Takashi Takahashi

In this study, we report for the first time reduced expression of the let-7 microRNA in human lung cancers. Interestingly, 143 lung cancer cases that had undergone potentially curative resection could be classified into two major groups according to let-7 expression in unsupervised hierarchical analysis, showing significantly shorter survival after potentially curative resection in cases with reduced let-7 expression (P = 0.0003). Multivariate COX regression analysis showed this prognostic impact to be independent of disease stage (hazard ratio = 2.17; P = 0.009). In addition, overexpression of let-7 in A549 lung adenocarcinoma cell line inhibited lung cancer cell growth in vitro. This study represents the first report of reduced expression of let-7 and the potential clinical and biological effects of such a microRNA alteration.

MicroRNAs (miRNAs) are small noncoding RNAs, thought to be involved in physiologic and developmental processes by negatively regulating expression of target genes. We have previously reported frequent down-regulation of the let-7 miRNA family in lung cancers and, in the present study, assessed alteration in a panel of 19 lung cancer cell lines. As a result, we found for the first time that the miR-17-92 cluster, which comprises seven miRNAs and resides in intron 3 of the C13orf25 gene at 13q31.3, is markedly overexpressed in lung cancers, especially with small-cell lung cancer histology. Southern blot analysis revealed the presence of increased gene copy numbers of the miRNA cluster in a fraction of lung cancer cell lines with overexpression. In addition, we were able to show predominant localization of C13orf25 transcripts within the nucleus and introduction of the expression construct of the miR-17-92 cluster, but not the putative open reading frame of C13orf25, enhancing lung cancer cell growth. These findings clearly suggest that marked overexpression of the miR-17-92 cluster with occasional gene amplification may play a role in the development of lung cancers, especially in their most aggressive form, small-cell lung cancer, and that the C13orf25 gene may well be serving as a vehicle in this regard.

Recently it has been reported that mutations in the tyrosine kinase domain of the epidermal growth factor receptor(EGFR) gene occur in a subset of patients with lung cancer showing a dramatic response to EGFR tyrosine kinase inhibitors. To gain further insights in the role of EGFR in lung carcinogenesis, we sequenced exons 18-21 of the tyrosine kinase domain using total RNA extracted from unselected 277 patients with lung cancer who underwent surgical resection and correlated the results with clinical and pathologic features. EGFR mutations were present in 111 patients (40%). Fifty-two were in-frame deletions around codons 746-750 in exon 19, 54 were point mutations including 49 at codon 858 in exon 21 and 4 at codon 719 in exon 18, and 5 were duplications/insertions mainly in exon 20. They were significantly more frequent in female (P < 0.001), adenocarcinomas (P = 0.0013), and in never-smokers (P < 0.001). Multivariate analysis suggested EGFR mutations were independently associated with adenocarcinoma histology (P = 0.0012) and smoking status (P < 0.001), but not with female gender (P = 0.9917). In adenocarcinomas, EGFR mutations were more frequent in well to moderately differentiated tumors (P < 0.001) but were independent of patient age, disease stages, or patient survival. KRAS and TP53 mutations were present in 13 and 41%, respectively. EGFR mutations never occurred in tumors with KRAS mutations, whereas EGFR mutations were independent of TP53 mutations. EGFR mutations define a distinct subset of pulmonary adenocarcinoma without KRAS mutations, which is not caused by tobacco carcinogens.

Allele loss is a hallmark of chromosome regions harboring recessive oncogenes. Lung cancer frequently demonstrates loss of heterozygosity on 17p. Recent evidence suggests that the p53 gene located on 17p13 has many features of such an antioncogene. The p53 gene was frequently mutated or inactivated in all types of human lung cancer. The genetic abnormalities of p53 include gross changes such as homozygous deletions and abnormally sized messenger RNAs along with a variety of point or small mutations, which map to the p53 open reading frame and change amino acid sequence in a region highly conserved between mouse and man. In addition, very low or absent expression of p53 messenger RNA in lung cancer cell lines compared to normal lung was seen. These findings, coupled with the previous demonstration of 17p allele loss in lung cancer, strongly implicate p53 as an anti-oncogene whose disruption is involved in the pathogenesis of human lung cancer.