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

Abstract

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.