Campbell and Moorehead BMC Cancer 2011, 11:480 http://www.biomedcentral.com/1471-2407/11/480 not correlated with any particular subtype of breast cancer [8] and therefore offers the opportunity for widespread use of targeted therapies. Based on these observations, a number of drugs targeting this pathway are currently in clinical trials [9,10], including small molecule kinase inhibitors that block downstream sig- nalling and monoclonal antibodies that block ligand receptor interactions [11]. While it is too early to determine the efficacy of such agents [9], it is of extreme importance to understand potential resistance mechanisms. Previously our lab has created an inducible transgenic mouse model (MTB-IGFIR) of IGF-IR overexpression in which mammary tumors were observed to develop [12]. IGF-IR transgene downregulation resulted in regression of almost all established palpable tumors after which a fraction recur in the absence of IGF-IR induction [13]. Thus, this model recapitulates treatment with IGF-IR- directed therapeutics and can be used to study potential resistance mechanisms. Previous work has provided initial characterization of IGF-IR-independence and has demonstrated the presence of an epithelial-mesenchymal transition (EMT) phenotype in these recurrent tumors [13]. Subsequent microarray data, comparing primary mammary tumors to IGF-IR-independent recurrent tumors, has since identified members of the platelet- derived growth factor family (PDGF) as being upregu- lated during this process [14]. The importance of PDGF receptors (PDGFRs) is well known in gliomas, sarcomas and gastrointestinal stro- mal tumors but has more recently been noted in such cancers as ovarian, prostate and neuroblastomas (reviewed in [15,16]). However, compared to other RTKs, little is known about the role of these proteins in breast cancer. Signalling through PDGFRs has been typically associated with proliferation and migration of mesenchymal cells and is important for these processes during mammalian development (reviewed in [17]). Through downstream mediators such as MAPK, FAK, PI-3K, Src and PLC-g, PDGFRs also govern processes such survival, proliferation and angiogenesis (reviewed in [15,18]). The best understood ligands for these receptors include PDGF-A and B which undergo homo- or heterodimerization to become active [16]. In breast cancer, expression of PDGFRs was initially shown to be restricted to the tumor stroma, while ligand was produced by the tumor cells [19,20]. How- ever, recent evidence suggests that expression of both PDGFRa and b, either in the tumor tissue or stroma, is correlated with invasive breast cancers and tumor progression [21-23]. In addition, PDGFRa was shown to be important for maintenance of EMT, evasion of apoptosis, and metastasis in transformed mesenchymal murine mammary cells [22]. Page 2 of 13 The purpose of this study was to examine the role of the PDGF-axis in mammary tumors that survive inde- pendent of IGF-IR signaling in MTB-IGFIR mice. Upon IGF-IR suppression, expression of PDGFRa and b were observed to increase, a process which occurred rapidly. In cell lines isolated from IGF-IR-independent recurrent tumors, PDGFRs were shown to facilitate motility and invasion with more modest effects on proliferation and apoptosis. Materials and methods Animal studies MTB-IGFIR transgenic mice are described elsewhere [12]. For IGF-IR transgene induction, mice were given a diet containing 2 mg/kg doxycycline (Bio-Serv Inc., Frenchtown, NJ). Tumors were allowed to grow to a maximum length of 17 mm (under the guidelines of the Canadian Council of Animal Care). IGF-IR-independent recurrent tumors were formed after the withdrawal of doxycycline, and are described in [13]. For IGF-IR downregulation studies, mice harboring mammary tumors induced through administration of doxycycline were given a normal diet and tissue was collected 24 and 48 h later. Animals were housed and cared for fol- lowing guidelines established by the Central Animal Facility at the University of Guelph and the guidelines established by the Canadian Council of Animal Care. Cell lines and culture conditions RM11A cells, a murine mammary tumor cell line derived from an MTB-IGFIR mammary tumor, were grown as described in [24]. Doxycycline (10 μg/mL) was added to the culture media for transgene induction and only omitted from the media for withdrawal studies. RJ345 cells were isolated from primary mammary tumors of MTB-IGFIR mice and cultured in media con- taining doxycycine as above while RJ348 and RJ423 cells were isolated from IGF-IR-independent spindle recur- rent tumors; all of these cell lines were isolated and cul- tured in media as described in [24]. Tumors formed from all cell lines were generated through injection of approximately 5 × 105 cells into the 4th mammary gland of syngeneic wild type FVB mice as described in [24]. Tumors were allowed to grow to 17 mm before the ani- mal was euthanized and tissue was collected. Western blotting Western blotting was performed as described in [24]. Briefly, 30 μg of protein lysate was subjected to SDS- PAGE with subsequent transfer onto nitrocellulose membranes. Primary antibodies used included anti- PDGFRa, anti-PDGFRb, anti-phospho-Akt, anti-Akt, anti-phospho-p42/44, anti-p42/44, anti-Cyclin D1 (all from Cell Signal technology, Danvers, MA, USA), and