A combined approach on the impact of EP300 overexpression on drug resistance, invasion and stemness in breast cancer

Abstract

Triple negative breast cancer, characterized by absence of estrogen and progesterone receptors and human epidermal growth factor receptor 2 (HER2), is very aggressive, and often progresses to metastasis. The absence of druggable targets implies that conventional chemotherapeutic agents do not work. Recent genomic studies show that epigenetics play a complicated role in maintaining healthy gene expression.

This thesis attempted to demonstrate a link between E1A binding protein P300 (EP300), epithelial to mesenchymal transition (EMT), drug resistance and cancer stem cell (CSC) traits by using breast cancer cell line models with EP300 overexpression (CAL51 and MDA-MB-231), short hairpin RNA (shRNA) lentiviral knockdown EP300 (T47D and MCF7), and EP300 knockout in colorectal cancer (HCT116). Techniques such as flow cytometry, migration/invasion assays and short/long term drug resistance assays were used to demonstrate these traits. This study also employed reverse transcription quantitative real-time PCR (RTqPCR) and RNA sequencing (RNA-seq) gene expression analysis for evaluating a gene signature and miR expression.

The study demonstrated positive regulation of EP300, forkhead box O3a (FOXO3a) and GATA binding protein 3 (GATA3) by miR-25 in minimally transformed mammary epithelial cancer (MTMEC) model. Neither RNA-seq nor RTqPCR demonstrated any correlation between cancer subtype and our gene signature. However, we confirmed that EP300 overexpression resulted in E-cadherin (CDH1) upregulation in CAL51 and MDA-MB-231. EP300 knockdown in T47D resulted in decreased expression of CDH1 and upregulated expression of FOXO3a. EP300 knockout in HCT116 downregulated CDH1. EP300 overexpression in MDA-MB-231 improved drug sensitivity to paclitaxel and doxorubicin. EP300 overexpression reduced aldehyde dehydrogenase + (ALDH) populations in MDA-MB-231 and CAL-51 cell lines, while EP300 modulation had a variable effect on CD44 antigen (CD44)high/CD24 antigen (CD24)low subpopulations in these cell line models. Finally, overexpression of EP300 in MDA-MB-231 demonstrated a decrease in motility and invasion.