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Cyclic AMP enhances TGF? responses of breast cancer cells by upregulating TGF? receptor I expression.

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

SUBMITTER: Oerlecke I

PROVIDER: S-EPMC3548810 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Cyclic AMP enhances TGFβ responses of breast cancer cells by upregulating TGFβ receptor I expression.

Oerlecke Ilka I Bauer Elke E Dittmer Angela A Leyh Benjamin B Dittmer Jürgen J

PloS one 20130118 1

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 p ...[more]

PMID: 23349840

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Project description:IntroductionBasal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells.MethodsCD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion.ResultsExpression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen.ConclusionHA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.

Dataset Information

Cyclic AMP enhances TGF? responses of breast cancer cells by upregulating TGF? receptor I expression.

Publications

Cyclic AMP enhances TGFβ responses of breast cancer cells by upregulating TGFβ receptor I expression.

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