The Role of Caveolin-1 Deficient Human Mammary Fibroblast on Chemotherapy Resistance in Breast Cancer Cells

Date of Award

5-2019

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Abigail Hielscher, PhD

Second Advisor

Francis Jenney, PhD

Third Advisor

Dennis Peffley, PhD, JD

Fourth Advisor

Richard White, PhD

Abstract

Chemotherapy resistance is a hurdle to achieving successful outcomes for the treatment of breast cancer. One mechanism which may contribute to chemotherapy resistance is the decreased expression of fibroblast caveolin-1 (cav-1), membrane invaginations which play a role in signal transduction. The decreased expression of cav-1 has been reported to contribute to a more aggressive breast tumor signature. As a result, it’s possible that decreased expression of cav-1 may promote breast cancer cell resistance to chemotherapy. To address this question, we sought to downregulate cav-1 in human mammary fibroblasts (HMFs), then co-culture these with breast cancer cells to determine whether the down-regulated expression of cav-1 imparted chemotherapy resistance. First, metastatic MDA-MB-231-GFP breast cancer cells were treated with various concentrations of doxorubicin for 24 and 48 hours. Using alamar blue and green fluorescent protein fluorescence, the results showed that 1.2ug/ml of doxorubicin resulted in viability of 63.69% at 24 hours and thus was selected for use in subsequent co-culture experiments. Non- metastatic MCF7 breast cancer cells were treated with various concentrations of tamoxifen for 24 and 48 hours. Analyzing results from the WST-1 viability assay showed that 7.5uM of tamoxifen resulted in viability at 86% at 24 hours and 68% at 48 hours. Next, HMFs were transfected with either a GFP plasmid, a control scrambled plasmid or the shRNA cav-1 plasmid. Following transfection, the HMFs were treated with puromycin to eliminate non-transfected cells. Control and cav-1 shRNA transfected HMFs were cultured and passaged a total of 5 times. It was determined that passages 2 and 3 of cav-1 shRNA treated HMFs exhibited the most down- regulation of cav-1. Next, co-cultures of cav-1 downregulated HMFs with MDA-MB-231-GFP breast cancer cells were set up in a 1:1 ratio and treated with doxorubicin for 24 hours. Preliminary results show that MDA-MB-231-GFP BCCs co-cultured with HMF-siCav1 demonstrated chemotherapy resistance in comparison to the controls. Overall, these results indicate that the downregulation of cav-1 may impart chemotherapy resistance in breast cancer cells.

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