Deregulation of Wnt/β-catenin signaling through genetic or epigenetic alterations in human neuroendocrine tumors

Carcinoid tumors are rare neuroendocrine tumors (NETs) that are increasing in incidence. Mutation and altered expression of Wnt/β-catenin signaling components have been described in many tumors but have not been well-studied in NETs. Here, we observed accumulation of β-catenin in the cytoplasm and/or nucleus in 25% of clinical NET tissues. By mutational analysis, the mutations of β-catenin (I35S) and APC (E1317Q, T1493T) were identified in NET cells and the tissues. Expression of representative Wnt inhibitors was absent or markedly decreased in BON, a human pancreatic carcinoid cell line; treatment with 5-aza-2′-deoxycytidine (5-aza-CdR) increased expression levels of the Wnt inhibitors. Methylation analyses demonstrated that CpG islands of SFRP-1 and Axin-2 were methylated, whereas the promoters of DKK-1, DKK-3 and WIF-1 were unmethylated in four NET cells. Aberrant methylation of SFRP-1 was particularly observed in most of clinical NET tissues. In addition, the repression of these unmethylated genes was associated with histone H3 lysine 9 dimethylation (H3K9me2) in BON cells. Together, 5-aza-CdR treatment inhibited cell proliferation and decreased the protein levels of H3K9me2 and G9a. Moreover, a novel G9a inhibitor, UNC0638, suppressed BON cell proliferation through inhibition of Wnt/β-catenin pathway. Overexpression of the inhibitory genes, particularly SFRP-1 and WIF-1 in BON cells, resulted in suppression of anchorage-independent growth and inhibition of tumor growth in mice. Our findings suggest that aberrant Wnt/β-catenin signaling, through either mutations or epigenetic silencing of Wnt antagonists, contributes to the pathogenesis and growth of NETs and have important clinical implications for the prognosis and treatment of NETs.