Cisplatin-resistance and aggressiveness are enhanced by a highly stable endothelin-converting enzyme-1c in lung cancer cells
Background: Lung cancer is the leading cause of cancer-related deaths globally. Elevated levels of endothelin-1 (ET-1), its receptor ETAR, and its activating enzyme, endothelin-converting enzyme-1 (ECE-1), have been reported in various cancers, including lung cancer. ECE-1 exists in four isoforms, which differ only in their cytoplasmic N-terminal region. Protein kinase CK2 phosphorylates the N-terminus of the ECE-1c isoform, enhancing its stability and promoting invasiveness in glioblastoma and colorectal cancer cells. This effect is thought to be mediated by Lys-6, a conserved putative ubiquitination site located near the CK2-phosphorylated residues Ser-18 and Ser-20. However, the role of Lys-6 in the acquisition of a cancer stem cell (CSC)-like phenotype and enhanced aggressiveness in human non-small cell lung cancer (NSCLC) cells has not been investigated.
Methods: To assess the role of Lys-6 in ECE-1c stability and lung cancer aggressiveness, we mutated this residue to a non-ubiquitinable arginine (ECE-1cK6R) and constitutively expressed both wild-type (ECE-1cWT) and mutant (ECE-1cK6R) proteins in A549 and H1299 human NSCLC cells via lentiviral transduction. We evaluated protein stability in these clones, with or without the CK2 inhibitor silmitasertib, and compared them to ECE-1cWT and mock-transduced cells. ET-1 secretion was measured by ELISA. Expression of stemness-related genes was assessed by Western blot and RT-qPCR. Chemoresistance to cisplatin was evaluated using MTS viability assays. Migration and invasion were measured using transwell and Matrigel assays, respectively, and the side population was determined by flow cytometry.
Results: ECE-1cK6R exhibited greater stability in NSCLC cells compared to ECE-1cWT-expressing cells, although no differences in ET-1 secretion were observed within 48 hours. Notably, ECE-1cK6R expression induced higher levels of stemness genes (c-Myc, Sox-2, Oct-4, CD44, and CD133), which are associated with enhanced self-renewal. Furthermore, ECE-1cK6R-expressing cells demonstrated increased chemoresistance to cisplatin, accompanied by a larger side population, reflecting higher expression of the ABCG2 efflux pump. In addition, ECE-1cK6R-expressing cells showed enhanced invasiveness, correlating with altered expression of epithelial-mesenchymal transition (EMT) markers.
Conclusions: Our findings highlight the significant role of ECE-1c in lung cancer, particularly in driving a non-canonical, ET-1-independent mechanism that promotes a CSC-like phenotype and enhances cancer aggressiveness. The stabilization of ECE-1c through CK2 phosphorylation, commonly upregulated in various cancers, is central to this process. Therefore, phospho-ECE-1c may serve as a novel prognostic biomarker for recurrence, and the CK2 inhibitor silmitasertib may offer a promising therapeutic strategy for lung cancer patients.