Date of Graduation

7-2021

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Bailey, Tameka A.

Committee Member

Pinto, Ines

Second Committee Member

Washington, Tyrone A.

Third Committee Member

Evans, Timothy A.

Keywords

Breast cancer subtypes; Metastatic cascade; Liver metastasis; Molecular biomarkers

Abstract

Triple Negative Breast Cancer (TNBC) is highly invasive and metastatic with approximately 15% of patients developing liver metastases. The primary treatment of metastatic TNBC is chemotherapy, however, there is an increased chance of resistance to this therapeutic technique. If Breast Cancer Liver Metastasis (BCLM) is left untreated most patients survive only 4 to 8 months with a very rare 5-year survival. Therefore, it is imperative to analyze markers and molecular pathways that TNBC cells use to progress, invade, and metastasize to the liver. The aim of this study was to examine the overexpression of angiopoietin-like 4 (ANGPTL4) in TNBC cells in vitro and in vivo.

Experimentally, ANGPTL4 mGFP was overexpressed in the MDA-MB-231(TNBC) cell line using lentiviral-mediated transduction. This expression was then confirmed using western blot analysis, ELISA, and fluorescence microscopy.

The intracellular localization of ANGPTL4, Tat-interactive protein p60 (Tip60), and Aurora Kinase A (AURKA) were evaluated by subcellular fractionation, western blot analysis, and immunofluorescence microscopy. This study showed that ANGPTL4 is localized in the nucleus and is a chromatin-bound protein. This implies that ANGPTL4 could potentially bind to DNA and affect transcriptional regulation. In addition, the findings of this study showed that TIP60 and AURKA, predictive interactors of ANGPTL4 are in the nucleus and compartmentalizes with ANGPTL4. This suggests that their association with ANGPTL4 could drive tumor progression and metastases. A mammosphere assay was performed to evaluate the “stemness” capability of TNBC cells expressing ANGPTL4. The results of the mammosphere assay promote the expansion of the cell colonies in the ANGPTL4 mGFP overexpression in the TNBC cell line. This indicates that the ANGPTL4 mGFP expressing cell line has stem-cell like renewal potential in vitro. In this study, the soft agar colony formation assay was performed to measure the prospective tumorigenic ability of the ANGPTL4 mGFP cell line. The ANGPTL4 mGFP cell line exhibited enhanced anchorage-independence which speaks to the possible tumorigenic capacity of the cells to be used in in vivo studies.

Subsequently, a mouse model was used to evaluate the metastatic ability of ANGPTL4 primed TNBC cells in vivo. The findings show that MDA-MB-231 ANGPTL4 mGFP cell line had bigger primary tumors compared to the mGFP cell line expressing endogenous ANGPTL4. Furthermore, ANGPTL4 mGFP cell line resulted in more metastatic lesions on the liver compared to the MDA-MB-231 mGFP cell line. Collectively, these findings imply that ANGPTL4 is a driver of TNBC proliferation and metastases and therefore could be used as a therapeutic target for TNBC treatment.

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