Abstract B13: Targeting the TGF-β pathway in breast cancer: Insights from preclinical studies
Authors: Yang Yu-an , Flanders Kathleen , Chen Jin-qui , Merchant Anand S. , Yang Howard , Lee Maxwell P. , Wakefield Lalage M. 
Published: May 2016

Abstract
Overexpression of transforming growth factor-βs (TGF-βs) correlates with metastasis and poor prognosis in many advanced cancers, and TGF-βs have pro-oncogenic effects on nearly every cell type in the ecosystem of advanced tumors. Based on these observations and encouraging results in preclinical models, strategies to block TGF-β signaling are in early phase clinical oncology trials. To date however, preclinical studies supporting the development of anti-TGF-β therapeutics in cancer have focused around a few well-characterized mouse models which do not capture the heterogeneity of the human disease. To address this issue for breast cancer, we have assembled a panel of transplantable mouse models of metastatic mammary cancer that can be used in fully immunocompetent hosts, so as to preserve any immune contributions to therapeutic efficacy. Hormone receptor status, histopathological characteristics and intrinsic subtype of the models were assessed. Using this panel of models, we tested the efficacy of a pan-TGF-β neutralizing antibody (1D11) in inhibiting lung metastasis. We observed therapeutic efficacy or trend to efficacy in 5 models (InhibMet), no effect in 2 models (NoEff) and an undesirable stimulation or trend to stimulation of metastasis in 4 models (StimMet). This heterogeneity in therapeutic responses suggests that it will be critical to develop good predictive biomarkers for patient selection in clinical trials using TGF-β antagonists. Since the model panel provides an excellent platform for biomarker discovery, we have performed transcriptomics on untreated primary tumors across the panel, as well as completing full exome gDNA sequencing and copy number variant analysis on the tumor cell lines. The relationship between response-to-therapy and mutation load will be presented. Analyses of the untreated primary tumors show that the tumor transcriptomes segregate by response to anti-TGF-β therapy in principal component analysis, after removal of mouse strain as a confounding factor. Encouragingly for biomarker development, this observation suggests that there are major pre-existing differences in the biology of InhibMet and StimMet primary tumors before they are treated. A gene signature generated from the differentially expressed gene list was strongly associated with outcome in a metaanalysis of human breast cancer datasets, suggesting human relevance. Ingenuity Pathway Analysis of differentially-expressed genes indicates that InhibMet models are characterized by higher TGF-β pathway activation, higher angiogenesis, poor immune cell infiltration/activation, and other markers of tumor aggressiveness such as higher tumor cell proliferation and survival. Interestingly, the higher TGF-β pathway activation that was strongly predicted in the InhibMet tumors by the transcriptomic analyses (p=5.8e-25) was not evident from a multipronged quantitative proteomics assessment of activation of canonical Smad signaling, or non-canonical signaling through Akt, Erk, Jnk or p38MAPK pathways. Thus steady-state signaling by TGF-β in tumors in vivo may represent the integrated sum of the activities of more downstream signaling pathways than were captured in this analysis, or additional information about the intracellular localization of the phospho-forms of the signaling proteins and/or the identity of interacting proteins may be necessary to fully assess the activation state of the pathway. Despite these caveats, the data suggest that TGF-β antagonists will have therapeutic efficacy in more aggressive, poor prognosis breast cancers, and suggest that it will be possible to generate molecular signatures that predict the therapeutic response.
Citation Format: Yu-an Yang, Kathleen Flanders, Jin-qui Chen, Anand S. Merchant, Howard Yang, Maxwell P. Lee, Lalage M. Wakefield. Targeting the TGF-β pathway in breast cancer: Insights from preclinical studies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B13.