Transient receptor potential melastatin 6 and transient receptor potential melastatin 6/7 antagonists suppress colon adenocarcinoma HT-29 cells

Background: Magnesium (Mg²⁺) plays a fundamental role in numerous cellular processes, including enzymatic reactions, DNA replication, oxidative stress response, and cytoskeletal dynamics. In fact, dysregulation of Mg²⁺ homeostasis has been increasingly associated with the development and progression of Cancer, particularly colorectal Cancer (CRC). Transient receptor potential melastatin (TRPM) channels, especially TRPM6 and TRPM7, are essential regulators of epithelial Mg²⁺ influx. While TRPM7 promotes CRC progression, the role of TRPM6 and TRPM6/7 channels remains unclear.

Aim: To investigate the role of membrane-localized TRPM6 and TRPM6/7 channels in Mg²⁺ influx, spheroid (SP) formation, stemness, and migration.

Methods: We used parental and SP-derived HT-29 cells at comparable passages as in vitro models. Mass spectrometry confirmed full-length sequences, phosphorylation, and methionine oxidation of TRPM6 and TRPM7. Mg²⁺ influx, total and free Mg²⁺ levels were measured by fluorescence imaging and biochemical assays. TRPM6 / TRPM7 expression and markers were analyzed by western blot. Functional assays, including secondary SP formation and wound healing, assessed stemness and migration. Cells were treated with Mg²⁺ transport inhibitors: Co(III)hexamine, 2-aminoethyl diphenylborinate (TRPM6/7 blocker), and Mesendogen (TRPM6 inhibitor).

Results: The expression of membrane-bound TRPM6, TRPM7, and TRPM6/7 was significantly higher in SP cells than in parental cells. Mass spectrometric analysis confirmed the presence of full-length TRPM6 and TRPM7 with increased phosphorylation and oxidation in SP cells. Enhanced Mg²⁺ influx and total intracellular Mg²⁺ levels were observed in SP cells. Free ionized intracellular Mg²⁺ levels remained comparable across all experimental groups. Pharmacological inhibition of TRPM6 and TRPM6/7 significantly reduced Mg²⁺ influx, decreased total Mg²⁺ content, compromised CRC SP stability, abolished Cancer stem-like properties, impaired cell migration, and downregulated pro-tumorigenic markers, including Nanog, cyclooxygenase-2, and matrix metalloproteinase-9.

Conclusion: Membrane-localized TRPM6 and TRPM6/7 channels regulate Mg²⁺ influx and promote CRC stemness, SP stability, and migration, highlighting their potential as therapeutic targets to inhibit CRC progression and metastasis.

Cancer stem cells; Cellular Mg2+ content; Colorectal cancer; Transient receptor potential melastatin 6; Transient receptor potential melastatin 6/7.