Abstract B57: Assessment of gene expression of calcium pumps, channels, and channel regulators in human breast cancer-associated fibroblasts

Abstract

Abstract Fibroblasts represent one of the predominant stromal cell populations present in the breast tumor microenvironment. Cancer-associated fibroblasts found growing in close proximity to the tumor site are thought to influence tumor behavior. While a number of protein and mRNA markers showing differential expression between cancer-associated and non-cancer associated fibroblasts have been proposed, current understanding of the phenotypic differences between these two cell populations remains incomplete. Calcium signaling plays an important role in each of the cancer hallmarks and is altered in a variety of disease states including cardiovascular disease. The aim of this study was to investigate the potential remodeling of the calcium signal and calcium channel and pump expression as a consequence of the transition from a normal to cancer-associated phenotype in human breast fibroblasts. To investigate expressional changes, RNA was isolated from primary cultures of matched normal and cancer-associated fibroblasts established from human breast cancer tissue specimens. Real time RT-PCR was used to assess mRNA levels of a total of 36 proteins involved in the movement of Ca2+ into and out of the cytoplasmic space, and included different classes of plasma membrane localized Ca2+ channels, as well as Ca2+ pumps of intracellular organelles. To further study potential differences in Ca2+ signaling between normal and cancer-associated fibroblast phenotypes, the immortalized human breast fibroblast line HMF3S was treated with transforming growth factor beta 1 (TGFβ1) (0, 0.01, 0.1, 1 and 10 ng/mL, 48 h), a growth factor implicated in the tumor microenvironment and myofibroblast differentiation. Successful TGFβ1-mediated activation of fibroblasts to a cancer-associated phenotype was confirmed by induction of smooth muscle alpha actin (SMαA) protein via immunoblotting. Real time RT-PCR was used to assess mRNA levels of classes of Ca2+ channels and pumps showing altered expression in patient matched normal and cancer-associated fibroblast pairs. In addition to assessing expression changes, potential functional differences in intracellular Ca2+ signaling profiles of HMF3S cells treated with TGFβ1 were assessed using a fluorometric imaging plate reader (FLIPRTETRA) and cells loaded with the Ca2+ sensitive indicator Fluo-4. Using both clinical samples, and an in vitro model of cancer-associated fibroblasts, our findings reveal a differential remodeling of plasma membrane calcium channels and organellar pumps of different classes in cells representative of cancer-associated relative to non-cancer associated fibroblasts. In addition to expression changes, there also appears to be a functional remodeling of the calcium signal in HMF3S fibroblasts induced to transition from a normal to cancer-associated phenotype by TGFβ1. This is the first study to assess changes in calcium pumps, channels and channel regulators, as well as remodeling of the calcium signal, in breast cancer-associated fibroblasts. This work may suggest that the breast cancer-associated fibroblast phenotype is associated with altered Ca2+ signaling. Citation Format: Teneale A. Stewart, Patsy S. Soon, Sarah J. Roberts-Thomson, Gregory R. Monteith. Assessment of gene expression of calcium pumps, channels, and channel regulators in human breast cancer-associated fibroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B57.