Date of Award

2015

Document Type

Thesis

Degree Name

Master of Biomedical Science

Department

Pathology and Laboratory Medicine

College

College of Graduate Studies

First Advisor

Amanda C. LaRue

Second Advisor

Victoria J. Findlay

Third Advisor

David P. Turner

Fourth Advisor

Dennis K. Watson

Fifth Advisor

Marvella E. Ford

Abstract

Breast cancer is a heterogeneous disease that is clinically classified based on the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Associated with the poorest prognosis of all subtypes, triple negative breast cancer (TNBC) is defined as ER-/PR-/HER2-; lacking these receptors, TNBCs are insensitive to effective targeted therapies and chemotherapy remains the mainstay of treatment. Recent research has shown that platinum chemotherapy agents are particularly active in TNBC, including a clinical trial that demonstrated single agent cisplatin alone can induce response in a subset of TNBC patients. However, identification of biomarkers is still needed to distinguish sensitive patients from resistant ones, whom may benefit from other forms of chemotherapy. Recent studies have revealed that cisplatin activates the c-Abl/TAp73 apoptotic pathway, specifically in TNBCs with p53 mutations (~60% of all TNBCs). Our lab has identified miR-510 to be overexpressed in human breast tissue samples, suggesting a role in breast cancer. Our lab has validated Prdx1 to be a direct target of miR-510. Prdx1 has been shown to protect cells from cisplatin and also inhibit the tyrosine kinase c-Abl. Therefore we wanted to determine the efficacy of miR-510 as a biomarker of cisplatin sensitivity and elucidate the mechanism of this sensitivity. Drug cytotoxicity assays indicate that miR-510 positively correlates with cisplatin sensitivity in TNBC cell lines. Furthermore, inhibition of miR-510 causes sensitive TNBC cell lines to become more resistant, while overexpression of miR-510 restores sensitivity in resistant TNBC cell lines in vitro. MicroRNAs are ideal biomarkers in cancer as they regulate many processes in cancer development and progression and are also stable in biological fluids. Analysis of samples from our in vivo study reveals a positive correlation of miR-510 levels in matched tumor and serum samples, suggesting that serum expression of miR-510 is indicative of its expression in tumor samples. Analysis of breast cancer patient serum samples demonstrates that varying levels of miR-510 can be detected in human samples as well. We then went on to explore the mechanism of miR-510 mediated cisplatin sensitivity. IHC analysis of in vivo tumors suggests that miR-510 mediated sensitivity may not be through decreased proliferation or increased cleaved-caspase 3 dependent apoptosis. Prdx1 IHC analysis, along with matched tumor miR-510 expression suggests that negative regulation of Prdx1 may play a role in miR-510 mediated cisplatin sensitivity. The Prdx1 rescue experiments found that the mechanism of miR-510 mediated sensitivity is dependent upon the negative regulation of Prdx1. Our mechanistic studies reveal that cisplatin treatment leads to upregulation and increased phosphorylation of TAp73 in miR-510 overexpressing cells. In cells overexpressing miR-510, we also show that cisplatin leads to dissociation of TAp73 from its repressor ΔNp63 and induction of downstream pro-apoptotic genes, PUMA and NOXA. Cumulatively, our data demonstrates that miR-510 is a potential, non-invasive biomarker of cisplatin sensitivity in TNBC. We also show this mechanism of sensitivity is attributed to, at least in part, the negative regulation of Prdx1 and the subsequent activation of TAp73 apoptotic pathway.

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