title: Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA.
abstract: 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.
authors.affiliation: Expression Regulation in Cancer Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal.
authors.forename: Renata
authors.initials: R
authors.lastname: Bordeira-Carriço
authors.affiliation: Expression Regulation in Cancer Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ferreira
authors.affiliation: Expression Regulation in Cancer Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal.
authors.forename: Denisa D
authors.initials: DD
authors.lastname: Mateus
authors.affiliation: Expression Regulation in Cancer Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal.
authors.forename: Hugo
authors.initials: H
authors.lastname: Pinheiro
authors.affiliation: 1] INEB, Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal [2] Universidade do Porto-Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Porto, Portugal [3] Universidade do Porto-Faculdade de Engenharia, Porto, Portugal.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Pêgo
authors.affiliation: RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal.
authors.forename: Manuel A S
authors.initials: MA
authors.lastname: Santos
authors.affiliation: 1] Expression Regulation in Cancer Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal [2] Faculty of Medicine of the University of Porto, Porto, Portugal.
authors.forename: Carla
authors.initials: C
authors.lastname: Oliveira
mesh_terms: D000818:Animals; D015703:Antigens, CD; D016466:CHO Cells; D015820:Cadherins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D018389:Codon, Nonsense; D006224:Cricetinae; D003412:Cricetulus; D006579:Heterozygote; D006801:Humans; D012343:RNA, Transfer / Q000235:genetics*; D013274:Stomach Neoplasms / Q000235:genetics*
chemical_list: D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; D018389:Codon, Nonsense; D012343:RNA, Transfer