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.

Role of Secreted Frizzled-Related Protein 1 in Early Mammary Gland Tumorigenesis and Its Regulation in Breast Microenvironment

“…This capacity of breast cells to develop osteomimetic properties were also reported in 2019 by O'Grady et al [103]. In fact, they demonstrated the ability of breast cancer cell lines to mineralize, and doing so, proved the capacity of breast cancer cells to produce de novo microcalcifications in osteogenic conditions [103]. Furthermore, microcalcifications are described as an indirect sign of pathological process, such as inflammation, and, as described previously, inflammation is also associated with breast involution in both post-lactation and peri-menopausal contexts, but also in tumorigenesis and tumoral growth.…”

“…Furthermore, they proved an osteoblast phenotype acquisition of the cells producing hydroxyapatite by performing immunohistochemical analysis. This capacity of breast cells to develop osteomimetic properties were also reported in 2019 by O'Grady et al [103]. In fact, they demonstrated the ability of breast cancer cell lines to mineralize, and doing so, proved the capacity of breast cancer cells to produce de novo microcalcifications in osteogenic conditions [103].…”

“…Most breast calcifications visible at mammography are made of a combination of calcium and phosphate, the exact composition of the mineral part of bones. Moreover, evidences of osteoblast-like cells in breast tissue suggest a potential for breast cells differentiation in a microcalcification environment [37,[95][96][97][98][99][100][101][102][103]. In 2005, Morgan et al [99] reported that microcalcifications were responsible for increasing the mitogenesis properties of breast cells, resulting in an amplification of the malignant process.…”

Osteogenic cocktail induces calcifications in human breast cancer cell line via placental alkaline phosphatase expression

“…In fact, O’Grady et al . showed that ALP enzyme activity was increased by OC in MDA-MB-231 and SKBR3 cells with calcium deposition 25 . However, the study did not completely unveil how breast cancer cells acquire ALP enzyme activity, although they showed that ALP inhibitor levamisole suppress mineralization in MDA-MB-231 cells cultured using OC.…”

Molecular Aspects of Thyroid Calcification

“…A high expression of ALP in breast cancer has been reported. Mineralizing cell lines (MDA-MB-231 and SKBR3) displayed higher levels of ALP activity that was further increased by the addition of mineralization-promoting media [ 117 ]. Additionally, elevated levels of ALP in the serum of breast cancer patients were detected when compared to controls [ 118 ], in patients with bone metastases in comparison to patients without bone metastases [ 119 , 120 ], and also in advanced stages of breast cancer as compared to early stages and/or healthy controls [ 121 , 122 ].…”