Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

This work is based on using suppressor-tRNAs as a possible therapeutic tool for inherited cancer syndromes, where about 10-20% of the mutations described in are nonsense mutations. A suppressor-tRNA is a tRNA that can be altered to recognize premature stop codons (nonsense mutations), promoting their readthrough to produce full-length proteins. As a model, we used hereditary diffuse gastric cancer (HDGC), which is associated with mutations in CDH1 gene, encoding the adhesion molecule E-cadherin. We demonstrate, for the first time, that a full length and functional E-cadherin can be efficiently recovered from a nonsense-mutated allele using a suppressor-tRNA in gastric cancer cells. Importantly, this strategy may be applied to other genetic diseases, being especially significant for inherited cancer syndromes. Further studies on this type of strategy might open new paths for cancer treatment.

Authors and affiliations: 

Bordeira-Carriço R¹, Ferreira D¹, Mateus DD¹, Pinheiro H¹, Pêgo AP², Santos MA³, Oliveira C⁴.

• ¹Expression Regulation in Cancer Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal.
• ²1] 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.
• ³RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal.
• ⁴1] 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.

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.

Journal:

European Journal of Human Genetics

Link:

http://www.nature.com/ejhg/journal/vaop/ncurrent/full/ejhg2013292a.html

Legend of Figure 1:

During translation of the nonsense-mutated mRNA, the suppressor-tRNA, whose anticodon is mutated to recognize the premature stop codon (PTC), is able to insert the correct amino acid in the mutation site, leading to the production of a wild-type protein.