Oncogene (2011) 30, 301–312 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Control of mammary tumor differentiation by SKI-606 (bosutinib) L Hebbard1,7,9, G Cecena1,7, J Golas2,8, J Sawada3,7, LG Ellies4, A Charbono1,7, R Williams1,7, RE Jimenez5, M Wankell1,7, KT Arndt2,8, SQ DeJoy2,8, RA Rollins2,8, V Diesl2,8, M Follettie2,8, LChen2,8, E Rosfjord2,8, RD Cardiff6, M Komatsu3,7, F Boschelli2,8 and RG Oshima1,7 1Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA; 2Center for Integrative Biology and Biotherapeutics, Pfizer Research, Pearl River, NY, USA; 3Sanford-Burnham Medical Research Institute, Lake Nona, FL, USA; 4Pathology Department, University of California, San Diego, CA, USA; 5Biomedical Science Program, University of California, San Diego, La Jolla, CA, USA and 6Center for Genomic Pathology University of California, Davis, CA, USA C-Src is infrequently mutated in human cancers but it Introduction mediates oncogenic signals of many activated growth factor receptors and thus remains a key target for cancer In breast cancer, Src is of particular interest as it is therapy. However, the broad function of Src in many cell activated by both steroid hormone receptors and ErbB types and processes requires evaluation of Src-targeted family growth factor receptors and activates transcrip- therapeutics within a normal developmental and immune- tion factors such as STAT3, STAT5 and b-catenin by competent environment. In an effort to understand the direct phosphorylation (Silva and Shupnik, 2007; Kim appropriate clinical use of Src inhibitors, we tested an Src et al., 2009). Cellular Src was discovered in 1978 as the inhibitor, SKI-606 (bosutinib), in the MMTV-PyVmT cellular counterpart of a viral gene, n-src, the transform- transgenic mouse model of breast cancer. Tumor forma- ing agent of Rous sarcoma virus (Spector et al., 1978). tion in this model is dependent on the presence of Src, but This non-receptor tyrosine kinase belongs to a family of the necessity of Src kinase activity for tumor formation proteins that mediate signaling by a large number of has not been determined. Furthermore, Src inhibitors have growth factor receptors, integrins, G-protein-coupled not been examined in an autochthonous tumor model that receptors, hormones and stress responses implicated in permits assessment of effects on different stages of tumor multiple types of cancer and vascular biology (Criscuoli progression. Here we show that oral administration of et al., 2005; Summy and Gallick, 2006; Kim et al., 2009). SKI-606 inhibited the phosphorylation of Src in mammary Src is over-expressed and activated in many human tumors and caused a rapid decrease in the Ezh2 Polycomb cancers, including breast cancer, and its activation group histone H3K27 methyltransferase and an increase generally correlates with poor prognosis. Nonetheless, in epithelial organization. SKI-606 prevented the appear- biomarkers predictive of a clinical response remain elusive. ance of palpable tumors in over 50% of the animals and The Src inhibitors dasatinib, saracatinib (AZD0530) stopped tumor growth in older animals with pre-existing and SKI-606 (bosutinib) are in phase II of clinical tumors. These antitumor effects were accompanied by development for the treatment of solid tumors of decreased cellular proliferation, altered tumor blood vessel metastatic breast and prostate cancer. SKI-606, a organization and dramatically increased differentiation multikinase inhibitor originally identified as an Src to lactational and epidermal cell fates. SKI-606 controls and Abl kinase inhibitor, is effective in vitro on the development of mammary tumors by inducing colorectal and breast tumor cells, chronic myelogenous differentiation. leukemia cells and also in multiple xenotransplantation Oncogene (2011) 30, 301–312; doi:10.1038/onc.2010.412; experiments (Boschelli et al., 2001; Jallal et al., 2007; published online 6 September 2010 Vultur et al., 2008). Src inhibitors generally have subtle effects on tumor cells under normal culture conditions Keywords: Src; SKI-606; mouse; breast cancer; Ezh2 (Golas et al., 2005; Vultur et al., 2008). The effects of SKI-606 in models of developing breast cancer have not been reported. The many functions of Src and Src family kinases in multiple signaling pathways suggest that the effects of inhibiting Src should be integrated into a whole- Correspondence: Professor RG Oshima, Tumor Development, Cancer animal context with a fully functional immune system Research Center, Sanford-Burnham Medical Research Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA. (Finn, 2008). The Src-dependent, immunocompetent, E-mails: [email protected] or [email protected] autochthonous FVB/N-Tg(MMTV-PyVmT)634Mul 7Formerly Burnham Institute for Medical Research. (MMTV-PyMT) breast cancer mouse model is an ideal 8Formerly Wyeth Oncology Discovery Research. tool for such a study because it permits the evaluation of 9Current address: Westmead Millennium Institute, Storr Liver Unit, Westmead NSW 2145, Australia. both early and late stages of luminal-type metastatic Received 26 April 2010; revised 28 July 2010; accepted 2 August 2010; carcinomas (Guy et al., 1992; Lin et al., 2003). This published online 6 September 2010 model mimics changes associated with human breast SKI-606 induced tumor differentiation L Hebbard et al 302 cancer progression, including upregulation of cyclinD1 SKI-606 accumulates in tumor tissue and inhibits Src and HER2, and infiltration of leukocytes. PyMT- activity induced mammary tumors are dependent on multiple Previously published data indicated that orally adminis- signaling molecules, including c-Src, phosphatidylinosi- tered SKI-606 was well distributed in the tissues of nude tol-3-kinase, Shc and insulin/insulin-like growth factor-1 mice and accumulated in human tumor xenografts. receptors (Ishizawar and Parsons, 2004). Analysis of plasma obtained from MMTV-PyMT Genetic inactivation of the c-src gene in MMTV- tumor-bearing mice 18 h after a single oral dose of PyMT mice limits the effect of PyMT transgene SKI-606 revealed plasma concentrations of 321 ng/ml expression on formation of hyperplastic lesions in (604 nM), well above the concentration effectively mammary tissues after a long latency period with inhibiting Src kinase activity (Boschelli et al., 2001) activated levels of c-Yes, a closely related member of and well within the range required to inhibit breast the Src family of kinases (Guy et al., 1994). c-Src cancer tumor cell growth and invasion in cell culture activation by PyMT is not the sole driver for tumor- (Jallal et al., 2007; Vultur et al., 2008) (Figure 1c). In igenesis, as expressing activated Src under the MMTV addition, SKI-606 accumulates in PyMT tumors to a promoter in the absence of PyMT leads to defective concentration of 2477 ng/g of tumor (Figure 1c). We mammary development and hyperplasia, but not in- determined the relative levels of active Src in these vasive tumor formation (Webster et al., 1995). Similarly, tumors by monitoring the phosphorylation of Y418 18 h when PyMT is mutated to block either SHC or PI3- following SKI-606 administration. SKI-606 treatment kinase association, only hyperplastic lesions are formed reduced P-Y418 levels in PyMT tumors by 75% of the (Webster et al., 1998), unless an angiogenic stimulus levels observed in tumors from vehicle-treated animals such as vascular endothelial growth factor (VEGF) is (Figure 1d), suggesting that SKI-606 inhibits the provided (Oshima et al., 2004). intended molecular target. Our studies show that SKI-606 treatment suppressed both early hyperplastic stages of the disease and overt tumor development. SKI-606 treatment stopped growth SKI-606 suppresses mammary tumor appearance of established tumors by inducing dysplastic differentia- PyMT expression occurs about 4 weeks after birth upon tion of tumor cells and altered vascular organization. mammary gland development at sexual maturity. The These responses were accompanied by downregulation mammary epithelial tree of MMTV-PyMT adolescent of the Polycomb repressor complex 2 subunit EZH2. females develops a small tumor-like mass beneath the The control of this aggressive model of breast cancer by nipple area concurrent with the emergence of the normal differentiation suggests that different clinical end points epithelial branching tubes (Neznanov et al., 1999; might be considered to evaluate drugs that control Maglione et al., 2001). Next focal hyperplastic lesions cancer through differentiation rather than cell death. arise among the normal epithelial structures, which then progress to mammary intraepithelial neoplasia and later to invasive cancer (Maglione et al., 2001; Lin et al., 2003). Results To test the effect of SKI-606 on both early hyperplastic lesions and mammary epithelial develop- SKI-606 inhibits cell growth in culture without inducing ment, SKI-606 (150 mg/kg) was administered by oral cell death gavage to female transgenic mice aged 30±2 days once Previous studies indicated that treatment with 1 mm daily for 14 days. SKI-606 treatment was very effective SKI-606 significantly reduced phosphorylation of the in limiting the growth of hyperplastic lesions and Y418 gatekeeper residue of c-Src in human tumor cells mammary intraepithelial neoplasia to less than 40% of (Golas et al., 2005; Vultur et al., 2008). To determine the size of lesions in the vehicle-treated control group whether SKI-606 affected in vitro growth of PyMT- (Supplementary Figures S1A and S1C). In contrast, transformed mammary tumor cells, Py-230 cells were the normal mammary epithelial ductal and endbud treated with SKI-606 at various concentrations. In a structures appeared fully extended to the distal limits 4-day assay, submicromolar concentrations of SKI-606 of the fat pad. This indicates that SKI-606 did not inhibited Py-230 cell proliferation (Figure 1a). However, interfere with the normal invasion and branching of Py-230 cells formed colonies from single cells in the the mammary gland while restricting abnormal hyper- presence of up to 750 nM SKI-606 (Figure 1a), but colony plastic growth.