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Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report

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Abstract

Purpose

In the past, anomalous estrogen receptor (ER) regulation has been associated with various lung pathologies, but so far its involvement in lung cancer initiation and/or progression has remained unclear. Here, we aimed at assessing in vivo and in vitro ER expression and its possible epigenetic regulation in non-small cell lung cancer (NSCLC) samples and their corresponding normal tissues and cells.

Methods

ERα and ERβ gene expression levels were assessed using real time quantitative PCR (RT-qPCR), whereas ERα and ERβ gene promoter methylation levels were assessed using DNA bisulfite conversion followed by pyrosequencing. We included NSCLC (n = 87) and adjacent histologically normal lung tissue samples from lung cancer patients (n = 184), primary normal bronchial epithelial-derived cell cultures (n = 11), immortalized bronchial epithelial-derived cell lines (n = 3) and NSCLC derived cell lines (n = 9).

Results

Using RT-qPCR we found significantly lower ERα and ERβ expression levels in the NSCLC tissue samples compared to their normal adjacent tissue samples. These lower ER expression levels were confirmed in vitro using primary normal bronchial epithelial-derived cell cultures, immortalized bronchial epithelial-derived cell lines and NSCLC-derived cell lines. By using this latter panel of cells, we found that ER gene promoter hypermethylation was associated with decreased ER expression. In addition we found that in tumor and normal lung tissues, smoking was associated with decreased ER expression and that normal lung tissues with a low ERβ expression level exhibited increased smoking-related DNA adducts.

Conclusions

Taken together, our results indicate that decreased ER expression mediated by DNA methylation may play a role in NSCLC development.

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Acknowledgments

This study was supported by grants from the Norwegian Cancer Society. We are grateful to Mrs. Tove Andreassen and Mrs. Elin Einarsdottir Thorner for excellent technical assistance. The authors gratefully acknowledge collaboration of Dr. Lodve Stangeland, Haukeland University Hospital, Bergen, for recruiting the lung cancer patients and collecting the tissue specimens.

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Authors and Affiliations

  1. Section for Toxicology and Biological Working Environment, Department of Biological and Chemical Working Environment, National Institute of Occupational Health, PO box 8149, Dep., Gydas vei 8, N-0033, Oslo, Norway

    Xavier Tekpli, Vidar Skaug, Rita Bæra, Aage Haugen & Steen Mollerup

  2. Department of Genetics, Institute for Cancer Research, Oslo University Hospital - The Norwegian Radium Hospital, Oslo, Norway

    Xavier Tekpli

  3. Analytical and Environmental Sciences, MRC-PHE Centre for Environment and Health, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK

    David H. Phillips

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  1. Xavier Tekpli
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  2. Vidar Skaug
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Correspondence to Xavier Tekpli.

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Electronic supplementary material

Supplementary Figure 1

ER expression detailed for each individual cell line a-b: ERα and ERβ gene expression was analyzed in 11 primary cultured human lung cells (NHBE), 3 immortalized human bronchial epithelial cell lines (HBEC), and 9 human lung adenocarcinoma cell lines (HLAC). (PPTX 225 kb)

Supplementary Figure 2

Schematic representation of the location of the different assays designed to study (a) ERα and (b) ERβ promoter DNA methylation. (PPTX 47 kb)

Supplementary Figure 3

DNA methylation of ER promoters detailed for each individual cell line: DNA methylation levels were obtained by analyzing the mean methylation of all the CpG sites present in the different regions of ERα (a) or ERβ (b) promoters. We used 11 primary cultured human lung cells (NHBE), 3 immortalized human bronchial epithelial cell lines (HBEC), and 9 human lung adenocarcinoma cell lines (HLAC). (PPTX 75 kb)

Supplementary Figure 4

Effect of AZA on ERs expression detailed for each individual cell line: a-b: AZA effects on ER expression in human lung cultured cells. Cells were exposed or not for 5 days during proliferation phase to the demethylating agent AZA (0.5 μm). Expression of ER mRNA was normalized to β-actin mRNA levels. AZA effects on gene expression are presented as fold induction of gene expression induced by AZA. (PPTX 72 kb)

Supplementary Table 1

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Supplementary Table 2

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Tekpli, X., Skaug, V., Bæra, R. et al. Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report. Cell Oncol. 39, 583–589 (2016). https://doi.org/10.1007/s13402-016-0295-3

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