Prevalence and specificity of LKB1 genetic alterations in lung cancers

Abstract

Germline LKB1 mutations cause Peutz-Jeghers syndrome, a hereditary disorder that predisposes to gastrointestinal hamartomatous polyposis and several types of malignant tumors. Somatic LKB1 alterations are rare in sporadic cancers, however, a few reports showed the presence of somatic alterations in a considerable fraction of lung cancers. To determine the prevalence and the specificity of LKB1 alterations in lung cancers, we examined a large number of lung cancer cell lines and lung adenocarcinoma (AdC) specimens for the alterations. LKB1 genetic alterations were frequently detected in the cell lines (21/70, 30%), especially in non-small cell lung cancers (NSCLCs) (20/51, 39%), and were significantly more frequent in cell lines with KRAS mutations. Point mutations were detected only in AdCs and large cell carcinomas, whereas homozygous deletions were detected in all histological types of lung cancer. Among lung AdC specimens, LKB1 mutations were found in seven (8%) of 91 male smokers but in none of 64 females and/or nonsmokers, and were significantly more frequent in poorly differentiated tumors. The difference in the frequency of LKB1 alterations between cell lines and tumor specimens was likely to be owing to masking of deletions by the contamination of noncancerous cells in the tumor specimens. These results indicate that somatic LKB1 genetic alterations preferentially occur in a subset of poorly differentiated lung AdCs that appear to correlate with smoking males.

Figure 1

Homozygous deletions of the LKB1 gene in lung cancer cell lines. DNA fragments for exons and upstream regions of the LKB1 gene amplified by genomic PCR were fractionated by a 3% agarose gel. Sizes of the fragments are shown on the right. A homozygous deletion in H2126, which was demonstrated in a previous report (Carretero et al., 2004), is not shown.

Figure 2

Expression of the LKB1 gene in lung cancer cell lines. RT–PCR was performed as described in Materials and methods. RT–PCR products were resolved on a 1% agarose gel. GAPDH amplification was used for a loading control.

Figure 3

(a) Deletion map of the LKB1 locus. Regions analysed by genomic PCR are shown by ovals. Results of the PCR analysis for the regions indicated by open ovals are shown in Figure 1. Distal and proximal breakpoints for deletions are shown by closed boxes, respectively. The bar put between the closed boxes indicates the extent of deletion in each cell line. In Ma29, the distal breakpoint was undetermined. (b) DNA sequences of breakpoint junctions for deletions in or upstream of the LKB1 gene. Junctions without insertion or overlap of nucleotides (A427), with overlaps of 2- or 3-bp nucleotides (Ma24, H2126 and Lu65) and with insertion of 2-bp nucleotides (H157) are shown, respectively. Overlapped nucleotides are boxed.