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.

Purpose: The role of transforming growth factor ␤ (TGF-␤) in breast cancer is ambiguous; it can display both tumor suppressing and enhancing effects. Activation of the TGF-␤ signal transduction system is subject to hormonal regulation. This study was conducted to further analyze the role of TGF-␤ receptors in breast cancer and to evaluate their significance as prognostic markers. Experimental Design: Expression of TGF-␤ receptor I (T␤RI) and TGF␤ receptor II (T␤RII) was retrospectively analyzed by immunohistochemistry in 246 breast cancer patients.Results: Expression of T␤RI 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, T␤RII was prognostic for overall survival in univariate analysis (P ‫؍‬ 0.0370). In estrogen receptor (ER) -negative patients T␤RII expression was correlated with highly reduced overall survival (P ‫؍‬ 0.0083). In multivariate analysis T␤RII proved to be an independent and highly significant prognostic marker with a hazard ratio of 6.8. Simultaneous loss of both ER and T␤RII was associated with longer overall survival times comparable with those of ER-positive patients.Conclusions: The results of this exploratory study show that T␤RII 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 TGF␤ takes place, whereas tumorpromoting aspects remain intact.

The excessive matrix deposition in lung fibrosis is thought to be due to enhanced formation and activity of TGFβ1, which stimulates synthesis and inhibits degradation of matrix proteins. The cellular mechanisms triggered by TGFβ1 are still incompletely understood. Recently, a novel transcriptional target of TGFβ1 has been identified, i.e. the human serum and glucocorticoid dependent kinase hSGK1. The present study has been performed to explore whether TGFβ1 stimulates hSGK1 transcription in lung fibroblasts and whether lung fibrosis is associated with enhanced hSGK1 expression. As evident from Northern Blotting, TGFβ1 strongly upregulates hSGK1 in human lung fibroblasts, an effect partially reversed by p38-kinase inhibitor SB203580. In situ hybridization experiments reveal that in intact lung tissue hSGK1 is expressed in single type II alveolar pneumocytes and macrophages. In contrast, in fibrotic lung tissue a dramatic upregulation of hSGK1 mRNA as well as a strong expression of hSGK1 protein is observed in epithelial cells and interstitial cells comprising macrophages and fibroblasts. In conclusion, in lung fibrosis, the serine/threonine kinase hSGK1 is upregulated, an effect at least partially accounted for by TGFβ1. The full effect of TGFβ1 requires the activation of p38 kinase.

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 β (TGFβ) in breast cancer cells may be relevant to the emergence of resistance, not only at the level of cell autonomous effects of TGFβ 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 TGFβ 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, antiestrogentreated 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 TGFβ-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 TGFβ neutralizing antibody. Our findings offer evidence that antiestrogen induces immunosuppression in the tumor microenvironment, through a TGFβ-dependent mechanism that may contribute to the development of antiestrogen resistance in breast cancer. Cancer Res; 70(4); 1314-22.

BackgroundAlthough transforming growth factor β (TGF-β) 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-β signaling, particularly involving the Smad-pathway, we investigated the role of Smad4-expression in breast cancer.MethodsSmad4 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.ResultsSmad4 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-β-receptor I (p < 0.001, n = 197) and TGF-β-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.ConclusionAccording 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-β signaling in tumor progression.

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.

Transforming growth factor-B (TGF-B), a multifunctional growth factor, plays an important role in breast cancer. There is increasing evidence that enhanced expression of TGF-B 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 À246 relative to the transcriptional start site (À246ins). Transient transfection experiments showed that the À246ins polymorphism significantly increased TGFB2 promoter activity in breast cancer cells. Electrophoretic mobility shift assays revealed binding of the transcription factor Sp1 to the À246ins allele. Overexpression of Sp1 enhanced promoter activity of the À246ins allele, demonstrating that Sp1 mediates transcriptional activation. Furthermore, the À246ins allele was associated with enhanced TGF-B 2 expression in breast cancer tissue (P = 0.0005). To evaluate the role of the polymorphism in breast cancer, frequency of the À246ins 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 À246ins allele was associated with lymph node metastasis (P = 0.003). The À246ins 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 À246ins polymorphism leads to enhanced TGF-B 2 expression levels in vivo and might thereby contribute to tumor progression and development of metastases. (Cancer Res 2006; 66(15): 7554-61)

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.