 and term. At embryonic day 15.5, their ventricular walls are extremely thin. Besides, at embryonic day 11.5, they demonstrate increased cell death of motoneurons in the medulla and the cervical spinal cord. Our results thus indicate K-Ras to be essential for normal development in mice and residual Ras composed of H-Ras and N-Ras cannot compensate for the loss of K-Ras function in the mutant mice.
Mammalian ras genes are thought to be critical in the regulation of cellular proliferation and differentiation and are mutated in approximately 30% of all human tumors. However, N-ras and H-ras are nonessential for mouse development. To characterize the normal role of K-ras in growth and development, we have mutated it by gene targeting in the mouse. On an inbred genetic background, embryos homozygous for this mutation die between 12 and 14 days of gestation, with fetal liver defects and evidence of anemia. Thus, K-ras is the only member of the ras gene family essential for mouse embryogenesis. We have also investigated the effect of multiple mutations within the ras gene family. Most animals lacking N-ras function and heterozygous for the K-ras mutation exhibit abnormal hematopoietic development and die between days 10 and 12 of embryogenesis. Thus, partial functional overlap appears to occur within the ras gene family, but K-ras provides a unique and essential function.
Nat. Genet. 34, 148–150 (2003). The subpanels in Figure 1 were labeled incorrectly. A corrected version appears below.
MULTIPLE lentigenes have been reported in association with a variety of abnormalities and a familial occurrence. It is likely, on the basis of the following literature review and additional case material, that multiple lentigenes commonly represent one facet of a more generalized hereditary syndrome.Zeisler and Becker1 described a 24-year-old woman who experienced a marked increase in number of lentigenes from birth to puberty. There were none on the face, but otherwise they were generally distributed. The patient also had pectus carinatum, ocular hypertelorism, and mandibular prognathism. Rosen2 reported three siblings (two male and one female) with large numbers of lentigenes, but no mention was made of other disorders. A large pedigree in which eight individuals in three generations had lentigenes was reported by Pipkin and Pipkin.3 Seven affected members had nystagmus, and photographs of some members gave evidence of mandibular prognathism. The pedigrees suggested an autosomal
Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kγ is strongly and directly activated by H-Ras G12V in vivo or by GTPγS-loaded H-Ras in vitro . We have determined a crystal structure of a PI3Kγ/Ras·GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kγ. Mutagenesis shows that interactions with both regions are essential for binding PI3Kγ. Ras also forms a direct contact with the PI3Kγ catalytic domain. These unique Ras/PI3Kγ interactions are likely to be shared by PI3Kα. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.
Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.
Initiation Factor-2 (eIF-2) mediates the early steps of the initiation of protein synthesis in eukaryotic cells. Over the last 15 years evidence has accumulated linking the phosphorylation of eIF-2 with the control of translation, but the mechanism by which phosphorylation of this protein affects its biological activity has only recently been established. It involves the exchange of guanine nucleotides bound to eIF-2 and resembles mechanisms involved in regulating other, quite diverse, biological processes.
A single-point mutation in exon 15 of the BRAF gene has recently been reported in a high percentage in cultured melanoma cells and in 6 of 9 primary melanomas examined. To evaluate the impact of the T1796A BRAF mutation, we screened primary melanomas, various types of nevi and lesions where a melanoma developed in an underlying nevus. We could detect the mutation in 28 of 97 (29%) melanomas and in 39 of 187 (21%) nevi, including blue nevi (0/20) and Spitz nevi (0/69), which did not carry the mutation. In melanomas with an underlying nevus, either the mutation was present in both the laser-microdissected nevus cells and the laser-microdissected melanoma cells (3/14) or both lesions were negative for the BRAF mutation except one case. In conclusion, mutations in exon 15 of the BRAF gene are nonspecific for progression of a nevus to a melanoma. Other so far unknown cofactors seem to be of importance.Keywords: b-raf, nevus, point mutation
Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLC, PLC and PLC, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLC, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While Gq, G or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras–effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.
The ras gene product (p21) is thought to transduce signals from various growth and differentiation factors. p21 is a GTP-binding protein, and its activity is regulated by the bound GDP/GTP ratio. We analysed p21-bound nucleotides in cell lysates of rat pheochromocytoma cell line PC12 cells stimulated with various factors. Nerve growth factors (NGF) rapidly increased the relative amount of active p21-GTP complex to as much as 20% of the total amount of p21 within 2 min. The amount of p21-GTP then declined to 8% after 10 min, and this level was sustained for at least 2 h. Epidermal growth factor (EGF) also stimulated a rapid accumulation of p21-GTP to the same extent as seen with NGF, but the amount of p21-GTP declined to 5% after 10 min and gradually returned to the basal level within 60 min. In contrast, basic fibroblast growth factor, interleukin 6 and dibutyryl cAMP, which induce neuronal differentiation of PC12 cells, did not stimulate the accumulation of p21-GTP at any time point examined. Phorbol 12-myristate 13-acetate also had no effect. Interestingly, the protein kinase inhibitor K-252a specifically suppressed the NGF-induced accumulation of p21-GTP, but did not suppress the EGF-induced response. These results strongly suggest that an active p21-GTP complex transduces the differentiation signal from NGF. It may also be suggested that the process of activating p21 is mediated by a K-252a-sensitive protein kinase(s).
Three new neurofibromatosis type 1 (NF1) mutations have been detected and characterized. Pulsed-field gel and Southern blot analyses reveal the mutations to be deletions of 190, 40, and 11 kb of DNA. The 11 kb deletion does not contain any of the previously characterized genes that lie between two NF1 translocation breakpoints, but it does include a portion of a rodent/human conserved DNA sequence previously shown to span one of the translocation breakpoints. By screening cDNA libraries with the conserved sequence, we identified a number of cDNA clones from the translocation breakpoint region (TBR), one of which hybridizes to an approximately 11 kb mRNA. The TBR gene crosses at least one of the chromosome 17 translocation breakpoints found in NF1 patients. Furthermore, the newly characterized NF1 deletions remove internal exons of the TBR gene. Although these mutations might act by compromising regulatory elements affecting some other gene, these findings strongly suggest that the TBR gene is the NF1 gene.
Von Recklinghausen neurofibromatosis, or type 1 neurofibromatosis (NF1), is a common autosomal dominant disorder characterized by abnormalities in multiple tissues derived from the embryonic neural crest. Portions of the gene have been recently identified by positional cloning, and sequence analysis has shown homology to the GTPase activating protein (GAP) family. In this report we present the results of an extensive cDNA walk resulting in the cloning of the complete coding region of the NF1 transcript. Analysis of the sequences reveals an open reading frame of 2818 amino acids, although alternatively spliced products may code for different protein isoforms. The gene extends for approximately 300 kb on chromosome 17, with its promoter in a CpG-rich island.
GTPases are conserved molecular switches, built according to a common structural design. Rapidly accruing knowledge of individual GTPases--crystal structures, biochemical properties, or results of molecular genetic experiments--support and generate hypotheses relating structure to function in other members of the diverse family of GTPases.
Overlapping cDNA clones from the translocation breakpoint region (TBR) gene, recently discovered at the neurofibromatosis type 1 locus and found to be interrupted by deletions and a t(17;22) translocation, have been sequenced. A 4 kb sequence of the transcript of the TBR gene has been compared with sequences of genomic DNA, identifying a number of small exons. Identification of splice junctions and a large open reading frame indicates that the gene is oriented with its 5' end toward the centromere, in opposition to the three known active genes in the region. PCR amplification of a subset of the exons, followed by electrophoresis of denatured product on native gels, identified six variant conformers specific to NF1 patients, indicating base pair changes in the gene. Sequencing revealed that one mutant allele contains a T----C transition changing a leucine to a proline; another NF1 allele harbors a C----T transition changing an arginine to a stop codon. These results establish the TBR gene as the NF1 gene and provide a description of a major segment of the gene.
cDNA walking and sequencing have extended the open reading frame for the neurofibromatosis type 1 gene (NF1). The new sequence now predicts 2485 amino acids of the NF1 peptide. A 360 residue region of the new peptide shows significant similarity to the known catalytic domains of both human and bovine GAP (GTPase activating protein). A much broader region, centered around this same 360 amino acid sequence, is strikingly similar to the yeast IRA1 product, which has a similar amino acid sequence and functional homology to mammalian GAP. This evidence suggests that NF1 encodes a cytoplasmic GAP-like protein that may be involved in the control of cell growth by interacting with proteins such as the RAS gene product. Mapping of the cDNA clones has confirmed that NF1 spans a t(1;17) translocation mutation and that three active genes lie within an intron of NF1, but in opposite orientation.
The neurofibromatosis type 1 (NF1) protein contains a region of significant sequence similarity to ras p21 GTPase-activating protein (GAP) and the yeast IRA1 gene product. A fragment of NF1 cDNA encoding the GAP-related domain (NF1 GRD) was expressed, immunoaffinity purified, and assayed for effects on N-ras p21 GTPase activity. The GTPase of wild-type ras p21 was stimulated by NF1 GRD, but oncogenic mutants of ras p21 (Asp-12 and Val-12) were unaffected, and the GTPase of an effector mutant (Ala-38) was only weakly stimulated. NF1 GRD also down-regulated RAS function in S. cerevisiae. The affinity of NF1 GRD for ras p21 was estimated to be 250 nM: this is more than 20-fold higher than the affinity of GAP for ras p21. However, its specific activity was about 30 times lower. These kinetic measurements suggest that NF1 may be a significant regulator of ras p21 activity, particularly at low ras p21 concentrations.
Proteins that bind and hydrolyse GTP are being discovered at a rapidly increasing rate. Each of these many GTPases acts as a molecular switch whose 'on' and 'off' states are triggered by binding and hydrolysis of GTP. Conserved structure and mechanism in myriad versions of the switch--in bacteria, yeast, flies and vertebrates--suggest that all derive from a single primordial protein, repeatedly modified in the course of evolution to perform a dazzling variety of functions.
Mutations in codon 12, 13, or 61 of one of the three ras genes, H-ras, K-ras, and N-ras, convert these genes into active oncogenes. Rapid assays for the detection of these point mutations have been developed recently and used to investigate the role mutated ras genes play in the pathogenesis of human tumors. It appeared that ras gene mutations can be found in a variety of tumor types, although the incidence varies greatly. The highest incidences are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%); in thyroid tumors (50%); and in myeloid leukemia (30%). For some tumor types a relationship may exist between the presence of a ras mutation and clinical or histopathological features of the tumor. There is some evidence that environmental agents may be involved in the induction of the mutations.
The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation
of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and
Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were
found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate
(GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic
protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12
p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts
from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive,
GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations
is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound
state.
Using site-directed mutagenesis, we have introduced mutations encoding 17 different amino acids at codon 61 of the human rasH gene. Fifteen of these substitutions increased rasH transforming activity. The remaining two mutants, encoding proline and glutamic acid, displayed transforming activities similar to the normal gene. Overall, these mutants vary over 1000-fold in transforming potency. Increased levels of p21 expression were required for transformation by weakly transforming mutants. The mutant proteins were unaltered in guanine nucleotide binding properties. However, all 17 different mutant proteins displayed equivalently reduced rates of GTP hydrolysis, 8- to 10-fold lower than the normal protein. There was no quantitative correlation between reduction in GTPase activity and transformation, indicating that reduced GTP hydrolysis is not sufficient to activate ras transforming potential.
Vertebrate genomes contain proto-oncogenes whose enhanced expression or alteration by mutation seems to be involved in the development of naturally occurring tumours. These activated genes, usually assayed by their ability to induce the malignant transformation of NIH 3T3 cells, are frequently related to the ras oncogene of Harvey (Ha-ras) or Kirsten (Ki-ras) murine sarcoma viruses, or a third member of this family (N-ras). Activation involves point mutation which often affect codon 12 (refs 16-26) of the encoded 21,000-molecular weight polypeptide (p21). To provide insight into structural requirements involved in p21 activation, we have now constructed 20 mutant c-Ha-ras1 genes by in vitro mutagenesis, each encoding a different amino acid at codon 12. Analysis of rat fibroblasts transfected with these altered genes demonstrates that all amino acids except glycine (which is encoded by normal cellular ras genes) and proline at position 12 activate p21, suggesting a requirement for an alpha-helical structure in this region of the polypeptide. The morphological phenotype of cells transformed by the activated genes can, however, depend on the particular amino acid at this position.
The 21-kilodalton protein (p21) encoded by normal cellular Harvey-ras has been expressed in Escherichia coli as a fusion protein by using the pUC8 vector and has been purified to greater than 95% homogeneity by ion-exchange chromatography and gel filtration. The purified protein molecules possess intrinsic GTPase activity on the basis of the following criteria: (i) elution of the GTPase activity with p21 GDP-binding activity in two different chromatography systems, (ii) parallel thermal inactivation of GTPase activity and p21 GTP-binding activity, and (iii) immunoprecipitation of the GTPase activity with monoclonal antibodies to p21. At 37 degrees C, the rate of GTP hydrolysis by the purified normal p21 assayed in solution was 5.3-6.6 mmol/min per mol of p21. The rate of GTP hydrolysis by a form of p21 [Val12] encoded by a human oncogene was significantly lower (1.4-1.9 mmol/min per mol of p21). The presence of a threonine phosphate acceptor site at residue 59 also decreased p21 GTPase activity. For regulatory proteins that use GTP as part of their biochemical mechanism, the hydrolysis of GTP to GDP reverses the biological activity of the respective proteins. The observation that oncogenic forms of p21 lose GTPase activity suggests that GTP hydrolysis may be a biochemical event that inactivates the growth-promoting effects of a p21 X GTP complex.
A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. The method uses an
interferon-responsive promoter to control the expression of Cre recombinase. Here, Cre was used to delete a segment of the
DNA polymerase beta gene flanked by IoxP recombinase recognition sites. Deletion was complete in liver and nearly complete
in lymphocytes within a few days, whereas partial deletion was obtained in other tissues. This method can be used for the
inducible inactivation of any other gene in vivo.
A number of different intracellular signaling pathways have been shown to be activated by receptor tyrosine kinases. These activation events include the phosphoinositide 3-kinase, 70 kDa S6 kinase, mitogen-activated protein kinase (MAPK), phospholipase C-gamma, and the Jak/STAT pathways. The precise role of each of these pathways in cell signaling remains to be resolved, but studies on the differentiation of mammalian PC12 cells in tissue culture and the genetics of cell fate determination in Drosophila and Caenorhabditis suggest that the extracellular signal-regulated kinase (ERK-regulated) MAPK pathway may be sufficient for these cellular responses. Experiments with PC12 cells also suggest that the duration of ERK activation is critical for cell signaling decisions.
We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.
The mutation rate of the neurofibromatosis type 1 (NF1) gene is one of the highest in the human genome, with about 50 percent of cases being due to new mutations. We describe a family in which neurofibromatosis type 1 occurred in two siblings with clinically normal parents, and we demonstrate germ-line mosaicism in the father.
We studied lymphocyte DNA from each member of the family and the father's spermatozoa for several polymorphic intragenic markers of the NF1 gene. Southern blots of DNA digested with several enzymes were hybridized with complementary DNA and individual NF1 exon probes to search for alterations in the gene.
The affected siblings, with a clinically severe form of neurofibromatosis type 1, showed no inheritance of paternal alleles for a marker in intron 38 of the NF1 gene, whereas they received alleles from both parents for other NF1 markers. Analysis with probes from this region of the NF1 gene showed a 12-kb deletion of the NF1 gene, involving exons 32 to 39, in the affected offspring. Ten percent of the father's spermatozoa carried the same NF1 deletion, but the abnormality was not detected in DNA from his lymphocytes.
The presence of the NF1 mutation in 10 percent of the clinically normal father's spermatozoa supports the hypothesis that most germ-line mutations occur in precursors of gametes. In cases of spontaneous mutation, analyzing the specific NF1 mutation in the father's sperm might help in the detection of mosaicism and thus facilitate genetic counseling about further pregnancies.
Protein-tyrosine-phosphatase SHPTP2 (Syp/PTP-1D/PTP2C) is the homologue of the Drosophila corkscrew (csw) gene product, which transmits positive signals from receptor tyrosine kinases. Likewise, SHPTP2 has been implicated in positive signaling from platelet-derived growth factor receptor beta (PDGFR). Upon PDGF stimulation, SHPTP2 binds to the PDGFR and becomes tyrosine-phosphorylated. We have identified tyrosine-542 (pY542TNI) as the major in vivo site of SHPTP2 tyrosine phosphorylation. The pY542TNI sequence conforms to the consensus binding site for the SH2 domain of Grb2, which, by association with Sos1, couples some growth factor receptors to Ras. Following PDGF stimulation, Grb2 binds tyrosine-phosphorylated SHPTP2. Moreover, a mutant PDGFR lacking its SHPTP2 binding site displays markedly reduced Grb2 binding. These data indicate that phosphorylation of SHPTP2 couples Grb2 to PDGFR in vivo, providing a mechanism for Ras activation by PDGFR and for positive signaling via SHPTP2 and Csw.
Genetic, physical and biochemical methods have been used successfully to identify discrete regions of the p21ras protein involved in protein-protein interactions. Of special interest are the effector residues of p21ras, which are essential for downstream signalling. This review details current understanding of what these residues are and how they bind and activate proteins essential to the ras pathway.
The three-dimensional structures and biochemical properties of two mutants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to the most frequent oncogenic and the only nononcogenic mutation of Gly-12, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the atomic model of the protein shows the most drastic changes of structure around the active site as compared to wild-type p21. This is due to the interactions of the aspartic acid side chain with Tyr-32, Gln-61, and the gamma-phosphate, which result in reduced mobility of these structural elements. The interaction between the carboxylate group of Asp-12 and the gamma-phosphate is mediated by a shared proton, which we show by 31P NMR measurements to exist in solution as well. The structure of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water. The pyrrolidine ring of Pro-12 points outward and seems to be responsible for the weaker affinity toward GAP (GTPase-activating protein) and the failure of GAP to stimulate GTP hydrolysis.
The RAS guanine nucleotide binding proteins activate multiple signaling events that regulate cell growth and differentiation. In quiescent fibroblasts, ectopic expression of activated H-RAS (H-RASV12, where V12 indicates valine-12) induces membrane ruffling, mitogen-activated protein (MAP) kinase activation, and stimulation of DNA synthesis. A mutant of activated H-RAS, H-RASV12C40 (where C40 indicates cysteine-40), was identified that was defective for MAP kinase activation and stimulation of DNA synthesis, but retained the ability to induce membrane ruffling. Another mutant of activated H-RAS, H-RASV12S35 (where S35 indicates serine-35), which activates MAP kinase, was defective for stimulation of membrane ruffling and induction of DNA synthesis. Expression of both mutants resulted in a stimulation of DNA synthesis that was comparable to that induced by H-RASV12. These results indicate that membrane ruffling and activation of MAP kinase represent distinct RAS effector pathways and that input from both pathways is required for the mitogenic activity of RAS.
The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.
Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematode Caenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras. To search for other effectors in C. elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210. PLC210 possesses two additional functional domains unseen in any known PI-PLCs. One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology to guanine nucleotide exchange proteins for Ras. These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras.