| Title | Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA |
| Publication Type | Journal Article |
| 2014 |
| Authors | Bordeira-Carriço, R, Ferreira, D, Mateus, DD, Pinheiro, H, Pêgo, AP, Santos, MAS, Oliveira, C |
| Journal | European Journal of Human Genetics |
| Volume | 22 |
| Issue | 9 |
| Pagination | 1085 - 1092 |
| Date Published | 2014 |
| hereditary diffuse gastric cancer (HDGC), nonsense suppression, Premature termination codon (PTC), Suppressor-tRNA |
| Hereditary diffuse gastric cancer (HDGC) syndrome, although rare, is highly penetrant at an early age, and is severe and incurable because of ineffective screening tools and therapy. Approximately 45% of HDGC families carry germline CDH1/E-cadherin alterations, 20% of which are nonsense leading to premature protein truncation. Prophylactic gastrectomy is the only recommended approach for all asymptomatic CDH1 mutation carriers. Suppressor-tRNAs can replace premature stop codons (PTCs) with a cognate amino acid, inducing readthrough and generating full-length proteins. The use of suppressor-tRNAs in HDGC patients could therefore constitute a less invasive therapeutic option for nonsense mutation carriers, delaying the development of gastric cancer. Our analysis revealed that 23/108 (21.3%) of E-cadherin-mutant families carried nonsense mutations that could be potentially corrected by eight suppressor-tRNAs, and arginine was the most frequently affected amino acid. Using site-directed mutagenesis, we developed an arginine suppressor-tRNA vector to correct one HDGC nonsense mutation. E-cadherin-deficient cell lines were transfected with plasmids carrying simultaneously the suppressor-tRNA and wild-type or mutant CDH1 mini-genes. RT-PCR, western blot, immunofluorescence, flow cytometry and proximity ligation assay (PLA) were used to establish the model, and monitor mRNA and protein expression and function recovery from CDH1 vectors. Cells expressing a CDH1 mini-gene, carrying a nonsense mutation and the suppressor-tRNA, recovered full-length E-cadherin expression and its correct localization and incorporation into the adhesion complex. This is the first demonstration of functional recovery of a mutated causative gene in hereditary cancer by cognate amino acid replacement with suppressor-tRNAs. Of the HDGC families, 21.3% are candidates for correction with suppressor-tRNAs to potentially delay cancer onset. © 2014 Macmillan Publishers Limited. All rights reserved. |
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