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After the publication of the original article [1], we were notified the upper panel of the Fig. 1, where the patients' codes are listed, was cropped by mistake so the patients 1-8 are repeated.
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BACKGROUND: Emerging mutations in the ESR1 gene that encodes for the estrogen receptor (ER) are associated with resistance to endocrine therapy. ESR1 mutations rarely exist in primary tumors (~ 1%) but are relatively common (10-50%) in metastatic, endocrine therapy-resistant cancers and are associated with a shorter progression-free survival. Little is known about the incidence and clinical implication of these mutations in early recurrence events, such as local recurrences or newly diagnosed metastatic disease. METHODS: We collected 130 archival tumor samples from 103 breast cancer patients treated with endocrine therapy prior to their local/metastatic recurrence. The cohort consisted of 41 patients having at least 1 sample from local/loco-regional recurrence and 62 patients with metastatic disease (of whom 41 newly diagnosed and 28 with advanced disease). The 5 most common ESR1 hotspot mutations (D538G, L536R, Y537S/N/C) were analyzed either by targeted sequencing or by droplet digital PCR. Progression-free survival (PFS), disease-free survival (DFS), and distant recurrence-free survival (DRFS) were statistically tested by Kaplan-Meier analysis. RESULTS: The prevalence of ESR1 mutations was 5/41 (12%) in newly diagnosed metastatic patients and 5/28 (18%) for advanced metastases, detected at allele frequency > 1%. All mutations in advanced metastases were detected in patients previously treated with both tamoxifen (TAM) and aromatase inhibitors (AI). However, in newly diagnosed metastatic patients, 4/5 mutations occurred in patients treated with TAM alone. PFS on AI treatment in metastatic patients was significantly shorter for ESR1 mutation carriers (p = 0.017). In the local recurrence cohort, ESR1 mutations were identified in 15/41 (36%) patients but only 4/41 (10%) were detected at allele frequency > 1%. Again, most mutations (3/4) were detected under TAM monotherapy. Notably, 1 patient developed ESR1 mutation while on neoadjuvant endocrine therapy. DFS and DRFS were significantly shorter (p = 0.04 and p = 0.017, respectively) in patients that had ESR1 mutations (> 1%) in their loco-regional recurrence tumor. CONCLUSIONS: Clinically relevant ESR1 mutations are prevalent in newly diagnosed metastatic and local recurrence of endocrine-treated breast cancer. Since local recurrences are amenable to curative therapy, these mutations may inform the selection of subsequent endocrine therapies.
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Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/mortalidade , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Mutação , Recidiva Local de Neoplasia/mortalidade , Neoplasias Hormônio-Dependentes/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Taxa de Sobrevida , Resultado do TratamentoRESUMO
Neural progenitor cells undergo somatic retrotransposition events, mainly involving L1 elements, which can be potentially deleterious. Here, we analyze the whole genomes of 20 brain samples and 80 non-brain samples, and characterized the retrotransposition landscape of patients affected by a variety of neurodevelopmental disorders including Rett syndrome, tuberous sclerosis, ataxia-telangiectasia and autism. We report that the number of retrotranspositions in brain tissues is higher than that observed in non-brain samples and even higher in pathologic vs normal brains. The majority of somatic brain retrotransposons integrate into pre-existing repetitive elements, preferentially A/T rich L1 sequences, resulting in nested insertions. Our findings document the fingerprints of encoded endonuclease independent mechanisms in the majority of L1 brain insertion events. The insertions are "non-classical" in that they are truncated at both ends, integrate in the same orientation as the host element, and their target sequences are enriched with a CCATT motif in contrast to the classical endonuclease motif of most other retrotranspositions. We show that L1Hs elements integrate preferentially into genes associated with neural functions and diseases. We propose that pre-existing retrotransposons act as "lightning rods" for novel insertions, which may give fine modulation of gene expression while safeguarding from deleterious events. Overwhelmingly uncontrolled retrotransposition may breach this safeguard mechanism and increase the risk of harmful mutagenesis in neurodevelopmental disorders.
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Encéfalo/fisiopatologia , Elementos Nucleotídeos Longos e Dispersos/genética , Transtornos do Neurodesenvolvimento/genética , Nucleotídeos de Adenina/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Bases de Dados Genéticas , Endonucleases/genética , Éxons , Regulação da Expressão Gênica , Genes/genética , Genômica/métodos , Humanos , MicroRNAs/genética , Mutação , Neurônios/metabolismo , Estatísticas não Paramétricas , Nucleotídeos de Timina/genética , Sequenciamento Completo do GenomaRESUMO
Adenosine deaminase acting on RNA 1 (ADAR1) is the master RNA editor, catalyzing the deamination of adenosine to inosine. RNA editing is vital for preventing abnormal activation of cytosolic nucleic acid sensing pathways by self-double-stranded RNAs. Here we determine, by parallel analysis of RNA secondary structure sequencing (PARS-seq), the global RNA secondary structure changes in ADAR1 deficient cells. Surprisingly, ADAR1 silencing resulted in a lower global double-stranded to single-stranded RNA ratio, suggesting that A-to-I editing can stabilize a large subset of imperfect RNA duplexes. The duplexes destabilized by editing are composed of vastly complementary inverted Alus found in untranslated regions of genes performing vital biological processes, including housekeeping functions and type-I interferon responses. They are predominantly cytoplasmic and generally demonstrate higher ribosomal occupancy. Our findings imply that the editing effect on RNA secondary structure is context dependent and underline the intricate regulatory role of ADAR1 on global RNA secondary structure.
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Adenosina Desaminase/genética , Conformação de Ácido Nucleico , Edição de RNA/genética , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Adenosina/metabolismo , Composição de Bases/genética , Linhagem Celular Tumoral , Desaminação , Células Hep G2 , Humanos , Inosina/metabolismo , Biossíntese de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transcriptoma/fisiologiaRESUMO
Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.
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Adenosina Desaminase/genética , Processamento Alternativo/genética , Precursores de RNA/genética , RNA Mensageiro/genética , Adenosina/genética , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Éxons/genética , Regulação da Expressão Gênica , Humanos , Inosina/genética , Edição de RNA/genética , Proteínas de Ligação a RNARESUMO
The ability to sequence nucleic acids at an unprecedented pace and decreased costs using massive parallel sequencing (MPS) strongly affects biomedical research. Here we applied MPS for the detection of rare, clinically relevant mutations in a chronic myeloid leukaemia (CML) patient. Tyrosine kinase inhibitors revolutionized CML therapy but in some patients the disease progresses due to resistance-conferring mutations. MPS was applied herein to monitor such mutations in BCR-ABL1 transcripts at different time points. The large volume of sequencing data increases sensitivity compared to direct sequencing and allows detection of marginally represented and previously uncharacterized mutations. We detected changes in the frequency of mutated clones including the emergence and disappearance of the resistance-associated ABL1 T315I mutation. We also observed correlation in appearance of adjacent mutations, and exploited this observation to demonstrate the existence of mutated clones at the time of diagnosis. A tool is provided for detection of low frequency single nucleotide variants/mutations from deep coverage MPS data, applicable to clinical translation of advanced sequencing technologies.
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Proteínas de Fusão bcr-abl/genética , Leucemia Mieloide de Fase Crônica/genética , Mutação/genética , Adulto , Análise Mutacional de DNA/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel approach, m(6)A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m(6)A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks--around stop codons and within long internal exons--and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m(6)A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a fundamental role in regulation of gene expression.
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Adenosina/análogos & derivados , Adenosina/genética , Metaboloma , RNA/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Sequência Conservada , Evolução Molecular , Células Hep G2 , Humanos , Metaboloma/genética , Metilação , Metiltransferases/deficiência , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , RNA/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genéticaRESUMO
ABC transporters are often found to be inherently expressed in a wide variety of stem cells, where they provide improved protection from toxins. We found a subpopulation of human melanoma cells expressing multidrug-resistance gene product 1 (MDR1). This fraction co-expresses the ABC transporters, ABCB5 and ABCC2 in addition to the stem cell markers, nanog and human telomerase reverse transcriptase (hTERT). The clonogenicity and self-renewal capacity of MDR1(+) melanoma cells were investigated in single cell settings using the limiting dilution assay. We found that the MDR1(+) cells, isolated by FACS sorting, demonstrated a higher self-renewal capacity than the MDR1(-) fraction, a key stem cell feature. Moreover, MDR1(+) cells had higher ability to form spheres in low attachment conditions, a hallmark of cancer. In conclusion, these novel findings imply that the MDR1(+) cells represent melanoma stem cells and thus should be considered as a unique cellular target for future anti-melanoma therapies.