NRAS
GTPase NRas precursor (EC 3.6.5.2) (Transforming protein N-Ras) [HRAS1]
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Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by peribiliary inflammation and fibrosis. Cholangiocyte senescence is a prominent feature of PSC. Here, we hypothesize that extracellular vesicles (EVs) from senescent cholangiocytes influence the phenotype of target cells. EVs were isolated from normal human cholangiocytes (NHCs), cholangiocytes from PSC patients and NHCs experimentally induced to senescence. NHCs, malignant human cholangiocytes (MHCs) and monocytes were exposed to 10 EVs from each donor cell population and assessed for proliferation, MAPK activation and migration. Additionally, we isolated EVs from plasma of wild-type and Mdr2 mice (a murine model of PSC), and assessed mouse monocyte activation. EVs exhibited the size and protein markers of exosomes. The number of EVs released from senescent human cholangiocytes was increased; similarly, the EVs in plasma from Mdr2 mice were increased. Additionally, EVs from senescent cholangiocytes were enriched in multiple growth factors, including EGF. NHCs exposed to EVs from senescent cholangiocytes showed increased NRAS and ERK1/2 activation. Moreover, EVs from senescent cholangiocytes promoted proliferation of NHCs and MHCs, findings that were blocked by erlotinib, an EGF receptor inhibitor. Furthermore, EVs from senescent cholangiocytes induced EGF-dependent Interleukin 1-beta and Tumour necrosis factor expression and migration of human monocytes; similarly, Mdr2 mouse plasma EVs induced activation of mouse monocytes. The data continue to support the importance of cholangiocyte senescence in PSC pathogenesis, directly implicate EVs in cholangiocyte proliferation, malignant progression and immune cell activation and migration, and identify novel therapeutic approaches for PSC.
Keywords
- biliary epithelial cell
- cellular senescence
- extracellular vesicles
- primary sclerosing cholangitis
- senescence-associated secretory phenotype
Melanoma patients carrying an oncogenic [i]NRAS[/i] mutation represent 20% of all cases and present worse survival, relapse rate and therapy response than patients with wild type [i]NRAS[/i] or with [i]BRAF[/i] mutations. Nevertheless, no efficient targeted therapy has emerged so far for this group of patients in comparison with the classical combination of BRAF and MEK inhibitors for the patient group carrying a [i]BRAF[/i] mutation. NRAS key downstream actors should therefore be identified for drug targeting, possibly in combination with MEK inhibitors. Here, we investigated the influence of different melanoma-associated [i]NRAS[/i] mutations (codon 12, 13 or 61) on several parameters such as oncogene-induced senescence, cell proliferation, migration or colony formation in immortalized melanocytes and in melanoma cell lines. We identified AXL/STAT3 axis as a main regulator of [i]NRASQ61[/i]-induced oncogene-induced senescence (OIS) and observed that [i]NRASQ61[/i] mutations are not only more tumorigenic than [i]NRASG12/13[/i] mutations but also associated to STAT3 activation. In conclusion, these data bring new evidence of the potential tumorigenic role of STAT3 in [i]NRAS[/i]-mutant melanomas and will help improving current therapy strategies for this particular patient group.
Keywords
- NRAS
- STAT3
- melanoma
- mutation
- oncogene-induced senescence
DNMT3A R882H, a frequent mutation in acute myeloid leukemia (AML), plays a critical role in malignant hematopoiesis. Recent findings suggest that DNMT3A mutant acts as a founder mutation and requires additional genetic events to induce full-blown AML. Here, we investigated the cooperation of mutant DNMT3A and NRAS in leukemogenesis by generating a double knock-in (DKI) mouse model harboring both Dnmt3a R878H and Nras G12D mutations. DKI mice with both Dnmt3a R878H and Nras G12D mutations were generated by crossing Dnmt3a R878H knock-in (KI) mice and Nras G12D KI mice. Routine blood test, flow cytometry analysis and morphological analysis were performed to determine disease phenotype. RNA-sequencing (RNA-seq), RT-PCR and Western blot were carried out to reveal the molecular mechanism. The DKI mice developed a more aggressive AML with a significantly shortened lifespan and higher percentage of blast cells compared with KI mice expressing Dnmt3a or Nras mutation alone. RNA-seq analysis showed that Dnmt3a and Nras mutations collaboratively caused abnormal expression of a series of genes related to differentiation arrest and growth advantage. Myc transcription factor and its target genes related to proliferation and apoptosis were up-regulated, thus contributing to promote the process of leukemogenesis. This study showed that cooperation of DNMT3A mutation and NRAS mutation could promote the onset of AML by synergistically disturbing the transcriptional profiling with Myc pathway involvement in DKI mice.
MeSH Terms
- Animals
- Apoptosis
- Carcinogenesis
- Cell Differentiation
- DNA (Cytosine-5-)-Methyltransferases
- Disease Models, Animal
- Disease Progression
- Gene Expression Regulation, Neoplastic
- Gene Knock-In Techniques
- Leukemia, Myeloid, Acute
- Longevity
- Mice
- Mice, Inbred C57BL
- Mice, Transgenic
- Monomeric GTP-Binding Proteins
- Mutation
- Phenotype
- Pilot Projects
- Proto-Oncogene Proteins c-myc
- RNA-Seq
- Transcription, Genetic
Keywords
- Acute myeloid leukemia
- DNMT3A mutation
- Myc activation
- Nras G12D
Recent progress in next-generation sequencing technologies allows us to discover frequent mutations throughout the coding regions of myelodysplastic syndromes (MDS), potentially providing us with virtually a complete spectrum of driver mutations in this disease. As shown by many study groups these days, such driver mutations are acquired in a gene-specific fashion. For instance, DDX41 mutations are observed in germline cells long before MDS presentation. In blood samples from healthy elderly individuals, somatic DNMT3A and TET2 mutations are detected as age-related clonal hematopoiesis and are believed to be a risk factor for hematological neoplasms. In MDS, mutations of genes such as NRAS and FLT3, designated as Type-1 genes, may be significantly associated with leukemic evolution. Another type (Type-2) of genes, including RUNX1 and GATA2, are related to progression from low-risk to high-risk MDS. Overall, various driver mutations are sequentially acquired in MDS, at a specific time, in either germline cells, normal hematopoietic cells, or clonal MDS cells.
MeSH Terms
- Aging
- DEAD-box RNA Helicases
- Genome, Human
- Humans
- Mutation
- Myelodysplastic Syndromes
- Prognosis
Keywords
- Germline mutations
- Myelodysplastic syndromes
- Secondary acute myeloid leukemia
- Somatic mutations
Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory cholangiopathy (disease of the bile ducts) of unknown pathogenesis. We reported that cholangiocyte senescence features prominently in PSC and that neuroblastoma RAS viral oncogene homolog (NRAS) is activated in PSC cholangiocytes. Additionally, persistent microbial insult ([i]e.g.[/i] LPSs) induces cyclin-dependent kinase inhibitor 2A ([[CDKN2A]]/p16 ) expression and senescence in cultured cholangiocytes in an NRAS-dependent manner. However, the molecular mechanisms involved in LPS-induced cholangiocyte senescence and NRAS-dependent regulation of [[CDKN2A]] remain unclear. Using our [i]in vitro[/i] senescence model, we found that LPS-induced [i][[CDKN2A]][/i] expression coincided with a 4.5-fold increase in [i]ETS1[/i] ([i]ETS proto-oncogene 1[/i]) mRNA, suggesting that ETS1 is involved in regulating [i][[CDKN2A]][/i] This idea was confirmed by RNAi-mediated suppression or genetic deletion of ETS1, which blocked [[CDKN2A]] expression and reduced cholangiocyte senescence. Furthermore, site-directed mutagenesis of a predicted ETS-binding site within the [i][[CDKN2A]][/i] promoter abolished luciferase reporter activity. Pharmacological inhibition of RAS/MAPK reduced ETS1 and [[CDKN2A]] protein expression and [i][[CDKN2A]][/i] promoter-driven luciferase activity by ∼50%. In contrast, constitutively active NRAS expression induced ETS1 and [[CDKN2A]] protein expression, whereas ETS1 RNAi blocked this increase. Chromatin immunoprecipitation-PCR detected increased ETS1 and histone 3 lysine 4 trimethylation (H3K4Me3) at the [i][[CDKN2A]][/i] promoter following LPS-induced senescence. Additionally, phospho-ETS1 expression was increased in cholangiocytes of human PSC livers and in the [i]Abcb4[/i] ([i]Mdr2[/i]) mouse model of PSC. These data pinpoint ETS1 and H3K4Me3 as key transcriptional regulators in NRAS-induced expression of [i][[CDKN2A]][/i], and this regulatory axis may therefore represent a potential therapeutic target for PSC treatment.
MeSH Terms
- Animals
- Cell Line
- Cellular Senescence
- Cholangitis, Sclerosing
- Cyclin-Dependent Kinase Inhibitor p16
- Humans
- Lipopolysaccharides
- Liver
- Mice
- Proto-Oncogene Protein c-ets-1
- RNA, Messenger
- Transcriptional Activation
- Up-Regulation
Keywords
- CDKN2A
- Cholangiocytes
- ETS1
- cell signaling
- epigenetics
- epithelial cell
- senescence
- transcription
Radiotherapy (RT) is used frequently in patients with melanoma, but results are suboptimal because the disease is often radioresistant. This may be due to constitutive activation of MAPK pathway signalling through mutations involving RAS/RAF. Thus, we studied whether trametinib, a potent and selective allosteric inhibitor of MEK1/2 could improve the efficacy of RT. Clonogenic survival assays were performed in human BRAF-mutant (A375), NRAS-mutant (D04, WM1631), KRAS-mutant (WM1791c) and wild-type (PMWK) melanoma cell lines. The effects of trametinib with and without radiation on protein levels of MEK effectors were measured by immunoblot analyses. Cell cycle effects, DNA damage repair, mitotic catastrophe and senescence were measured using flow cytometry, γH2Ax staining, nuclear fragmentation and β-galactosidase staining, respectively. Additionally, athymic mice with D04 flank tumours were treated with fractionated RT after gavage with trametinib and monitored for tumour growth. All cell lines, except PMWK, exhibited enhanced cytotoxicity when RT was combined with trametinib compared to either agent alone. Sensitiser enhancement ratios were 1.70, 1.32, 1.10, and 1.70 for A375, D04, WM1361 and WM1791c, respectively. Trametinib efficiently blocked RT-induced phosphorylation of ERK at nanomolar concentrations. Increased radiosensitivity correlated with prolonged G1 arrest and reduction in the radioresistant S phase up to 48 h following RT. A larger population of senescence-activated β-galactosidase-positive cells was seen in the trametinib pretreated group, and this correlated with activation of two of the major mediators of induced senescence, p53 and pRb. Mice receiving the combination treatment (trametinib 1mg/kg and RT over 3 days) showed a reduced mean tumour volume compared with mice receiving trametinib alone (p=0.016), or RT alone (p=0.047). No overt signs of drug toxicity were observed. Trametinib radiosensitised RAS-/RAF-mutated melanoma cells by inducing prolonged G1 arrest and premature senescence. In this pre-clinical study we demonstrate that combining trametinib and RT is well tolerated, and reduces tumour growth in vivo.
MeSH Terms
- Aging
- Animals
- Cell Cycle
- Female
- MAP Kinase Signaling System
- Melanoma, Experimental
- Mice
- Mice, Nude
- Mutation
- Protein Kinase Inhibitors
- Proto-Oncogene Proteins B-raf
- Pyridones
- Pyrimidinones
- Radiation-Sensitizing Agents
- ras Proteins
Keywords
- GSK 1120212
- MEK inhibitor
- Radiosensitization
- Senescence
- Trametinib
The Kang et al. (2011), published in Nature in 2011. The experiments that will be replicated are those reported in Figures 3B, 3C, 3E, and 4A. In these experiments, Kang et al. (2011) demonstrate the phenomenon of oncogene-induced cellular senescence and immune-mediated clearance of senescent cells after intrahepatic injection of NRAS (Figures 2I, 3B, 3C, and 3E). Additionally, Kang et al. (2011) show the specific necessity of CD4 T cells for immunoclearance of senescent cells (Figure 4A). The Reproducibility Project: Cancer Biology is a collaboration between the eLife.
MeSH Terms
- Analysis of Variance
- Animals
- Carcinogenesis
- Cellular Senescence
- Female
- Hepatocytes
- Humans
- Liver Neoplasms
- Mice, Inbred C57BL
- Reproducibility of Results
Keywords
- CD4 T-cells
- Reproducibility Project: Cancer Biology
- cell biology
- human biology
- immune-mediated clearance
- medicine
- methodology
- mouse
- oncogene-induced senescence