Activation of Wnt signaling has been implicated in gastric tumorigenesis, although mutations in APC (adenomatous polyposis coli), CTNNB1 (b-catenin) and AXIN are seen much less frequently in gastric cancer (GC) than in colorectal cancer. In the present study, we investigated the relationship between activation of Wnt signaling and changes in the expression of secreted frizzled-related protein (SFRP) family genes in GC. We frequently observed nuclear b-catenin accumulation (13/15; 87%) and detected the active form of b-catenin in most (12/16; 75%) GC cell lines. CpG methylation-dependent silencing of SFRP1, SFRP2 and SFRP5 was frequently seen among GC cell lines (SFRP1, 16/16, 100%; SFRP2, 16/ 16, 100%; SFRP5, 13/16, 81%) and primary GC specimens (SFRP1, 42/46, 91%; SFRP2, 44/46, 96%; SFRP5, 30/46, 65%), and treatment with the DNA methyltransferase inhibitor 5-aza-2 0 -deoxycytidine rapidly restored SFRP expression. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor transcriptional activity, suppressed cell growth and induced apoptosis in GC cells. Analysis of global expression revealed that overexpression of SFRP2 repressed Wnt target genes and induced changes in the expression of numerous genes related to proliferation, growth and apoptosis in GC cells. It thus appears that aberrant SFRP methylation is one of the major mechanisms by which Wnt signaling is activated in GC.

Although mutation of APC or CTNNB1 (b-catenin) is rare in breast cancer, activation of Wnt signalling is nonetheless thought to play an important role in breast tumorigenesis, and epigenetic silencing of Wnt antagonist genes, including the secreted frizzled-related protein (SFRP) and Dickkopf (DKK) families, has been observed in various tumours. In breast cancer, frequent methylation and silencing of SFRP1 was recently documented; however, altered expression of other Wnt antagonist genes is largely unknown. In the present study, we found frequent methylation of SFRP family genes in breast cancer cell lines (SFRP1, 7 out of 11, 64%; SFRP2, 11 out of 11, 100%; SFRP5, 10 out of 11, 91%) and primary breast tumours (SFRP1, 31 out of 78, 40%; SFRP2, 60 out of 78, 77%; SFRP5, 55 out of 78, 71%). We also observed methylation of DKK1, although less frequently, in cell lines (3 out of 11, 27%) and primary tumours (15 out of 78, 19%). Breast cancer cell lines express various Wnt ligands, and overexpression of SFRPs inhibited cancer cell growth. In addition, overexpression of a b-catenin mutant and depletion of SFRP1 using small interfering RNA synergistically upregulated transcriptional activity of T-cell factor/lymphocyte enhancer factor. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in breast cancer, and suggest that their loss of function contributes to activation of Wnt signalling in breast carcinogenesis. British Journal of Cancer (2008) Wnt ligands are secreted proteins that bind to transmembrane receptors in the Frizzled (Fz) family. During normal developmental processes, the resultant Wnt signalling plays essential roles in the regulation of cell proliferation, patterning and fate determination (Cadigan and Nusse, 1997). The binding of Wnt to Fz leads to dephosphorylation and stabilisation of b-catenin, enabling it to be translocated into the nucleus, where it interacts with members of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family of transcription factors to stimulate the expression of target genes. This signalling pathway is strongly implicated in tumorigenesis; indeed, the first mammalian Wnt isoform was identified based on its ability to promote mouse mammary tumorigenesis (Polakis, 2000). In addition, aberrant nuclear and cytoplasmic localisation of b-catenin is frequently observed in human breast cancer (Lin et al, 2000;Ryo et al, 2001;Chung et al, 2004). In contrast to colorectal cancer (CRC), however, mutation of APC, AXIN or CTNNB1 (b-catenin) is rare in breast cancer, indicating that other mechanisms are responsible for the activation of b-catenin. These mechanisms could include increased expression of Wnt ligand (Huguet et al, 1994;Dale et al, 1996;Bui et al, 1997) and/or the loss of Wnt antagonists.Several classes of secreted Wnt antagonists are known, including the Cerberus, Wnt inhibitory factor 1, secreted frizzled-related protein (SFRP) and the Dickkopf (DKK) families (Kawano and Kypta, 2003). The SFRP family is comprised of five secreted glycopr...

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ObjectiveThe objective of this study was to identify new causes of Charcot–Marie–Tooth (CMT) disease in patients with autosomal‐recessive (AR) CMT.MethodsTo efficiently identify novel causative genes for AR‐CMT, we analyzed 303 unrelated Japanese patients with CMT using whole‐exome sequencing and extracted recessive variants/genes shared among multiple patients. We performed mutation screening of the newly identified membrane metalloendopeptidase (MME) gene in 354 additional patients with CMT. We clinically, genetically, pathologically, and radiologically examined 10 patients with the MME mutation.ResultsWe identified recessive mutations in MME in 10 patients. The MME gene encodes neprilysin (NEP), which is well known to be one of the most prominent beta‐amyloid (Aβ)‐degrading enzymes. All patients had a similar phenotype consistent with late‐onset axonal neuropathy. They showed muscle weakness, atrophy, and sensory disturbance in the lower extremities. All the MME mutations could be loss‐of‐function mutations, and we confirmed a lack/decrease of NEP protein expression in a peripheral nerve. No patients showed symptoms of dementia, and 1 patient showed no excess Aβ in Pittsburgh compound‐B positron emission tomography imaging.InterpretationOur results indicate that loss‐of‐function MME mutations are the most frequent cause of adult‐onset AR‐CMT2 in Japan, and we propose that this new disease should be termed AR‐CMT2T. A loss‐of‐function MME mutation did not cause early‐onset Alzheimer's disease. Identifying the MME mutation responsible for AR‐CMT could improve the rate of molecular diagnosis and the understanding of the molecular mechanisms of CMT. Ann Neurol 2016;79:659–672

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