Cisplatin-resistance and aggressiveness are enhanced by a highly stable endothelin-converting enzyme-1c in lung cancer cells

• Published in: Biological research 2024-10, Vol.57 (1), p.74-12, Article 74
• Main Authors: Almarza, Cristopher, Villalobos-Nova, Karla, Toro, María A, González, Manuel, Niechi, Ignacio, Brown-Brown, David A, López-Muñoz, Rodrigo A, Silva-Pavez, Eduardo, Gaete-Ramírez, Belén, Varas-Godoy, Manuel, Burzio, Verónica A, Jara, Lilian, Aguayo, Francisco, Tapia, Julio C
• Format: Article
• Language: English
• Subjects: Amino acids; Antineoplastic Agents - pharmacology; Arginine; Blotting, Western; c-Myc protein; Cancer; Cancer cells; Cancer therapies; Carcinoma, Non-Small-Cell Lung - genetics; Carcinoma, Non-Small-Cell Lung - metabolism; Carcinoma, Non-Small-Cell Lung - pathology; Casein kinase II; CD44 antigen; Cell Line, Tumor; Cell self-renewal; Chemoresistance; Chemotherapy; Cisplatin; Cisplatin - pharmacology; CK2; Cloning; Colorectal cancer; Colorectal carcinoma; Drug Resistance, Neoplasm - genetics; Endothelin; Endothelin 1; Endothelin-Converting Enzymes - genetics; Endothelin-Converting Enzymes - metabolism; Enzyme-linked immunosorbent assay; Enzymes; Flow cytometry; Genes; Glioblastoma; Gliomas; Health aspects; Humans; Invasiveness; Isoforms; Kinases; Lung cancer; Lung cancer, Non-small cell; Lung Neoplasms - genetics; Lung Neoplasms - metabolism; Lung Neoplasms - pathology; Medical equipment and supplies industry; Medical prognosis; Medical test kit industry; Mortality; Myc protein; N-Terminus; Neoplastic Stem Cells - metabolism; Non-small cell lung carcinoma; Oct-4 protein; Oncology, Experimental; Peptides; Phenotypes; Phosphorylation; Protection and preservation; Protein kinase C; Protein kinases; Proteins; Small cell lung carcinoma; Stem cells; Stemness; Ubiquitin; Ubiquitination; United States
• ISSN: 0717-6287; 0716-9760; 0717-6287
• DOI: 10.1186/s40659-024-00551-9

Lung cancer constitutes the leading cause of cancer mortality. High levels of endothelin-1 (ET-1), its cognate receptor ET R and its activating enzyme, the endothelin-converting enzyme-1 (ECE-1), have been reported in several cancer types, including lung cancer. ECE-1 comprises four isoforms, which only differ in their cytoplasmic N-terminus. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE-1c, increasing its stability and leading to enhanced invasiveness in glioblastoma and colorectal cancer cells, which is believed to be mediated by the amino acid residue Lys-6, a conserved putative ubiquitination site neighboring the CK2-phosphorylated residues Ser-18 and Ser-20. Whether Lys-6 is linked to the acquisition of a cancer stem cell (CSC)-like phenotype and aggressiveness in human non-small cell lung cancer (NSCLC) cells has not been studied. In order to establish the role of Lys-6 in the stability of ECE-1c and its involvement in lung cancer aggressiveness, we mutated this residue to a non-ubiquitinable arginine and constitutively expressed the wild-type (ECE-1c ) and mutant (ECE-1c ) proteins in A549 and H1299 human NSCLC cells by lentiviral transduction. We determined the protein stability of these clones alone or in the presence of the CK2 inhibitor silmitasertib, compared to ECE-1c and mock-transduced cells. In addition, the concentration of secreted ET-1 in the growth media was determined by ELISA. Expression of stemness genes were determined by Western blot and RT-qPCR. Chemoresistance to cisplatin was studied by MTS viability assay. Migration and invasion were measured through transwell and Matrigel assays, respectively, and the side-population was determined using flow cytometry. ECE-1c displayed higher stability in NSCLC cells compared to ECE-1c -expressing cells, but ET-1 secreted levels showed no difference up to 48 h. Most importantly, ECE-1c promoted expression of the stemness genes c-Myc, Sox-2, Oct-4, CD44 and CD133, which enhance cellular self-renewal capability. Also, the ECE-1c -expressing cells showed higher cisplatin chemoresistance, correlating with an augmented side-population abundance due to the increased expression of the ABCG2 efflux pump. Finally, the ECE-1c -expressing cells showed enhanced invasiveness, which correlated with the regulated expression of known EMT markers. Our findings suggest an important role of ECE-1c in lung cancer. ECE-1c is key in a non-canonical ET-1-independent mechanism which triggers a CSC-