Análise em larga escala da senescência desencadeada por FGF-2 em células tumorais de camundongo da linhagem Y1

Análise em larga escala da senescência desencadeada por FGF-2 em células tumorais de camundongo da linhagem Y1

Author Lopes, Mariana De Camargo Autor UNIFESP Google Scholar
Advisor Cunha, Julia Pinheiro Chagas Da Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Graduate program Microbiologia e Imunologia
Abstract Despite being a growth factor, FGF-2 has anti-proliferative and tumor suppressive functions in some cellular contexts. In a murine adrenocortical tumor cell line that has the proto-oncogene K-Ras constitutively amplified and overexpressed (lineage Y1), FGF-2 triggers an initial mitogenic response through ERK1/2, PKC and AKT signaling that ultimately results in cell cycle arrest at the G2/M transition through RhoA/GTP-src signaling pathways. To better understand the dual phenotypic response (mitogenic versus cell cycle arrest) induced by FGF-2, we employed quantitative mass spectrometry to serially analyze histone PTMs and general protein phosphorylation in Y1 cells over time after treatment FGF-2 or serum alone. Despite their relevance to understanding the regulation of gene expression, large-scale analyses that connect signaling pathways to histone post-translational modification dynamics are lacking in the literature. We report that treatment of Y1 cells with FGF-2 induces effects that are in agreement with the phenomenon of oncogene-induced cell senescence, based on detection of heterochromatin foci formation with histone H1 depletion, silencing of cyclins and cyclin dependent kinase genes, DNA damage foci formation, lamin-B1 depletion and induction of a secretory phenotype containing interleukins and other soluble factors, such as IL-11 and PAI-1. Specifically, we observed a global decrease in markers of active chromatin regions, including histone H4 N-terminal acetylation and H3K27ac, and an increase in the abundance of the inactive chromatin marker H3K27me3, starting 3h after FGF-2 treatment. The hypothesis that FGF-2 induces alterations in chromatin structure, increasing the number of heterochromatin regions, was confirmed by electron microscopy. Subsequently, we integrated the data to obtain a systems biology perspective of the signaling pathways and epigenetic changes induced by serum and/or FGF-2. Moreover, our results indicate that FGF-2 treatment affects the phosphorylation dynamics of nuclear proteins related to transcription, as well as the abundance of proteins related to certain XVI biological processes, such as mitochondrial activity and translation. Having established that FGF-2 can alter transcriptional activity, we performed RNA-seq to identify genes and pathways affected by this growth factor, which may serve as candidates for future knockdown and overexpression studies to better understand the induction of senescence in tumor cells. We conclude that FGF-2 negatively affects pathways related to the cell cycle and reduces the abundance of the active chromatin marker H3K27ac near transcription start sites (TSS) of genes such as cyclin dependent kinase, cyclin A2 and cyclin B2, all of which are important for cell cycle progression. In addition, we observed that FGF-2 can induce a negative regulation of the MAPK pathway, increasing H3K27ac abundance near the TSS of the Dusp5 and Dusp6 genes, which are also upregulated transcriptionally and may relate to the phenotype discussed here. We believe that our work will shed light on how signaling pathways may interact with histone PTMs in both proliferative (serum stimulated cells) and non-proliferative (FGF-2 stimulated cells) responses.

O fator de crescimento de fibroblastos 2 (FGF-2) é conhecido por ser um agente mitogênico, mas pode apresentar funções anti-proliferativas e supressoras de tumor em alguns contextos. Em uma linhagem de tumor adrenocortical de camundongos que possui o proto-oncogone K-Ras constitutivamente amplificado e superexpresso (linhagem Y1) FGF- 2 desencadeia uma resposta mitogênica inicial, pelas vias de sinalização de ERK1/2, PKC e AKT, mas resulta em um bloqueio do ciclo celular em G2/M pela via de sinalização de RhoA/GTP-src. Para melhor entendermos essa dualidade de respostas induzidas por esse fator de crescimento (mitogênica versus parada no ciclo celular), realizamos uma análise do proteoma e do fosfoproteoma das células Y1 em uma curva de tempo após tratamento com FGF-2, utilizando espectrometria de massas e ferramentas de bioinformática. Análises em larga escala de vias de sinalização integradas com análises de dinâmicas de modificações pós traducionais em histonas são pouco exploradas na literatura, apesar de sua importância na regulação gênica. Observamos que o tratamento com FGF-2 induz alguns efeitos que estão de acordo com o fenômeno da senescência induzida por oncogenes como formação de focos de heterocromatina com depleção de histona H1, silenciamento de genes de ciclinas, formação de focos de dano no DNA e de formação de um fenótipo secretório contendo interleucinas como IL- 11 e outros fatores solúveis, como PAI-1, além da depleção de lamina-B1. Mais especificamente, observamos uma diminuição global em marcadores de regiões de cromatina ativa, como acetilação no N-terminal da histona H4 e H3K27ac, e um aumento na abundância de marcadores de regiões de cromatina inativa, como H3K27me3, iniciando-se após 3h de tratamento com FGF-2. A hipótese de que FGF-2 induz alterações na estrutura da cromatina, aumentado a incidência de regiões de biological processes, such as mitochondrial activity and translation. Having established that FGF-2 can alter transcriptional activity, we performed RNA-seq to identify genes and pathways affected by this growth factor, which may serve as candidates for future knockdown and overexpression studies to better understand the induction of senescence in tumor cells. We conclude that FGF-2 negatively affects pathways related to the cell cycle and reduces the abundance of the active chromatin marker H3K27ac near transcription start sites (TSS) of genes such as cyclin dependent kinase, cyclin A2 and cyclin B2, all of which are important for cell cycle progression. In addition, we observed that FGF-2 can induce a negative regulation of the MAPK pathway, increasing H3K27ac abundance near the TSS of the Dusp5 and Dusp6 genes, which are also upregulated transcriptionally and may relate to the phenotype discussed here. We believe that our work will shed light on how signaling pathways may interact with histone PTMs in both proliferative (serum stimulated cells) and non-proliferative (FGF-2 stimulated cells) responses.
Keywords FGF-2
Senescence
Histones
PTM
Mass Spectrometry
RNAseq
ChiP-seq
FGF-2
Senescência
Histonas
MPT
Espectrometria De Massas
RNAseq
ChiP-seq
Language Portuguese
Date 2019-08-29
Research area Estudos De Epidemiologia Molecular, Biologia Molecular/Regulação, Diversidade Genética E Evolução Em Patógenos Humanos Do Tipo Vírus, Bactérias, Fungos E Tripanosomatídeos
Knowledge area Biologia Molecular
Publisher Universidade Federal de São Paulo (UNIFESP)
Extent 181 p.
Origin https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=8038489
Access rights Closed access
Type Thesis
URI https://repositorio.unifesp.br/handle/11600/59232

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