Graduation Date

Fall 12-20-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Melissa Teoh-Fitzgerald

Abstract

The tumor microenvironment (TME) is a key determining factor in breast cancer, especially the more aggressive subtype triple negative breast cancer (TNBC). The activated fibroblasts and macrophages within the TME have many tumor promoting functions. Therefore, targeting their activation presents a novel therapeutic approach in TNBC. My work studied the role of reactive oxygen species (ROS) during fibroblast and macrophage activation in breast cancer.

My studies showed that expression of the secreted antioxidant enzyme, EcSOD, is silenced in breast cancer samples, in part, via increased promoter methylation. The re-expression of EcSOD inhibited c-Met activation in the TNBC cell line, MDA-MB231. HGF, the ligand for c-Met, is secreted by surrounding fibroblasts in breast cancer. Due to its extracellular localization, EcSOD significantly inhibited the paracrine HGF/c-Met signaling during co-culture of MDA-MB231 with HGF overexpressing mammary fibroblast, RMF-HGF. EcSOD and a SOD mimetic, MnTE-2-PyP, inhibited the HGF-mediated 3D growth and invasion of MDA-MB231 in co-culture with RMF-HGF. EcSOD also inhibited RMF-HGF-stimulated orthotopic tumor growth of MDA-MB231. Interestingly, long-term co-culture showed that EcSOD expression in MDA-MB231 inhibited the aggressive phenotype in RMF-HGF. Additionally, upregulation of the ROS generating enzyme, Nox4, increased the aggressiveness of RMF-HGF over control RMF cells.

Tumor associated macrophages (TAM), which resemble the M2 type, enhance the aggressiveness of breast cancer. M2 polarization was selectively inhibited by MnTE-2-PyP. MnTE-2-PyP reduced M2 markers and inhibited the macrophage-mediated cancer cell growth. Additionally, MnTE-2-PyP inhibited M2-mediated T cell suppression, in part, via decreased PD-L2 levels. This study also determined that M2 macrophages have lower levels of reactive oxygen species (ROS) and lower production of extracellular hydrogen peroxide compared to the anti-tumor M1 macrophages, due to reduced levels of pro-oxidants enzymes and higher levels of antioxidant enzymes. Despite lower ROS levels, M2 macrophages require ROS for proper polarization as MnTE-2-PyP reduced ROS levels. Additionally, inhibition of Nox-derived ROS with DPI inhibited M2 markers, while adding hydrogen peroxide increased M2 markers. Mechanistically, MnTE-2-PyP and DPI inhibited M2 polarization via decreased Stat3 activation.

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