Doxorubicin in Combination with a Small TGFb Inhibitor: A Potential Novel Therapy for Metastatic Breast Cancer in Mouse Models Abhik Bandyopadhyay1, Long Wang1, Joseph Agyin2, Yuping Tang1, Shu Lin1, I-Tien Yeh3, Keya De 1 , 1 Lu-Zhe Sun * 1 Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, United States of America, 2 Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, United States of America, 3 Department of Pathology, University of Texas Health Science Center, San Antonio, Texas, United States of America Abstract Background: Recent studies suggested that induction of epithelial-mesenchymal transition (EMT) might confer both metastatic and self-renewal properties to breast tumor cells resulting in drug resistance and tumor recurrence. TGFb is a potent inducer of EMT and has been shown to promote tumor progression in various breast cancer cell and animal models. Principal Findings: We report that chemotherapeutic drug doxorubicin activates TGFb signaling in human and murine breast cancer cells. Doxorubicin induced EMT, promoted invasion and enhanced generation of cells with stem cell phenotype in murine 4T1 breast cancer cells in vitro, which were significantly inhibited by a TGFb type I receptor kinase inhibitor (TbRI-KI). We investigated the potential synergistic anti-tumor activity of TbR1-KI in combination with doxorubicin in animal models of metastatic breast cancer. Combination of Doxorubicin and TbRI-KI enhanced the efficacy of doxorubicin in reducing tumor growth and lung metastasis in the 4T1 orthotopic xenograft model in comparison to single treatments. Doxorubicin treatment alone enhanced metastasis to lung in the human breast cancer MDA-MB-231 orthotopic xenograft model and metastasis to bone in the 4T1 orthotopic xenograft model, which was significantly blocked when TbR1-KI was administered in combination with doxorubicin. Conclusions: These observations suggest that the adverse activation of TGFb pathway by chemotherapeutics in the cancer cells together with elevated TGFb levels in tumor microenvironment may lead to EMT and generation of cancer stem cells resulting in the resistance to the chemotherapy. Our results indicate that the combination treatment of doxorubicin with a TGFb inhibitor has the potential to reduce the dose and consequently the toxic side-effects of doxorubicin, and improve its efficacy in the inhibition of breast cancer growth and metastasis. Citation: Bandyopadhyay A, Wang L, Agyin J, Tang Y, Lin S, et al. (2010) Doxorubicin in Combination with a Small TGFb Inhibitor: A Potential Novel Therapy for Metastatic Breast Cancer in Mouse Models. PLoS ONE 5(4): e10365. doi:10.1371/journal.pone.0010365 Editor: Kurt Anderson, The Beatson Institute for Cancer Research, United Kingdom Received December 10, 2009; Accepted April 1, 2010; Published April 28, 2010 Copyright: 2010 Bandyopadhyay et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by NIH Grants R01CA079683, R01CA075253, P01CA040035, and P30CA054174, and by Shelby Rae Tang Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sunl@uthscsa.edu Introduction Breast cancer is the leading cause of cancer death in women with more than a million newly diagnosed cases annually worldwide [1]. It is estimated that 30–75% of patients undergoing surgery and adjuvant treatment will develop recurrent metastatic disease. Metastatic breast cancer (MBC) is essentially incurable with standard therapy and patients with MBC have a median survival of about 2 years after metastasis have been detected [2]. Doxorubicin is an anthracycline drug widely used in chemother- apy regimen for patients with MBC [3] and shown overall response rates of between 35 and 50% in patients with MBC who have not previously received chemotherapy [4]. Despite its excellent anti-tumor activity, doxorubicin has a relatively low therapeutic index and its clinical utility is limited due to acute and chronic toxicities such as myelosuppression, immunosuppression and dose-cumulative cardiotoxicity [5]. Therefore, combination treatment with another highly effective novel non-toxic drug which can lower the dose of chemotherapeutic agents would be desirable. Transforming growth factor beta (TGFb) has been shown to be overly produced during progression of various types of carcinomas including breast cancer [6,7] and to accelerate metastatic progression [8–10]. Several mechanisms are believed to mediate TGFb’s tumor-promoting activity. TGFb produced by tumor cells can act in a paracrine fashion to stimulate myofibroblast differentiation [11] and tumor angiogenesis [12], and to suppress host immune surveillance [13]. Acting in an autocrine fashion, TGFb signaling has been shown to be necessary for the survival of breast cancer cells [14,15] and to induce epithelial-mesenchymal transition (EMT) and cell migration [16]. Due to its oncogenic role, various components of TGFb pathway are being evaluated as therapeutic targets [17–19]. TGFb type I receptor (TbRI) kinase is one potential target for the blockade of TGFb signaling [20]. PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10365