Abstract
Endothelin‐1 is a mitogenic peptide that activates several proliferation, survival, and invasiveness pathways. The effects of endothelin‐1 rely on its activation by endothelin‐converting enzyme‐1 (ECE1), which is expressed as four isoforms with different cytoplasmic N termini. Recently, isoform ECE1c has been suggested to have a role in cancer aggressiveness. The N terminus of ECE1c is phosphorylated by protein kinase CK2 (also known as casein kinase 2), and this enhances its stability and promotes invasiveness in colorectal cancer cells. However, it is not known how phosphorylation improves stability and why this is correlated with increased aggressiveness. We hypothesized that CK2 phosphorylation protects ECE1c from N‐terminal ubiquitination and, consequently, from proteasomal degradation. Here, we show that lysine 6 is the bona fide residue involved in ubiquitination of ECE1c and its mutation to arginine (ECE1cK6R) significantly impairs proteasomal degradation, thereby augmenting ECE1c stability, even in the presence of the CK2 inhibitor silmitasertib. Furthermore, colorectal cancer cells overexpressing ECE1cK6R displayed enhanced cancer stem cell (CSC) traits, including increased stemness gene expression, chemoresistance, self‐renewal, and colony formation and spheroid formation in vitro, as well as enhanced tumor growth and metastasis in vivo. These findings suggest that CK2‐dependent phosphorylation enhances ECE1c stability, promoting an increase in CSC‐like traits. Therefore, phospho‐ECE1c may be a biomarker of poor prognosis and a potential therapeutic target for colorectal cancer.
Highlights
Colorectal cancer (CRC) is the third-leading cause of cancer-related deaths in developed countries
We demonstrate that Lys-6 is crucial for the stability of endothelin-converting enzyme isoform 1c (ECE1c) and that its mutation to arginine significantly increases the stability of this protein, even in the presence of the specific CK2 inhibitor silmitasertib
We assessed whether our various DLD-1 cell clones overexpressing either super-stable ECE1cK6R or ECE1cWT expressed differential levels of mRNA for genes traditionally associated with stemness, such as Nanog, Stat3, c-Myc, and Lgr5, as well as genes coding for stem cell surface markers, such as CD133 and CD44
Summary
Colorectal cancer (CRC) is the third-leading cause of cancer-related deaths in developed countries. Half of CRC patients die from liver, lung, or brain metastases (Brenner et al, 2014; Marley and Nan, 2016). There is a need for greater knowledge on the molecular and cellular bases of the aggressiveness. Abbreviations CRC, colorectal cancer; CSC, cancer stem cell; ECE1c, endothelin-converting enzyme isoform 1c; EMT, epithelial–mesenchymal transition; ET-1, endothelin-1. Molecular Oncology 14 (2020) 347–362 a 2019 The Authors.
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