ABSTRACT
Notch signaling is a conserved signaling pathway that participates in many aspects of mammary gland development and homeostasis, and has extensively been associated with breast tumorigenesis. Here, to unravel the as yet debated role of Notch3 in breast cancer development, we investigated its expression in human breast cancer samples and effects of its loss in mice. Notch3 expression was very weak in breast cancer cells and was associated with good patient prognosis. Interestingly, its expression was very strong in stromal cells of these patients, though this had no prognostic value. Mechanistically, we demonstrated that Notch3 prevents tumor initiation via HeyL-mediated inhibition of Mybl2, an important regulator of cell cycle. In the mammary glands of Notch3-deficient mice, we observed accelerated tumor initiation and proliferation in a MMTV-Neu model. Notch3-null tumors were enriched in Mybl2 mRNA signature and protein expression. Hence, our study reinforces the anti-tumoral role of Notch3 in breast tumorigenesis.
Subject(s)
Breast Neoplasms , Cell Transformation, Neoplastic , Animals , Female , Humans , Mice , Basic Helix-Loop-Helix Transcription Factors , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Cycle , Cell Cycle Proteins , Cell Division , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Homeostasis , Receptor, Notch3/genetics , Repressor Proteins , Trans-ActivatorsABSTRACT
Understanding drug target engagement and the relationship to downstream pharmacology is critical for drug discovery. Here we have evaluated target engagement of Chk1 by the small-molecule inhibitor V158411 using two different target engagement methods (autophosphorylation and cellular thermal shift assay [CETSA]). Target engagement measured by these methods was subsequently related to Chk1 inhibitor-dependent pharmacology. Inhibition of autophosphorylation was a robust method for measuring V158411 Chk1 target engagement. In comparison, while target engagement determined using CETSA appeared robust, the V158411 CETSA target engagement EC50 values were 43- and 19-fold greater than the autophosphorylation IC50 values. This difference was attributed to the higher cell density in the CETSA assay configuration. pChk1 (S296) IC50 values determined using the CETSA assay conditions were 54- and 33-fold greater than those determined under standard conditions and were equivalent to the CETSA EC50 values. Cellular conditions, especially cell density, influenced the target engagement of V158411 for Chk1. The effects of high cell density on apparent compound target engagement potency should be evaluated when using target engagement assays that necessitate high cell densities (such as the CETSA conditions used in this study). In such cases, the subsequent relation of these data to downstream pharmacological changes should therefore be interpreted with care.
