Purpose: Transforming growth factor-h (TGF-h) suppresses tumor development by inhibiting cellular proliferation, inducing differentiation and apoptosis, and maintaining genomic integrity. However, once tumor cells escape from the tumor-suppressive effects of TGF-h, they often constitutively overexpress and activateTGF-h, which may promote tumor progression by enhancing invasion, metastasis, and angiogenesis and by suppressing antitumor immunity. The purpose of this study was to test this hypothesis usingTGF-h pathway antagonists. Experimental Design: We examined the effects of selectiveTGF-h type I receptor kinase inhibitors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3Tand 4T1) in vitro and in vivo. Results: Both agents blocked TGF-h-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC 50 between 20 and 80 nmol/L. TGF-h failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal transdifferentiation, migration, and invasiveness into Matrigel in vitro. These effects were inhibited by SD-093, indicating that these processes are partly driven byTGF-h. Treatment of syngeneic R3T or 4T1tumor-bearing mice with orally given SD-208 inhibited primary tumor growth as well as the number and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth or metastasis in athymic nude mice. Moreover, in vitro anti-4T1 cell cytotoxic T-cell responses of splenocytes from drug-treated animals were enhanced compared with cells from control animals. In addition, SD-208 treatment resulted in a decrease in tumor angiogenesis. Conclusion: TGF-h type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.

No abstract

The 1029 series of mammary epithelial cell lines (D6, GP+E, r3 and r3T) are progressively more transformed: the latter two by val 12 ras. These cell lines respond to TGFβ by undergoing early events of epithelial-mesenchymal transition (EMT), including morphological changes and redistribution of E-cadherin. Tumors formed by r3T cells in the choroid of the eye express vimentin, a late marker of EMT, possibly in response to TGFβ. In vitro, vimentin expression is induced in all the cell lines by TGFβ treatment, while cytokeratin expression is only slightly affected. Surprisingly, ras transformation results in a 10-fold suppression of vimentin expression. Neither suppression of vimentin by ras transformation nor induction by TGFβ are mediated by the vimentin promoter in r3T cells. In transient transfection assays, several human vimentin promoter constructs are more active in the low-expressing r3T cell line than in the vimentin-expressing mesenchymal cell line NIH3T3. In the r3T cells, there is no effect of TGFβ treatment for 9 days on the activity of either promoter. Azacytidine treatment does not affect vimentin expression in either NIH3T3 or r3T, suggesting that promoter methylation is not the mechanism of suppression by ras. Finally, the half life of the vimentin mRNA is similar in both the r3T cells and NIH3T3 cells. We conclude that the suppression of vimentin expression by ras, and the relief of this suppression by TGFβ, occurs in a promoter-independent fashion, possibly through sequences in the first or second intron.

Supplementary Figure 1 from Inhibition of Growth and Metastasis of Mouse Mammary Carcinoma by Selective Inhibitor of Transforming Growth Factor-β Type I Receptor Kinase <i>In vivo</i>

<div>Abstract<p><b>Purpose:</b> Transforming growth factor-β (TGF-β) suppresses tumor development by inhibiting cellular proliferation, inducing differentiation and apoptosis, and maintaining genomic integrity. However, once tumor cells escape from the tumor-suppressive effects of TGF-β, they often constitutively overexpress and activate TGF-β, which may promote tumor progression by enhancing invasion, metastasis, and angiogenesis and by suppressing antitumor immunity. The purpose of this study was to test this hypothesis using TGF-β pathway antagonists.</p><p><b>Experimental Design:</b> We examined the effects of selective TGF-β type I receptor kinase inhibitors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3T and 4T1) <i>in vitro</i> and <i>in vivo</i>.</p><p><b>Results:</b> Both agents blocked TGF-β-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC₅₀ between 20 and 80 nmol/L. TGF-β failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal transdifferentiation, migration, and invasiveness into Matrigel <i>in vitro</i>. These effects were inhibited by SD-093, indicating that these processes are partly driven by TGF-β. Treatment of syngeneic R3T or 4T1 tumor-bearing mice with orally given SD-208 inhibited primary tumor growth as well as the number and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth or metastasis in athymic nude mice. Moreover, <i>in vitro</i> anti-4T1 cell cytotoxic T-cell responses of splenocytes from drug-treated animals were enhanced compared with cells from control animals. In addition, SD-208 treatment resulted in a decrease in tumor angiogenesis.</p><p><b>Conclusion:</b> TGF-β type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.</p></div>