Characterization of Mg-independent and Mg-dependent Ecto-ATPase activities in luminal a breast cancer MCF-7 cells

Abstract

Breast cancer remains the most prevalent malignancy among women, characterized by molecular heterogeneity and complex tumor microenvironment interactions. Purinergic signaling, particularly through extracellular adenosine triphosphate (ATP) hydrolysis, plays a significant role in regulating cancer progression. Ectonucleotidases and ectophosphatases modulate ATP and inorganic phosphate (Pi) metabolism, thereby influencing tumor-associated processes such as migration and adhesion. In this study, we biochemically characterized ecto-ATPase activity in luminal breast cancer cells (MCF-7) and compared it to non-tumorigenic breast epithelial cells (MCF10-A). We identified distinct Mg²⁺-dependent and independent ecto-ATPase activities, with MCF-7 cells exhibiting a significant Mg²⁺-dependent increase in ATP hydrolysis. Kinetic analyses revealed that Mg²⁺-independent activity presents higher substrate affinity, whereas the Mg²⁺-dependent enzyme displays greater catalytic capacity. Substrate specificity assays demonstrated preferential hydrolysis of ATP and ADP, highlighting the potential role of ecto-ATPase in extracellular nucleotide homeostasis. Pharmacological inhibition with ARL67156 reduced Mg²⁺-dependent ecto-ATPase activity, leading to a significant decrease in cell migration and adhesion, indicating a functional role in breast cancer progression. These findings underscore the relevance of ecto-ATPase in luminal breast cancer and its potential as a therapeutic target. Future investigations should explore its interplay with purinergic signaling and oncogenic pathways to develop novel strategies for targeted breast cancer treatment.