NCX1 interacts with TRPA1 to promote cell proliferation and tumor growth of colon cancer via disruption of calcium homeostasis

Introduction: Aberrant CA²⁺ signaling plays a critical role in the hallmark of Cancer, but its regulatory mechanisms in tumorigenesis remain largely unclear. Na⁺/CA²⁺ exchanger 1 (NCX1) functions as a bidirectional Na⁺ and CA²⁺ transporter, operating in either CA²⁺ entry or exit mode, while the transient receptor potential ankyrin 1 (TRPA1) serves as a CA²⁺-permeable channel. Both play crucial roles in maintaining normal homeostasis of cytosolic CA²⁺ ([CA²⁺]_cyt). Although each of them has been implicated in some tumorigenesis, the potential coordination between NCX1 and TRPA1 in the pathogenesis of colon Cancer (CC) remains unexplored.

Objectives: We investigated the impact of NCX1- and TRPA1-mediated CA²⁺ signaling on CC and the underlying mechanisms.

Methods: The cell experiments were conducted using the human normal colonic epithelial cell line (HCoEpiC) and human colon Cancer cell lines (Caco-2, SW620, and DLD-1). We performed stable transfection to knock down NCX1 or TRPA1 genes and employed CCK8, colony formation, and flow cytometry assays to assess cell proliferation. We employed RT-qPCR, Western blotting, immunofluorescence and co-immunoprecipitation assays to explore the expression and regulatory relationship between NCX1 and TRPA1. Calcium and sodium assays were used to determine [CA²⁺]_cyt and [Na⁺]_cyt. Finally, we used the xenografted tumor model to verify their impact on CC development in vivo.

Results: NCX1 and TRPA1 were parallelly over-expressed, co-localized, and bound, and their functional activities were enhanced in human CC cells. NCX1 functions in CA²⁺ exit mode to expel [CA²⁺]_cyt, in which TRPA1 function was clearly verified as well. Moreover, when the CA²⁺ exit mode of NCX1 was inhibited, TRPA1 activation resulted in a larger amount of [CA²⁺]_cyt to suppress cell proliferation through inhibiting ERK1/2 and β-catenin phosphorylation. NCX1 or TRPA1 knockdown significantly diminished tumor growth in vivo.

Conclusion: TRPA1 channels couple with the CA²⁺ exit mode of NCX1 to maintain a moderate increase in [CA²⁺]_cyt in CC cells, thereby promoting CC cell proliferation and tumor growth through ERK1/2 and β-catenin phosphorylation. Consequently, the NCX1/TRPA1 coupling may serve as an innovative target for preventing and treating CC.

Ca(2+) signaling; Na(+)/Ca(2+) exchanger 1; Proliferation; TRPA1 channel; Tumor growth.