RESUMO
Fluxes in human copper levels recently garnered attention for roles in cellular signaling, including affecting levels of the signaling molecule cyclic adenosine monophosphate. We herein apply an unbiased temporal evaluation of the signaling and whole genome transcriptional activities modulated by copper level fluctuations to identify potential copper sensor proteins responsible for driving these activities. We find that fluctuations in physiologically relevant copper levels modulate EGFR signal transduction and activation of the transcription factor CREB. Both intracellular and extracellular assays support Cu1+ inhibition of the EGFR phosphatase PTPN2 (and potentially PTPN1)-via ligation to the PTPN2 active site cysteine side chain-as the underlying mechanism. We additionally show i) copper supplementation drives weak transcriptional repression of the copper importer CTR1 and ii) CREB activity is inversely correlated with CTR1 expression. In summary, our study reveals PTPN2 as a physiological copper sensor and defines a regulatory mechanism linking feedback control of copper stimulated EGFR/CREB signaling and CTR1 expression.
Assuntos
Transportador de Cobre 1 , Cobre , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Receptores ErbB , Proteína Tirosina Fosfatase não Receptora Tipo 2 , Transdução de Sinais , Receptores ErbB/metabolismo , Receptores ErbB/genética , Cobre/metabolismo , Humanos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Transportador de Cobre 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Methylation-based liquid biopsies show promises in detecting cancer using circulating cell-free DNA; however, current limitations impede clinical application. Most assays necessitate substantial DNA inputs, posing challenges. Additionally, underrepresented tumor DNA fragments may go undetected during exponential amplification steps of traditional sequencing methods. Here, we report linear amplification-based bisulfite sequencing (LABS), enabling linear amplification of bisulfite-treated DNA fragments in a genome-wide, unbiased fashion, detecting cancer abnormalities with sub-nanogram inputs. Applying LABS to 100 patient samples revealed cancer-specific patterns, copy number alterations, and enhanced cancer detection accuracy by identifying tissue-of-origin and immune cell composition.
Assuntos
Metilação de DNA , Neoplasias , Análise de Sequência de DNA , Sulfitos , Humanos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Ácidos Nucleicos Livres , Técnicas de Amplificação de Ácido Nucleico/métodos , Variações do Número de Cópias de DNA , DNA de Neoplasias/genética , DNA Tumoral Circulante/genéticaRESUMO
Fluxes in human intra- and extracellular copper levels recently garnered attention for roles in cellular signaling, including affecting levels of the signaling molecule cyclic adenosine monophosphate (cAMP). We herein applied an unbiased temporal evaluation of the whole-genome transcriptional activities modulated by fluctuations in copper levels to identify the copper sensor proteins responsible for driving these activities. We found that fluctuations in physiologically-relevant copper levels rapidly modulate EGFR/MAPK/ERK signal transduction and activation of the transcription factor cAMP response element-binding protein (CREB). Both intracellular and extracellular assays support Cu 1+ inhibition of the EGFR-phosphatase PTPN2 (and potentially the homologous PTPN1)-via direct ligation to the PTPN2 active site cysteine side chain-as the underlying mechanism of copper-stimulated EGFR signal transduction activation. Depletion of copper represses this signaling pathway. We additionally show i ) copper supplementation drives transcriptional repression of the copper importer CTR1 and ii ) CREB activity is inversely correlated with CTR1 expression. In summary, our study reveals PTPN2 as a physiological copper sensor and defines a regulatory mechanism linking feedback control of copper-stimulated MAPK/ERK/CREB-signaling and CTR1 expression, thereby uncovering a previously unrecognized link between copper levels and cellular signal transduction.
