Global signalling network analysis of luminal T47D breast cancer cells in response to progesterone

Background: Breast cancer cells enter into the cell cycle following progestin exposure by the activation of signalling cascades involving a plethora of enzymes, transcription factors and co-factors that transmit the external signal from the cell membrane to chromatin, ultimately leading to a change of the gene expression program. Although many of the events within the signalling network have been described in isolation, how they globally team up to generate the final cell response is unclear. Methods: In this study we used antibody microarrays and phosphoproteomics to reveal a dynamic global signalling map that reveals new key regulated proteins and phosphor-sites and links between previously known and novel pathways. T47D breast cancer cells were used, and phospho-sites and pathways highlighted were validated using specific antibodies and phenotypic assays. Bioinformatic analysis revealed an enrichment in novel signalling pathways, a coordinated response between cellular compartments and protein complexes. Results: Detailed analysis of the data revealed intriguing changes in protein complexes involved in nuclear structure, epithelial to mesenchyme transition (EMT), cell adhesion, as well as transcription factors previously not associated with breast cancer cell proliferation. Pathway analysis confirmed the key role of the MAPK signalling cascade following progesterone and additional hormone regulated phospho-sites were identified. Full network analysis shows the activation of new signalling pathways previously not associated with progesterone signalling in T47D breast cancer cells such as ERBB and TRK. As different post-translational modifications can mediate complex crosstalk mechanisms and massive PARylation is also rapidly induced by progestins, we provide details of important chromatin regulatory complexes containing both phosphorylated and PARylated proteins. Conclusions: This study contributes an important resource for the scientific community, as it identifies novel players and connections meaningful for breast cancer cell biology and potentially relevant for cancer management. This research was supported by European Research Council (Project “4D Genome” 609989), the Ministerio de Economía y Competitividad (Project G62426937) and the Generalitat de Catalunya (Project AGAUR SGR 575 and AGAUR 2019PROD00115/IU68-016733), European Research Council -Proof Of Concept (Project “Impacct” 825176).