In FY09, we have continued to focus primarily on elucidating the tumor cell-autonomous components of the switch in activity of TGF-beta from tumor suppressor to pro-progression factor. There have been two main areas of effort on this project. In the first we are addressing the hypothesis that TGF-beta is a key regulator of cancer stem cell dynamics, and that this activity is central to its role as a tumor suppressor in the early stages of carcinogenesis. In the second, we are using integrated genomic approaches to identify molecular determinants that cause the TGF-beta response to switch during cancer progression, with a particular focus on the signal transduction component Smad3 as a key mediator of this process. For both parts of this study, we our main discovery platform is a xenograft model of breast cancer progression based on the MCF10A human breast epithelial cell line. We have previously demonstrated that TGF-beta switches from tumor suppressor to pro-progression factor in this model, and the high degree of genetic relatedness between the different cell lines of the progression series gives us an exceptionally high signal-to-noise system in which to address mechanisms underlying the TGF-beta switch. However, key findings from the model are validated in additional mouse models and human clinical material where possible. Following up on our previously published demonstration that TGF-beta may regulate cancer stem cell dynamics, we have focused over the past year on developing functional imaging approaches that will allow us to identify the minority cancer stem cell (CSC) population, and thus analyze its behavior in the context of interactions with other cellular and acellular components of the microenvironment. Starting with the assumption that most CSCs will express one or more of the constellation of transcription factors that can induce or maintain stemness/ pluripotency in normal cells, we have generated constructs that report on the simultaneous presence of the stem-related transcription factors Oct4 and Sox2, and we have shown that these reporters identify cells that are enriched for CSC activities, including an enhanced ability to initiate tumorigenesis in vivo. Using a TGF-beta pathway reporter expressing a different fluorescent protein, we can now simultaneously visualize TGF-beta pathway activation and stemness in vitro and in vivo. Furthermore, we are developing and/or optimizing sensitive Firefly-based microproteomic techniques and single cell gene expression analysis tools to address molecular mechanisms in this minority population. With these tools in hand, we are poised to address our hypothesis that TGF-beta may regulate CSC dynamics by inducing or maintaining CSC quiescence, or by influencing the balance between self-renewal and commitment to differentiation. Understanding how CSCs are regulated will be critical to development of more effective cancer therapies as these cells are largely resistant to existing therapeutic approaches, leading ultimately to relapse. In the second part of this project, we are addressing the molecular mechanisms that underlie the switch in TGF-beta response from tumor suppression to tumor promotion. Data from our lab and others implicates the downstream signal transduction component Smad3 in both these activities. We hypothesize that genetic or epigenetic changes that occur during cancer progression may alter the Smad3-mediated readout of the TGF-beta signal so that tumor promoting activities dominate. To address this question, we have performed genome-wide chromatin immunoprecipitation to identify Smad3 target genes in response to TGF-beta treatment in the MCF10 model. Specifically, we have focused on two closely related cell lines;MCF10Ca1h in which TGF-beta functions as tumor suppressor, and MCF10Ca1a in which TGF-beta functions as a pro-progression factor. Integration of the promoter occupancy data with global gene expression data has yielded core Smad3-based gene signatures that are associated with the two different outcomes. These signatures should yield important insights into mechanisms underlying the switch process, and will be exploited in gene expression based screens to find novel compounds that might reverse the switch and restore the tumor suppressor activities of TGF-beta.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005785-15
Application #
7965077
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2009
Total Cost
$548,681
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Keysar, Stephen B; Le, Phuong N; Miller, Bettina et al. (2017) Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2. J Natl Cancer Inst 109:
Sato, Misako; Kadota, Mitsutaka; Tang, Binwu et al. (2014) An integrated genomic approach identifies persistent tumor suppressive effects of transforming growth factor-? in human breast cancer. Breast Cancer Res 16:R57
Bae, Eunjin; Sato, Misako; Kim, Ran-Ju et al. (2014) Definition of smad3 phosphorylation events that affect malignant and metastatic behaviors in breast cancer cells. Cancer Res 74:6139-49
Wakefield, Lalage M; Hill, Caroline S (2013) Beyond TGF?: roles of other TGF? superfamily members in cancer. Nat Rev Cancer 13:328-41
Kohn, Ethan A; Yang, Yu-an; Du, Zhijun et al. (2012) Biological responses to TGF-? in the mammary epithelium show a complex dependency on Smad3 gene dosage with important implications for tumor progression. Mol Cancer Res 10:1389-99
Figueroa, Jonine D; Flanders, Kathleen C; Garcia-Closas, Montserrat et al. (2010) Expression of TGF-beta signaling factors in invasive breast cancers: relationships with age at diagnosis and tumor characteristics. Breast Cancer Res Treat 121:727-35
Mendoza, Arnulfo; Hong, Sung-Hyeok; Osborne, Tanasa et al. (2010) Modeling metastasis biology and therapy in real time in the mouse lung. J Clin Invest 120:2979-88
Kadota, Mitsutaka; Yang, Howard H; Gomez, Bianca et al. (2010) Delineating genetic alterations for tumor progression in the MCF10A series of breast cancer cell lines. PLoS One 5:e9201
Kohn, Ethan A; Du, Zhijun; Sato, Misako et al. (2010) A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGF? signaling in the mammary gland. Breast Cancer Res 12:R83
Stuelten, Christina H; Busch, Johanna I; Tang, Binwu et al. (2010) Transient tumor-fibroblast interactions increase tumor cell malignancy by a TGF-Beta mediated mechanism in a mouse xenograft model of breast cancer. PLoS One 5:e9832

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