Background: Previous familial linkage studies have identified a tumor suppressor locus at 6q23-25 in lung cancer (LC). However, no single gene has yet been implicated within this 30 Mb region. The identification of two-hit targets — genes disrupted by two mechanisms at the DNA level, such as copy number loss and hypermethylation — involved in LC risk and development will lead to better methods for risk assessment, early detection and treatment.

Methods: EYA4 was identified as a two-hit target using multidimensional genome-wide analysis combining gene dosage, DNA methylation, and gene expression data in non-small cell lung cancer (NSCLC) and early pre-malignant lesions with matched adjacent non-malignant tissue. The association of allelic variants with LC risk was assessed in data collected by the Genetic Epidemiology of Lung Cancer Consortium (GELCC), and the association of gene expression with prognosis was investigated in public data using statistical analyses. Bronchial epithelial cells of LC patients and high risk individuals were examined for early hypermethylation of EYA4. The role of EYA4 in apoptosis, and the association of EYA4 with the DNA repair genes GADD45a and γ-H2AX was assayed using stable EYA4 mRNA knock-downs in lymphoblast cell lines. Cells were either serum-starved for 12 and 24 hours followed by Annexin5/propidium iodide staining and GADD45a qRT-PCR, or irradiated, and levels of γ-H2AX protein were measured over time.

Results: Integration of NSCLC gene dosage, DNA methylation and mRNA expression showed EYA4 to be frequently affected by two-hits and significantly down-regulated. Numerous EYA4 variants were associated with familial LC, and low EYA4 expression was significantly associated with poor prognosis in multiple independent data sets. Congruent with EYA family member function, in vitro assays revealed that EYA4 knock-down cells displayed a decrease in the number of apoptotic cells following serum starvation. Further investigations led to the discovery of EYA4 hypermethylation not only in the bronchial epithelial brushings of NSCLC patients, but also in the brushings of individuals at a high risk of LC development. EYA4 knock-down led to attenuation of the GADD45a DNA damage repair response, as well as increased levels of γ-H2AX following DNA damage repair.

Conclusions: EYA4 is a gene disrupted early and frequently that maps to a locus previously associated with cancer risk. It is implicated in somatic as well as familial cancers, and is likely a tumor suppressor gene with apoptotic and DNA repair functions. The direct association of EYA4 with risk and survival as well as its early disruption underscores its relevance on a clinical level.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A29.