Abstract
Background: Lung cancer is the leading and second cause of cancer deaths among women and men in Taiwan, respectively. However, the molecular mechanisms involved in lung tumorigenesis in Taiwan remain poorly defined. Previous studies have shown the β-catenin plays an important role in the wingless (Wnt) signaling pathway. The β-catenin protein overexpressed in many tumors but only a few patients showed mutations in the β-catenin gene. It is possible that β-catenin protein accumulation results from alternative mechanisms other than mutation. Aim: The present study investigated the possibility of alterations of AXIN2, BTRCP, and, ICAT which are putative tumor suppressor genes in regulating the Wnt signaling pathway, leading to β-catenin protein stabilization. Patients and Methods: The alterations including promoter hypermethylation, loss of heterozygosity (LOH), low mRNA and protein expression of the AXIN2, BTRCP, and, ICAT genes were examined by the methylation specific-PCR, LOH, reverse-transcriptase PCR, and immunohistochemistry analyses, respectively, in 78 non-small cell lung cancer (NSCLC) tumorigenesis in Taiwan. Results: We found that the frequency of AXIN2 LOH and promoter hypermethylation was 32% (22/68) and 38% (30/78), respectively. In addition, 35% (27/78) and 44% (34/78) NSCLC patients had decreased or loss of AXIN2 protein and mRNA expression, respectively. The abnormal AXIN2 protein and mRNA expression was found more frequently in early-staged patients (P=0.047; P=0.028). With regard to BTRCP gene alteration analyses, the frequency of BTRCP LOH and promoter hypermethylation was 26% (14/53) and 50% (39/78), respectively. The BTRCP promoter hypermethylation was found more frequently in squamous carcinoma (SQ) and smoking patients (P=0.001; P=0.082). Note that 32% (25/78) and 29% (23/78) NSCLC patients had decreased or loss of BTRCP protein and mRNA expression, respectively. With regard to ICAT gene alteration analyses, the frequency of ICAT LOH and promoter hypermethylation was 42% (33/71) and 32% (23/78), respectively. The ICAT promoter hypermethylation was found more frequently in elderly patients (P=0.017). The ICAT LOH was found more frequently in female patients (P=0.007). There were 35% (27/78) and 29% (23/78) of NSCLC patients had decreased or loss of ICAT protein and mRNA expression, respectively. Overall, 67% (52/78) of NSCLC tumors had alteration in at least one of the three Wnt upstream effectors. The β-catenin deregulation was significantly attributable to low mRNA/protein expression of AXIN2 (P = 0.004) and BTRCP (P = 0.013). In addition, β-catenin overexpression and low expression of AXIN2/BTRCP/ICAT were frequent in SQ patients (P = 0.014). A high concordance was observed between low protein/mRNA expression and promoter hypermethylation (P < 0.001) for the AXIN2, BTRCP, and ICAT genes. Conclusion: The study was the first report which comprehensively examined the alteration of the Wnt signaling pathway genes, AXIN2, BTRCP, and ICAT at DNA, RNA and protein levels in lung cancer. Our data suggest that deregulation of the β-catenin/AXIN2/BTRCP/ICAT pathway is important in the pathogenesis of a subset of NSCLC and that promoter hypermethylation is the predominant mechanism in AXIN2, BTRCP, and ICAT alterations.