RSK3 switches cell fate: from stress-induced senescence to malignant progression

Background TGF beta induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGF beta but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGF beta-induced senescence escape.Methods In vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-beta-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections.Results A screen with active kinases in HMECs upon TGF beta treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGF beta-induced senescence. Interestingly, RSK3 expression decreased in response to TGF beta in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGF beta-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-kappa Beta pathway through the decrease in proteasome-mediated I kappa B alpha degradation. Strikingly, senescent TGF beta-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-kappa Beta in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland.Conclusions We conclude that RSK3 switches cell fate from senescence to malignancy in response to TGF beta signaling.

Automated summary

The study revealed that RSK3 can promote escape from TGF beta-induced senescence by inhibiting the NF-kappa Beta pathway, shifting the cell fate from senescence to malignancy. This finding provides a novel insight into the understanding of cell fate switching in TGF beta signaling, which may be relevant to tumor progression.