Live tumor imaging shows macrophage induction and TMEM-mediated enrichment of cancer stem cells during metastatic dissemination.

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites.

Aging and CNS Myeloid Cell Depletion Attenuate Breast Cancer Brain Metastasis.

PURPOSE: Breast cancer diagnosed in young patients is often aggressive. Because primary breast tumors from young and older patients have similar mutational patterns, we hypothesized that the young host microenvironment promotes more aggressive metastatic disease. EXPERIMENTAL DESIGN: Triple-negative or luminal B breast cancer cell lines were injected into young and older mice side-by-side to quantify lung, liver, and brain metastases. Young and older mouse brains, metastatic and naïve, were analyzed by flow cytometry. Immune populations were depleted using antibodies or a colony-stimulating factor-1 receptor (CSF-1R) inhibitor, and brain metastasis assays were conducted. Effects on myeloid populations, astrogliosis, and the neuroinflammatory response were determined. RESULTS: Brain metastases were 2- to 4-fold higher in young as compared with older mouse hosts in four models of triple-negative or luminal B breast cancer; no age effect was observed on liver or lung metastases. Aged brains, naïve or metastatic, contained fewer resident CNS myeloid cells. Use of a CSF-1R inhibitor to deplete myeloid cells, including both microglia and infiltrating macrophages, preferentially reduced brain metastasis burden in young mice. Downstream effects of CSF-1R inhibition in young mice resembled that of an aged brain in terms of myeloid numbers, induction of astrogliosis, and Semaphorin 3A secretion within the neuroinflammatory response. CONCLUSIONS: Host microenvironmental factors contribute to the aggressiveness of triple-negative and luminal B breast cancer brain metastasis. CSF-1R inhibitors may hold promise for young brain metastasis patients.

Peptidylarginine Deiminase IV Regulates Breast Cancer Stem Cells via a Novel Tumor Cell-Autonomous Suppressor Role.

Peptidylarginine deiminases (PADI) catalyze posttranslational modification of many target proteins and have been suggested to play a role in carcinogenesis. Citrullination of histones by PADI4 was recently implicated in regulating embryonic stem and hematopoietic progenitor cells. Here, we investigated a possible role for PADI4 in regulating breast cancer stem cells. PADI4 activity limited the number of cancer stem cells (CSC) in multiple breast cancer models in vitro and in vivo. Mechanistically, PADI4 inhibition resulted in a widespread redistribution of histone H3, with increased accumulation around transcriptional start sites. Interestingly, epigenetic effects of PADI4 on the bulk tumor cell population did not explain the CSC phenotype. However, in sorted tumor cell populations, PADI4 downregulated expression of master transcription factors of stemness, NANOG and OCT4, specifically in the cancer stem cell compartment, by reducing the transcriptionally activating H3R17me2a histone mark at those loci; this effect was not seen in the non-stem cells. A gene signature reflecting tumor cell-autonomous PADI4 inhibition was associated with poor outcome in human breast cancer datasets, consistent with a tumor-suppressive role for PADI4 in estrogen receptor-positive tumors. These results contrast with known tumor-promoting effects of PADI4 on the tumor stroma and suggest that the balance between opposing tumor cell-autonomous and stromal effects may determine net outcome. Our findings reveal a novel role for PADI4 as a tumor suppressor in regulating breast cancer stem cells and provide insight into context-specific effects of PADI4 in epigenetic modulation. SIGNIFICANCE: These findings demonstrate a novel activity of the citrullinating enzyme PADI4 in suppressing breast cancer stem cells through epigenetic repression of stemness master transcription factors NANOG and OCT4.

The Outcome of TGFß Antagonism in Metastatic Breast Cancer Models In Vivo Reflects a Complex Balance between Tumor-Suppressive and Proprogression Activities of TGFß.

PURPOSE: TGFßs are overexpressed in many advanced cancers and promote cancer progression through mechanisms that include suppression of immunosurveillance. Multiple strategies to antagonize the TGFß pathway are in early-phase oncology trials. However, TGFßs also have tumor-suppressive activities early in tumorigenesis, and the extent to which these might be retained in advanced disease has not been fully explored. EXPERIMENTAL DESIGN: A panel of 12 immunocompetent mouse allograft models of metastatic breast cancer was tested for the effect of neutralizing anti-TGFß antibodies on lung metastatic burden. Extensive correlative biology analyses were performed to assess potential predictive biomarkers and probe underlying mechanisms. RESULTS: Heterogeneous responses to anti-TGFß treatment were observed, with 5 of 12 models (42%) showing suppression of metastasis, 4 of 12 (33%) showing no response, and 3 of 12 (25%) showing an undesirable stimulation (up to 9-fold) of metastasis. Inhibition of metastasis was immune-dependent, whereas stimulation of metastasis was immune-independent and targeted the tumor cell compartment, potentially affecting the cancer stem cell. Thus, the integrated outcome of TGFß antagonism depends on a complex balance between enhancing effective antitumor immunity and disrupting persistent tumor-suppressive effects of TGFß on the tumor cell. Applying transcriptomic signatures derived from treatment-naïve mouse primary tumors to human breast cancer datasets suggested that patients with breast cancer with high-grade, estrogen receptor-negative disease are most likely to benefit from anti-TGFß therapy. CONCLUSIONS: Contrary to dogma, tumor-suppressive responses to TGFß are retained in some advanced metastatic tumors. Safe deployment of TGFß antagonists in the clinic will require good predictive biomarkers.

SOD2 acetylation on lysine 68 promotes stem cell reprogramming in breast cancer.

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.

Epigenetic re-wiring of breast cancer by pharmacological targeting of C-terminal binding protein.

The C-terminal binding protein (CtBP) is an NADH-dependent dimeric family of nuclear proteins that scaffold interactions between transcriptional regulators and chromatin-modifying complexes. Its association with poor survival in several cancers implicates CtBP as a promising target for pharmacological intervention. We employed computer-assisted drug design to search for CtBP inhibitors, using quantitative structure-activity relationship (QSAR) modeling and docking. Functional screening of these drugs identified 4 compounds with low toxicity and high water solubility. Micro molar concentrations of these CtBP inhibitors produces significant de-repression of epigenetically silenced pro-epithelial genes, preferentially in the triple-negative breast cancer cell line MDA-MB-231. This epigenetic reprogramming occurs through eviction of CtBP from gene promoters; disrupted recruitment of chromatin-modifying protein complexes containing LSD1, and HDAC1; and re-wiring of activating histone marks at targeted genes. In functional assays, CtBP inhibition disrupts CtBP dimerization, decreases cell migration, abolishes cellular invasion, and improves DNA repair. Combinatorial use of CtBP inhibitors with the LSD1 inhibitor pargyline has synergistic influence. Finally, integrated correlation of gene expression in breast cancer patients with nuclear levels of CtBP1 and LSD1, reveals new potential therapeutic vulnerabilities. These findings implicate a broad role for this class of compounds in strategies for epigenetically targeted therapeutic intervention.

The transcription factor CBFB suppresses breast cancer through orchestrating translation and transcription.

Translation and transcription are frequently dysregulated in cancer. These two processes are generally regulated by distinct sets of factors. The CBFB gene, which encodes a transcription factor, has recently emerged as a highly mutated driver in a variety of human cancers including breast cancer. Here we report a noncanonical role of CBFB in translation regulation. RNA immunoprecipitation followed by deep sequencing (RIP-seq) reveals that cytoplasmic CBFB binds to hundreds of transcripts and regulates their translation. CBFB binds to mRNAs via hnRNPK and enhances translation through eIF4B, a general translation initiation factor. Interestingly, the RUNX1 mRNA, which encodes the transcriptional partner of CBFB, is bound and translationally regulated by CBFB. Furthermore, nuclear CBFB/RUNX1 complex transcriptionally represses the oncogenic NOTCH signaling pathway in breast cancer. Thus, our data reveal an unexpected function of CBFB in translation regulation and propose that breast cancer cells evade translation and transcription surveillance simultaneously through downregulating CBFB.

Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3.

Thousands of long noncoding RNAs (lncRNAs) have been discovered, yet the function of the vast majority remains unclear. Here, we show that a p53-regulated lncRNA which we named PINCR (p53-induced noncoding RNA), is induced ~100-fold after DNA damage and exerts a prosurvival function in human colorectal cancer cells (CRC) in vitro and tumor growth in vivo. Targeted deletion of PINCR in CRC cells significantly impaired G1 arrest and induced hypersensitivity to chemotherapeutic drugs. PINCR regulates the induction of a subset of p53 targets involved in G1 arrest and apoptosis, including BTG2, RRM2B and GPX1. Using a novel RNA pulldown approach that utilized endogenous S1-tagged PINCR, we show that PINCR associates with the enhancer region of these genes by binding to RNA-binding protein Matrin 3 that, in turn, associates with p53. Our findings uncover a critical prosurvival function of a p53/PINCR/Matrin 3 axis in response to DNA damage in CRC cells.

Immunocompetent mouse allograft models for development of therapies to target breast cancer metastasis.

Effective drug development to combat metastatic disease in breast cancer would be aided by the availability of well-characterized preclinical animal models that (a) metastasize with high efficiency, (b) metastasize in a reasonable time-frame, (c) have an intact immune system, and (d) capture some of the heterogeneity of the human disease. To address these issues, we have assembled a panel of twelve mouse mammary cancer cell lines that can metastasize efficiently on implantation into syngeneic immunocompetent hosts. Genomic characterization shows that more than half of the 30 most commonly mutated genes in human breast cancer are represented within the panel. Transcriptomically, most of the models fall into the luminal A or B intrinsic molecular subtypes, despite the predominance of an aggressive, poorly-differentiated or spindled histopathology in all models. Patterns of immune cell infiltration, proliferation rates, apoptosis and angiogenesis differed significantly among models. Inherent within-model variability of the metastatic phenotype mandates large cohort sizes for intervention studies but may also capture some relevant non-genetic sources of variability. The varied molecular and phenotypic characteristics of this expanded panel of models should aid in model selection for development of antimetastatic therapies in vivo, and serve as a useful platform for predictive biomarker identification.

Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2.

Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. Conclusions: The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.

Quantitation of TGF-ß proteins in mouse tissues shows reciprocal changes in TGF-ß1 and TGF-ß3 in normal vs neoplastic mammary epithelium.

Transforming growth factor-ßs (TGF-ßs) regulate tissue homeostasis, and their expression is perturbed in many diseases. The three isoforms (TGF-ß1, -ß2, and -ß3) have similar bioactivities in vitro but show distinct activities in vivo. Little quantitative information exists for expression of TGF-ß isoform proteins in physiology or disease. We developed an optimized method to quantitate protein levels of the three isoforms, using a Luminex® xMAP®-based multianalyte assay following acid-ethanol extraction of tissues. Analysis of multiple tissues and plasma from four strains of adult mice showed that TGF-ß1 is the predominant isoform with TGF-ß2 being ~10-fold lower. There were no sex-specific differences in isoform expression, but some tissues showed inter-strain variation, particularly for TGF-ß2. The only adult tissue expressing appreciable TGF-ß3 was the mammary gland, where its levels were comparable to TGF-ß1. In situ hybridization showed the luminal epithelium as the major source of all TGF-ß isoforms in the normal mammary gland. TGF-ß1 protein was 3-8-fold higher in three murine mammary tumor models than in normal mammary gland, while TGF-ß3 protein was 2-3-fold lower in tumors than normal tissue, suggesting reciprocal regulation of these isoforms in mammary tumorigenesis.

Immune-mediated pathology in Duchenne muscular dystrophy.

Immunological and inflammatory processes downstream of dystrophin deficiency as well as metabolic abnormalities, defective autophagy, and loss of regenerative capacity all contribute to muscle pathology in Duchenne muscular dystrophy (DMD). These downstream cascades offer potential avenues for pharmacological intervention. Modulating the inflammatory response and inducing immunological tolerance to de novo dystrophin expression will be critical to the success of dystrophin-replacement therapies. This Review focuses on the role of the inflammatory response in DMD pathogenesis and opportunities for clinical intervention.

Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics.

There is an emerging demand for the use of molecular profiling to facilitate biomarker identification and development, and to stratify patients for more efficient treatment decisions with reduced adverse effects. In the past decade, great strides have been made to advance genomic, transcriptomic and proteomic approaches to address these demands. While there has been much progress with these large scale approaches, profiling at the protein level still faces challenges due to limitations in clinical sample size, poor reproducibility, unreliable quantitation, and lack of assay robustness. A novel automated capillary nano-immunoassay (CNIA) technology has been developed. This technology offers precise and accurate measurement of proteins and their post-translational modifications using either charge-based or size-based separation formats. The system not only uses ultralow nanogram levels of protein but also allows multi-analyte analysis using a parallel single-analyte format for increased sensitivity and specificity. The high sensitivity and excellent reproducibility of this technology make it particularly powerful for analysis of clinical samples. Furthermore, the system can distinguish and detect specific protein post-translational modifications that conventional Western blot and other immunoassays cannot easily capture. This review will summarize and evaluate the latest progress to optimize the CNIA system for comprehensive, quantitative protein and signaling event characterization. It will also discuss how the technology has been successfully applied in both discovery research and clinical studies, for signaling pathway dissection, proteomic biomarker assessment, targeted treatment evaluation and quantitative proteomic analysis. Lastly, a comparison of this novel system with other conventional immuno-assay platforms is performed.

Differential Proteome Analysis Identifies TGF-ß-Related Pro-Metastatic Proteins in a 4T1 Murine Breast Cancer Model.

Transforming growth factor-ß (TGF-ß) has a dual role in tumorigenesis, acting as either a tumor suppressor or as a pro-oncogenic factor in a context-dependent manner. Although TGF-ß antagonists have been proposed as anti-metastatic therapies for patients with advanced stage cancer, how TGF-ß mediates metastasis-promoting effects is poorly understood. Establishment of TGF-ß-related protein expression signatures at the metastatic site could provide new mechanistic information and potentially allow identification of novel biomarkers for clinical intervention to discriminate TGF-ß oncogenic effects from tumor suppressive effects. In the present study, we found that systemic administration of the TGF-ß receptor kinase inhibitor, SB-431542, significantly inhibited lung metastasis from transplanted 4T1 mammary tumors in Balb/c mice. The differentially expressed proteins in the comparison of lung metastases from SB-431542 treated and control vehicle-treated groups were analyzed by a quantitative LTQ Orbitrap Velos system coupled with stable isotope dimethyl labeling. A total of 36,239 peptides from 6,694 proteins were identified, out of which 4,531 proteins were characterized as differentially expressed. A subset of upregulated proteins in the control group was validated by western blotting and immunohistochemistry. The eukaryotic initiation factor (eIF) family members constituted the most enriched protein pathway in vehicle-treated compared with SB-43512-treated lung metastases, suggesting that increased protein expression of specific eIF family members, especially eIF4A1 and eEF2, is related to the metastatic phenotype of advanced breast cancer and can be down-regulated by TGF-ß pathway inhibitors. Thus our proteomic approach identified eIF pathway proteins as novel potential mediators of TGF-ß tumor-promoting activity.

A flexible reporter system for direct observation and isolation of cancer stem cells.

Many tumors are hierarchically organized with a minority cell population that has stem-like properties and enhanced ability to initiate tumorigenesis and drive therapeutic relapse. These cancer stem cells (CSCs) are typically identified by complex combinations of cell-surface markers that differ among tumor types. Here, we developed a flexible lentiviral-based reporter system that allows direct visualization of CSCs based on functional properties. The reporter responds to the core stem cell transcription factors OCT4 and SOX2, with further selectivity and kinetic resolution coming from use of a proteasome-targeting degron. Cancer cells marked by this reporter have the expected properties of self-renewal, generation of heterogeneous offspring, high tumor- and metastasis-initiating activity, and resistance to chemotherapeutics. With this approach, the spatial distribution of CSCs can be assessed in settings that retain microenvironmental and structural cues, and CSC plasticity and response to therapeutics can be monitored in real time.

Definition of smad3 phosphorylation events that affect malignant and metastatic behaviors in breast cancer cells.

Smad3, a major intracellular mediator of TGFß signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFß, the TGFß receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFß-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFß responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer.

Selective targeting of KRAS-mutant cells by miR-126 through repression of multiple genes essential for the survival of KRAS-mutant cells.

MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors.

Brightfield proximity ligation assay reveals both canonical and mixed transforming growth factor-ß/bone morphogenetic protein Smad signaling complexes in tissue sections.

Transforming growth factor-ß (TGF-ß) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-ß signaling occurs through Smad2/3-Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-ß may activate a novel mixed Smad complex (Smad2/3-Smad1/5/9), which is required for some of the pro-oncogenic activities of TGF-ß. To determine if mixed Smad complexes are evident in vivo, we developed antibodies that can be used with a proximity ligation assay to detect either canonical or mixed Smad complexes in formalin-fixed paraffin-embedded sections. We demonstrate high expression of mixed Smad complexes in the tissues from mice genetically engineered to express high levels of TGF-ß1. Mixed Smad complexes were also prominent in 15-16 day gestation mouse embryos and in breast cancer xenografts, suggesting important roles in embryonic development and tumorigenesis. In contrast, mixed Smad complexes were expressed at extremely low levels in normal adult mouse tissue, where canonical complexes were correspondingly higher. We show that this methodology can be used in archival patient samples and tissue microarrays, and we have developed an algorithm to quantitate the brightfield read-out. These methods will allow quantitative analysis of cell type-specific Smad signaling pathways in physiological and pathological processes.

Synergistic antitumor effects of a TGFß inhibitor and cyclophosphamide.

In a mouse model of breast carcinoma, the combination of cyclophosphamide and transforming growth factor ß1,2,3 (TGFß1,2,3)-targeting antibody achieved superior antineoplastic effects. This novel paradigm of synergistic chemoimmunotherapy promises to improve the clinical outcome of cancer patients with micrometastases, and thus deserves further investigation.

An integrated genomic approach identifies persistent tumor suppressive effects of transforming growth factor-ß in human breast cancer.

INTRODUCTION: Transforming growth factor-ßs (TGF-ßs) play a dual role in breast cancer, with context-dependent tumor-suppressive or pro-oncogenic effects. TGF-ß antagonists are showing promise in early-phase clinical oncology trials to neutralize the pro-oncogenic effects. However, there is currently no way to determine whether the tumor-suppressive effects of TGF-ß are still active in human breast tumors at the time of surgery and treatment, a situation that could lead to adverse therapeutic responses. METHODS: Using a breast cancer progression model that exemplifies the dual role of TGF-ß, promoter-wide chromatin immunoprecipitation and transcriptomic approaches were applied to identify a core set of TGF-ß-regulated genes that specifically reflect only the tumor-suppressor arm of the pathway. The clinical significance of this signature and the underlying biology were investigated using bioinformatic analyses in clinical breast cancer datasets, and knockdown validation approaches in tumor xenografts. RESULTS: TGF-ß-driven tumor suppression was highly dependent on Smad3, and Smad3 target genes that were specifically enriched for involvement in tumor suppression were identified. Patterns of Smad3 binding reflected the preexisting active chromatin landscape, and target genes were frequently regulated in opposite directions in vitro and in vivo, highlighting the strong contextuality of TGF-ß action. An in vivo-weighted TGF-ß/Smad3 tumor-suppressor signature was associated with good outcome in estrogen receptor-positive breast cancer cohorts. TGF-ß/Smad3 effects on cell proliferation, differentiation and ephrin signaling contributed to the observed tumor suppression. CONCLUSIONS: Tumor-suppressive effects of TGF-ß persist in some breast cancer patients at the time of surgery and affect clinical outcome. Carefully tailored in vitro/in vivo genomic approaches can identify such patients for exclusion from treatment with TGF-ß antagonists.