In FY10, 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. Our main experimental 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. Our work in FY10 has involved three main areas of emphasis: 1. THE ROLE OF TGF-BETA IN REGULATING CANCER STEM CELL DYNAMICS. During FY10 we have continued to develop and refine functional imaging approaches that will allow us to identify the minority cancer stem cell (CSC) population in situ, and thus analyze its behavior in the context of interactions with other tumor cells and the microenvironment. Existing stem cell reporters contain TGF-beta response elements and thus do not allow stemness to be reported on independently of the activation status of the TGF-beta pathway. To overcome this problem, we have generated a novel reporter construct consisting of concatemerized binding sites for the stem-related transcription factors Oct4 and Sox2, and we have shown that this reporter identifies 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. We have successfully visualized cells expressing both reporters in tumorspheres in vitro, and ex vivo in tumors following surgical excision. Our data confirm that there is a sub-population of cancer stem cells that shows active TGF-beta signaling. The functional significance of this subpopulation is currently under study. 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. 2. MOLECULAR MECHANISMS THAT UNDERLIE THE SWITCH IN TGF-BETA RESPONSE FROM TUMOR SUPPRESSION TO TUMOR PROMOTION. Smad3 is a key transducer of the TGF-beta signal. We previously hypothesized 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 a number of genome-wide analyses in the MCF10-based xenograft model of breast cancer progressioin. In collaboration with Dr. Maxwell Lee (LPG, CCR, NCI), global analysis of gene copy number changes in this model identified the transcription factor Runx1 as a potential novel tumor suppressor gene whose loss may contribute to the switch in TGF-beta response from tumor suppressor to tumor promoter. Validation experiments are ongoing. In addition, we have used genome-wide chromatin immunoprecipitation approaches to identify changes in Smad3 target genes during tumor progression in the model. Our data suggest a progressive reduction in TGF-beta-regulatable Smad3 target genes with cancer progression. Integration of the promoter occupancy data with global gene expression data has yielded a core Smad3-based gene signature that is associated with tumor suppression. This signature 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. 3. CHANGES IN TGF-BETA PATHWAY SIGNALING AND ASSOCIATION WITH CLINICAL PARAMETERS IN LARGE BREAST CANCER COHORTS. To address the relevance of the preclinical findings in human disease, in a collaboration with Dr. Mark Sherman (HREB, DCEG, NCI) we have immunostained a breast cancer tissue microarray from a large cohort of Polish breast cancer patients for various key markers of the TGF-beta pathway. The results in this very large cohort have demonstrated a more complex relationship between TGF-beta pathway activation and breast cancer progression than was previously inferred from the literature. The most striking finding from this study was a strong inverse correlation between type II TGF-beta receptor (TGFBR2) expression and age of onset of breast cancer, with younger breast cancer patients showing much higher levels of TGFBR2 expression and TGF-beta pathway activation as assessed by Smad2 phosphorylation. This difference in TGFBR2 expression may contribute to the known differences in breast cancer biology in young and old patients. Work is ongoing to develop new tools (eg. proximity ligation assays) that will allow us to reliably address more subtle aspects of TGF-beta signaling. These would include assessment of C-terminal vs. linker phosphorylation of Smads and the formation of mixed TGF-beta/BMP Smad complexes. We anticipate that these additional tools will help clarify the relationship between TGF-beta pathway activation and breast cancer progression.
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