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

Abstract

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ 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 TGFβ-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 TβRI (TGFβ receptor 1), were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI 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 TGFβ 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 TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected.

The Effect of cAMP-PKA Activation on TGF-β1-Induced Profibrotic Signaling

“…A recent study suggested that cAMP can promote TGF-β/-Smad3-mediated expression in breast cancer cells by upregulating the expression of the TGF-β receptor TβRI, and a 3 h or 6 h duration of forskolin treatment is sufficient to significantly raise TβRI expression indicating that TβRI levels rose early in response to forskolin [16]. These imply that in MDCK cells, forskolin-promoted TGF-β/ Smad2-mediated expression may also by regulating TβRI expression.…”

“…One study suggested that cAMP can promote TGF-β/-Smad3-mediated expression in breast cancer cells by up-regulating the expression of the TGF-β receptor TβRI [16]. Another study found that cAMP-PKA can inhibit the enhancement of collagen type I mRNA expression caused by TGF-β, which was reversed by pre-treatment with H89 in human peritoneal mesothelial cells (HPMC) [17].…”

Expression of transmembrane protein 26 (TMEM26) in breast cancer and its association with drug response

“…RNA isolation, cDNA synthesis and quantitative (Q) PCR were performed as described [48]. The RNA isolation kit was purchased from Roche and the dNTP mix was from Qiagen.…”

“…Protein extractions and Western blot analysis were carried out as described [48]. Briefly, adherent cells were scraped off, pelleted and resuspended in 400 μl buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA).…”

Mesenchymal Stem Cells Relevance in Multicellular Bioengineered 3D In Vitro Tumor Models

“…Several subsequent studies, using scaffold‐based technologies for 3D models assembly provide evidence that BM‐MSCs exert a positive effect in cancer cell survival and migratory capacity. In fact, cells co‐cultured in tissue mimetic scaffolds including those based on: i) collagen hydrogels; ii) silk‐fibroin scaffolds; or iii) porous chitosan scaffolds containing hydroxyapatite organotypic as bone mimetics laden with undifferentiated or differentiated MSCs, evidence their capability to up‐regulate the expression of EMT‐related genes in cancer cells, to induce higher migratory capacity (Figure ), and to enhance proliferation through different signaling factors.…”