IDENTIFICATION AND CHARACTERIZATION OF NOVEL REGULATORS OF MAMMARY STEM CELL AND BREAST CANCER

Abstract

The mammary gland epithelium is a hierarchically organized tissue with multipotent mammary stem cells (MaSCs) capable of self-renewal and differentiation. It is fundamentally important to understand how stemness and differentiation of MaSCs are properly maintained, as deregulation of MaSC signaling pathways has been linked to breast cancer development. In the present dissertation, combining molecular biology and genomics tools with animal models and CRSIPR/Cas9 genome editing technologies, I provide two examples in identifying and characterizing novel regulators of MaSCs and breast cancer progression.

In the first study, I performed gene expression profiling analysis of different populations of cells isolated from the mouse mammary gland. I next sought to functionally characterize phospholipid phosphatase 1 (Plpp1), a candidate MaSC regulator predominantly expressed in the MaSC-enriched basal cells. Mammary glands from virgin Plpp1 knockout mice revealed significant developmental defects. Further studies confirmed the functional importance of Plpp1 in maintaining mammary stem cell activities. Mechanistically, Plpp1 protects the stem cell populations from lysophosphatidic acid sensitivity. During breast cancer progression, Plpp1 serves as a tumor suppressor and is gradually lost due to DNA hypermethylation. Clinically, lower PLPP1 expression is associated with poorer prognosis in breast cancer patients. This study shed light on a potentially new class of genes in the mammary epithelium that mediate dual functions in maintaining stem cell properties while suppressing uncontrolled proliferation during cancer progression.

Second, I aimed to establish innovative CRISPR/Cas9-based screening strategies to systematically identify novel MaSC and breast cancer regulators. In the first design, MaSCs were isolated from Cas9-expressing transgenic mice, followed by lentiviral transduction with a customized library targeting candidate MaSC regulators. These cells were injected into the cleared mammary fat pad of recipient mice or cultured in vitro in mammosphere media, both providing a selective condition that requires MaSC activity to support cellular outgrowth. In the second model, human breast cancer cell lines were transduced with genome-wide CRISPR libraries and subsequently selected in three-dimensional (3D) tumorsphere cultures, proving a selective condition that enriches for positively selected genes. I obtained a handful of novel candidate regulators from NGS analysis and an ongoing effort is made to validate their biological functions in vitro and in vivo.