Abstract
Endothelin-converting enzyme-1 (ECE1) activates the endothelin-1 peptide, which upregulates pathways that are related to diverse hallmarks of cancer. ECE1 is expressed as four isoforms differing in their N-terminal domains. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE1c, enhancing its stability and promoting invasiveness of colorectal cancer cells. However, the specific residues in ECE1c that are phosphorylated by CK2 and how this phosphorylation promotes invasiveness was unknown. Here we demonstrate that Ser-18 and Ser-20 are the bona fide residues phosphorylated by CK2 in ECE1c. Thus, biphospho-mimetic ECE1cDD and biphospho-resistant ECE1cAA mutants were constructed and stably expressed in different colorectal cancer cells through lentiviral transduction. Biphospho-mimetic ECE1cDD displayed the highest stability in cells, even in the presence of the specific CK2 inhibitor silmitasertib. Concordantly, ECE1cDD-expressing cells showed enhanced hallmarks of cancer, such as proliferation, migration, invasiveness, and self-renewal capacities. Conversely, cells expressing the less-stable biphospho-resistant ECE1cAA showed a reduction in these features, but also displayed an important sensitization to 5-fluorouracil, an antineoplastic agent traditionally used as therapy in colorectal cancer patients. Altogether, these findings suggest that phosphorylation of ECE1c at Ser-18 and Ser-20 by CK2 promotes aggressiveness in colorectal cancer cells. Therefore, phospho-ECE1c may constitute a novel biomarker of poor prognosis and CK2 inhibition may be envisioned as a potential therapy for colorectal cancer patients.
Highlights
The Endothelin-1 (ET-1) axis is involved in several cancers by promoting tumor development and progression
Augmented proliferation was observed in both colorectal cancer (CRC) cell lines expressing ECE1cWT and ECE1cDD in comparison to mock cells, which was significant from 48 h of growth
In DLD-1 cells, significantly larger spheres were obtained only with ECE1cDD-expressing cells (Figure 7C), while in HT-29 cells spheres were significantly larger compared only with ECE1cAA-expressing and mock cells (Figure 7E). These findings indicate that increased ECE1c stability promoted by CK2 phosphorylation at Ser-18 and Ser-20 leads to enhanced aggressiveness of CRC cells through improved self-renewal capacity
Summary
The Endothelin-1 (ET-1) axis is involved in several cancers by promoting tumor development and progression. ET-1 activation involves processing of its precursor big-ET-1, which is catalyzed by the endothelin-converting enzyme-1 (ECE1). ECE1 overexpression has been observed to increase invasiveness of ovarian cancer cells, the identity of the isoform(s) responsible for this effect is unclear [3]. Overexpression or silencing of ECE1c in prostate cancer cells was able to either increase or reduce invasiveness, respectively [4, 5]. Similar findings were reported when the ECE1c isoform was overexpressed in colorectal cancer (CRC) cells, which showed significantly enhanced migration and invasiveness [6]. Recently ECE1c has been suggested as an emerging potential target due its role in promoting aggressiveness of several cancers [7]
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