Abstract 1121: LKB1 mutation correlates with a unique gene expression profile in NSCLC

Abstract

Abstract Background: Germline mutations in LKB1 gene cause the Peutz-Jegher syndrome, an elevated cancer risk condition. LKB1 somatic mutations are present in ∼30% of non-small cell lung cancers (NSCLC). LKB1 encodes a serine-threonine kinase involved in the regulation of cell growth, metabolism and epithelial cell polarity. Importantly, downstream proteins and events in LKB1 pathway remain to be determined in NSCLC. Aims: 1) Characterize the mutational status of LKB1 in a panel of NSCLC cell lines; 2) Correlate mutations of LKB1, EGFR and KRAS, and 3) Identify differentially expressed genes in NSCLC cell lines harboring LKB1 inactivating mutations. Methods: 42 lung adenocarcinoma cell lines derived from never or light smokers with or without EGFR mutations (n=20) and from smokers with or without KRAS mutations (n=22), were screened for LKB1: 1) gene mutations (by exon sequencing); 2) gene copy number variations (by qPCR) and 3) promoter hypermethylation (by methylation-specific PCR). Gene expression profiling (Illumina microarray) was performed in LKB1 mutant cell lines. NSCLC cell lines wild-types for LKB1 gene were selected for comparison. Candidate gene expression levels were verified by qRT-PCR. Protein levels were determined by immunobloting. Results: LKB1 gene mutations were present in 33% of the cell lines. Specifically, frameshift (7/14), nonsense (4/14), and missense (2/14) mutations were detected. Homozygous deletion was found in only one cell line. EGFR activating mutation and LKB1 inactivating mutations were mutually exclusive, whereas LKB1 mutational status was not associated with KRAS mutations nor smoker status. LKB1 protein was undetectable in all the cell lines with LKB1 gene inactivating mutations (12/14). No gain in LKB1 gene copy number was found and gene promoter hypermethylation was detected in only five cell lines. Importantly, no correlations between LKB1 promoter hypermethylation and LKB1 mRNA and protein levels were found. Significance Analysis of Microarrays and False Discovery Rate (FDR) analysis identified differentially expressed genes in the LKB1 mutant versus LKB1 wild type NSCLC cell lines. With a FDR tolerance of < 5%, 465 genes were differentially expressed in LKB1 mutant cell lines. A FDR tolerance <1% still identified 195 genes. The identified genes are involved in cell proliferation, motility, invasion as well as angiogenesis. A Multi-Dimensional Scaling analysis revealed that LKB1 mutant and LKB1 wild-type NSCLC cell lines are distinguishable based solely on their gene expression profile. Conclusion: 1) LKB1 mutations are a frequent finding in NSCLC cell lines and they are mutually exclusive with EGFR mutations; 2) there is a good concordance between LKB1 inactivating mutations and absence of LKB1 protein expression, suggesting that LKB1 protein expression is a good proxy for LKB1 mutational status in NSCLC and 3) LKB1 mutations correlates with a unique gene expression profile in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1121.