The channel-kinase TRPM7 (transient receptor potential melastatin 7) is a bifunctional protein consisting of a cation channel that is permeable to Mg2+, Ca2+ and Mn2+ ions fused to a C-terminal kinase domain. A growing number of studies with clinical significance suggest that TRPM7 is linked to adhesion and migration of breast cancer cells and promotes breast tumor metastasis. While the channel properties of TRPM7 have been studied extensively, little is known about the function of its kinase activity. To understand the functions of the kinase domain we identified a cell-permeable inhibitor of the kinase domain (TRPM7-IN-1) and developed MDA-MB-231 breast cancer cell lines in which TRPM7 is knocked out by CRISPR/Cas9 (KO), and in which various forms of TRPM7 were stably re-expressed. These were wild type TRPM7 (WT), a kinase-inactive mutant of TRPM7 (KD), and TRPM7 truncated at the kinase domain (KT). Knock out of TRPM7 significantly inhibited MDA-MB-231 cell migration. Only expression of the wild type TRPM7 (WT) rescued the migration phenotype, supporting a role for the kinase domain in the regulation of cell migration. Magnesium deprivation, which promotes TRPM7 kinase activity, induces phosphorylation of eEF2, presumably to impede protein synthesis. Treatment of magnesium-deprived HEK293 cells with TRPM7-IN-1 decreased eEF2 phosphorylation, consistent with suppression of TRPM7 kinase activity in-cells. TRPM7-IN-1 decreased the binding of Myosin IIB to TRPM7 in HEK293 and MDA-MB-231 cells. And when MDA-MB-231 cells were treated with increasing doses of TRPM7-IN-1, TRPM7 phosphorylation of Ser-1569 and its downstream substrate myosin IIa (Ser1943) were completely abrogated at a concentration of 5 µM. TRPM7-IN-1 inhibited MDA-MB-231 cell migration and invasion, while treatment of the KO cells with TRPM7-IN-1 showed no further inhibition of migration. Electrophysiological assessment of the TRPM7 channel revealed that the inhibitor did not affect the channel function in MD-MB-231 cells, supporting the notion that the inhibitor affects the migration exclusively through the inhibition of the TRPM7 kinase domain. Finally, in an experimental metastasis model, TRPM7-IN-1 significantly impeded metastasis to the lung. As a conclusion, inhibition of TRPM7 kinase activity may reduce or block breast tumor progression and/or metastasis.

The Trpm7 kinase domain regulates breast cancer cell migration and tumor metastasis

255th National Meeting (2018)

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