High calcium inducible genes in triple negative breast cancer cells

Malignant breast cancer is often associated with skeletal metastasis and co-morbidities such as cancer-induced hypercalcemia. Although circulating calcium has been linked to aggressive and larger breast tumors, the mechanisms underlying the potential oncogenic effects of high calcium remain poorly understood. Here, we assessed the effects of high extracellular calcium on the modulation of gene expression in TNBC cells. This study showed that chronic high calcium attenuates the activity of the calcium sensing receptor but promotes the growth and migration of triple negative breast cancer cells. We also show that in TNBC cells, high extracellular calcium triggered the expression of early response genes such as FOS/FOSB and subsequently, mailgnancy assoicated genes including the cancer testis antigen MAGEC2. This study provides novel mechanistic insights into the hitherto unappreciated oncogenic effects of high extracellular calcium especially in malignant or late-stage breast cancers. MDA-MB-231 cells were cultured in complete medium or complete medium supplemented with 3.0 mM or 5.0 mM calcium for 48 h (n=4) and differential gene expression was assessed by gene expression profiling on Human Gene 2.0ST Affimetrix Genechip. After hybridization, the cartridge arrays were washed, and stained per standard Affymetrix protocols using an Affymetrix Fluidics Station 450. The arrays were then scanned in an Affymetrix 7G plus scanner and the resulting data were analyzed by Affymetrix Expression Console v.1.2 using an Robust Multi-array Aver-age (RMA) normalization algorithm producing log base 2 results. Differential gene expression was assessed between replicate groups (n=4) using a moderated t-test and Benjamini-Hochberg multiple testing correction, with significance determined by adjusted p-value <0.05 and absolute value fold change >2.0. Hierarchical clustering was performed in GeneSpring on both averaged and non-averaged normalized expression values.