Abstract 2363: Transcription factors of keratin 18 gene in MDA-MB-231Br (brain-colonizing) breast cancer cells
Authors: Johnson Anissa Elice , Singh Sagardeep , Lockman Paul , Liu Tuoen , Szalai Gabor 
Published: June 2022

Abstract
Among all cancer types, breast cancer has the highest incidence rate and second highest mortality rate in females in the United States. Breast cancer patients with brain metastasis have a poor prognosis, with a median survival time of less than 1 year despite treatment. Decreasing the prevalence of brain metastasis would improve the overall prognosis of patients. We previously built a novel mouse model of breast cancer brain metastasis by intracardiac injection of the MDA-MB-231 (triple-negative breast cancer cell line) cells in mice. The brain colonizing counterpart of the parental cells primarily metastasizes to the brain, which is called the MDA-MB-231Br cell line. We have also shown that keratin 18 (an epithelial marker) plays a crucial role in the brain metastasis process as the MDA-MB-231Br cells have lower expression of the keratin 18 gene compared to their parental MDA-MBA-231 cells. Therefore, it is important to understand the transcriptional control of the keratin 18 gene, in order to fully understand the role of keratin 18 in regulating brain metastasis of breast cancer. We hypothesize that keratin 18 gene expression differs between these two cell lines due to the differential occupation of the gene by transcription factors such as Fli-1 or ETS-1. In this study, we analyzed the pattern of chromatin occupation of transcription factors ETS-1 and Fli-1 to determine whether they are responsible for the altered expression of the keratin 18 gene in MDA-MB-231Br cells.The Chromatin Immunoprecipitation (ChIP) was performed using the Pierce Magnetic ChIP Kit to analyze the presence of transcription factors Fli-1 and ETS-1 with enzymatic and mechanical shearing combined. The ChIP Kit included the positive control anti-RNA Polymerase II Antibody and GAPDH control primers. We optimized the experimental conditions including ChIP analysis for MDA-MB-231Br cells, detection of expected positive control results, and sonication settings to break apart the chromatin. We measured the ChIP mediated enrichment of the intron 1 region of the keratin 18 gene, as the DNA is hypermethylated in this region in MDA-MB-231Br cells. The primers were designed and used as follows: forward: 5′-GATCATCGAGGACCTGAGGG-3′ and reverse: 5′-GGGGAGCAGATCCTTCTTAGC-3′. Our results indicated that an ETS transcription factor occupies intron 1 of the keratin 18 gene in the MDA-MB-231Br cells, also showing that ChIP is an effective method to examine transcription factor binding. For future studies, we plan to perform similar experiments with the parental MDA-MB-231 cells. Then a quantitative PCR will be used to measure DNA binding sites of the transcription factors and determine if there is a significant difference in the level of chromatin occupation between the regular and brain-colonizing breast cancer cells. Our studies are necessary to understand the role of the keratin 18 gene in regulating tumor metastasis at the molecular level.
Citation Format: Anissa Elice Johnson, Sagardeep Singh, Paul Lockman, Tuoen Liu, Gabor Szalai. Transcription factors of keratin 18 gene in MDA-MB-231Br (brain-colonizing) breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2363.