Advisory Committee Chair
Selvarangan Ponnazhagan
Advisory Committee Members
Chenbei Chang
Xu Feng
Rosa Serra
Gene Seigal
Jaideep Thottassery
Document Type
Dissertation
Date of Award
2008
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Overexpresssion of transforming growth factor (TGF)-β has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers including carcinoma of the breast. Thus, targeted downregulation of TGF-β1 expression in breast cancer in situ and determination of its implications potentially could provide new treatment approaches for disease management. siRNA constructs targeting TGF-β1 were validated and used to develop clonal derivatives of the MDA-MB-435 metastatic breast cancer cell line. Inhibition of TGF-β1 expression in MDA-MB-435 cells showed decrease in migration and invasion in vitro with an increase in proliferation. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases. Analysis of TGF-β signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKT and ERK activation. Analysis of TGF-β signaling in the tumor microenvironment was performed using co-culture assays consisting of both MDA-MB 435 tumor cells and fibroblasts. Early experiments involving analysis of gene expression in a breast cancer xenograft model showed an increase in TGF-β1 and MMP expression in the tumor stroma over tumor ii progression. Co-culture experiments using Boyden chambers indicated increased expression of TGF-β1, MMP-9, MMP-2, TβR1 and TβR2 in the tumor cells. Interestingly, there was a significant decrease in TGF-β1, MMP-2, TβR1 and TβR2 expression in fibroblasts that were co-cultured with tumor cells. Experiments using conditioned media also showed a decrease in fibroblasts and tumor cell differentiation through decreases in alpha-smooth muscle actin and cytokeratin 18, respectively. Therapeutic efficacy was determined using rAAV to administer TGF-β1 siRNA intra-tumorally. Results confirmed that the AAV6 serotype was effectively able to transduce the tumor cells both in vitro and in vivo. RT-PCR data confirmed that TGF-β1, MMP-9 and MMP-2 were effectively decreased in the tumor injected with rAAV-TGFβsi when compared to the rAAV-GFP injected tumors. Macroscopic lung metastases were decreased in the treated group; however microscopic metastases were still present. Lung weights also show a decrease, however, the differences were not statistically significant. Better understanding of the molecular pathways involved in TGF-β signaling will help to advance the development of targeted therapeutics along with improvements in delivery mechanics. Through our studies we have shown that inhibition of TGF-β can effectively reduce cancer metastasis; we have identified secondary pathways active in mammary cancer neoplastic progression which may be targeted in combination to give a synergistic effect and tested alternative methods of delivery.
Recommended Citation
Moore, Lakisha Dionne, "Modulation Of Transforming Growth Factor (Tgf)-ß1 And Its Implications In Breast Cancer Metastasis" (2008). All ETDs from UAB. 3763.
https://digitalcommons.library.uab.edu/etd-collection/3763