Diagnostic role of Wnt pathway gene promoter methylation in non small cell lung cancer

Abstract

Wnt signal pathway genes are known to be involved with cancer development. Here we tested the hypothesis whether DNA methylation of genes part of the Wnt signaling pathway could help the diagnosis of non-small cell lung cancer (NSCLC). The methylation levels of SFRP1, SFRP2, WIF1 and PRKCB in 111 NSCLC patients were evaluated by quantitative methylation-specific PCR (qMSP). Promoter methylation levels of four candidate genes were significantly higher in tumor tissues compared with the adjacent tissues. SFRP1, SFRP2 and PRKCB genes were all shown to be good predictors of NSCLC risk (SFRP1: AUC = 0.711; SFRP2: AUC = 0.631; PRKCB: AUC = 0.650). The combined analysis showed that the methylation status of the four genes had a sensitivity of 70.3% and a specificity of 73.9% in the prediction of NSCLC risk for study cohort. A higher diagnostic value with an AUC of 0.945 (95% CI: 0.923-0.967, sensitivity: 90.6%, specificity: 93.0%) was found in TCGA cohort. In addition, SFRP1 and SFRP2 hypermethylation events were specific to male patients. Further TCGA data mining analysis suggested that SFRP1_cg15839448, SFRP2_cg05774801, and WIF1_cg21383810 were inversely associated with the host gene expression. Moreover, GEO database analysis showed that 5'-Aza-deoxycytidine was able to upregulate gene expression in several lung cancer cell lines. Subsequent dual-luciferase reporter assay showed a crucial regulatory function of PRKCB promoter. In summary, our study showed that a panel of Wnt signal pathway genes (SFRP1, SFRP2, WIF1 and PRKCB) had the potential as methylation biomarkers in the diagnosis of NSCLC.

Keywords: DNA methylation; Wnt pathway; diagnosis; non-small cell lung cancer; quantitative methylation-specific PCR.

Figure 1. Comparisons of methylation levels of (A) SFRP1, (B) SFRP2, (C) PRKCB and (D) WIF1 genes between tumor tissues and paired adjacent non-tumor tissue

The error bars of PMR data were described as median (interquartile range). PMR: the percentage of methylated reference; LUAD: lung adenocarcinoma; LUSC: lung squamous carcinoma.

Figure 2. Receiver operating characteristic (ROC) curves for the methylation panel in (A) NSCLC, (B) LUAD and (C) LUSC of our study cohort and (D) NSCLC, (E) LUAD and (F) LUSC of TCGA cohort

AUC: area under the ROC curve; CI: confidence interval; NSCLC: non-small cell lung cancer; LUAD: lung adenocarcinoma; LUSC: lung squamous carcinoma; TCGA: The Cancer Genome Atlas.

Figure 3. Correlation between 6 Illumina Human Methylation 450K CpG probes [(A) SFRP1_cg15839448, (B) SFRP2_cg05874561, (C) SFRP2_cg05774801, (D) WIF1_cg21383810, (E) PRKCB_cg24250393 and (F) PRKCB_cg08406370] and corresponding gene expression in 818 NSCLC from TCGA data portal

NSCLC: non-small cell lung cancer; TCGA: The Cancer Genome Atlas.

Figure 4. The expression value changes of SFRP1, SFRP2, WIF1 and PRKCB genes before and after 5′-Aza-deoxycytidine (0.5 μM for 6 days) treatment in three NSCLC cell lines (A549, H1993 and H2073)

P value was evaluated by moderated t test. P > 0.10 was not shown in the figure.

Figure 5. Luciferase activity determined by a dual-luciferase assay system

The reporter gene plasmids containing different gene promoter regions were constructed, including pGL3-SFRP1: +464 to +863, pGL3-SFRP2: +261 to +660, pGL3-WIF1: −511 to −111, and pGL3-PRKCB: −580 to −180. HEK-293T cells were transfected with each reporter gene plasmid, together with renilla luciferase reporter plasmid. Empty pGL3-Basic and pGL3-Promoter vectors were used as the negative and positive control, respectively. **P < 0.01; ****P < 0.0001.