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

Date of Award

5-2017

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Abigail Hielscher, PhD

Second Advisor

Francis Jenney, PhD

Third Advisor

Harold Komiskey, PhD

Fourth Advisor

Lori Redmond, PhD

Abstract

Chemotherapy is one of the most widely used treatments of many types of breast cancer, however, it has been shown that carcinoma-associated fibroblasts (CAFs) induce chemotherapy resistance in breast cancer cells. Previous research has shown that the downregulation of caveolin-1 (cav-1), a structural component of membrane caveolae, promotes a CAF-like phenotype in stromal cells. This study was performed to evaluate the effect that downregulation of cav-1 in mammary fibroblasts imparts on chemotherapy resistance in breast cancer cells (BCCs). To determine the half maximal inhibitory concentration (IC50) of chemotherapy agents on breast cancer cells, MDA-MB-231 and MCF-7 BCCs were treated for 24 and 48 hours with doxorubicin or tamoxifen, then cell viability was measured. The WST-1 cell viability assay revealed that there was significant cytotoxicity after 48 hours for both cell lines and treatment types. Specifically, the MDA-MB-231 BCCs showed significant cell death following treatment with 0.9 ug/mL of doxorubicin and 7 uM of tamoxifen. For the MCF-7 BCC’s results, significant cell death was observed at 0.92 ug/mL for doxorubicin and 7.5 uM for tamoxifen. Multiple attempts were made to isolate mammary gland fibroblasts from cav-1 -/- and cav1 +/+ mice with no success. As an alternative, human mammary fibroblasts (HMFs) were i transfected with cav-1 shRNA to induce downregulation of cav-1 gene expression. HMFs transfected with cav-1 shRNA were positively selected with puromycin, then trypsinized and subcultured to ensure only transfected cells remained adherent. Following a growth period of 72 hours, protein lysate from whole transfected HMF cells was collected and a western blot was performed to assess cav-1 expression. Results showed a significant downregulation of cav-1 in cav-1 shRNA treated samples compared to HMFs plated in transfection medium only. Transfected HMFs were then co-cultured with MCF-7 and MDA-MB-231 BCCs for 24 hours then treated with tamoxifen for 24 and 48 hours. WST-1 assays showed there was no significant increase in cell proliferation of 24 and 48 hour tamoxifen treated MCF-7 BCCs co-cultured with cav-1 shRNA transfected HMFs or 24 hour tamoxifen treated MDA-MB-231 BCCs co-cultured with cav-1 shRNA transfected HMFs. However, significant increase in cell proliferation was revealed in the 48 hour tamoxifen treated MDA-MB-231 BCCs co-cultured with cav-1 shRNA transfected HMFs through a WST-1 assay. Overall, these results indicate that tamoxifen is a strong cytotoxic agent against MDA-MB-231 and MCF-7 BCCs, while doxorubicin is more effective against MDA-MB-231 BCCs. Additionally, the use of shRNA transfection proved to be a beneficial technique for downregulation of cav-1 in HMFs. Lastly, cav-1 shRNA transfected HMFs co-cultured with MDA-MB-231 BCCs was associated with a decrease in the cytotoxic effects of tamoxifen against the MDA-MB231 BCCs after 48 hours tamoxifen treatment.

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