Background & Aims:Hereditary diffuse gastric cancer (HDGC) families carry CDH1 heterozygous germline mutations; their tumors acquire complete CDH1 inactivation through "2nd-hit" mechanisms. Most frequently, this occurs via promoter hypermethylation (epigenetic modification), and less frequently via CDH1 mutations and loss of heterozygosity (LOH). We quantified the different 2nd hits in CDH1 occurring in neoplastic lesions from HDGC patients. Methods: Samples were collected from 16 primary tumors and 12 metastases from 17 patients among 15 HDGC families; CDH1 mutations, LOH, and promoter hypermethylation were analyzed. E-cadherin protein expression and localization were determined by immunohistochemistry. Results: Somatic CDH1 epigenetic and genetic alterations were detected in lesions from 80% of HDGC families and in 75% of all lesions analyzed (21/28). Of the 28 neoplastic lesions analyzed, promoter hypermethylation was found in 32.1%, LOH in 25%, both alterations in 17.9%, and no alterations in 25%. Half of the CDH1 2nd hits in primary tumors were epigenetic modifications, whereas a significantly greater percentage of 2nd hits in metastases were LOH (58.3%; P ‫؍‬ .0274). Different neoplastic lesions from the same patient frequently displayed distinct 2nd-hit mechanisms. Different 2nd-hit mechanisms were also detected in the same tumor sample. Conclusion: The 2nd hit in CDH1 frequently occurs via epigenetic changes in HDGC primary tumors and LOH in metastases. Because of the concomitance and heterogeneity of these alterations in neoplastic lesions and the plasticity of hypermethylated promoters during tumor initiation and progression, drugs targeting only epigenetic alterations might not be effective, particularly in patients with metastatic HDGC.

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer susceptibility syndrome characterized by early-onset diffuse gastric cancer (DGC) and lobular breast cancer. E-cadherin (CDH1) heterozygous germline mutations and deletions are found in 40% of families. Independent of CDH1 alterations, most HDGC tumours display mislocalized or absent E-cadherin immunoexpression, therefore undetected defects at the CDH1 locus may still be involved. We aimed at determining whether CDH1 mutation-negative probands display germline CDH1 allele-specific expression (ASE) imbalance, using a single-nucleotide primer extension-based procedure and tried to uncover the underlying molecular defect. CDH1 ASE analysis was performed using three intragenic SNPs in RNA extracted from the blood of 21 cancer-free individuals and 22 HDGC probands (5 CDH1 mutation carriers and 17 CDH1 negative). Germline promoter methylation, deletions and haplotype-related susceptibility at the CDH1 locus were analysed. Both CDH1 alleles from cancer-free individuals displayed equivalent expression levels, whereas monoallelic CDH1 expression or high allelic expression imbalance (AI) was present in 80% of CDH1 mutant and 70.6% (n = 12) of CDH1-negative HDGC probands. Germline deletions and promoter hypermethylation were found in 25% of probands displaying high CDH1 AI. No particular haplotype was found to be associated with CDH1 high AI. Germline CDH1 AI is highly frequent among CDH1 mutation-negative probands but was not seen in cancer-free individuals. This implicates the CDH1 locus in the majority of mutation-negative HDGC families.

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Deregulation of tRNAs, aminoacyl-tRNA synthetases and tRNA modifying enzymes are common in cancer, raising the hypothesis that protein synthesis efficiency and accuracy (mistranslation) are compromised in tumors. We show here that human colon tumors and xenograft tumors produced in mice by two epithelial cancer cell lines mistranslate 2- to 4-fold more frequently than normal tissue. To clarify if protein mistranslation plays a role in tumor biology, we expressed mutant Ser-tRNAs that misincorporate Ser-at-Ala (frequent error) and Ser-at-Leu (infrequent error) in NIH3T3 cells and investigated how they responded to the proteome instability generated by the amino acid misincorporations. There was high tolerance to both misreading tRNAs, but the Ser-to-Ala misreading tRNA was a more potent inducer of cell transformation, stimulated angiogenesis and produced faster growing tumors in mice than the Ser-to-Leu misincorporating tRNA. Upregulation of the Akt pathway and the UPR were also observed. Most surprisingly, the relative expression of both misreading tRNAs increased during tumor growth, suggesting that protein mistranslation is advantageous in cancer contexts. These data highlight new features of protein synthesis deregulation in tumor biology.