DNA Methylation of the Keratin 18 Gene Regulates Brain Metastasis of Triple Negative Breast Cancer
Authors: Liu Tuoen , Kohler William , Wolff Benjamin , Butler Christopher , Szalai Gabor , Straight Benjamin , Lockman Paul 
Published: April 2020

Background Introduction

Breast cancer patients with brain metastasis have a poor prognosis and current chemotherapeutic agents are largely ineffective, which led us to seek new chemotherapeutic agents for the treatment. Our preliminary results showed the hypomethylating agent azacitidine (abbreviated as AZA) is effective in treating brain metastasis breast cancer using in vitro cell and in vivo preclinical mouse models. In the present study, we explored the molecular mechanism of action of AZA for this treatment. Keratin 18 is a member of the cytokeratin family proteins (epithelial marker). We observed that the keratin 18 gene is present in both parental regular MDA‐MB‐231 triple negative breast cancer cells (“231”) and their brain metastasized counterpart cells (“231Br”). However, mRNA and protein levels of keratin 18 are significantly decreased in 231Br cells compared to 231 cells.

Hypothesis

Based on our preliminary results, we hypothesize that decreased expression of the keratin 18 gene in 231Br cells is due to DNA hypermethylation, which contributes to the brain metastatic characteristics and sensitivity to AZA treatment in these cells.

Research Method and Results

The study by Umezawa et al. showed that DNA methylation of intron 1 in the keratin 18 gene is important in regulating its expression. In our study, we compared the DNA methylation status of the intron 1 region (737 bp) of the keratin 18 gene between the regular 231 and brain metastasized 231Br breast cancer cells.

We found that: (1) The DNA sequence of intron 1 of the keratin 18 gene is identical between 231 and 231Br cells. We did not detect DNA mutation or deletion of the intron 1 region of the keratin 18 gene between both cell lines, suggesting the decreased expression of keratin 18 in 231Br cells may due to DNA hypermethylation. (2) The DNA methylation status is different in intron 1 of the keratin 18 gene between 231 and 231Br cells. We fully sequenced and compared the DNA methylation of the intron 1 region of the keratin 18 gene between both cell lines by using the bisulfite conversion method. In this method, treating DNA with bisulfite chemically modifies non‐methylated cytosines into uracil (appears as thymine in DNA sequencing results), but methylated cytosines remain unchanged. We found three cytosines were converted into uracil in 231 cells while they remained unchanged in 231Br cells, suggesting the DNA methylation status of the keratin 18 gene is different between both cell lines.

Conclusion

In this study, we found that the keratin 18 gene is hypermethylated in brain metastasized breast cancer cells, which is important in regulating its expression and response to hypomethylating agent treatment. Based on our findings, the hypermethylated keratin 18 gene can be a potential drug target used for the development of novel targeted therapy drugs in treating patients with brain metastasis breast cancer.

Support or Funding Information

The research was supported by the National Institute of General Medical Sciences of NIH (Award Number 5U54GM104942‐03) and WVSOM intramural fund