Transient mechanical stimuli elicit rapid mechano-chemical signal transduction in non-tumorigenic and malignant mammary epithelial cells

Abstract

Changes in are observed during breast tumor formation and progression, which promote malignant phenotypes in both normal mammary epithelial cells and breast cancer cells. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. This work applied time-lapse confocal microscopy and quantitative methods to define the rapid mechanically-stimulated calcium signaling mechanisms that occur in breast epithelial cells. While most tumor cell studies focus on long-term effects of mechanical stimulation (>24h), the current approach detected an immediate initiation of cytosolic calcium signals within 2 seconds after transient mechanical stimulation. Two novel methods were developed to describe this response and underlying mechanisms: a) the real-time scratch assay and b) scratch on low elastic dishes (SLED). The real-time scratch assay revealed an ATP/P2Y2/Ca2+ signaling axis in response to scratch with implications as a path toward EMT. The second method developed was SLED, a non-damaging application of mechanical stress to breast epithelial cells with physiologic implications as the elasticity of the cell substrate better matched that of the mammary gland in vivo than the typical glass or plastic experimental dishes. New data obtained using this novel approach revealed that normal breast epithelial cells are mechanically sensitive, responding to mechanical stimuli through a two-part calcium signaling mechanism. An immediate, robust rise in intracellular calcium (within seconds) was observed followed by a persistent extracellular calcium influx (up to 30 minutes). This persistent calcium was sustained via microtubule-dependent mechano-activation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on TRPM8 channels to prolong calcium signaling. Disruption of this conserved mechanobiology mechanism was possible through oncogenic activation such that, among common oncogenic mutations, constitutively-active KRas suppressed this signaling pathway. Therefore, certain oncogenes render cells mechanically unresponsive which could have interesting implications for the evolution of cancer cell mechanosensing in the tumor microenvironment as cells acquire new mutations. In addition, altered expression of the ROS generator (NOX2) and the ROS-responsive channel (TRPM8) are indicators of reduced overall survival in ER-negative breast cancer, suggesting that this mechano-pathway identified in breast epithelial cells may also be modified in patients in vivo.