Resistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ER?)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells.
Reversible ubiquitylation of proteins contributes to their integrity, abundance and activity. The RE1-silencing transcription factor (REST) plays key physiological roles and is dysregulated in a spectrum of disease. It is rapidly turned over and is phosphorylated, polyubiquitylated and degraded en masse during neuronal differentiation and cell cycle progression. Through siRNA screening we identified the deubiquitylase USP15 as a key regulator of cellular REST. Both antagonism of REST polyubiquitylation and rescue of endogenous REST levels are dependent on the deubiquitylase activity of USP15. However, USP15 depletion does not destabilize pre-existing REST, but rather specifically impairs de novo REST synthesis. Indeed, we find that a small fraction of endogenous USP15 is associated with polysomes. In accordance with these findings, USP15 does not antagonize the degradation of phosphorylated REST at mitosis. Instead it is required for the rapid accumulation of newly synthesized REST on mitotic exit, thus playing a key role in its cell cycle oscillations. Importantly, this study reveals a novel role for a DUB in specifically promoting new protein synthesis.
The scattering response of epithelial cells to activation of the Met receptor tyrosine kinase represents one facet of an "invasive growth" program. It is a complex event that incorporates loss of cell-cell adhesion, morphological changes, and cell motility. Ubiquitination is a reversible posttranslational modification that may target proteins for degradation or coordinate signal transduction pathways. There are approximately 79 active deubiquitinating enzymes (DUBs) predicted in the human genome. Here, via a small interfering RNA (siRNA) library approach, we have identified 12 DUBs that are necessary for aspects of the hepatocyte growth factor (HGF)-dependent scattering response of A549 cells. Different phenotypes are evident that range from full loss of scattering, similar to receptor knockdown (e.g., USP30, USP33, USP47), to loss of cell-cell contacts even in the absence of HGF but defective motility (e.g., USP3, ATXN3L). The knockdowns do not incur defective receptor, phosphatidylinositol 3-kinase, or MAP kinase activation. Our data suggest widespread involvement of the ubiquitin system at multiple stages of the Met activation response, implying significant crosstalk with phosphorylation-based transduction pathways. Development of small-molecule inhibitors of particular DUBs may offer a therapeutic approach to contain metastasis.
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