Y-box Binding Protein-1 Enhances Oncogenic Transforming Growth Factor ß Signaling in Breast Cancer Cells via Triggering Phospho-Activation of Smad2.

BACKGROUND/AIM: Transforming growth factor ß (TGFß) plays a role in diverse oncogenic pathways including cell proliferation and cell motility and is regulated by the pleiotropic factor Y-box binding protein-1 (YB-1). In breast cancer, Sma/Mad related protein 2 (Smad2) represents the most common downstream transducer in TGFß signaling. MATERIALS AND METHODS: Here, YB-1's impact on Smad2 phospho-activation was characterized by incubation of the breast cancer cell line MCF-7 with or without TGFß1 in the absence or presence of overexpressed YB-1 protein. The phospho-status of Smad2 was assessed via western blotting. RESULTS: Analysis of MCF-7 cells revealed no induction of total Smad2 neither in the presence of TGFß1, nor during YB-1 overexpression. In contrast, incubation with TGFß1 led to an increase of phosphorylated Smad2 forms which was significantly amplified by simultaneously overexpressed YB-1 (2.8±0.2-fold). CONCLUSION: Oncogenic YB-1 indirectly enhances TGFß signaling cascades via Smad2 phospho-activation and may represent a promising factor for future diagnosis and therapy of breast cancer.

Antiestrogens suppress effects of transforming growth factor-ß in breast cancer cells via the signaling axis estrogen receptor-α and Y-box Binding Protein-1.

BACKGROUND: Multifunctional Y-box Binding Protein-1 (YB1) is correlated with a poor outcome in breast cancer. We found YB1 expression to be regulated by antiestrogens commonly used in the hormonal therapy of breast cancer and known as activators of Transforming Growth Factor-ß (TGFß). Thus, a putative influence of YB1 on TGFß signaling should be investigated. MATERIALS AND METHODS: The effect of YB1 on TGFß signaling was monitored by expression analysis and reporter gene assays in breast cancer cells overexpressing YB1 and treated with antiestrogens. RESULTS: Antiestrogen-mediated inhibition of estrogen receptor-α led to a suppression of YB1 protein synthesis. On the other hand, YB1 was found to be an enhancer of TGFß signaling. CONCLUSION: High levels of YB1 expression lead to a stimulation of TGFß pathways, thereby counteracting antihormonal breast cancer therapy and representing a putative resistance mechanism.

Microsomal epoxide hydrolase expression in the endometrial uterine corpus is regulated by progesterone during the menstrual cycle.

We have shown previously that high expression levels of microsomal epoxide hydrolase (mEH) correlate with a poor prognosis of breast cancer patients receiving tamoxifen, suggesting that enhanced mEH expression could lead to antiestrogen resistance (Fritz et al. in J Clin Oncol 19:3-9, 2001). Thus, the purpose of this study was to gain insights into the role of mEH in hormone-responsive tissues. We analyzed biopsy samples of the endometrium by immunohistochemical staining, pointing to a regulation of mEH during the menstrual cycle: during the first half mEH expression was low, increased during the second half and reached highest levels during pregnancy. Additionally, the progesterone receptor (PR) positive human endometrial cell lines IKPRAB-36 (estrogene receptor alpha [ERalpha] negative) and ECC1-PRAB72 (ERalpha positive) were chosen to further investigate the hormonal regulation of mEH expression. Western Blot and quantitative RT-PCR analysis revealed an increase of mEH expression after treatment with medroxy-progesterone 17-acetate (MPA) in the ERalpha containing ECC1-PRAB72 cells. In contrast our results suggest that MPA had no influence on the mEH protein level in the ERalpha- IKPRAB-36 cells. In conclusion, mEH expression is regulated by progesterone in the presence of both PRs and ERalpha.

Antiestrogens induce transforming growth factor beta-mediated immunosuppression in breast cancer.

Antiestrogens are universally used to treat estrogen receptor--positive breast cancer, but relapses occur commonly due to the development of drug resistance. The ability of antiestrogen to induce transforming growth factor beta (TGFbeta) in breast cancer cells may be relevant to the emergence of resistance, not only at the level of cell autonomous effects of TGFbeta on cancer progression but also at the level of its effects on the host immune system. To evaluate the potential role of tumor-derived, antiestrogen-induced TGFbeta as an immune suppressor, we established in vitro mixed lymphocyte tumor reactions (MLTR) using MCF-7 cells and peripheral blood mononuclear cells (PBMC), as well as tumor tissue and autologous tumor infiltrating lymphocytes (TIL) obtained from primary breast cancer biopsies. In allogeneic MLTR, antiestrogen-treated MCF-7 cells caused downregulation of the effector molecules granzyme B, perforin, and Fas ligand in CD8(+) T cells, and suppressed the generation of cytotoxic effector cells in a TGFbeta-dependent manner. Furthermore, we documented induction of regulatory T cells in CD4(+) T cells, based on Foxp3 expression and T-cell activation in cocultures. In autologous MLTR, antiestrogen treatment gave rise to enhanced Foxp3 expression of TIL/PBMC and decreased the number of apoptotic tumor cells. These effects were reversed by addition of a TGFbeta neutralizing antibody. Our findings offer evidence that antiestrogen induces immunosuppression in the tumor microenvironment, through a TGFbeta-dependent mechanism that may contribute to the development of antiestrogen resistance in breast cancer.

Estrogen receptor alpha attenuates transforming growth factor-beta signaling in breast cancer cells independent from agonistic and antagonistic ligands.

To investigate a presumed crosstalk between estrogen receptor alpha (ERalpha) and the TGF-beta signaling pathway in breast cancer, we analyzed the TGF-beta-induced expression of the plasminogen activator inhibitor 1 (PAI-1) gene in ER-positive MCF-7 cells. After siRNA-mediated knock-down of endogenous ERalpha, the transcription level of PAI-1 was upregulated, pointing to an attenuation of TGF-beta signaling by the presence of ERalpha. We verified these findings by a vice versa approach using a primary ER-negative cell model transiently overexpressing either ERalpha or ERbeta. We found that ERalpha, but not ERbeta, led to a strong inhibition of the TGF-beta1 signal, monitored by TGF-beta reporter assays. This attenuation was completely independent of receptor stimulation by beta-estradiol (E2) or inhibition by the pure antagonist ICI 182.780 (ICI). Our results indicate a permanent repression of PAI-1 by ERalpha and suggest a ligand-independent crosstalk between ERalpha and TGF-beta signaling in breast cancer cells.

Functional characterization of the DnaK chaperone system from the archaeon Methanothermobacter thermautotrophicus DeltaH.

We characterized the biochemical and functional properties of the DnaK system from the archaeon Methanothermobacter thermautotrophicus DeltaH. In contrast to the eubacterial chaperone components the archaeal Hsp70 system shows thermal transitions only slightly above the optimal environmental temperature (65 degrees C). Nevertheless, it prevents aggregation of luciferase in the physiological temperature range of the organism, but is also fully functional at 30 degrees C in luciferase refolding. Additionally, GrpE(M.th.) and DnaJ(M.th.) substitute their eubacterial counterparts whereas DnaK(M.th.) is only functional with its native cochaperones which could be attributed to a functional specialization of the eubacterial chaperones during evolution.