Molecular Pathogenic Mechanisms Of Primary Non-Small Cell Lung Cancer Unveiled From TGF-beta/Smad Signaling Pathway

Transforming growth factor-β (TGF-β) inhibits the growth of multiple epithelial cells in general. Many malignant tumor cells including non-small cell lung cancer (NSCLC) cells are frequently resistant to TGF-β-mediated signal transduction, suggesting that alterations of the TGF-β signaling pathway may be involved in NSCLC The TGF-β/Smad signal is transduced through a pair of transmembrane serine-threonine kinase receptors named receptor type II and I (TGFBRII and TGFBRI) and Smad proteins. Alterations in these key effectors of the TGF-p/Smad signaling pathway have been detected in many human tumors and account for loss of growth inhibition by TGF-p. Whether and how TGF-p signaling is disrupted in NSCLC. however, remains unclear.To investigate whether genetic alterations of these key effectors, including TGFBRII, TGFBRI and Smad2, contribute to inactivation of TGF-p/Smad signaling and NSCLC tumorigenesis, the author undertook polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) or polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by sequencing to scan for mutations in the entire coding regions and SNPs in flanking intron sequences of the three genes in 53 primary NSCLC patients. No pathogenic mutation other than a SNP at the fourth-to-last base in intron 3 of the TGFBRII gene was identified. Two silent mutations and a polymorphism were found in the TGFBRI gene, and the two silent mutations located at codon 344 (AAT to AAC) and codon 406 (TTA to CTA) respectively and the polymorphism was at the 24th base of intron 7 (G to A) Interestingly, we found that the subjects with homozygous genotype A/A displayed more than 3-fold increased risk of developing NSCLC than the common wild genotype G/G. No mutation was detected in the Smad2 gene.Meanwhile, immunohistochemical analysis was performed to examine their expressions using the corresponding antibodies. As a result, immunohistochemicai staining of TGFBRII and TGFBRI has been shown to be positive in all the normal paracarcinomas tissues and an overwhelming majority of tumor tissues from NSCLCpatients, whereas Smad2 expression has been shown to be negative. Moreover, according to the data, reduced expressions of TGFBRII and TGFBRI proteins were founded in 44.2% and 37.2% of the tumor tissues respectively.Furthermore, to examine whether epigenetic alterations responsible for reduced expressions of TGFBRII and TGFBRI exists, methylation-specific PCR (MSP) was carried out to identify aberrant CpG mehylation of the promoters of TGFBRII and TGFBRI genes. In this part of study, only 3 (12.5%) of 24 preserved TGFBRII-expressing tumor tissues were methylated, whereas methylation was found in 15 (78.9%) of 19 reduced TGFBRII-expressing tumor tissues. In the majority of cases, the corresponding paracarcinoma tissue was completely unmethylated. No methylation evidence was observed in the TGFBRI gene.Finally, semi-quantitative RT-PCR was performed to evaluate the role of TGFBRII promoter methylation in TGFBRII expression. Sixteen of 19 reduced TGFBRII-expressing tumor tissues demonstrated reduced or loss mRNA expression for TGFBRII in comparison with the corresponding normal tissues.In conclusion, the results suggest that defect expressions of TGFBRII and TGFBRI play important roles in development of NSCLC, although mutation of the two genes is a rare event in primary NSCLC. Decreased expression of TGFBRII could be caused by down-regulation of the gene expression at transcriptional level due to an aberrant 5' CpG methylation.