TGF-β signaling regulation in breast cancer metastasis

Abstract

The majority of cancer fatality is caused by tumor metastasis which is underlined by Epithelial-Mesenchymal-Transition (EMT) and its opposing process Mesenchymal-Epithelial-Transition (MET) at the cellular level. Transforming Growth Factor-β (TGF-β) is one of the main molecular driving forces for EMT. Therefore, more than 20 phase I/II/III clinical trials targeting TGF-β signaling have been developed with mixed outcomes, highlighting the complex nature of TGF-β signaling in driving EMT and metastasis.

Bioactive vesicles, exosomes, have recently been an exciting focus of research. An increasing number of publications have shown that exosomes play an essential role in both primary tumor growth and metastatic evolution. However the mechanisms of how exosomes promote cancer malignancy are poorly understood. In this thesis, exosomes were isolated from highly-invasive breast cancer cells MDA-MB-231 and fibroblast NIH3T3 cells by differential centrifugation. We demonstrated that exosomes mediated amplification of TGF-β signaling in highly-invasive breast cancer cells, as well as phenocopying of invasive/metastatic behavior. Targeting exosomes trafficking, secretion and uptake significantly reduced TGF-β responses and consequent cellular functions, indicating a new therapeutic strategy for cancer treatment.

Another Transforming Growth Factor-β superfamily member bone morphogenetic proteins (BMPs) inhibit TGF-β-induced EMT and promote MET. However, the mechanism regarding the antagonism and interaction between these two signaling pathways in breast cancer remains unclear. TGF-β abolished BMP-induced Smad1/5 activation in the highly-invasive MDA-MB-231 cells, but to a less extent in the non-invasive MCF7 cells, and even lesser in the normal MCF10A cells. This suggests TGF-β signaling acts in a double whammy fashion in driving cancer invasion and metastasis. We discovered that restored BMP signaling, by combining suboptimal concentration of UO126 and FK506, significantly reduced breast cancer cell self-seeding, liver and bone metastasis in vivo. Surprisingly, no effect on lung metastasis was observed, suggesting a differential role played by BMP signaling in organ specific metastasis. Consequently, there is a potential for them or their analogs to be developed for clinical use.

Opposite to EMT, MET is the process whereby disseminated cancer cells re-establish epithelial-like cells and reinitiate proliferative programs. Thus, MET maybe represents a rate-limiting step of metastasis. While a plethora of studies in EMT have been published, more research is needed regarding the MET process and its regulation. In this thesis, a novel modified transwell assay was designed for detecting cell migration as well as detaching-reseeding. Meanwhile, TGF-β signaling activity per cell was measured by infecting MDA-MB-231 cells with Ad-CMV-Gaussia-luc and TGF-β-Smad3 reporter Ad-CAGA-luc. We illustrated that TGF-β plays a positive role in inducing cell migration but a negative one in detaching-reseeding, which may shed new lights for future cancer treatment. Taken together, this study demonstrates that multiple mechanisms may account for the TGF-β-mediated breast cancer cell migration/metastasis, providing substantial evidences for the development of new cancer therapeutics.