Promoter Methylation of Selected Genes in Non-Small-Cell Lung Cancer Patients and Cell Lines

Abstract

Specific gene promoter DNA methylation is becoming a powerful epigenetic biomarker in cancer diagnostics. Five genes (CDH1, CDKN2Ap16, RASSF1A, TERT, and WT1) were selected based on their frequently published potential as epigenetic markers. Diagnostic promoter methylation assays were generated based on bisulfite-converted DNA pyrosequencing. The methylation patterns of 144 non-small-cell lung cancer (NSCLC) and 7 healthy control formalin-fixed paraffin-embedded (FFPE) samples were analyzed to evaluate the applicability of the putative diagnostic markers. Statistically significant changes in methylation levels are shown for TERT and WT1. Furthermore, 12 NSCLC and two benign lung cell lines were characterized for promoter methylation. The in vitro tests involved a comparison of promoter methylation in 2D and 3D cultures, as well as therapeutic tests investigating the impact of CDH1/CDKN2Ap16/RASSF1A/TERT/WT1 promoter methylation on sensitivity to tyrosine kinase inhibitor (TKI) and DNA methyl-transferase inhibitor (DNMTI) treatments. We conclude that the selected markers have potential and putative impacts as diagnostic or even predictive marker genes, although a closer examination of the resulting protein expression and pathway regulation is needed.

Keywords: DNA methylation; NSCLC; epigenetic marker; epigenetics; precision medicine.

Figure 1

Developments of CDH1/CDKN2Ap16/RASSF1A/TERT/WT1 methylation levels. (A) Average promoter methylation levels for each gene of interest for tumor-free control samples (n_CDH1 = 2; n_CDKN2Ap16 = 7; n_RASSF1A = 1; n_TERT = 7; n_WT1 = 3), wildtype (n_CDH1 = 34; n_CDKN2Ap16 = 89; n_RASSF1A = 22; n_TERT = 78; n_WT1 = 23) and EGFR/KRAS mutated tumor samples (n_CDH1 = 22; n_CDKN2Ap16 = 46; n_RASSF1A = 19; n_TERT = 39; n_WT1 = 16). Significant changes are measured for TERT methylation; i.e., G0 vs. mutated, p = 0.050 and mutated vs. wild-type p = 0.046. Student’s t-test. (B) Average methylation of each gene in non-tumor/G0, low grade (G1-G2) (n_CDH1 = 14; n_CDKN2Ap16 = 43; n_RASSF1A = 11; n_TERT = 33; n_WT1 = 18), and high grade tumor samples (G3-G4) (n_CDH1 = 42; n_CDKN2Ap16 = 92; n_RASSF1A = 30; n_TERT = 85; n_WT1 = 21). A statistically significant increase of TERT methylation was found comparing tumor-free control to low-grade tumors (p = 0.022) and to high grade tumors (p = 0.021), the steady increase with tumor progression is a significant correlation (p = 0.003) Spearman correlation. WT1 methylation decreased significantly comparing tumor-free control to low grade tumor samples (p = 0.043) Student’s t-test. (C) Methylation of target genes in the patient samples grouped by gender and age. A Spearman correlation analysis matrix showed no significant correlation between overall age and promoter methylation for any of the analyzed genes. * p < 0.05.

Figure 2

DNA methylation in cell lines. (A) The methylation levels over all analyzed CpG sites in 2D (top) and 3D (bottom) culture. Each line represents one female (pink) or male (blue) cell line. Within CGIs, the CpG sites show a fluctuation in methylation levels. This observation especially pertains to RASSF1A, TERT, and WT1 in both culture conditions, as well as for CDKN2Ap16 in 3D culture. (B) The average DNA methylation levels with standard deviation of grouped cell lines in the five marker genes analyzed in 2D and 3D cultures, shown as pooled values as well as pooled female and pooled male. Furthermore, average values of male, female, and all FFPE samples were added for reference.

Figure 3

Dose-response curves of NSCLC-cell lines treated with the DNMTIs decitabine and zebularine; shading discriminates male (blue) and female (pink) cell lines. EGFR/KRAS mutation is indicated by an X between the two curves of a cell line.

Figure 4

EC₅₀ values of wild-type versus mutant cancerous cell lines treated with decitabine or zebularine. Averages and standard deviations are shown (n_{mut LC} = 5; n_{wt LC} = 3). A statistically significant difference between the decitabine EC₅₀ values of mut versus wt cell lines was found (p = 0.01, Student’s t-test). No statistical significance was reached for zebularine (p = 0.12; Students t-test).