Poster - #VH-28129 - Keywords: DNA Damage Mammary Gland Development Epithelial Cell Differentiation

Exploring the Link Between the DNA Damage Response Functionality of KAP1 and Its Impact on Mammary Epithelial Cell Differentiation

Zuhaib Rana1; Nayantara Govindrajan1 Ph.D.; Maximilian Brant1, Alexandria Bartlett2, Nargis Khan2 Ph.D.; Kay-Uwe Wagner3 Ph.D.; Carrie Shemanko Ph.D. 1,4
Zuhaib Rana1; Nayantara Govindrajan1 Ph.D.; Maximilian Brant1, Nargis Khan2 Ph.D.; Kay-Uwe Wagner3 Ph.D.; Carrie Shemanko Ph.D. 1,4 1Department of Biological Science, University of Calgary, Calgary, Alberta, Canada 2Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, USA 4Arnie Charbonneau Cancer Institute Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

ABSTRACT:

Abstract

The DNA damage response (DDR) is a fundamental process that ensures genomic integrity by repairing endogenous and exogenous DNA damage, preventing oncogenic transformation and cell death. Beyond maintaining genome stability, the DDR also plays a pivotal role in regulating cell fate decisions and tissue development. A key regulator of the DDR is KRAB-associated protein 1 (KAP1), a multifunctional protein that interacts with the Krüppel-Associated Box Zinc Finger Protein (KRAB-ZFP) family of transcriptional repressors. KAP1 orchestrates chromatin remodelling and transcriptional regulation, influencing gene expression programs critical for epithelial-to-mesenchymal transition (EMT), stem cell pluripotency, and DDR activation. However, its role in mammary epithelial cell (MEC) differentiation remains largely unexplored. Therefore, the objective of this study is to elucidate the connection between KAP1’s DDR function and lactogenic differentiation of mammary epithelial cells (MEC). To achieve this, we utilized an MMTV-Cre conditional KAP1 KO mouse model and employed a CRISPR-Cas9 KAP1 knockout (KO) in the murine MEC cell line HC11. KO P10 glands exhibited significantly decreased expression of KAP1 at the protein and transcript level, as evidenced by immunofluorescence and RNAscope analysis. Importantly, our findings demonstrate that KAP1 is indispensable for alveolar formation, as KAP1-null alveoli fail to develop. Single-cell RNA sequencing revealed a significantly decreased expression of Csn2 in an alveolar progenitor cluster, while RNAscope analysis of P10 KO tissue demonstrated a non-significant decrease in Csn2 expression relative to control. Furthermore, loss of KAP1 leads to the accumulation of unresolved DNA damage, elevated reactive oxygen species (ROS) levels, and increased mitochondrial content. Moreover, KAP1 KO cells exhibited impaired mitochondrial respiration and glycolytic capacity relative to WT, as evidenced by Seahorse metabolic analysis. Additionally, KO cells exhibit elevated polyploidization, which is further exacerbated upon treatment with 50 nM doxorubicin, suggesting impaired cell cycle regulation in response to DNA damage and KAP1 loss. Additionally, P10 KO glands exhibited decreased expression of Cdkn1a and Gadd45a, which are key KAP1 target genes that regulate cell cycle progression in response to DNA damage. Moreover, bulk RNA sequencing on HC11 KO cells revealed elevated expression of genes implicated in cell cycle progression and the DNA damage response, including notable upregulation of Ccnd1, Bcl2, Mbd1, and Bard1 relative to WT. Lastly, loss of KAP1 resulted in a decrease in the CD61 positive luminal progenitor population in HC11. Collectively, our findings underscore the pivotal role of the functionality of KAP1 in preserving genomic integrity and regulating cell fate decisions and tissue development within the mammary gland.