TGF-βs play a central regulatory role in maintaining homeostasis and coordinating response to injury in the adult animal. Consequently, dysregulation of TGF-β signaling has been implicated in many pathological states, including cancer. Several TGF-β pathway antagonists are now in early phase clinical oncology trials. However, surprisingly little is known about when and where the TGF-β pathway is activated in the adult animal. To address this need, we have generated a TGF-β pathway reporter mouse in which the expression of eGFP is driven by an artificial enhancer element consisting of 6 repeats of a strong Smad3 binding element from the distal region of the JunB promoter (S3 × 6>GFP reporter). This reporter was >10x more sensitive in vitro than the commonly used CAGA12-based reporter. The reporter construct was knocked into the mouse ROSA26 locus using CRISPR technology and a founder line was derived. Whole body fluorescent imaging of an adult female mouse highlighted TGF-β pathway activation in the gastrointestinal tract, lymph nodes, costal cartilage, brown adipose tissue, brain ventricles and choroid plexi, among other tissues. Since high dose pharmacologic inhibition of the TGF-β pathway has previously been associated with cardiac valvulopathy in preclinical toxicology studies, we immunostained heart sections from the reporter mouse for GFP and observed high endogenous TGF-β pathway activation in the atrioventricular valve leaflets. Quantitative IVIS fluorescent imaging of isolated organs confirmed TGF-β pathway activation in many different tissues in the normal adult mouse, with the pancreas showing the highest level of endogenous activation. Interestingly, the level of TGF-β pathway activation in different tissues was highly correlated with the frequency of inactivating mutations in TGF-β pathway components in tumors from the corresponding tissue in humans. This observation suggests that a high level of TGF-β pathway activation in normal tissues may reflect a non-redundant tumor suppressor role for the TGF-β pathway in maintaining homeostasis in those tissues. To specifically address the activation state of the TGF-β pathway during tumorigenesis, we intercrossed the S3 × 6>GFP reporter mouse with the MMTV-PyVT mouse model of metastatic breast cancer. Reporter activity was strongly upregulated in mammary tumors when compared with the surrounding mammary gland. Using cells cultured from the primary tumors, we confirmed that the reporter signal in the tumor cells can be blocked by small molecule antagonists of the TGF-β pathway. This reporter mouse should be a useful tool to assess the cellular location and extent of TGF-β pathway activation during tumor development, and the impact of TGF-β antagonists on TGF-β signaling in tumors and normal tissues.
Citation Format: Yu-an Yang, Christina Stuelten, Youngjae Bahn, Ji-Hyeon Lee, Madhu Gargesha, Hibret Adissu, Mark Simpson, Caroline S. Hill, Sushil G. Rane, Lalage M. Wakefield. A new TGF-b pathway reporter mouse for analysis of TGF-β signaling in normal homeostasis and cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1645.