G·A·B·B·A

E-cadherin is an adhesion molecule that acts as a tumor suppressor protein by inhibiting tumor cell invasion and metastasis as a result of its ability to mediate cell-to-cell adhesion. Germline mutations of the E-cadherin gene (CDH1) are the underlying genetic defect responsible for hereditary diffuse gastric cancer (HDGC), with or without cleft lip (OMIM 192090). An unusually high percentage (almost 80%) of CDH1 mutations generate premature termination codons (PTCs). We hypothesized that transcripts from such mutant genes are degraded by nonsense-mediated decay (NMD), an mRNA quality-control mechanism that recognizes and degrades transcripts harboring PTCs.
To test this hypothesis, we examined whether PTC-containing CDH1 transcripts are downregulated by NMD. Using an allele-specific expression (ASE) assay to differentiate between mutated and wild type CDH1 alleles, we observed that PTC-containing CDH1 mRNAs are strongly downregulated in normal gastric tissue from several asymptomatic CDH1 mutation carriers. We definitively demonstrated that NMD is responsible for this robust downregulation, as CDH1 transcripts harboring PTCs in the KATO-III gastric cancer cell line were upregulated in response to protein synthesis inhibitors (as NMD depends on translation) or by depletion of the NMD factors UPF1, SMG1, or eIF4AIII.
Evidence suggests that NMD modulates the phenotype of numerous diseases. In some diseases, NMD function appears to reduce the severity of the phenotype by downregulating the expression of truncated proteins with dominant-negative activity. In other diseases, NMD appears to increase disease severity by degrading transcripts encoding truncated proteins which retain residual function. We examined the role of NMD in determining the phenotype of HDGC patients caused by truncating mutations in the CDH1 gene. Analysis of clinical data from 264 HDGC patients harboring CDH1 alleles with PTCs at a wide variety of different positions indicated an association of their predicted ability to induce NMD and an earlier age of onset of gastric cancer. Furthermore, we demonstrated that haploinsufficiency due to NMD and inactivation of the second CDH1 allele by promoter methylation may be necessary for diffuse gastric cancer development. Additionally, we evaluated the use of CDH1 ASE in a HDGC family under chromoendoscopic surveillance as a potential diagnostic method. CDH1 ASE was able to detect both downregulation of PTC-containing alleles in asymptomatic carries, as well as CDH1 promoter methylation of the wild type allele in a diffuse gastric tumor. Altogether, this dissertation provides compelling evidence that NMD may be detrimental for HDGC patients and therefore NMD is a potentially useful diagnostic and therapeutic target in CDH1 mutation carriers.