In FY13, 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, this year we have also established a number of new breast cancer cell strains from freshly excised patient tumors to extend our findings to primary cells. Our work in FY13 has focused in two main areas: 1. THE ROLE OF TGF-BETA IN REGULATING CANCER STEM CELL DYNAMICS. During FY13 we have continued to validate and employ a novel functional imaging approach for the identification of the putative cancer stem cell (CSC) population. Our CSC reporter responds to the presence of the stem cell master transcription factors Oct4 and Sox2, and marks cells that are enriched for CSC activities, including the ability to divide asymmetrically, to resist cell killing by conventional chemotherapeutics, and to initiate tumorigenesis and metastasis in vivo. We confirmed that the approach works in primary human breast cancer cells as well as in established cell lines, and we have made a number of advances in the lentiviral vector design to increase sensitivity. We are now exploiting this reporter using a variety of approaches including timelapse fluorescence microscopy in vitro and confocal microscopy on tumors ex vivo to address factors that regulate CSC localization, plasticity and behavior, and to screen for therapeutics that target the CSC population. A major focus is the role of TGF-beta superfamily members in CSC biology. Using a xenograft breast cancer model in which TGF-beta functions as a tumor suppressor, we have combined our stem cell reporter with a TGF-beta pathway reporter and shown that endogenous TGF-beta signaling is activated more highly in the CSCs compared with the bulk population, suggesting a particularly important role for TGF-betas in the stem cell compartment. CSCs with endogenous TGF-beta signaling active were intrinsically less proliferative than CSCs that had not activated the pathway. Furthermore, we showed that TGF-beta selectively inhibited asymmetric self-renewing mitoses in the CSC compartment, and that it specifically inhibited the invasion of CSCs through basement membrane, while having little effect on the invasion of the bulk tumor cell population. These observations show that TGF-beta is an important modulator of CSC biology, and identify new mechanisms that could underlie the tumor suppressive effects of TGF-beta. To extend our imaging approach from transplanted to spontaneously arising tumors, we have successfully generated transgenic mice expressing the stem cell reporter. 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. INSIGHTS INTO TGF-BETA-MEDIATED EFFECTS ON TUMORIGENESIS FROM GENOMIC APPROACHES. TGF-beta antagonists are being developed as cancer therapeutics. However, the complex role of TGF-beta in cancer progression makes it imperative to avoid treating patients whose tumors still have intact tumor suppressive responses to TGF-beta. Currently it is not clear whether the tumor suppressive responses to TGF-beta are still retained by any breast cancers at the time of diagnosis and surgery. To address this question, we chose to develop a TGF-beta response signature that specifically reflects the tumor suppressive effects, since published TGF-beta signatures were not designed a priori to distinguish the tumor suppressive responses from the neutral or tumor promoting responses. Using the MCF10-based model of human breast cancer progression, we applied integrated genome-wide chromatin immunoprecipitation and transcriptomic approaches in vitro and in vivo to specifically dissect out a core gene signature that is associated with TGF-beta/Smad3-mediated tumor suppression. In a meta-analysis of more than 1300 human breast cancers, high expression of this signature associated with good distant metastasis-free survival in women with estrogen receptor positive (ER+) breast cancer, suggesting that the tumor suppressor effects of TGF-beta are still active and affecting disease outcome in a subset of patients. We have continued to probe the underlying biological and molecular mechanisms, and we demonstrated that TGF-beta-induced inhibition of cell proliferation and induction of cellular differentiation both contribute to tumor suppression. We have further identified a functional role for Ephrin signaling in mediating the tumor suppressive effects of TGF-beta. However, we have shown that the TGF-beta-regulated transcriptome is highly context-dependent, since the downstream transcriptional mediator, Smad3, appears only to bind into regions of chromatin that are already transcriptionally active. As a result, we believe that it will be necessary to build tailored TGF-beta signatures for different tumor types, and that there will be no single signature that will serve as a biomarker of TGF-beta tumor suppression in all cancer types. This finding has important implications for the development of signature-based biomarkers to use for patient inclusion/exclusion in clinical oncology trials with TGF-beta antagonists. We are also addressing effects of TGF-beta on the miRome in the same MCF-10-based model system to build an integrated molecular mechanistic picture of the changing role of TGF-beta in breast cancer progression. To this end, we have performed Next-Gen sequencing of the basal and TGF-beta-regulated miRomes of four of the MCF10-derived cell lines representing normal, premalignant, low-grade malignant and high-grade malignant stages in breast cancer progression. Analysis of these datasets is ongoing.
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