Copper chelation inhibits TGF-βpathways and suppresses epithelial-mesenchymal transition in cancer

Abstract

Copper is a trace element essential to cellular function with elevated levels implicated in cancer progression. Clinical trials using copper chelators are associated with improved patient survival, however, the molecular mechanisms by which copper depletion inhibits tumor progression are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Epithelial-mesenchymal transition (EMT) is often exploited by malignant cells to promote growth and metastasis. Transforming growth factor (TGF)-β is a master regulator of EMT and facilitates cancer progression through changes in the tumor and its microenvironment. Herein, we report that a reduction of copper with the chelating agent tetraethylenepentamine (TEPA) inhibited EMT in vitro in three diverse cancer cell types; human triple-negative breast cancer (TNBC), neuroblastoma (NB), and diffuse intrinsic pontine glioma (DIPG) cell lines. Single-molecule imaging demonstrated EMT markers including Vimentin, β-catenin, ZEB1, and p-SMAD2 had increased expression with copper treatment and this pro-mesenchymal shift was rescued by the addition of TEPA. Moreover, SNAI1, ZEB1, and p-SMAD2 demonstrated increased accumulation in the cytoplasm after treating with TEPA. Transcriptomic analyses revealed a significant downregulation of the EMT pathway, including canonical (TGF-β/SMAD2&3) and non-canonical (TGF-β/PI3K/AKT and TGF-β/RAS/RAF/MEK/ERK) TGF signaling pathways. Matrix metalloproteinases MMP-9 and MMP-14 proteins which activate latent TGF-β complexes were also downregulated by TEPA treatment. These molecular changes are consistent with reduced plasma levels of TGF-β we observed in cancer models treated with TEPA. Importantly, copper chelation reduced metastasis to the lung in a TNBC orthotopic syngeneic mouse model. Our studies suggest copper chelation therapy can be used to inhibit EMT-induced metastasis by targeting TGF-β signalling. Because on-target anti-TGF-β therapies are failing in the clinic, copper chelation presents itself as a potential therapy for targeting TGF-β in cancer.