Abstract B029: Dissecting out the tumor suppressor aspect of TGF-β in breast cancer using integrated genomics

The purpose of our study was to identify a gene expression signature that specifically reflects the tumor suppressive effects of transforming growth factor-β (TGF-β), for use as a negative selective biomarker in clinical trials using TGF-β antagonists. TGF-β effects are highly contextual, and the dogma is that tumor suppressive effects are active in the early stages of carcinogenesis, but that pro-progression effects come to dominate later on. Since TGF-β antagonists are in early phase clinical trials in cancer, it is important to know whether the tumor suppressive effects of TGF-β are still intact in any tumors at the time of diagnosis and treatment, as this would be a contraindication for anti-TGF-β therapy. Existing TGF-β-related gene expression signatures were not designed a priori to discriminate the tumor suppressive from the tumor promoting activities. To address this question, we applied integrated ChIP-chip and transcriptomic approaches in the MCF10A-based model of breast cancer progression. We have previously shown that TGF-β has tumor suppressor activity in the less malignant cell lines of the series (MCF10AT1k, MCF10Ca1h), but that this effect is lost in the most malignant cell line (MCF10Ca1a). Here we showed that the tumor suppressor activity in this model system is specifically dependent on the downstream signaling component Smad3 and not Smad2. Using promoter-wide ChIP-chip, we found that the genomic landscape of TGF-β induced Smad3 binding differed dramatically between the four cell lines, despite their close genetic relatedness and similar Smad3 levels and activation profiles. Interestingly, TGF-β induced Smad3 binding only at loci that were already transcriptionally active, suggesting that TGF-βs may primarily play a modulator rather than an instigator role in regulating transcription. This feature probably contributes importantly to the known contextuality of TGF-β activity. By focusing on the two malignant cell lines (MCF10CA1h and MCF10CA1a), we identified a core signature of 26 TGF-β/Smad3 regulated genes that was specifically associated with the tumor suppressor activity of TGF-β. Unexpectedly, the direction of regulation of 25% of these genes by TGF-β differed in vitro and in vivo, highlighting a further novel contribution to TGF-β contextuality. The in vivo weighted form of the TGF-β/Smad3 tumor suppressor signature (TSTSS) was associated with good outcome specifically in estrogen-receptor positive (ER+) breast cancer patients, suggesting that TGF-β tumor suppressive pathways are still active and influencing disease outcome in a subset of patients at the time of surgery. TGF-β is a potent growth inhibitor for most epithelial cells and the TSTSS was weakly inversely correlated with proliferation in ER+ breast cancer, but the signature prognosticated independently of proliferation in multivariate analysis. Instead, the TSTSS was enriched for genes involved in cellular differentiation and movement, and ephrin was the top enriched signaling pathway. Ephrin knockdown in MCF10Ca1h cells led to increased migration, reduced apoptosis and differentiation and enhanced tumorigenesis. Our demonstration that a subset of breast cancer patients still has an active TGF-β tumor suppressor pathway at the time of surgery has important implications for patient stratification in ongoing clinical trials with TGF-β antagonists. Citation Format: Misako Sato, Mitsutaka Kadota, Binwu Tang, Yu-an Yang, Mengge Shan, Jia Weng, Michael Welsh, Yoshiko Nagano, Aleksandra Michalowski, Howard Yang, Robert Clifford, Maxwell Lee, Lalage Wakefield. Dissecting out the tumor suppressor aspect of TGF-β in breast cancer using integrated genomics. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B029.