Estratégias de regulação de genes subjacentes a atrofia do músculo esquelético na cachexia associada ao câncer
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Data
2018-03-19
Autores
Fernandez Garcia, Geysson Javier
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A caquexia associada ao câncer é uma síndrome caracterizada pela grave perda de tecido musculo esquelético; que se estima que afeta mais de 50% de todos os pacientes com câncer e resulta em menor qualidade de vida devido a fadiga, fraqueza, redução da função imune, resistência à insulina e baixa tolerância e resposta à quimioterapia. Notavelmente, 20% das mortes relacionadas ao câncer são diretamente causadas pela caquexia. A principal limitação de que atualmente não há terapia direcionada, é o uso de abordagens tradicionais que não tratam a complexidade em sistemas biológicos, caracterizada por interações não-lineares de redes de regulação genética (GRN, do inglês Gene Regulatory Networks). Por esse motivo, ainda é necessária uma identificação dos componentes da GRN e uma compreensão quantitativa de sua integração temporal no controle das respostas celulares. Adquirir tal conhecimento é fundamental para capturar detalhes mecanicistas essenciais para direcionar estratégias terapêuticas para uma doença complexa, como a caquexia do câncer. Neste trabalho, examinamos a expressão genética do músculo esquelético em dois abordagens metodológicos diferentes: usando dados de expressão de genes estáticos e dinâmicos. Estruturamos nosso trabalho da seguinte maneira: o Capítulo 1 apresenta uma caracterização quantitativa das vias de sinalização e uma reconstrução de GRN no tecido musculo esquelético em ratos portadores de carcinoma de pulmão de Lewis (LLC, do inglês Lewis lung carcinoma) através da integração de perfis de expressão de mRNAs e miRNAs em um tempo. Os resultados mostram que os camundongos LLC reduziram o peso corporal em 20% e apresentaram perda de tecido muscular e adiposo após 23 dias de indução tumoral. Além disso, encontramos 1008 mRNAs expressos diferencialmente (487 induzidos e 521 reprimidos) e 18 miRNAs desregulados (13 induzidos e 5 reprimidos). Nossos dados sugerem a ativação do fator transcricional NF-κB e Stat3, que foram descritos na ativação de programas genéticos atróficos no musculo esquelético. Além disso, identificamos a potencial regulação pós-transcricional por miRNAs de três processos biológicos importantes: organização da matriz extracelular, migração celular e ligação do fator de transcrição. Em geral, nossos resultados identificam um conjunto de caminhos de sinalização que podem contribuir na perda de tecido musculo esquelético na caquexia associada ao câncer, entre eles genes da matriz extracelular com potencial mecanismo regulatório mediado por miRNAs. O Capítulo 2 fornece uma dissecção da via de sinalização de NF-κB em células musculares atrofiadas. Neste capitulo, examinamos quantitativamente os efeitos dinâmicos na expressão genética pela ativação de NF-κB após exposição ao fator de necrose tumoral alfa (TNF-α) nas células do músculo esquelético (C2C12). Caracterizamos as estratégias reguladoras de indução e repressão de genes, medindo as taxas de transcrição e degradação para cada mRNA e conectando esses processos através de modelagem matemática. Nossos dados apontam para um papel dominante da transcrição na regulação dos programas de indução e repressão de genes em resposta ao TNF; e revela uma diminuição da taxa de degradação de mRNA como estratégia para genes de resposta tardia para aumentar suas concentrações intracelulares. Além disso, nossa análise mostra a degradação constitutiva como uma característica intrínseca dos genes que determina a ordem temporal dos perfis de expressão gênica. Usando uma forma não degradável do inibidor kappa B alfa e um knockout de RelA, descobrimos que o fator NF-κB é responsável por indução de genes e repressão de genes durante a atrofia de células musculares induzida por TNF. Nossos dados destacam a importância da dinâmica de sinalização na mediação dos efeitos do TNF nas células do músculo esquelético e revela uma interação crítica entre o controle de síntese e degradação, que regula os programas dinâmicos de expressão gênica.
Cancer cachexia is a syndrome characterized by the severe skeletal muscle wasting tissue; that affects more than 50% of all cancer patients and results in lower quality of life due to compromised fatigue, weakness, decreased immune function, insulin resistance and poor tolerance and response to radio and chemotherapy. Remarkably, approximately 20% of cancer-related deaths are estimated to be directly caused by cachexia. There is currently no effective targeted therapy and the main limitation lays on the traditional approaches that not deal with the inherent complexity, characterized by non-linear interactions, of gene regulatory networks (GRN). Thus, a clear identification of the components of gene regulation, and a quantitative understanding of their temporal integration to control cellular responses is fundamental for capture essential mechanistic details that will ultimately enable the development of direct therapeutic strategies for the treatment of cancer cachexia. Here, we examine genome-wide gene expression of muscle wasting under two different frameworks, using static and dynamic gene expression data. We structure this approach as follow: Chapter 1 presents a quantitative characterization of the signaling pathways and a GRN reconstruction of muscle wasting in Lewis Lung Carcinoma (LLC) tumor-bearing mice by integrating static mRNAs and microRNAs expression profiles. The results show that LLC mice reduced body weight in 20% and presented muscle and fat tissue wasting after 23 days of tumor induction. In addition, we found 1008 differential expressed mRNAs (487 up-regulated and 521 down-regulated) and eighteen deregulated miRNAs (13 up-regulated and 5 down-regulated). Our data suggest activation of the transcriptional factor NF-κB and Stat3, which have been described in the activation of atrophic gene programs. Moreover, we ident potential posttranscriptional regulation by miRNAs of three important biological process: extracellular matrix organization, cell migration and transcription factor binding. Overall our results identify a set of signaling pathways that may contribute to muscle wasting in cancer cachexia, between them extracellular matrix genes with potential regulatory mechanism mediated by miRNAs. Chapter 2 provides further dissection of the NF-κB signaling pathway in atrophying muscle cells. Here, we examine quantitatively the genome-wide dynamic gene expression effects of the activation of NF-κB by the exposure of tumor necrosis factor – alpha (TNF-α) on skeletal muscle cells (C2C12). We characterize the regulatory strategies of gene induction and repression by measuring both mRNA transcription and degradation rates and connecting these processes via mathematical modeling. Our data points to a dominant role of transcription dynamics in the regulation of both gene induction and repression programs in response to TNF; and unveils a decrease in mRNA degradation rate as strategy for genes of late response to increase their intracellular concentrations. Furthermore, our analysis shows constitutive degradation as an intrinsic characteristic of genes that determines most of temporal ranks of gene expression profiles. Using a non-degradable form of inhibitor kappa B alpha and RelA knockout C2C12 cells we found that NF-κB is responsible for both gene induction and gene repression during muscle cell atrophy induced by TNF-α. Our fine-grained data highlights the importance of signaling dynamics in mediating the TNF-α effects on skeletal muscle cells and reveals a critical interplay between synthesis and degradation control in that regulates dynamic gene expression programs.
Cancer cachexia is a syndrome characterized by the severe skeletal muscle wasting tissue; that affects more than 50% of all cancer patients and results in lower quality of life due to compromised fatigue, weakness, decreased immune function, insulin resistance and poor tolerance and response to radio and chemotherapy. Remarkably, approximately 20% of cancer-related deaths are estimated to be directly caused by cachexia. There is currently no effective targeted therapy and the main limitation lays on the traditional approaches that not deal with the inherent complexity, characterized by non-linear interactions, of gene regulatory networks (GRN). Thus, a clear identification of the components of gene regulation, and a quantitative understanding of their temporal integration to control cellular responses is fundamental for capture essential mechanistic details that will ultimately enable the development of direct therapeutic strategies for the treatment of cancer cachexia. Here, we examine genome-wide gene expression of muscle wasting under two different frameworks, using static and dynamic gene expression data. We structure this approach as follow: Chapter 1 presents a quantitative characterization of the signaling pathways and a GRN reconstruction of muscle wasting in Lewis Lung Carcinoma (LLC) tumor-bearing mice by integrating static mRNAs and microRNAs expression profiles. The results show that LLC mice reduced body weight in 20% and presented muscle and fat tissue wasting after 23 days of tumor induction. In addition, we found 1008 differential expressed mRNAs (487 up-regulated and 521 down-regulated) and eighteen deregulated miRNAs (13 up-regulated and 5 down-regulated). Our data suggest activation of the transcriptional factor NF-κB and Stat3, which have been described in the activation of atrophic gene programs. Moreover, we ident potential posttranscriptional regulation by miRNAs of three important biological process: extracellular matrix organization, cell migration and transcription factor binding. Overall our results identify a set of signaling pathways that may contribute to muscle wasting in cancer cachexia, between them extracellular matrix genes with potential regulatory mechanism mediated by miRNAs. Chapter 2 provides further dissection of the NF-κB signaling pathway in atrophying muscle cells. Here, we examine quantitatively the genome-wide dynamic gene expression effects of the activation of NF-κB by the exposure of tumor necrosis factor – alpha (TNF-α) on skeletal muscle cells (C2C12). We characterize the regulatory strategies of gene induction and repression by measuring both mRNA transcription and degradation rates and connecting these processes via mathematical modeling. Our data points to a dominant role of transcription dynamics in the regulation of both gene induction and repression programs in response to TNF; and unveils a decrease in mRNA degradation rate as strategy for genes of late response to increase their intracellular concentrations. Furthermore, our analysis shows constitutive degradation as an intrinsic characteristic of genes that determines most of temporal ranks of gene expression profiles. Using a non-degradable form of inhibitor kappa B alpha and RelA knockout C2C12 cells we found that NF-κB is responsible for both gene induction and gene repression during muscle cell atrophy induced by TNF-α. Our fine-grained data highlights the importance of signaling dynamics in mediating the TNF-α effects on skeletal muscle cells and reveals a critical interplay between synthesis and degradation control in that regulates dynamic gene expression programs.
Descrição
Palavras-chave
Cancer Cachexia, gene regulation, miRNA, TNF signaling, Muscle atrophy, Transcriptional regulation