Doxorubicin and TGFb Inhibitor Figure 7. Treatment with TbRI-KI blocked doxorubicin-induced spontaneous lung metastasis of human breast cancer cells. A. EGFP- expressing sub confluent MDA-MB-231 cells (16106) were inoculated into both inguinal mammary fat pads of female athymic nude mice. Tumor volume was measured externally and calculated as described in the legend of Figure 5. Tumors were allowed to reach 80–100 mm3 in size and the animals were then ranked according to their tumor burden and divided into four groups such that the mean and the median of the tumor volume of the groups are closely matched. Animals were treated with PBS (control), TbRI-KI every alternative day, doxorubicin once every week, and with the combination of TbRI-KI and doxorubicin respectively. The experiment was terminated after 3 weeks and the final tumor volume (sum of two tumors per mouse) was used for calculating mean tumor volume6SEM from 4 mice in control and TbRI-KI groups and 5 mice in the other two groups. Asterisk ‘‘*’’ denotes significant difference (P,0.05) of the terminal tumor volumes between control and the combination treatment group by ANOVA. B. Representative Pictures of lung metastasis colonies formed by EGFP-expressing MDA-MB-231 cells detected by fluorescence microscopy (6200 magnification). C. Green fluorescent micrometastatic colonies in the whole lung were detected and counted. The percent of mice with lung metastasis in each group is expressed as total lung metastasis incidence. Incidence as percent of mice with numbers of lung metastasis colonies ranging between 1 and 20, between 20 and 50, and .50 is also presented. doi:10.1371/journal.pone.0010365.g007 upregulation of TGFb expression, several chemotherapeutic agents including doxorubicin were shown to stimulate TGFb1 expression and secretion from xenograft tumors [31]. Surprisingly, in a recent study, it was shown that doxorubicin treatment caused increased circulating levels of TGFb as well as increased circulating tumor cells and lung metastases in the MMTV/ PyVmT transgenic animal model [32]. We have observed that the presence of doxorubicin induced the migration and invasion of murine 4T1 and human MDA-MB-231 breast cancer cells similar to the TGFb1 treated cells in a Boyden chamber assay. Further investigation revealed that doxorubicin stimulated the phosphor- ylation of Smad2 and Smad3 in both 4T1 and MDA-MB-231 cells. Consistent with the stimulation of Smad2 and Smad3 phosphorylation, doxorubicin also stimulated the TGFb-respon- sive PAI-1 promoter activity in a dose dependent manner. Our results strongly indicate that although doxorubicin can induce cytostatic and/or cytocidal effects, it can also induce cancer cell migration/invasion by activating TGFb pathway. EMT is a latent embryonic program by which some cells within a tumor during cancer progression can reactivate mesenchymal traits to disseminate to metastatic sites [33]. Induction of EMT by TGFb has been shown to increase motility and invasiveness via the disassembly of cell-to-cell contacts, loss of cell polarity and significant cytoskeletal reorganization in normal and malignant mammary epithelial cell types. We observed in the murine breast cancer 4T1 cell model that TGFb and surprisingly also doxorubicin can induce EMT as evidenced by the mesenchymal morphology, reorganization of the actin cytoskeleton and nuclear localization of the embryonic transcription factor Snail. Emerging evidence has shown that certain type of tumors including breast tumors contain a subset of cells that initiate and propagate the tumors with high efficiency. These cells are PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e10365