Stromal Stiffness Increases YAP-Dependent Mediated Cell Survival of Breast Cancer Cells

Abstract

The Extracellular Matrix (ECM) provides biochemical and biophysical cues that activate key molecular pathways that direct cell fate in cancer cell development. These cues are primarily transduced through the integrin family of receptor ECM stiffness, which are known to enhance integrin activity and enhance the malignant behavior of tumors by activating the YAP-TAZ transcription factors. The stroma of cancerous tissues is a potent and active mitigator of malignant transformation. The mechanism by which stromal tension acts to promote resistance has yet to be identified. In the present study, we investigate whether ECM stiffness mediates therapy resistance in premalignant invasive breast cancer tumors by regulating integrin-dependent YAP activity. We used 3D organotypic cultures of invasive breast cancer cells (T4-2 WT, and T4-2 YAP-KO cells) cultured on polyacrylamide gels of varying stiffness (Low [400 Pa], and High [4K Pa]). To determine whether stromal stiffness increases YAP-mediated cell survival, we monitored proliferation (Ki-67) and cell survival (TUNEL) post-irradiation treatment (IR, 8 Gy). Additionally, we looked at the direct effect of YAP activation on tumor grow by injecting T4-2 WT or T4-2 YAP-KO breast cancer cells in Neu mice treated either with a YAP-inhibitor verteporfin (100mg/kg) or a control (IPTG, 10mM/M) for two weeks (5 treatments). Our study shows that irradiated breast cancer cells grown on stiff ECM in vitro lead to a significant increase in YAP nuclear localization. Furthermore, using ANOVA analysis, we observed a significant elevated cell proliferation (P < 0.05) and enhanced cell survival (P < 0.05) leading to the activation of the YAP-TAZ signaling pathways. Our results also showed that inhibition of YAP signaling in stiff ECM cultures treated with IR resulted in a significant reduction of proliferation of breast cancer cells cultured in vitro as well as a reduction of tumor growth in mice (P < 0.05). Overall, these results provide evidence that stiff micro-environmental conditions of breast cancer cells are a potent mitigator of therapy resistance whose effects are mediated through YAP activity.