Secretory pathway Ca²⁺‐ATPases promote in vitro microcalcifications in breast cancer cells

Abstract

Calcification of the breast is often an outward manifestation of underlying molecular changes that drive carcinogenesis. Up to 50% of all non-palpable breast tumors and 90% of ductal carcinoma in situ present with radiographically dense mineralization in mammographic scans. However, surprisingly little is known about the molecular pathways that lead to microcalcifications in the breast. Here, we report on a rapid and quantitative in vitro assay to monitor microcalcifications in breast cancer cell lines, including MCF7, MDA-MB-231 and Hs578T. We show that the Secretory Pathway Ca2+-ATPases SPCA1 and SPCA2 are strongly induced under osteogenic conditions that elicit microcalcifications. SPCA gene expression is significantly elevated in breast cancer subtypes that are associated with microcalcifications. Ectopic expression of SPCA genes drives microcalcifications and is dependent on pumping activity. Conversely, knockdown of SPCA expression significantly attenuates formation of microcalcifications. We propose that high levels of SPCA pumps may initiate mineralization in the secretory pathway by elevating luminal Ca2+. Our new findings offer mechanistic insight and functional implications on a widely observed, yet poorly understood radiographic signature of breast cancer.

A Ca2+-ATPase Regulates E-cadherin Biogenesis and Epithelial-Mesenchymal Transition in Breast Cancer Cells

“…whereas high SPCA1 is associated with basal subtypes, SPCA2 was found to be significantly elevated in luminal A/B and HER2+ subtypes (19). To further distinguish between their tumor expression pattern and gain insight into isoform-specific functions, we compared the expression of SPCA1 (ATP2C1) and SPCA2 (ATP2C2) genes in breast cancer patient specimens from TCGA (n=526) with a "benchmark" gene set, consisting of 18 epithelial signature genes (ESG) and 13 mesenchymal signature genes (MSG), described previously (24).…”

“…FUGW overexpression constructs and pLK0.1 shRNA and lentiviral construction of both SPCA isoforms was packaged and transfected according to previous methods using pCMV-Δ8.9 and PMDG at a ratio using 9:8:1 in HEK293T cells. A mixture of two shRNA constructs for SPCA2 gave the same results as individual constructs and have been previously validated, as reported (16,19). Virus was collected after 48 hours and concentrated with Lenti-X Concentrator (Clontech, Cat#631231) (16).…”

“…Therefore, low SPCA2 may be a useful prognostic marker of the malignant invasive phenotype. In contrast, high SPCA2 is characteristic of luminal A/B and HER2+ subtypes where it contributes to the formation of breast microcalcifications associated with tumorigenesis (19). We propose a model to summarize the role of SPCA2 in breast cancer.…”

“…Down-regulation of SPCA2 is associated with basal and claudin-low breast cancer subtypes (19) that are characterized by hormone receptor negativity, and a high degree of invasion and metastasis. Therefore, low SPCA2 may be a useful prognostic marker of the malignant invasive phenotype.…”

“…Gene expression correlations offer unique insight on disease pathophysiology, based on the premise that genes expressed together are likely to function together in a common pathway (24,25). Both SPCA isoforms have been implicated in breast cancer oncogenesis and formation of breast microcalcifications linked to malignancy (16,19,26). Previously, we have evaluated subtype specific gene expression in breast tumors:…”