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.

TGFbeta2 and TbetaRII are valid molecular biomarkers for the antiproliferative effects of tamoxifen and tamoxifen metabolites in breast cancer cells.

Response to treatment with the antiestrogen tamoxifen is variable and at least partially due to its highly complex metabolism. Tamoxifen is transformed by polymorphic and inducible cytochrome P450 enzymes to a large number of metabolites with varying biological activities. The estrogen receptor dependent growth inhibitory effect of antiestrogens is mediated by activation of antiproliferative Transforming Growth Factor beta (TGFbeta) signal transduction pathways. The aim of the present study was to establish if TGFbeta2 or TGFbeta receptor II (TbetaRII), could be used as markers to assess the pharmacological potency of tamoxifen and its metabolites. Consequently, we analyzed the growth inhibitory effect of tamoxifen and its major metabolites and explored whether it correlated with their capacity to induce TGFbeta2 and TbetaRII expression. Human breast cancer cells (MCF-7 and T47D) were treated with tamoxifen and tamoxifen metabolites and mRNA expression of TGFbeta2 and TbetaRII was analyzed by quantitative RT-PCR. Only two metabolites 4-hydroxytamoxifen and N-desmethyl-4-hydroxytamoxifen had significant antiproliferative activity and were able to induce TGFbeta2 and TbetaRII. Plasma concentrations of these metabolites are usually very low in patients. However, even minor growth inhibitory effects at concentrations which are below the limit of quantification in plasma samples resulted in clearly discernible effects on expression of TGFbeta2 and TbetaRII. Taken together, our data demonstrate that TGFbeta2 and TbetaRII are very specific and sensitive biomarkers for the antiestrogenic activity of tamoxifen metabolites in breast cancer.

A novel functional polymorphism in the transforming growth factor-beta2 gene promoter and tumor progression in breast cancer.

Transforming growth factor-beta (TGF-beta), a multifunctional growth factor, plays an important role in breast cancer. There is increasing evidence that enhanced expression of TGF-beta promotes breast cancer progression contributing to metastasis and invasiveness of the tumor. We identified a functional polymorphism in the TGFB2 promoter, a 4-bp insertion at position -246 relative to the transcriptional start site (-246ins). Transient transfection experiments showed that the -246ins polymorphism significantly increased TGFB2 promoter activity in breast cancer cells. Electrophoretic mobility shift assays revealed binding of the transcription factor Sp1 to the -246ins allele. Overexpression of Sp1 enhanced promoter activity of the -246ins allele, demonstrating that Sp1 mediates transcriptional activation. Furthermore, the -246ins allele was associated with enhanced TGF-beta(2) expression in breast cancer tissue (P = 0.0005). To evaluate the role of the polymorphism in breast cancer, frequency of the -246ins allele was determined in breast cancer patients (n = 78) and healthy female controls (n = 143). No significant differences were found. However, the presence of the -246ins allele was associated with lymph node metastasis (P = 0.003). The -246ins allele was a significant predictor for lymph node metastasis independent of estrogen and progesterone receptor status in a multivariate logistic regression analysis (P = 0.0118, odds ratio, 5.18; 95% confidence interval, 1.44-18.62). We provide evidence that the TGFB2 -246ins polymorphism leads to enhanced TGF-beta(2) expression levels in vivo and might thereby contribute to tumor progression and development of metastases.

Smad4-expression is decreased in breast cancer tissues: a retrospective study.

BACKGROUND: Although transforming growth factor beta (TGF-beta) typically inhibits proliferation of epithelial cells, consistent with a tumor suppressor activity, it paradoxically also exhibits pro-metastatic activity in the later stages of carcinogenesis. Since tumors often display altered TGF-beta signaling, particularly involving the Smad-pathway, we investigated the role of Smad4-expression in breast cancer. METHODS: Smad4 expression was investigated by immunohistochemistry in formalin-fixed, paraffin-embedded tissue from 197 samples of primary breast cancer obtained between 1986 and 1998. The prognostic value of Smad4-expression was analyzed. RESULTS: Smad4 expression was found to be reduced in lobular and ductal breast carcinoma as compared to surrounding uninvolved lobular and ductal breast epithelia (p < 0.001, n = 50). Smad4-expression correlated positively with expression of TGF-beta-receptor I (p < 0.001, n = 197) and TGF-beta-receptor II (p < 0.001, n = 197), but showed no significant correlation with tumor size, metastases, nodal status, histological grade, histological type, or estrogen receptor expression. While not achieving statistical significance, there was a trend towards longer survival times in patients with Smad4 negative tumors. CONCLUSION: According to the suggested role of Smad4 as a tumor suppressor we observed that expression of Smad4 is lower in human breast cancer than in surrounding breast epithelium. However, we also observed a trend towards longer survival times in Smad4-negative patients, indicating the complex role of TGF-beta signaling in tumor progression.

TGF-beta signaling in breast cancer.

The antiestrogen tamoxifen is one of the most successful drugs in the endocrine treatment of breast cancer and significantly reduces the risk of recurrence and death. Antiestrogens act by inhibiting the production of growth-stimulatory factors as well as by activating peptides with growth-inhibitory effects like transforming growth factor- beta (TGF-beta). In hormone-responsive breast cancer cells treatment with antiestrogens leads to the conversion of TGF-beta1 into a biologically active form. Expression of TGF-beta2 and TGF-beta receptor (TbetaR) II is induced via a transcriptional mechanism involving p38 MAP kinase. Inhibition of p38 abolishes antiestrogen-dependent growth inhibition. However, the role of TGF-beta in breast cancer progression is ambiguous, as it was shown to display both tumor-suppressing and -enhancing effects. A polymorphism in the promoter of TGF-beta2 that enhances expression of the protein was associated with lymph node metastasis in breast cancer patients, pointing to a role of TGF-beta2 in the process of invasion. An immunohistochemical study on TbetaRI and TbetaRII expression in breast cancer tissues indicates that the estrogen receptor (ER) status of a tumor is an important marker and a potential mediator of the transition of TGF-beta from tumor suppressor to tumor promoter. In ER-negative tumors, expression of TbetaRII was associated with a subset of tumors that appeared to be highly aggressive, leading to strongly reduced overall survival times. Further characterization of the influence of ER expression on TGF-beta signal transduction shows that ER-alpha plays a crucial role in TGF-beta signaling.

Antiestrogens induce growth inhibition by sequential activation of p38 mitogen-activated protein kinase and transforming growth factor-beta pathways in human breast cancer cells.

Antiestrogens are successfully used in the treatment of breast cancer. The purpose of this study was to investigate the role of different signal transduction pathways in antiestrogen-induced growth inhibition to gain insights into mechanisms of antiestrogen resistance. We used specific MAPK inhibitors and MCF-7 carcinoma cells as a model to demonstrate that p38 MAPK is an important mediator of antiestrogen growth inhibition in breast cancer. A kinase assay showed that antiestrogens (4-hydroxytamoxifen and ICI 182.780) rapidly induce p38 activity. Overexpression of kinase-deficient mutants of p38 reduced the antiestrogen suppression of Cyclin A transcription. TGFbeta, a negative regulator of breast cancer cell growth, is induced by antiestrogens; therefore, activation of p38 could have been mediated by TGFbeta. We used a TGFbeta and antiestrogen-sensitive reporter gene assay to show that p38 activation precedes TGFbeta activation. These results were further confirmed by quantitative RT-PCR analysis of the antiestrogen-induced transcription of TGFbeta2 and TGFbeta receptor II. Inhibition of p38 reduced the induction of both genes. Finally, Western blot analysis shows that antiestrogens induce phosphorylation of Smad (mothers against decapentaplegic homolog) 2 via p38. Promoter assays with the Smad-dependent reporter p6SBE confirm participation of Smad3 and Smad4 in antiestrogen action. Taken together, our data delineate an antiestrogen signal transduction pathway involving sequential activation of p38 and TGFbeta pathways to mediate growth inhibition.

Prognostic significance of transforming growth factor beta receptor II in estrogen receptor-negative breast cancer patients.

PURPOSE: The role of transforming growth factor beta (TGF-beta) in breast cancer is ambiguous; it can display both tumor suppressing and enhancing effects. Activation of the TGF-beta signal transduction system is subject to hormonal regulation. This study was conducted to further analyze the role of TGF-beta receptors in breast cancer and to evaluate their significance as prognostic markers. EXPERIMENTAL DESIGN: Expression of TGF-beta receptor I (TbetaRI) and TGFbeta receptor II (TbetaRII) was retrospectively analyzed by immunohistochemistry in 246 breast cancer patients. RESULTS: Expression of TbetaRI was strongly correlated with tumor size (P < 0.001) and nodal status (P = 0.012) but only weakly with overall survival (P = 0.056). In contrast, TbetaRII was prognostic for overall survival in univariate analysis (P = 0.0370). In estrogen receptor (ER) -negative patients TbetaRII expression was correlated with highly reduced overall survival (P = 0.0083). In multivariate analysis TbetaRII proved to be an independent and highly significant prognostic marker with a hazard ratio of 6.8. Simultaneous loss of both ER and TbetaRII was associated with longer overall survival times comparable with those of ER-positive patients. CONCLUSIONS: The results of this exploratory study show that TbetaRII is an independent, highly significant prognostic indicator for overall survival in ER-negative patients. In addition our results are supportive of a mechanism of breast cancer progression in which a selective loss of the tumor inhibitory action of TGFbeta takes place, whereas tumor- promoting aspects remain intact.