Methylation of the Claudin 1 Promoter Is Associated with Loss of Expression in Estrogen Receptor Positive Breast Cancer

Francescopaolo Di Cello,Cynthia A Zahnow,Stephen B Baylin,Wei Wang,Huili Li,Leslie Cope,Jana Jeschke,Robert Dante

doi:10.1371/journal.pone.0068630

Francescopaolo Di Cello, Cynthia A Zahnow + Show 6 more

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https://doi.org/10.1371/journal.pone.0068630

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Abstract

Downregulation of the tight junction protein claudin 1 is a frequent event in breast cancer and is associated with recurrence, metastasis, and reduced survival, suggesting a tumor suppressor role for this protein. Tumor suppressor genes are often epigenetically silenced in cancer. Downregulation of claudin 1 via DNA promoter methylation may thus be an important determinant in breast cancer development and progression. To investigate if silencing of claudin 1 has an epigenetic etiology in breast cancer we compared gene expression and methylation data from 217 breast cancer samples and 40 matched normal samples available through the Cancer Genome Atlas (TCGA). Moreover, we analyzed claudin 1 expression and methylation in 26 breast cancer cell lines. We found that methylation of the claudin 1 promoter CpG island is relatively frequent in estrogen receptor positive (ER+) breast cancer and is associated with low claudin 1 expression. In contrast, the claudin 1 promoter was not methylated in most of the ER-breast cancers samples and some of these tumors overexpress claudin 1. In addition, we observed that the demethylating agents, azacitidine and decitabine can upregulate claudin 1 expression in breast cancer cell lines that have a methylated claudin 1 promoter. Taken together, our results indicate that DNA promoter methylation is causally associated with downregulation of claudin 1 in a subgroup of breast cancer that includes mostly ER+ tumors, and suggest that epigenetic therapy to restore claudin 1 expression might represent a viable therapeutic strategy in this subtype of breast cancer.

Highlights

Tight junctions are responsible for some of the defining characteristics of epithelial cells and tissues
The HumanMethylation450 microarray includes 13 probes for claudin 1. Six of these probes are located within a CpG island that extends across the transcription start site (TSS), one of the remaining seven probes is upstream of the CpG island and six are downstream (Figure 1)
Methylation of the claudin 1 CpG island is generally low in all the normal breast tissue samples with very few outliers, which is indicative of a normal methylation pattern for an expressed gene

Summary

Introduction

Tight junctions are responsible for some of the defining characteristics of epithelial cells and tissues. Several studies have analyzed the role of claudin 1 (CLDN1) in epithelial physiology as well as in cancer development and metastasis (reviewed by Gupta and Ryan [1] and Myal, Leygue and Blanchard [2]) Both downregulation and overexpression of claudin 1 have been associated with tumorigenesis, suggesting that claudin 1 may alternatively function as a tumor suppressor or as an oncogene. To investigate if claudin 1 downregulation has an epigenetic etiology in human breast cancer we compared gene expression and methylation data from 217 patient samples available through TCGA [23] and from 26 breast cancer cell lines analyzed in our laboratory. We demonstrated the causality of this link by showing that treating human breast cancer cell lines with the DNA demethylating drugs azacitidine and decitabine results in increased claudin 1 expression and in its localization to the cell surface, which can potentially lead to the restoration of normal polarized growth [4,5] or to tumor suppression via apoptosis [17]

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