Abstract 3123: The Notch pathway inhibits TGF-β signaling in breast cancer through HEYL-mediated crosstalk

Acquired resistance to Transforming growth factor-β (TGF-β) is a key step in the early stages of tumorigenesis. Mutations of TGF-β signaling components, often found in other cancers, are rare in breast cancer, and little is known about the development of this resistance in breast cancer. On the other hand, activation of Notch pathway is known to play a substantial role in promoting breast cancer development. We hypothesized that crosstalk between these two pathways occurs through HEYL, a basic helix-loop-helix (bHLH) transcription factor, and a known direct target of Notch. We found that HEYL is overexpressed in 40% of breast cancers. Expression of Notch increased HEYL expression while knockdown of RBP-J, a critical mediator of Notch, reduced HEYL expression in HS578T and MDAMB-231 breast cancer cells. Taking into account the contradictory biological effects of Notch and TGF-β signaling, we sought to examine whether HEYL inhibits the TGF-β pathway. TGF-β treatment or Smad3 overexpression significantly increased the luciferase activity of the TGF-β responsive reporter vector, p3TP-Luc, and the P15 (CDKN2B) gene promoter, but this transactivation was strongly inhibited by HEYL. Bimolecular fluorescence complementation assays confirmed the interaction between Smad 3 and HEYL; immunoprecipitation assays with deletion constructs showed that the Basic domain of HEYL interacts with the MH2 domain of Smad3, and that their interaction is necessary for HEYL to inhibit TGF-β signaling. In addition, using a tet-off inducible breast cancer cell model, HEYL was shown to transcriptionally up-regulate many genes that drive metastasis and tumor angiogenesis. Supporting this concept, HEYL/HER2 double transgenic mice showed nearly 50% increase in lung metastasis (20% vs 46%) compared to HER2/neu mice. Similarly, lung colonization occurred in 100% of mice injected with MDAMB231 while HEYL-shRNAs reduced incidence to 50%. Therefore, HEYL promotes breast cancer growth and metastasis in Smad-dependent and Smad-independent mechanisms. Citation Format: Han Liangfeng, Adam Diehl, Nguyen K. Nguyen, Preethi Korangath, Teo Weiwen, Zhe Zhang, Scott Kominsky, Pedram Argani, Goran Landberg, Saraswati V. Sukumar. The Notch pathway inhibits TGF-β signaling in breast cancer through HEYL-mediated crosstalk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3123. doi:10.1158/1538-7445.AM2013-3123