Deposition of calcium in an in vitro model of human breast tumour calcification reveals functional role for ALP activity, altered expression of osteogenic genes and dysregulation of the TRPM7 ion channel

Abstract

Microcalcifications are vital mammographic indicators contributing to the early detection of up to 50% of non-palpable tumours and may also be valuable as prognostic markers. However, the precise mechanism by which they form remains incompletely understood. Following development of an in vitro model using human breast cancer cells lines cultured with a combination of mineralisation-promoting reagents, analysis of calcium deposition, alkaline phosphatase (ALP) activity and changes in expression of key genes was used to monitor the calcification process. Two cell lines were identified as successfully mineralising in vitro, MDA-MB-231 and SKBR3. Mineralising cell lines displayed higher levels of ALP activity that was further increased by addition of mineralisation promoting media. qPCR analysis revealed changes in expression of both pro- (RUNX2) and anti- (MGP, ENPP1) mineralisation genes. Mineralisation was suppressed by chelation of intracellular Ca²⁺ and inhibition of TRPM7, demonstrating a functional role for the channel in formation of microcalcifications. Increased Mg²⁺ was also found to effectively reduce calcium deposition. These results expand the number of human breast cancer cell lines with a demonstrated in vitro mineralisation capability, provide further evidence for the role of an active, cellular process of microcalcification formation and demonstrate for the first time a role for TRPM7 mediated Ca²⁺ transport.

Figure 1

Establishment of a human in vitro model of microcalcification formation. Representative Alizarin Red S staining of MCF7 (A), MCF7-HER2 (B), SKBR3 (C) and MDA-MB-231 (D) cells over time, shown at 10X magnification (n = 3). Control (regular growth media), OC (osteogenic cocktail) (50 µg/ml ascorbic acid and 10 mM β-glycerophosphate) Dex (100 nM dexamethasone). Scale bar represents 50 µm. Calcium content of SKBR3 (E) and MDA-MB-231 (F) cells as determined by o-cresolphthalein calcium assay and normalised to protein. Each point represents the mean + SD (n = 3), two-way ANOVA. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2

ALP activity as a key determinant of cellular mineralisation potential in human breast cancer cell lines. ALP activity was analysed in MDA-MB-231 cells (A) every 7 days by incubation with the ALP substrate p-nitrophenyl phosphate (PNPP), monitoring absorbance at 405 nm. ALP activity was normalised to protein content. Each point represents the mean + SD (n = 3), two-way ANOVA. *p < 0.05, **p < 0.01, ****p < 0.0001. ALP activity was compared between three cell lines (MDA-MB-231, SKBR3 & MCF7) by measuring “Control” values (3 replicates at 4 time points for a total of n = 12 for each cell line) (b). Alizarin Red S staining of MCF7 cells grown in control (regular growth media), OC (osteogenic cocktail) (50 µg/ml ascorbic acid and 10 mM β-glycerophosphate) or OC + Dex (OC + 100 nM dexamethasone) media supplemented with 1 U/mL exogenous bovine ALP or vehicle control (C), and MDA-MB-231 cells grown in control, OC or OC + Dex media supplemented with 100 µM levamisole or vehicle control (D). Representative images (n = 3) were captured at 10X magnification. Scale bar represents 50 µm.

Figure 3

Altered expression of mineralisation regulating factors in MDA-MB-231 cells. Expression of RUNX2 (A), MGP (B) and ENPP1 (C) was analysed by qPCR in MDA-MB-231 cells cultured in control (regular growth media) or OC + Dex (50 µg/ml ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone) media at the indicated time-points. Each time point represents the mean + SD (n = 3). qPCR results normalised to control values at each time point. Statistical significance was determined by two-way ANOVA, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

Relationship between extracellular and intracellular Ca²⁺ levels and development of MDA-MB-231 mineralisations. Representative images (10X magnification) of Alizarin Red S stained MDA-MB-231 cells in either OC (osteogenic cocktail) (50 µg/ml ascorbic acid and 10 mM β-glycerophosphate) or OC + Dex (OC + 100 nM dexamethasone) media at the indicated Ca²⁺ concentration (A). Scale bar represents 50 µm. Calcium content, as measured by OCP assay normalised to protein content revealed increased mineralisation in OC + Dex cultured cells at 2.7 mM Ca²⁺ (B). Each point represents mean + SD (n = 4). Usage of the intracellular Ca²⁺ chelator BAPTA-AM significantly decreased both calcification (C) and ALP activity (D). Each point represents mean + SD (n = 3). Statistical significance was determined by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Role of TRPM7 channel in microcalcification formation. Expression of TRPM7 was analysed by qPCR in MDA-MB-231 cells cultured in control (regular growth media) or OC + Dex (50 µg/ml ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone) at the indicated time-points (A). Each time point represents the mean + SD (n = 3). qPCR results normalised to control values at each time point. Calcium content of MDA-MB-231 cells cultured in OC + Dex media with 2-APB (B) or NS8593 (C) at the indicated concentration. Each value represents mean + SD (n = 3). ALP activity of MDA-MB-231 cells treated with 150 µM with 2-APB or 10 µM NS8593 (D). Decreased TRPM7 expression (E), Alizarin Red S (F) and calcium content (G) of MDA-MB-231 cells treated with TRPM7-targeting siRNA. Each point represents mean + SD (n = 5). Representative images shown at 10X magnification. Scale bar represents 50 µm. Statistical significance was determined by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

Anti-mineralisation effect of Mg²⁺. Representative Alizarin Red S staining of MDA-MB-231 cells cultured in OC + Dex media (50 µg/ml ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone) with or without 0.7 mM Mg²⁺ supplementation (A). Alizarin Red S staining (B) and calcium content (C) of MDA-MB-231 cells cultured in OC + Dex media with 0.7 mM Mg²⁺ supplementation and NS8593 at the indicated concentration. Each point represents mean + SD (n = 3). Representative images shown at 10X magnification. Scale bar represents 50 µm. Statistical significance was determined by one-way ANOVA.

Figure 7

Proposed model of microcalcification formation. Extracellular nucleotide triphosphates (primarily ATP) are hydrolysed by activity of ENPP1 to release inhibitory pyrophosphate (PPi) which is further degraded by ALP. Free phosphate (Pi) enters the cell via the sodium-dependent phosphate transporter Pit-1, where it can combine with Ca²⁺ entering, at least in part, via the TRPM7 channel to begin nucleation of hydroxyapatite. The OC + Dex media formulation used in our studies enhances several aspects of this process including upregulation of the pro-mineralisation ALP and RUNX2, and downregulation of the anti-mineralisation ENPP1.