En esta tesis informamos del crecimiento de células T47D que responden a hormonas como esferoides 3D en Matrigel imitando la MEC. Una caracterización detallada de las principales diferencias físicas y bioquímicas entre las células cultivadas en 2D y como esferoides 3D, centrándose en las vías particulares reguladas a través de la señalización mecano-transduccional. En los dos primeros capítulos se detallan las principales diferencias físicas y genómicas. Las células cultivadas en 3D tienen un núcleo más redondo con cromatina menos accesible y más compactada, lo que influye en la regulación de más de 2000 genes. La topología génica analizada por HiC, indica que las células 3D exhiben una tendencia sutil hacia el enriquecimiento en las regiones que pertenecen al compartimento B, con una disminución de unión en la proteína CTCF y mayor fusión de TAD. En el tercer capítulo discutimos la participación de la vía Hippo, involucrada en la mecanosensibilidad en células cultivadas en 3D. La fosforilación de CTCF mediada por p-LATS promueve su desplazamiento de la cromatina. Con respecto a la regulación hormonal, las células cultivadas en 3D responden de manera más eficiente y exhiben un aumento en los sitios de unión de PR que conducen a la remodelación de la cromatina y a un mayor número de genes regulados. Nuestros resultados sugieren que la MEC ejerce efectos importantes sobre la estructura y función del núcleo de las células del cáncer de mama.
The cellular processes required for the development and behavior of cancer cells are not only defined by their genome but also by their epigenome, which is largely influenced by the cell microenvironment. Our motivation arises from the fact that routinely, cancer cells are studied in isolation, grown in 2D as monolayers on plastic surfaces. However, recent studies highlight the impact of the 3D environment on multiple cellular functions. Therefore, cancer cells should be studied in contact with other cells and with the extracellular matrix (ECM).
In this Thesis, we report results divided into four different chapters, the successful growth of hormone responsive T47D cells as 3D spheroids in Matrigel mimicking the ECM. By using a genome-wide approach we performed a detailed characterization of the main physical and biochemical differences between cells cultured in conventional 2D and as 3D spheroids, focusing on the particular pathways regulated through mechano-transductional signaling.
Compared to cells cultured in 2D, 3D grown cells have a rounder nucleus with less accessible and more compacted chromatin, impacting on the regulation of over 2000 genes. We assessed how growing the cells in 3D affects the genome topology, as analyzed by HiC. Our results indicate that 3D cells exhibit a subtle trend towards enrichment in regions belonging to the B compartment (inactive chromatin), a decrease on chromatin bound architectural protein CTCF and increased fusion of Topologically Associating Domains (TADs).
Interestingly, the Hippo pathway kinase LATS involved in mechanosensing, is activated in 3D grown cells. In line with the observed p-LATS mediated phosphorylation of CTCF promoting its displacement from chromatin.
Regarding hormone regulation, 3D grown cells respond more efficiently and exhibit an increase in PR binding sites leading to chromatin remodeling and to a higher number of regulated genes. The increased activation of p-LATS promotes a cytoplasmic retention of YAP allowing binding of PR to new sites and thus regulating new genes. In conclusion, the Hippo pathway through p-LATS acts at least at two levels in 3D cells: i) through phosphorylation of CTCF and ii) through phosphorylation of YAP . Whether these two events are connected remains to be investigated.
Our results suggest that the ECM exerts important effects on the structure and function of the breast cancer cell nucleus.
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