Several studies showed that treatment with TbRI kinase inhibitors (TbRI-KI) can inhibit malignant properties of cancer cells in vitro and in vivo [21–25]. Recent studies have demonstrated that EMT induced by TGFb and other factors is associated with the expression of many stem cell markers and phenotypes in transformed human mammary epithelial cells [26,27]. These studies suggest that TGFb-induced EMT may result in the maintenance and formation of stem-like breast cancer cells. This notion is consistent with a recent report demonstrating an enhanced TGFb isoform expression and pathway activity in CD44+ breast cancer cells [28]. Although TGFb-associated drug resistance has been described previously [29], these recent findings would suggest that TGFb-induced drug resistance may be in a large part due to its induction of stem-like features in carcinoma cells. Thus, TGFb inhibitors should potentiate the efficacy of chemotherapeutic agents, especially for those agents that can adversely activate TGFb pathway. It was found that various anti-tumor drugs induced TGFb1 production in tumor cells [29,30], which could increase the potential of recurrent disease. In our study, we observed that chemotherapeutic drug doxorubicin activates TGFb signaling in human breast cancer MDA-MB-231 and murine breast cancer 4T1 cells. Doxorubicin induced EMT, promoted invasion and enhanced generation of cells with stem cell phenotype in murine 4T1 breast cancer cell line in vitro, which were significantly inhibited by TbRI-KI. Our in vivo studies showed for the first time the benefit of simultaneous treatment of doxorubicin with a TGFb antagonist to improve the inhibition of tumor growth and lung/bone metastasis in a syngeneic immunocompetent murine xenograft model and in a nude mice xenograft model of human breast cancer. Results Doxorubicin stimulated both motility and invasion of murine and human breast cancer cells Cell motility and invasion are integral processes in the metastatic dissemination of tumor cells. Doxorubicin has been used for the treatment of metastatic breast cancer. Therefore, we Doxorubicin and TGFb Inhibitor tested the effect of doxorubicin on the migration and invasion of murine 4T1 and human MDA-MB-231 cells in a Boyden chamber assay. Surprisingly, instead of inhibition, treatment with doxoru- bicin significantly enhanced both 4T1 and MDA-MB-231 tumor cell migration (Figure 1A and Figure 1B). Enhanced invasion through matrigel was also observed with both 4T1 and MDA-MB- 231 cells after treatment with doxorubicin (Figure S1A and Figure S1B respectively). As expected, TGFb1 treatment significantly induced both motility and invasion in both cell lines under similar conditions. These results indicated that doxorubicin could act like TGFb and might have the potential to promote tumor metastasis by stimulating tumor cell motility and invasion. Doxorubicin enhanced TGFb signaling in murine and human breast cancer cells Doxorubicin has been shown to induce circulating TGFb and lung metastasis in xenograft and transgenic animal models [31,32]. To examine whether activation of TGFb signaling has any role in the doxorubicin induced migration/invasion, we examined the effect of doxorubicin treatment on TGFb-mediated phosphoryla- tion of Smad2 and Smad3 in both murine 4T1 and human MDA- MB-231 cells. After treatment for 6 days, we found that similar to TGFb1 treatment (2 ng/ml), doxorubicin treatment (25 nM) also stimulated phosphorylation of Smad2 and Smad3 proteins in both 4T1 and MDA-MB-231 (Figure 2) cells, which was inhibited by the addition of the TGFb inhibitor TbRI-KI. Similar results were also obtained during a shorter duration of treatments (24 hours) with TGFb3 or doxorubicin in the presence or absence of TbRI- KI (Figure S2A). Like TGFb1, doxorubicin could also induce the transcriptional activation of a TGFb-responsive PAI-1 promoter in a dose-dependent manner (Figure S2B). Thus, our results indicate that doxorubicin can activate the TGFb pathway, which can be antagonized by a kinase inhibitor of TbRI in both breast cancer cell lines in vitro. TbRI-KI reversed Doxorubicin- or TGFb-induced EMT Acquisition of mesenchymal phenotype through EMT has been shown to be associated with aggressive breast cancer subtypes and Figure 1. Stimulation of migration by doxorubicin and TGFb treatment in murine and human breast cancer cells. In a Boyden Chamber migration assay, 40,000 murine breast cancer 4T1 cells (A) and human breast cancer MDA-MB-231 cells (B) in serum free medium were placed inside the insert of an invasive chamber and treated with doxorubicin (100 nM) or TGFb1 (5 ng/ml in A and 1 ng/ml in B) for 6 hours (4T1 cells) and 24 hours (MDA-MB-231 cells). Lower chamber contained the medium with 1% serum as chemoattractant. Cells were removed from the upper surface of the chamber membrane and the membrane was then stained for the migratory cells, which were counted under a microscope. Results are the mean of the five fields of observation at 1006 magnification and expressed as mean+SEM. ‘‘*’’ indicates significant difference from the control value with ANOVA test, P,0.05. doi:10.1371/journal.pone.0010365.g001 PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10365