Campbell and Moorehead BMC Cancer 2011, 11:480 http://www.biomedcentral.com/1471-2407/11/480 regression of most of the mammary tumors. However approximately 11% of the tumors resumed growth fol- lowing IGF-IR transgene downregulation and these tumors no longer expressed high levels of IGF-IR [13]. Analysis at both the mRNA and protein level revealed that PDGFRa and PDGFRb were consistently upregu- lated in tumor cells and tumor tissue following loss of IGF-IR expression. Therefore, one change that occurs during IGF-IR independent tumor growth is the upregu- lation of PDGFR signaling. Although this reciprocal expression pattern has not been described previously in breast cancer, it has been observed in a rhabdomyosarcoma cell line selected for resistance to BMS-754807 (an IGF-IR small molecule inhibitor) [27]. In this study, administration of increas- ing concentrations of BMS-75807 to Rh41 cells for approximately 5 months resulted in the generation of a clone resistant to BMS-754807. The BMS-754907-resis- tant clone expressed lower levels of IGF-IR and elevated levels of PDGFRa compared to the parental cells. In addition, the investigators found that the combination of an anti-IGF-IR agent with an anti-PDGFRa agent resulted in synergistic cell kill [27]. A reciprocal rela- tionship between IGF-IR and PDGFRa/c-kit expression has also been observed in gastrointestinal stromal tumors [28]. Therefore, it appears that PDGFR signaling is enhanced following the loss of IGF-IR signaling and vice versa, an important concept to considering when designing therapeutic strategies targeting either of these receptors. To determine the functional consequence of elevated PDGFRa and PDGFR b, cell survival, proliferation, apoptosis and migration/invasion were evaluated follow- ing RNAi-mediated knockdown of PDGFRa and PDGFR b alone or concomitant knockdown of both receptors. The most dramatic effect was the decrease in migration and invasion following concomitant knockdown of both PDGFRs. In other systems, PDGFRb has previously been shown to promote cell migration, while PDGFRa has been shown to inhibit this process [29-31]. However, more recently, it was shown that both receptors could promote migration in fibroblasts with additive effects when both were present [18]. Similar to these results, the greatest inhibitory effect on migration was observed in this study when both isoforms were knocked down compared to either one individually. Analogous to our findings, in human breast cancer cell lines, PDGF ligands have also previously been shown to mediate invasion or upregulate markers of invasion [32,33]. These processes are important for metastasis and it will be interesting to determine if the PDGF-axis is involved in metastasis in our model, especially during IGF-IR- independent recurrence. Previously, in a model of ras- Page 11 of 13 transformed mammary epithelial cells undergoing EMT, PDGFR inhibition was shown to impede metastasis [22]. Somewhat surprisingly, downregulation of both PDGFRs resulted in a modest increase in proliferation and a decrease in apoptosis. These results are not in accordance with conventional models of PDGFR signal- ing. Typically this pathway is involved in survival and proliferation for mesenchymal cells including a number of cancers of mesenchymal origin such as dermatofibro- sarcoma protuberans, gastrointestinal stromal tumors, chronic myeloid leukemias and gliomas [15,34]. How- ever, the mitogenic and transforming effect of PDGFRs in other tumor types of epithelial origin, including breast cancer, remains largely unknown. While targeting the PDGFR usually results in inhibition of cell survival [35], similar results to ours have been reported in other models; PDGFRa overexpression was shown to inhibit melanoma tumor cell growth in vitro and in vivo [36], while overexpression of PDGF-BB in human pancreatic and colorectal cells resulted in a reduction in tumor growth [37]. Interestingly, the levels of phosphorylated Akt were elevated in both cell lines following downregulation of PDGFRs. This observation is in contrast to published reports indicating that PDGFRs signal via Akt [38], but would explain why an increase in proliferation and a decrease in apoptosis was observed following PDGFR downregulation. Phosphorylated Akt initiates signals that promote cell proliferation and inhibits apoptosis [39-41]. Therefore, activation of a signaling pathway other than the PDGFRs must compensate for the loss of IGF-IR signaling and maintain high levels of Akt phos- phorylation in our model. One logical receptor family would be the EGFR/ErbB family as these receptors have been shown to mediate resistance to IGF-IR targeting agents in some models [42-44]. However, western blot- ting of IGF-IR-dependent and IGF-IR-independent mammary tumors did not show any consistent differ- ences in the levels of EGFR, ErbB2 or ErbB3 or the phosphorylated forms of these receptors (data not shown). Recently we have shown that mammary tumors that become independent of IGF-IR signaling in MTB-IGFIR transgenic mice and the RJ348 cell line have characteris- tics of claudin-low breast tumors [14,45]. The claudin- low breast cancer subtype was identified in 2007 and is characterized by low levels of claudins 3, 4 and 7 as well as other tight junction proteins [46,47]. Claudin-low tumors also express high levels of markers associated with epithelial-to-mesenchymal transition (EMT) such as Twist1/2, Zeb1/2, Slug and Snail while expressing lit- tle or no markers of luminal differentiation [46]. The prevalence of claudin-low tumors is reported to be