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.

中文翻译：

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内皮素转化酶-1c促进大肠癌细胞的干细胞特性和侵袭性。

内皮素-1是一种有丝分裂肽，可激活多种增殖，存活和侵袭性途径。内皮素-1的作用依赖于内皮素转化酶-1（ECE1）的激活，该酶表达为具有不同胞质N末端的四种同工型。最近，已提出同工型ECE1c在癌症侵袭性中起作用。ECE1c的N末端被蛋白激酶CK2（也称为酪蛋白激酶2）磷酸化，这增强了它的稳定性并促进了对结直肠癌细胞的侵袭性。但是，尚不清楚磷酸化如何提高稳定性以及为什么将其与增加的攻击性相关联。我们假设CK2磷酸化可以保护ECE1c免受N端泛素化的影响，从而防止蛋白酶体降解。这里，我们显示，赖氨酸6是参与ECE1c泛素化的真正残基，其突变为精氨酸（ECE1cK6R）显着损害蛋白酶体降解，从而增强了ECE1c的稳定性，即使存在CK2抑制剂silmitasertib时也是如此。此外，过表达ECE1cK6R的结直肠癌细胞表现出增强的癌症干细胞（CSC）特性，包括体外增加干基因表达，化学抗性，自我更新，菌落形成和球体形成，以及体内肿瘤生长和转移增强。这些发现表明，依赖CK2的磷酸化增强了ECE1c的稳定性，促进了CSC样性状的增加。因此，磷酸ECE1c可能是预后不良的生物标志物，并且可能是结直肠癌的潜在治疗靶标。