NALCN Promoter Methylation as a Biomarker for Metastatic Risk in a Cohort of Non-Small Cell Lung Cancer Patients

Abstract

Liquid biopsy enables real-time monitoring of tumor development and response to therapy through the analysis of CTCs and ctDNA. NALCN is a sodium leak channel that is frequently involved in tumor evolution and immunity and acts as a tumor suppressor. Deletion of NALCN has been shown to increase cancer metastasis and the number of CTCs in peripheral blood. In this study, we investigated for the first time NALCN promoter methylation in (a) Aza-treated cell lines (A549, TE671, BT20, and MDA-MB-468), (b) paired NSCLC tissues (n = 22), and (c) plasma cell-free DNA (ctDNA) from patients with NSCLC (early stage n = 39, metastatic n = 39) and DNA from 10 healthy donors (HD) using a newly developed highly specific and sensitive real-time MSP method. Treatment with 5'-aza-dC induced the expression of NALCN only in the A549 cell line, suggesting that DNA methylation regulates its expression in certain cancers. The mRNA expression levels of NALCN were quantified in non-small cell lung cancer (NSCLC) and adjacent non-cancerous tissues, and it was found to be underexpressed in 54.5% of tumor tissues, with significantly higher expression in recurrence-free patients (p = 0.009) than in patients who relapsed. The NALCN methylation level was not statisticallysignificantlycorrelated with the corresponding expression (p = 0.439), while Kaplan-Meier analysis showed an association between NALCN promoter hypermethylation and worse disease-free intervals (DFIs) (p = 0.017). Evaluation of NALCN methylation in ctDNA revealed that it was detected in 5.1% of early and 10.2% of advanced cases. Our results strongly suggest that epigenetic inactivation of NALCN may be a predictor of metastasis in NSCLC. Our results should be validated in further studies based on a larger patient cohort to further investigate whether DNA methylation of the NALCN promoter could serve as a potential prognostic DNA methylation biomarker and predictor of metastasis in NSCLC.

Keywords: NALCN; NSCLC; metastasis; methylation; prognostic biomarker.

Figure 1

Outline of the experimental procedure.

Figure 2

mRNA NALCN expression levels in aza-treated cell lines. Cell lines (A549, TE671, BT20, and MDA-MB-468) were treated with 20 µmol/L 5-Aza-dC for one, two, and three days. Each sample was subjected to qRT-PCR for detection of NALCN and B2M expression. Relative quantification was based on the ΔΔCq method.

Figure 3

Correlation analysis of NALCN DNA methylation and mRNA gene expression in NLCLC pair tissues. (a) Relative expression levels of NALCN mRNA in paired adjacent normal tissues and cancerous tissues from NSCLC patients that were relapsed or were relapsed-free using quantitative PCR. Each number representsa pair of tissues of the same patient, whereas star representsthe overexpression of NALCN. The relative expression data were analyzed by the 2^−ΔΔCq method. B2M was used as an internal control. (b) Correlation analysis of NALCN mRNA expression and NALCN promoter methylation was performed in the same pairs of tissuesamples. mRNA expression of NALCN and hypermethylation of NALCN were evaluated using fold change and threshold was defined as a fold-change > 2. (c) Methylation and mRNA expression status of NALCN in NSCLC pair tissues. The red color represents the hypermethylation or overexpression of NALCN, while the green color represents hypomethylation or underexpression.

Figure 3

Correlation analysis of NALCN DNA methylation and mRNA gene expression in NLCLC pair tissues. (a) Relative expression levels of NALCN mRNA in paired adjacent normal tissues and cancerous tissues from NSCLC patients that were relapsed or were relapsed-free using quantitative PCR. Each number representsa pair of tissues of the same patient, whereas star representsthe overexpression of NALCN. The relative expression data were analyzed by the 2^−ΔΔCq method. B2M was used as an internal control. (b) Correlation analysis of NALCN mRNA expression and NALCN promoter methylation was performed in the same pairs of tissuesamples. mRNA expression of NALCN and hypermethylation of NALCN were evaluated using fold change and threshold was defined as a fold-change > 2. (c) Methylation and mRNA expression status of NALCN in NSCLC pair tissues. The red color represents the hypermethylation or overexpression of NALCN, while the green color represents hypomethylation or underexpression.

Figure 4

Kaplan–Meier estimates of (a) disease-free interval (DFI) in months for NSCLC patients withrespect to NALCN promoter methylation status in tumor tissues (p = 0.017), (b) overall survival (OS) in months for early NSCLC patients withrespect to NALCN promoter methylation status in tumor tissues (p = 0.077).

Figure 5

DNA methylation of NALCN gene promoter in plasma cfDNA of patients diagnosed with advanced NSCLC, early-stage NSCLC, and healthy donors. Each cycle represents a patient sample while red cycle represents the hypermethylation of NALCN.