BRAF
Serine/threonine-protein kinase B-raf (EC 2.7.11.1) (Proto-oncogene B-Raf) (p94) (v-Raf murine sarcoma viral oncogene homolog B1) [BRAF1] [RAFB1]
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The conditional reprogramming cell culture method was developed to facilitate growth of senescence-prone normal and neoplastic epithelial cells, and involves co-culture with irradiated fibroblasts and the addition of a small molecule Rho kinase (ROCK) inhibitor. The aim of this study was to determine whether this approach would facilitate the culture of compact low grade gliomas. We attempted to culture 4 pilocytic astrocytomas, 2 gangliogliomas, 2 myxopapillary ependymomas, 2 anaplastic gliomas, 2 difficult-to-classify low grade neuroepithelial tumors, a desmoplastic infantile ganglioglioma, and an anaplastic pleomorphic xanthoastrocytoma using a modified conditional reprogramming cell culture approach. Conditional reprogramming resulted in robust increases in growth for a majority of these tumors, with fibroblast conditioned media and ROCK inhibition both required. Switching cultures to standard serum containing media, or serum free neurosphere conditions, with or without ROCK inhibition, resulted in decreased proliferation and induction of senescence markers. ROCK inhibition and conditioned media both promoted Akt and Erk1/2 activation. Several cultures, including one derived from a NF1-associated pilocytic astrocytoma (JHH-NF1-PA1) and one from a BRAF p.V600E mutant anaplastic pleomorphic xanthoastrocytoma (JHH-PXA1), exhibited growth sufficient for preclinical testing in vitro. In addition, JHH-NF1-PA1 cells survived and migrated in larval zebrafish orthotopic xenografts, while JHH-PXA1 formed orthotopic xenografts in mice histopathologically similar to the tumor from which it was derived. These studies highlight the potential for the conditional reprogramming cell culture method to promote the growth of glial and glioneuronal tumors in vitro, in some cases enabling the establishment of long-term culture and in vivo models.
Keywords
- BRAFV600E
- Conditional reprogramming
- NF1
- Senescence
- low grade glioma
The crosstalk between Notch and MAPK pathway plays a role in MEK inhibitor resistance in BRAF metastatic melanoma (MM) and promotes migration in GNAQ uveal melanoma (UM) cells. We determined the cytotoxicity of combinatorial inhibition of MEK and Notch by cobimetinib and γ-secretase inhibitor (GSI) nirogacestat, in BRAF and BRAF wt MM and GNAQ UM cells displaying different Erk1/2 and Notch activation status, with the aim to elucidate the impact of Notch signaling in the response to MEK inhibitor. Overall the combination was synergic in BRAF MM and GNAQ UM cells and antagonistic in BRAF wt one. Focusing on UM cells, we found that cobimetinib resulted in G0/G1 phase arrest and apoptosis induction, whereas the combination with GSI increased treatment efficacy by inducing a senescent-like state of cells and by blocking migration towards liver cancer cells. Mechanistically, this was reflected in a strong reduction of cyclin D1, in the inactivation of retinoblastoma protein and in the increase of p27 expression levels. Of note, each drug alone prevented Notch signaling activation resulting in inhibition of c-jun(Ser63) and Hes-1 expression. The combination achieved the strongest inhibition on Notch signaling and on both c-jun(Ser63) and Erk1/2 activation level. In conclusion we unveiled a coordinate action of MAPK and Notch signaling in promoting proliferation of BRAF MM and GNAQ UM cells. Remarkably, the simultaneous inhibition of MEK and Notch signaling highlighted a role for the second pathway in protecting cells against senescence in GNAQ UM cells treated with the MEK inhibitor.
Keywords
- Cobimetinib (PubChem CID: 16222096)
- MEK
- Nirogacestat (PubChem CID:46224413)
- Notch
- Senescence
- Uveal melanoma
Detailed catalog of lung cancer-associated gene mutations provides valuable information for lung cancer diagnosis and treatment. In China, there has never been a wide-ranging study cataloging lung cancer-associated gene mutations. This study aims to reveal a comprehensive catalog of lung cancer gene mutations in china, focusing on EGFR, ALK, KRAS, HER2, PIK3CA, MET, BRAF, HRAS, and CTNNB1 as major targets. Additionally, we also aim to correlate smoking history, gender, and age distribution and pathological types with various types of gene mutations. A retrospective data acquisition was conducted spanning 6 years (2013-2018) among all patients who underwent lung cancer surgeries not bronchial or percutaneous lung biopsy at three major tertiary hospitals. Finally, we identified 1,729 patients who matched our inclusion criteria. 1081 patients (62.49%) harbored EGFR mutation. ALK ([i]n[/i] = 42, 2.43%), KRAS ([i]n[/i] = 201, 11.62%), CTNNB1 ([i]n[/i] = 28, 1.62%), BRAF ([i]n[/i] = 31, 1.79%), PIK3CA ([i]n[/i] = 51, 2.95%), MET ([i]n[/i] = 14, 0.81%), HER2 ([i]n[/i] = 47, 2.72%), HRAS ([i]n[/i] = 3, 0.17%), and other genes([i]n[/i] = 232, 13.4%). Females expressed 55.38% vs. males 44.62% mutations. Among subjects with known smoking histories, 32.82% smokers, 67.15% non-smokers were observed. Generally, 51.80% patients were above 60 years vs. 48.20% in younger patients. Pathological types found includes LUADs 71.11%, SQCCs 1.68%, ASC 0.75%, LCC 0.58%, SCC 0.35%, ACC 0.17%, and SC 0.06%, unclear 25.19%. We offer a detailed catalog of the distribution of lung cancer mutations. Showing how gender, smoking history, age, and pathological types are significantly related to the prevalence of lung cancer in China.
Keywords
- China
- aging
- gene mutation
- lung cancer
- pathology
- tobacco smoking
In addition to genetic changes, post-transcriptional events strongly contribute to the progression of malignant tumors. The RNA-binding protein HuR ([i]ELAVL1[/i]) is able to bind and stabilize a large group of target mRNAs, which contain AU-rich elements (ARE) in their 3'-untranslated region. We found HuR to be upregulated in malignant melanoma in vitro and in vivo, significantly correlating with progression in vivo. Additionally, we could show that miR-194-5p can regulate HuR expression level. HuR knockdown in melanoma cells led to the suppression of proliferation and the induction of cellular senescence. Interestingly, HuR overexpression was sufficient to inhibit senescence in [i]BRAF [/i]-expressing melanocytes and to force their growth. Here, MITF (Microphthalmia-associated transcription factor), a key player in suppressing senescence and an ARE containing transcript, is positively regulated by HuR. Our results show for the first time that the overexpression of HuR is an important part of the regulatory pathway in the development of malignant melanoma and functions as a switch to overcome oncogene-induced senescence and to support melanoma formation. These newly defined alterations may provide possibilities for innovative therapeutic approaches.
Keywords
- HuR
- MITF
- Microphthalmia-associated transcription factor
- malignant melanoma
- oncogene induced senescence
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
Aberrations in DNA methylation are widespread in colon cancer (CC). Understanding origin and progression of DNA methylation aberrations is essential to develop effective preventive and therapeutic strategies. Here, we aimed to dissect CC subtype-specific methylation instability to understand underlying mechanisms and functions. We have assessed genome-wide DNA methylation in the healthy normal colon mucosa (HNM), precursor lesions and CCs in a first comprehensive study to delineate epigenetic change along the process of colon carcinogenesis. Mechanistically, we used stable cell lines, genetically engineered mouse model of mutant BRAF and molecular biology analysis to establish the role of BRAF -mediated-TET inhibition in CpG-island methylator phenotype (CIMP) inititation. We identified two distinct patterns of CpG methylation instability, determined either by age-lifestyle (CC-neutral CpGs) or genetically (CIMP-CpGs). CC-neutral-CpGs showed age-dependent hypermethylation in HNM, all precursors, and CCs, while CIMP-CpGs showed hypermethylation specifically in sessile serrated adenomas/polyps (SSA/Ps) and CIMP-CCs. BRAF -mutated CCs and precursors showed a significant downregulation of TET1 and TET2 DNA demethylases. Stable expression of BRAF in nonCIMP CC cells and in a genetic mouse model was sufficient to repress TET1/TET2 and initiate hypermethylation at CIMP-CpGs, reversible by BRAF inhibition. BRAF -driven CIMP-CpG hypermethylation occurred at genes associated with established CC pathways, effecting functional changes otherwise achieved by genetic mutation in carcinogenesis. Hence, while age-lifestyle-driven hypermethylation occurs generally in colon carcinogenesis, BRAF -driven hypermethylation is specific for the "serrated" pathway. This knowledge will advance the use of epigenetic biomarkers to assess subgroup-specific CC risk and disease progression.
MeSH Terms
- Animals
- Caco-2 Cells
- Cell Line, Tumor
- Colonic Neoplasms
- DNA Methylation
- DNA-Binding Proteins
- Down-Regulation
- Epigenesis, Genetic
- Female
- Gene Regulatory Networks
- HT29 Cells
- Humans
- Male
- Mice
- Mixed Function Oxygenases
- Mutation
- Neoplasms, Experimental
- Proto-Oncogene Proteins
- Proto-Oncogene Proteins B-raf
Keywords
- Aging
- BRAF V600E
- CIMP
- Colon cancer
- DNA methylation
- TET
Senescence is defined as irreversible cell cycle arrest and constitutes a major driving force in diseases related to aging or premature aging. Recent studies indicate that activation of the serine/threonine protein kinase B-raf (BRAF) plays important roles in oncogene-induced senescence. However, it remains elusive whether BRAF inhibition might be effective for abrogating senescence. In this study, we assessed several BRAF inhibitors to identify compounds that ameliorate senescence and revealed SB590885 as an effective agent. Senescence-ameliorating effect upon BRAF inhibition was evident from the observation that SB590885 treatment increased cellular proliferation but diminished senescent phenotypes. Moreover, BRAF inhibition induced the mitochondrial functional recovery along with the metabolic reprogramming, which comprises two salient features that are altered in senescent cells. Furthermore, mitochondrial metabolic reprogramming via BRAF inhibition was a prerequisite for senescence amelioration. Taken together, our data revealed a novel mechanism in which senescence amelioration is mediated by mitochondrial metabolic reprogramming upon BRAF inhibition.
MeSH Terms
- Cell Proliferation
- Cells, Cultured
- Cellular Reprogramming
- Cellular Senescence
- Drug Evaluation, Preclinical
- Humans
- Mitochondria
- Proto-Oncogene Proteins B-raf
Keywords
- BRAF
- Metabolic reprogramming
- Mitochondrial function
- SB590885
- Senescence
- Hairy cell leukemia (HCL) is a rare, indolent B-cell neoplasm. The classical variant of the disease is characterized by the [i]BRAF[/i] V600E mutation, which is present in virtually all cases. How this mutation leads to the signs and symptoms of the disease is currently not known. : This review explores the genetic background of HCL, especially the [i]BRAF[/i] V600E driver mutation, but passenger mutations and their effects are also included. The clinical significance of [i]BRAF[/i] mutations in other cancer types is discussed, as well as [i]BRAF[/i]- induced senescence. An overview of the major forms of treatment of HCL (cytostatic drugs, specific BRAF inhibitors, B cell-specific antibodies) is given. Finally, possible mechanisms of the monocytopenia and hairy morphology so typical of this disease are discussed. : Although being a rare disease, HCL and its pathogenesis can yield important information about [i]BRAF[/i]-related cancer metabolism. Many aspects of the disease are still unclear, but with the right resources, this could change. This can lead to a more efficient and specific treatment, thus leading to decreased morbidity.
MeSH Terms
- B-Lymphocytes
- Cytostatic Agents
- Genetic Association Studies
- Genotype
- Humans
- Leukemia, Hairy Cell
- Mutation
- Phenotype
- Proto-Oncogene Proteins B-raf
- Rare Diseases
Keywords
- V600E mutation
- mutations
- -induced senescence
- epidemiology
- genomic instability
- hairy cell leukemia
- hairy morphology
- immunophenotype
- monocytopenia
Recent developments in molecular-targeted therapies have improved the clinical outcome of cancer patients; however, the issue of adverse effects due to treatments has often gone unconsidered. We herein report the results of a clinical trial of dual genomic analyses for healthy longevity in a postoperative cancer patient. We performed dual genomic analyses for a representative 79-year-old rectal cancer patient who relapsed with liver metastasis. First, we determined single-nucleotide polymorphisms according to the constitution and disease risk in the genomic DNA from the patient's saliva by referring to the data of 10,000 Japanese patients obtained from Yahoo Japan Corporation. Second, we conducted whole-exome sequencing to detect druggable mutations in the primary tumour. Forty of 59 determinable characters related to the constitution were consistent with the clinical phenotype. Several diseases classified as 'high risk' diseases actually occurred during the patient's clinical course. Of the 129 significant mutations, we identified somatic mutations in BRAF, PIK3CA, and SMAD4 as targets. The dual genomic examination will improve the follow-up observation system to support primary care doctors in the social community for taking care of postoperative cancer patients.
MeSH Terms
- Aged
- Class I Phosphatidylinositol 3-Kinases
- Clinical Trials as Topic
- DNA
- Genome, Human
- Humans
- Liver Neoplasms
- Longevity
- Male
- Mutation
- Neoplasm Recurrence, Local
- Polymorphism, Single Nucleotide
- Postoperative Period
- Proto-Oncogene Proteins B-raf
- Quality of Life
- Rectal Neoplasms
- Risk
- Survival
- Whole Exome Sequencing
Keywords
- Disease risk
- Healthy longevity
- Quality of life
- Single nucleotide polymorphism
- Somatic mutation
We addressed the precursor role of aging-like spontaneous promoter DNA hypermethylation in initiating tumorigenesis. Using mouse colon-derived organoids, we show that promoter hypermethylation spontaneously arises in cells mimicking the human aging-like phenotype. The silenced genes activate the Wnt pathway, causing a stem-like state and differentiation defects. These changes render aged organoids profoundly more sensitive than young ones to transformation by Braf , producing the typical human proximal BRAF -driven colon adenocarcinomas characterized by extensive, abnormal gene-promoter CpG-island methylation, or the methylator phenotype (CIMP). Conversely, CRISPR-mediated simultaneous inactivation of a panel of the silenced genes markedly sensitizes to Braf -induced transformation. Our studies tightly link aging-like epigenetic abnormalities to intestinal cell fate changes and predisposition to oncogene-driven colon tumorigenesis.
MeSH Terms
- Adenocarcinoma
- Age Factors
- Aging
- Animals
- Cell Transformation, Neoplastic
- Colonic Neoplasms
- DNA Methylation
- Gene Expression Regulation, Neoplastic
- Gene Silencing
- Genetic Predisposition to Disease
- Humans
- Mice, Inbred NOD
- Mice, Mutant Strains
- Mice, SCID
- Mutation
- Phenotype
- Proto-Oncogene Proteins B-raf
- Stem Cells
- Time Factors
- Tissue Culture Techniques
- Wnt Signaling Pathway
Keywords
- BRAF(V600E)
- CIMP
- CpG-island DNA methylation
- aging
- cancer risk
- colon adenocarcinomas
- epigenetic silencing
- transformation
- tumor predisposition
- tumorigenesis
Targeting MAPK pathway in mutant BRAF melanoma with the specific BRAF inhibitor vemurafenib showed robust initial responses in the majority of patients followed by relapses due to acquired resistance to the drug. In BRAF melanoma cell lines, senescence-associated β-galactosidase activity is often encountered in a constitutive manner or induced after MAPK inhibition. However, the link between the senescence-like phenotype and the resistance to BRAF inhibition is not fully understood yet. Our data validate a senescence-like phenotype (low cell proliferation, high cell volume, and high β-Gal activity) in mutant BRAF cells. Vemurafenib increased β-Gal activity in 4 out of 5 sensitive lines and in 2 out of 5 lines with intrinsic resistance to the drug. Interestingly, the 3 lines with acquired resistance to vemurafenib became depending on the drug for proliferation. In absence of drug, these lines showed a lower cell proliferation rate together with a substantial increase of β-Gal activity both [i]in vitro[/i] and [i]in vivo[/i]. In all settings, the senescence-like phenotype was significantly associated with an inhibition of pRB and cyclin D1, explaining the inhibition of cell proliferation. In conclusion, β-Gal activity is increased by BRAF inhibition in the majority of sensitive and intrinsically resistant melanoma cells. Acquired resistance to vemurafenib is associated with a dependence to the drug for cell proliferation and tumor growth, and, in this case, drug removal stimulate β-Gal activity suggesting that the senescence-like phenotype could contribute to the acquired resistance to BRAF inhibition.
Keywords
- V600EBRAF
- acquired resistance
- melanoma
- senescence
- vemurafenib
Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAF in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other [i]in vivo[/i] systems.
MeSH Terms
- Aging
- Animals
- Animals, Genetically Modified
- CRISPR-Cas Systems
- Carcinogenesis
- Disease Models, Animal
- Disease Progression
- Electroporation
- Embryo, Nonmammalian
- Gene Transfer Techniques
- Melanoma
- Plasmids
- Promoter Regions, Genetic
- Transgenes
- Zebrafish
Keywords
- Cancer
- Electroporation
- Melanoma
- Metastasis
- Zebrafish
Tissue-specific effects of 17β-estradiol are delivered via both estrogen receptors and microRNAs (miRs). Menopause is known to affect the whole-body fat distribution in women. This investigation aimed at identifying menopause- and hormone replacement therapy (HRT)-associated miR profiles and miR targets in subcutaneous abdominal adipose tissue and serum from the same women. A discovery phase using array technology was performed in 13 women, including monozygotic twin pairs discordant for HRT and premenopausal young controls. Seven miRs, expressed in both adipose tissue and serum, were selected for validation phase in 34 women from a different cohort. An age/menopause-related increase of miRs-16-5p, -451a, -223-3p, -18a-5p, -19a-3p,-486-5p and -363-3p was found in the adipose tissue, but not in serum. MiR-19a-3p, involved in adipocyte development and estrogen signaling, resulted to be higher in HRT users in comparison with non-users. Among the identified targets, AKT1, BCL-2 and BRAF proteins showed lower expression in both HRT and No HRT users in comparison with premenopausal women. Unexpectedly, ESR1 protein expression was not modified although its mRNA was lower in No HRT users compared to premenopausal women and HRT users. Thus, both HRT and menopause appear to affect adipose tissue homeostasis via miR-mediated mechanism.
Keywords
- adipose tissue
- aging
- estrogen therapy
- miR-19a-3p
- microRNAs
Monoclonal antibodies (mAb) targeting PD-1/PD-L1 have revolutionized melanoma treatment, yet data regarding effectiveness and tolerability across age groups is limited. We sought to determine the impact of age on overall survival (OS), progression-free survival (PFS), and rates of immune-mediated toxicities in patients treated with anti-PD-1/anti-PD-L1 mAb at two academic medical centers. We retrospectively collected data on all patients with metastatic melanoma treated with anti-PD-1/PD-L1 mAb between May 2009 and April 2015. We used Kaplan-Meier and Cox regression analyses to assess OS and PFS and identify factors associated with these outcomes. We also compared rates of autoimmune toxicity across age groups. Of 254 patients, 57 (22.4%) were <50 years old, 85 (33.5%) were age 50-64, 65 (25.6%) were age 65-74, and 47 (18.5%) were ≥75 years. Across age groups, no differences existed in median OS (age <50: 22.9 months, age 50-64: 25.3 months, age 65-74: 22.0 months, age ≥75: 24.3 months) or PFS (age <50: 4.1 months, age 50-64: 6.5 months, age 65-74: 5.4 months, age ≥75: 7.9 months). The presence of liver metastases and elevated pre-treatment lactate dehydrogenase (LDH) were associated with reduced OS. Presence of liver metastasis, pretreatment LDH, BRAF mutation, and type of melanoma correlated with PFS. Overall, 110 patients (43.3%) experienced immune-mediated toxicities; 25 (9.8%) had colitis and 26 (10.2%) had endocrine toxicity. Rates of colitis, hepatitis, and pneumonitis did not differ across age groups. We demonstrated that patients could safely tolerate anti-PD1/PDL-1 mAb therapy and achieve similar outcomes regardless of their age. Immunotherapy has revolutionized treatment for patients with metastatic melanoma, yet data are lacking regarding the effectiveness and tolerability of these treatments for older patients. In this study, we demonstrated that patients with melanoma safely tolerate immunotherapy and achieve similar outcomes regardless of their age. Specifically, we utilized data from two academic cancer centers and found no significant difference in overall survival, progression free survival, or immune-related toxicities, other than arthritis, across age groups. As the population ages, studies such as this will become critical to help us understand how best to treat older adults with cancer.
MeSH Terms
- Age Factors
- Aged
- Antibodies, Monoclonal
- Antineoplastic Agents
- B7-H1 Antigen
- Disease-Free Survival
- Female
- Humans
- Immunotherapy
- Male
- Melanoma
- Middle Aged
- Programmed Cell Death 1 Receptor
- Treatment Outcome
Keywords
- Geriatrics
- Immunotherapy
- Melanoma
- Toxicity
Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo, preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and [[CDKN2A]]/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, [[CDKN2A]] (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro. Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.
MeSH Terms
- Antigens, Polyomavirus Transforming
- Astrocytoma
- Blotting, Western
- Brain Neoplasms
- Cell Culture Techniques
- Cell Line, Tumor
- Cell Proliferation
- Cellular Senescence
- Child, Preschool
- Drug Screening Assays, Antitumor
- Gene Expression Profiling
- Humans
- Male
- Oncogene Proteins, Fusion
- Polymerase Chain Reaction
- Proto-Oncogene Proteins B-raf
- Transcriptome
- Transduction, Genetic
Keywords
- KIAA1549:BRAF-fusion
- MAPK-inhibitors
- oncogene-induced senescence (OIS)
- pediatric low grade glioma
- pilocytic astrocytoma
Reactive oxygen species (ROS) derive from molecular oxygen and present higher reactivity. ROS designation comprehends free radicals such as superoxide radical (O ° ), hydroxyl radical (OH° ); but also nonradical molecule like hydrogen peroxide (H O ). ROS play a critical role in several physiological functions like proliferation and signalling pathways. Thanks to cellular (oxidant/antioxidant) systems, ROS level is tightly regulated to avoid excessive damage to biological macromolecules (proteins, lipids, and DNA). An imbalance of redox equilibrium can lead to persistent oxidative stress favoring senescence, inflammation, and carcinogenesis.Oncogene activation can induce severe or irreparable DNA damage and causes proliferative arrest named senescence. Senescence acts as a tumorigenesis barrier, and its bypass can promote transition between normal homeostasis and neoplastic transformation. The mechanisms through which oncogenes induce senescence remain unclear but involve increased cellular level of Reactive Oxygen Species. Among ROS, H O is of particular interest because the hydrogen peroxide is more stable, can diffuse actively or freely through the cellular membranes, and can generate locally the hydroxyl radicals by iron-mediated Fenton reaction. Interestingly, growing data support the role of H O in the propagation of the stressful effects of senescent cells to their neighbors through the bystander effect. In this protocol, we present our routinely used methodology to detect extracellular H O using the Amplex red/horseradish peroxidase assay. This highly sensitive method detects specifically H O , and offers the possibility to quantify it using the H O standard curve.To illustrate this method of detection of extracellular H O in cells undergoing oncogene-induced senescence, we compare two human cell lines: BCPAP (from human papillary thyroid carcinomas carrying BRAF mutation) and HTori-3.1 cell line (immortalized human thyroid epithelial cells) because expression of BRAF in human thyroid cells triggers senescence.
MeSH Terms
- Cell Line
- Cellular Senescence
- Extracellular Space
- Humans
- Hydrogen Peroxide
- Oncogenes
- Oxidation-Reduction
- Reactive Oxygen Species
- Spectrometry, Fluorescence
Keywords
- Amplex red
- Hydrogen peroxide
- Reactive oxygen species
- Senescence
Geriatric glioblastoma (GBM) patients have a poorer prognosis than younger patients, but IDH1/2 mutations (more common in younger patients) confer a favorable prognosis. We compared key GBM molecular alterations between an elderly (age ≥ 70) and younger (18 < = age < = 45) cohort to explore potential therapeutic opportunities. Alterations more prevalent in the young GBM cohort compared to the older cohort (P < 0.05) were: overexpression of ALK, RRM1, TUBB3 and mutation of ATRX, BRAF, IDH1, and TP53. However, PTEN mutation was significantly more frequent in older patients. Among patients with wild-type IDH1/2 status, TOPO1 expression was higher in younger patients, whereas MGMT methylation was more frequent in older patients. Within the molecularly-defined IDH wild-type GBM cohort, younger patients had significantly more mutations in PDGFRA, PTPN11, SMARCA4, BRAF and TP53. GBMs from 178 elderly patients and 197 young patients were analyzed using DNA sequencing, immunohistochemistry, in situ hybridization, and MGMT-methylation assay to ascertain mutational and amplification/expressional status. Significant molecular differences occurred in GBMs from elderly and young patients. Except for the older cohort's more frequent PTEN mutation and MGMT methylation, younger patients had a higher frequency of potential therapeutic targets.
MeSH Terms
- Adult
- Age Factors
- Aged
- Aging
- Biomarkers, Tumor
- Brain Neoplasms
- Cohort Studies
- DNA Methylation
- DNA Mutational Analysis
- ErbB Receptors
- Gene Expression Regulation, Neoplastic
- Glioblastoma
- Humans
- Mutation
- Tumor Suppressor Protein p53
Keywords
- DNA sequencing
- GBM
- mutational analysis
Cancer is driven by mutations in genes whose products participate in major signaling pathways that fuel cell proliferation and survival. It is easy to assume that the more of these so-called driver mutations a tumor accumulates, the faster it progresses. However, this does not appear to be the case: Data from large-scale genome sequencing studies indicate that mutations in driver oncogenes often are mutually exclusive. The mechanisms underlying the mutual exclusivity of oncogenes are not completely understood, but recent reports suggest that the mechanisms may depend on the tumor type, and the nature of interacting oncogenes. Here we discuss our recent findings that the oncogenes KRAS and BRAF are mutually exclusive in lung cancer in mouse models because their coexpression leads to oncogene-induced senescence.
MeSH Terms
- Animals
- Cell Death
- Cellular Senescence
- Gene Expression Regulation, Neoplastic
- Humans
- Mutation
- Neoplasms
- Oncogenes
Keywords
- RAF
- RAS
- mutual exclusivity
- oncogene
- senescence
Oncogene activation induces DNA damage responses and cell senescence. We report a key role of type I interferons (IFNs) in oncogene-induced senescence. IFN signaling-deficient melanocytes expressing activated Braf do not exhibit senescence and develop aggressive melanomas. Restoration of IFN signaling in IFN-deficient melanoma cells induces senescence and suppresses melanoma progression. Additional data from human melanoma patients and mouse transplanted tumor models suggest the importance of non-cell-autonomous IFN signaling. Inactivation of the IFN pathway is mediated by the IFN receptor IFNAR1 downregulation that invariably occurs during melanoma development. Mice harboring an IFNAR1 mutant, which is partially resistant to downregulation, delay melanoma development, suppress metastatic disease, and better respond to BRAF or PD-1 inhibitors. These results suggest that IFN signaling is an important tumor-suppressive pathway that inhibits melanoma development and progression and argue for targeting IFNAR1 downregulation to prevent metastatic disease and improve the efficacy of molecularly target and immune-targeted melanoma therapies.
MeSH Terms
- Adult
- Aged
- Aged, 80 and over
- Animals
- Cell Line, Tumor
- Cellular Senescence
- Down-Regulation
- Female
- HEK293 Cells
- Humans
- Interferon Type I
- Male
- Melanocytes
- Melanoma
- Mice
- Mice, Inbred C57BL
- Middle Aged
- Mutation
- Proto-Oncogene Proteins B-raf
- Receptor, Interferon alpha-beta
- Signal Transduction
Keywords
- BRAF
- interferon receptor
- melanoma
- metastasis
- senescence
- type I interferon
Oncogenic triggering of the MAPK pathway in melanocytes results in senescence, and senescence escape is considered as one critical step for melanocytic transformation. In melanoma, induction of a senescent-like state by BRAF-inhibitors (BRAFi) in a fraction of treated cells - instead of killing - contributes to the repression of tumor growth, but may also provide a source for relapse. Here, we demonstrate that NOTCH activation in melanocytes is not only growth-promoting but it also protects these cells against oncogene-induced senescence. In turn, treatment of melanoma cells with an inhibitor of the NOTCH-activating enzyme γ-secretase led to induction of a senescent-like status in a fraction of the cells but overall achieved only a moderate inhibition of melanoma cell growth. However, combination of γ-secretase inhibitor (GSI) with BRAFi markedly increased the treatment efficacy particularly in long-term culture. Moreover, even melanoma cells starting to regrow after continuous BRAFi treatment - the major problem of BRAFi therapy in patients - can still be affected by the combination treatment. Thus, combining GSI with BRAFi increases the therapeutic efficacy by, at least partially, prolonging the senescent-like state of treated cells.
MeSH Terms
- Amyloid Precursor Protein Secretases
- Antineoplastic Combined Chemotherapy Protocols
- Cell Cycle Checkpoints
- Cell Line, Tumor
- Cell Proliferation
- Cellular Senescence
- Cyclin-Dependent Kinase 6
- Drug Resistance, Neoplasm
- Humans
- Melanocytes
- Melanoma
- Mutation
- Phosphorylation
- Protease Inhibitors
- Protein Kinase Inhibitors
- Proto-Oncogene Proteins B-raf
- Receptor, Notch1
- Retinoblastoma Protein
- Skin Neoplasms
- Time Factors
- Transfection
Keywords
- BRAF inhibition
- Melanoma
- Notch
- Senescence
The activity of the M isoform of microphthalmia-associated transcription factor (MITF-M) has been attributed to regulation of differentiation, proliferation, survival and senescence of melanoma cells. MITF expression was shown to be antagonized by the activation of transcription factor NF-κB. Parthenolide, an inhibitor of NF-κB, has not been yet reported to affect MITF-M expression. Our results obtained in patient-derived melanoma cell populations indicate that parthenolide efficiently decreases the MITF-M level. This is neither dependent on p65/NF-κB signaling nor RAF/MEK/ERK pathway activity as inhibition of MEK by GSK1120212 (trametinib) and induction of ERK1/2 activity by parthenolide itself do not interfere with parthenolide-triggered depletion of MITF-M in both wild-type BRAF and BRAF(V600E) melanoma populations. Parthenolide activity is not prevented by inhibitors of caspases, proteasomal and lysosomal pathways. As parthenolide reduces MITF-M transcript level and HDAC1 protein level, parthenolide-activated depletion of MITF-M protein may be considered as a result of transcriptional regulation, however, the influence of parthenolide on other elements of a dynamic control over MITF-M cannot be ruled out. Parthenolide induces diverse effects in melanoma cells, from death to senescence. The mode of the response to parthenolide is bound to the molecular characteristics of melanoma cells, particularly to the basal MITF-M expression level but other cell-autonomous differences such as NF-κB activity and MCL-1 level might also contribute. Our data suggest that parthenolide can be developed as a drug used in combination therapy against melanoma when simultaneous inhibition of MITF-M, NF-κB and HDAC1 is needed.
MeSH Terms
- Antineoplastic Agents
- Cell Proliferation
- Cell Survival
- Down-Regulation
- Extracellular Signal-Regulated MAP Kinases
- Histone Deacetylase 1
- Humans
- MAP Kinase Signaling System
- Melanoma
- Microphthalmia-Associated Transcription Factor
- Myeloid Cell Leukemia Sequence 1 Protein
- Protein Kinase Inhibitors
- Proto-Oncogene Proteins B-raf
- Pyridones
- Pyrimidinones
- Sesquiterpenes
- Transcription Factor RelA
- Tumor Cells, Cultured
Keywords
- MITF
- NF-κB
- cellular senescence
- melanoma
- parthenolide
Encorafenib (LGX818) is a new-generation BRAF inhibitor that is under evaluation in clinical trials. However, the underlying mechanism remains to be elucidated. Here we show that LGX818 potently decreased ERK phosphorylation and inhibited proliferation in BRAFV600E melanoma cell lines. Moreover, LGX818 downregulated CyclinD1 in a glycogen synthase kinase 3β-independent manner and induced cell cycle arrest in the G1 phase, Surprisingly, LGX818 triggered cellular senescence in BRAFV600E melanoma cells, as evidenced by increased β-galactosidase staining, while no appreciable induction of apoptosis was detected, as determined by Annexin V and propidium iodide staining and immunoblot analysis of caspase-3 processing and poly (ADP-ribose) polymerase cleavage. Increased p27KIP1 expression and retinoblastoma protein activation were detected during LGX818-induced senescence. Additionally, inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 1B by AZ191 reversed LGX818-induced CyclinD1 turnover and senescence. Interestingly, autophagy is triggered through inhibition of the mTOR/70S6K pathway during LGX818-induced senescence. Moreover, autophagy inhibition by pharmacological and genetic regulation attenuates LGX818-induced senescence. Notably, combining LGX818 with autophagy modulators has anti-proliferative effect in LGX818-resistant BRAF mutant melanoma cells. Altogether, we uncovered a mechanism by which LGX818 exerts its anti-tumor activity in BRAFV600E melanoma cells.
MeSH Terms
- Autophagy
- Carbamates
- Cell Line, Tumor
- Cellular Senescence
- Cyclin D1
- Cyclin-Dependent Kinase Inhibitor p27
- Glycogen Synthase Kinase 3
- Glycogen Synthase Kinase 3 beta
- Humans
- MAP Kinase Signaling System
- Melanoma
- Protein-Serine-Threonine Kinases
- Protein-Tyrosine Kinases
- Proto-Oncogene Proteins B-raf
- Retinoblastoma Protein
- Sulfonamides
- TOR Serine-Threonine Kinases
Keywords
- Autophagy
- BRAF
- Encorafenib (LGX818)
- Melanoma
- Senescence
Cutaneous melanoma is the most aggressive form of skin cancer. With age as a risk factor, melanoma is projected to become a substantial healthcare burden. The clinical course of melanoma in older patients is different from that in middle-aged and younger patients: melanomas are thicker, have higher mitotic rates and are more likely to be ulcerated. Older patients also have a higher mortality rate, yet, paradoxically, have a lower rate of lymph node metastases. After decades of no significant progress in the treatment of this devastating disease, novel insights into the mechanisms underlying the pathophysiology of metastatic melanoma have led to new and remarkably efficient therapeutic opportunities. The discovery that about half of all melanomas carry BRAF mutations led to the introduction of targeted therapy with significant improvements in clinical outcomes. Although these drugs appear to be equally effective in older patients, specific considerations regarding adverse events are required. Besides targeted therapy, immunotherapy has emerged as an alternative therapeutic option. Antibodies that block cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) can induce responses with high durability. Despite an aging immune system, older patients seem to benefit to the same degree from these treatments, apparently without increased toxicity. In this review, we focus on the epidemiology, clinicopathological features, and recent developments of systemic treatment in cutaneous melanoma with regard to older patients.
MeSH Terms
- Adult
- Aged
- Aging
- Antineoplastic Agents
- Humans
- Immunotherapy
- Lymphatic Metastasis
- Melanoma
- Middle Aged
- Molecular Targeted Therapy
- Skin Neoplasms
Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that around 80% of colorectal tumors carrying a mutation in BRAF also overexpress splice variant Rac1b. We used normal NCM460 colonocytes as a model to express oncogenic B-Raf-V600E in the presence or absence of co-transfected Rac1b and then analyzed the effect on expression of senescence markers. When oncogenic B-Raf-V600E was expressed we observed the induction of the senescence-associated β-galactosidase and of the cell-cycle inhibitors p14, p15 and p21 whereas proliferation marker Ki67 was suppressed. Upon co-expression of splice variant Rac1b, but not of Rac1, the B-Raf-induced senescence phenotype was reverted and expression of the cell-cycle inhibitors downregulated in a reactive oxygen-species dependent manner. We thus provide evidence that co-expression of splice variant Rac1b counteracts B-Raf-induced senescence, indicating the selection for increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.
MeSH Terms
- Cell Line, Tumor
- Cellular Senescence
- Colorectal Neoplasms
- Disease Progression
- Humans
- Proto-Oncogene Proteins B-raf
- Signal Transduction
- Transfection
- rac1 GTP-Binding Protein
Keywords
- B-Raf
- Colorectal cancer
- Oncogene-induced senescence
- Rac1b
- Tumor progression
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 activation of oncogenes in primary cells blocks proliferation by inducing oncogene-induced senescence (OIS), a highly potent in vivo tumor-suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near-genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF(V) (600E) -induced arrest. With genome-wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence-associated (SA)-β-galactosidase activity, interleukins, and tumor suppressor p15(INK) (4B) . Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.
MeSH Terms
- Amino Acid Substitution
- Cell Cycle Checkpoints
- Cell Line, Tumor
- Cellular Senescence
- Humans
- Melanoma
- Mutation, Missense
- PTEN Phosphohydrolase
- Proto-Oncogene Proteins B-raf
- RNA Interference
- Tumor Suppressor Proteins
- ras Guanine Nucleotide Exchange Factors
Keywords
- BRAF
- RASEF
- melanoma
- methylation
- screen
- senescence
The clonal/neoplastic nature of Langerhans cell histiocytosis (LCH) has recently been demonstrated by a high prevalence of BRAF mutations, including pulmonary LCH (PLCH). We hypothesized that BRAF-induced senescence, as demonstrated in nevi and melanoma, is involved in the pathogenesis of LCH and PLCH. In a series of pulmonary (19 cases) and non-pulmonary LCH (19 cases), including five aggressive cases, we investigated occurrence of the BRAF V600E mutation by molecular analysis and/or immunohistochemistry using a validated antibody (VE1). The expression of cell-senescence markers p16(INK4a) and p21(CIP1/WAF1) was also immunohistochemically investigated. We demonstrated that 6/19 cases of LCH and 12/19 cases of PLCH were VE1 positive, matching with molecular analysis, and in all cases both p16(INK4a) and p21(CIP1/WAF1) were expressed, irrespective of BRAF mutation status. Interestingly, all the aggressive cases did not express p16(INK4a), thus suggesting that loss of senescence control could be related to clinical aggressiveness of LCH, as in melanoma.
MeSH Terms
- Adolescent
- Adult
- Aged
- Cellular Senescence
- Child, Preschool
- Cyclin-Dependent Kinase Inhibitor p16
- Cyclin-Dependent Kinase Inhibitor p21
- DNA Mutational Analysis
- Female
- Histiocytosis, Langerhans-Cell
- Humans
- Immunohistochemistry
- Infant
- Lung
- Male
- Middle Aged
- Mutation, Missense
- Proto-Oncogene Proteins B-raf
- Young Adult
Keywords
- BRAF mutation
- PLCH
- p16INK4a
- p21CIP1/WAF1
- senescence in LCH
Whether and how autophagy is involved in tumorigenesis is poorly understood. We approached this question by investigating a relatively large cohort of patients with mostly early primary melanoma for their expression of 2 markers for autophagy, the protein ATG5 (autophagy-related 5) and MAP1LC3B/LC3 (microtubule-associated protein 1 light chain 3B). Surprisingly, we discovered that both ATG5 and LC3 levels are decreased in patients with melanomas as compared with those with benign nevi. We wondered why reduced autophagy should facilitate early tumor development. Using an in vitro model of melanoma tumorigenesis, in which a mutated oncogene, BRAF (v-raf murine sarcoma viral oncogene homolog B), had been introduced into normal human melanocytes, we were able to show that downregulation of ATG5 promoted the proliferation of melanocytes because it facilitated bypassing oncogene-induced senescence (OIS). Our work supports previous reports that had argued that autophagy actually suppresses tumorigenesis and explains the possible mechanism. Furthermore, our findings suggest that the status of ATG5 and autophagy could serve as a diagnostic marker for distinguishing benign from malignant tumors of melanocytes.
MeSH Terms
- Autophagy
- Autophagy-Related Protein 5
- Carcinogenesis
- Cell Line, Tumor
- Cell Proliferation
- Cell Transformation, Neoplastic
- Cellular Senescence
- Humans
- Melanoma
- Microtubule-Associated Proteins
Keywords
- ATG5
- autophagy
- epigenetics
- melanoma
- promoter methylation
- senescence