RESUMEN
BACKGROUND: During breast cancer progression, the epithelial to mesenchymal transition has been associated with metastasis and endocrine therapy resistance; however, the underlying mechanisms remain elusive. To gain insight into this process, we studied the transition undergone by MCF7-derived cells, which is driven by the constitutive nuclear expression of a MKL1 variant devoid of the actin-binding domain (MKL1 ΔN200). We characterized the adaptive changes that occur during the MKL1-induced cellular model and focused on regulation of translation machinery and metabolic adaptation. METHODS: We performed a genome-wide analysis at the transcriptional and translational level using ribosome profiling complemented with RNA-Seq and analyzed the expression of components of the translation machinery and enzymes involved in energy metabolism. NGS data were correlated with metabolomic measurements and quantification of specific mRNAs extracted from polysomes and western blots. RESULTS: Our results reveal the expression profiles of a luminal to basal-like state in accordance with an epithelial to mesenchymal transition. During the transition, the synthesis of ribosomal proteins and that of many translational factors was upregulated. This overexpression of the translational machinery appears to be regulated at the translational level. Our results indicate an increase of ribosome biogenesis and translation activity. We detected an extensive metabolic rewiring occurring in an already "Warburg-like" context, in which enzyme isoform switches and metabolic shunts indicate a crucial role of HIF-1α along with other master regulatory factors. Furthermore, we detected a decrease in the expression of enzymes involved in ribonucleotide synthesis from the pentose phosphate pathway. During this transition, cells increase in size, downregulate genes associated with proliferation, and strongly upregulate expression of cytoskeletal and extracellular matrix genes. CONCLUSIONS: Our study reveals multiple regulatory events associated with metabolic and translational machinery adaptation during an epithelial mesenchymal-like transition process. During this major cellular transition, cells achieve a new homeostatic state ensuring their survival. This work shows that ribosome profiling complemented with RNA-Seq is a powerful approach to unveil in-depth global adaptive cellular responses and the interconnection among regulatory circuits, which will be helpful for identification of new therapeutic targets.
RESUMEN
Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.