Development of a Breast Cancer Stem Cell Model and the Inhibitory Regulation of Small Molecule Phytochemicals on Various Stages of Human Breast Cancer Cells

The development of clinical breast cancer is a multistep process that manifests itself as a disease with various distinct phenotypes. At present, it is believed that a neoplastically-transformed cell undergoes many heterogeneous changes and mutations before evolving into a tumorous or invasive breast cancer. Due to the heterogeneity of breast cancer, a single therapeutic strategy is rarely completely effective for all patients. Current therapeutic options such as hormone antagonists, radiation and chemotherapy have many deleterious side effects which demonstrates a need for a more efficacious therapy alternative that can not only target varying phenotypes but also ameliorates harsh side effects. In addition, it is essential to identify compounds that can slow the promotion of the disease from preneoplastic lesions to invasive breast cancer. This thesis details the generation of a novel breast cancer stem cell model and the molecular mechanism of two small molecule phytochemicals, indole-3-carbinol (I3C), and artemisinin, target breast cancer stem cells and luminal A, estrogen sensitive breast cancer, respectively. We show that ectopic expression of HER2, a member of the epidermal growth factor receptor family, in MCF-10AT preneoplastic mammary epithelial cells induces a phenotype with the molecular markers of breast cancer stem cells expression: CD44+/CD24-/ALDH-1+ with highly expressing levels of stem cell marker, nucleostemin. These cells are capable of forming tumorspheres in 3-D cultures, indicative of a population highly enriched with stem cells. Furthermore, as few as 30,000 cells are able to form viable mammary tumors in athymic mouse xenograft models. In breast cancer stem cells, I3C induces apoptosis by selectively targeting nucleostemin and activating the p53 apoptotic pathway. I3C induces the proteolytic degradation of Akt1 and thereby functionally inactivates MDM2. Loss of MDM2 phosphorylation and its subsequent inactivation frees p53 to induce apoptosis. Coupled with the activation of nucleostemin by I3C, nucleostemin sequesters MDM2 away from p53 further enhancing the apoptotic effect. Knockdown of nucleostemin prevents the I3C apoptotic effects, suggesting the selective role of I3C on breast cancer stem cells. Also, expressing constitutively active Akt1 or a dominant negative form of p53 overrides the I3C induced apoptotic effect, highlighting the specific Akt1/MDM2/p53 pathway modulated by I3C. The preclinical results implicate I3C as a novel anti-cancer agent that can selectively target cancer stem cells especially given that I3C also increases MDM2-nucleostemin interactions and can reduce tumors volumes in vivo. We also show that artemisinin, derived from the sweet wormwood, Artemisia annua, ablates key G1 cell cycle regulators to induce growth arrest in luminal A, estrogen sensitive breast cancer as well as inhibit in vivo xenograft growth in athymic mice. Artemisinin is selective towards malignant cells, such as the MCF-7 breast cancer cell line since it is ineffective in arresting growth in nontumorigenic breast cell lines. Artemisinin exposed MCF-7 cells displayed ablated levels of cyclin-dependent kinases 2 and 4 (CDK), cyclin E, cyclin D1 as well as E2F1 at the protein and mRNA level. Promoter deletion mapping and subsequent chromatin immunoprecipitation analyses revealed that downregulation of E2F1 resulted in inhibition of CDK2 of promoter activity. Additionally, constitutive expression of E2F1 reversed the growth arrest, CDK2 and cyclin E downregulation induced by artemisinin.

Advisor: Firestone, Gary L