Cyclic AMP Enhances TGFb Responses of Breast Cancer Cells by Upregulating TGFb Receptor I Expression

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFb and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFb-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein) and TbRI (TGFb receptor 1), were responsive to cAMP. While YAP had little effect on TGFb-dependent expression and Smad3 phosphorylation, a constitutively active form of TbRI mimicked the cAMP effect on TGFb signaling. In 3D-cultured cells, which show much higher levels of TbRI and cAMP, TbRI was unresponsive to cAMP. Upregulation of TbRI expression by cAMP was dependent on transcription. A proximal TbRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TbRI expression at least partially by activating TbRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TbRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TbRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFb on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFb pathways. In summary, these data suggest that combined effects of cAMP and TGFb, as e.g. induced by mesenchymal stem cells, involve the upregulation of TbRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected. Citation: Oerlecke I, Bauer E, Dittmer A, Leyh B, Dittmer J (2013) Cyclic AMP Enhances TGFb Responses of Breast Cancer Cells by Upregulating TGFb Receptor I Expression. PLoS ONE 8(1): e54261. doi:10.1371/journal.pone.0054261 Editor: Aamir Ahmad, Wayne State University School of Medicine, United States of America Received September 14, 2012; Accepted December 10, 2012; Published January 18, 2013 Copyright: 2013 Oerlecke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by 1. Deutsche Krebshilfe grant no. 109271 (external funding)http://www.krebshilfe.de/wir-foerdern.html),and 2. Land Sachsen-Anhalt (Wilhelm-Roux-funding) grant FKZ VF1/A8 (internal funding of the Medical Faculty of the University of Halle, no URL available, because the Wilhelm-Roux-Funding-Program is not accessible for the public outside the University). The salary for Ilka Oerlecke was payed by the University grant FKZ VF1/A8. The salary for Benjamin Leyh was payed by the Deutsche Krebshilfe grant 109271. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: juergen.dittmer@medizin.uni-halle.de