Abstract P6-06-15: Remodelling of calcium influx pathways in breast cancer associated fibroblasts

Abstract

Abstract Disruption of Ca2+ signaling during breast cancer tumorigenesis is now well-established. Despite the increasing recognition of the role of the tumour microenvironment, and cancer associated fibroblasts (CAFs), in tumorigenesis, very few studies have assessed alterations in Ca2+ influx pathways in this context. The aims of this study were to identify changes in the nature of Ca2+ influx in breast CAFs and to define the role of Ca2+ signaling in the induction of CAFs. We first developed a model for CAF induction using an immortalized human mammary fibroblast cell line, HMF3S. These cells were treated with transforming growth factor beta (TGFβ) to induce a CAF phenotype (HMF3S-CAF) as confirmed by the induction established CAF markers, including alpha smooth muscle actin. Fluorescence Imaging Plate Reader Ca2+ assays demonstrated that store operated Ca2+ entry (SOCE) was reduced as a consequence of TGFβ stimulated CAF induction in HMF3S-CAFs. As a potential mechanism, mRNA levels of the SOCE components, ORAI1, STIM1 and STIM2 were assessed. STIM2 was downregulated and ORAI1 was slightly upregulated suggesting that the decrease in SOCE in HMF3S-CAF cells were independent of mRNA expression changes of these genes. Voltage gated calcium channel (VGCC) mRNA expression was measured to determine whether other Ca2+ influx pathways were altered during CAF differentiation. Here, we found that Cav 1.2 and Cav 3.2 were significantly upregulated in HMF3S-CAF cells. Assessment of mRNA levels of SOCE components and VGCC in paired normal and CAF patient samples showed no change in SOCE components while, of the VGCCs, only Cav 1.2 and Cav 3.2 were upregulated, suggesting that increased expression of these channels may be a feature of breast cancer CAFs. The importance of Cav 1.2 and Cav 3.2 in CAF induction was assessed by pharmacological inhibition using nimodipine (Cav 1.2) or ML218 (Cav 3.2), both of which decreased TGFβ-mediated CAF activation. Our preliminary data suggest that siRNA against specific VGCCs may also reduce CAF activation. Our data indicate that Ca2+ signaling is remodelled in CAFs and that VGCC Ca2+ signal is important for breast CAF induction. Citation Format: Francisco Sadras, Teneale A Stewart, Melanie Robitaille, Amelia A Peters, Priyakshi Kalita-de Croft, Patsy Soon, Jodi M Saunus, Sunil R Lakhani, Sarah J Roberts-Thomson, Gregory R Monteith. Remodelling of calcium influx pathways in breast cancer associated fibroblasts [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-15.