Advisor(s)
Selvarangan Ponnazhagan
Committee Member(s)
Chenbei Chang
Xu Feng
Rosa Serra
Gene Seigal
Jaideep Thottassery
Document Type
Dissertation
Date of Award
2008
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.
ProQuest Publication Number
ISBN
978-0-549-77075-6
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
