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.

在肿瘤进展、恶变和转移过程中观察到机械微环境和机械信号的变化。在这种情况下，了解机械转导信号传导的分子细节可能会提供独特的治疗靶点。在这里，我们报告正常乳腺上皮细胞对机械敏感，通过两部分钙信号传导机制对瞬时机械刺激做出反应。我们观察到细胞内钙立即、强劲上升（几秒钟内），随后细胞外钙持续流入（长达 30 分钟）。这种持久的钙通过 NADPH 氧化酶 2 (NOX2) 产生的活性氧 (ROS) 的微管依赖性机械激活来维持，ROS 作用于瞬时受体电位阳离子通道亚家族 M 成员 8 (TRPM8) 通道以延长钙信号传导。相比之下，引入组成型活性致癌 KRas 突变抑制了初始钙信号传导的强度，并严重削弱了持续的钙流入。致癌 KRas 在 ROS 水平上抑制机械诱导的钙的鉴定，为 KRas 如何改变细胞对肿瘤微环境力学的反应提供了一种机制，并可能揭示癌症的化疗靶点。此外，我们发现 NOX2 和 TRPM8 mRNA 的表达变化预示着雌激素受体 (ER) 阴性乳腺癌患者的临床结果不佳，而该人群的可用治疗选择有限。 临床和机械数据表明，乳腺癌患者中这种机械激活的钙途径被破坏，并且通过 KRas 激活揭示了信号传导的改变，可能影响癌细胞对肿瘤机械微环境的反应并影响患者的生存。