Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas
Menée sur des cellules épithéliales mammaires non tumorigènes, cette étude démontre que les espèces réactives de l'oxygène, générées par l'activation mécanique de la NADPH oxydase 2, prolongent la signalisation calcique des canaux TRPM8 et que cette signalisation est inhibée par l'oncogène KRAS
In breast cancer, the chronically stiffening mechanical microenvironment promotes tumor growth/invasion. However, the molecular details and integration of cancer mechanotransduction signaling pathways are not well understood. We find that nontumorigenic breast epithelial cells are mechanically sensitive and respond to acute mechanical stimuli with the generation of ROS-stimulated calcium signaling in a microtubule-dependent manner. These results establish that epithelial cells conserve a mechanotransduction pathway from muscle and bone (X-ROS). Using common breast cancer genetic mutations, we further report that oncogene activation reduces X-ROS pathway mechanoresponsiveness at the level of ROS sensitivity. This definition of how oncogene activation disrupts highly conserved mechanotransduction signaling mechanisms could improve the understanding of altered tumor cell responses to the mechanical microenvironment and reveal new therapeutic opportunities. Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.There are no restrictions on the availability of materials or information. Data are provided in this manuscript as well as in Dataset S1. All MATLAB analysis code used in this manuscript is publicly available in a GitHub repository (DOI: 10.5281/zenodo.3986143).