Treatment of adolescent MMTV-PyMT females for four weeks effectively prevented palpable tumor formation in half of the SKI-606 treated animals. Instead of residual solid tumors, distended, dysplastic mammary cysts with accumulated casein protein filled the fat pads. The differentiation-inducing effect of SKI-606 was most dramatically demonstrated by the 2-week treatment of tumor-bearing animals. This resulted in enrichment of organized K14-positive basal epithelia juxtaposed with luminal epithelial cells in cystic structures, the accumulation of milk protein and sporadic epidermal differentiation. Strikingly similar observations of cystic buildup of milk proteins were reported for Src null mice, which were ascribed to a defect in secretory activation during pregnancy (Watkin et al., 2008
The relatively small list of differentially expressed genes in SKI-606 treated tumors is likely because of the variation of the differentiation of individual tumors that decreased the probability of passing multiple statistical thresholds. Genes whose expression was increased in all SKI-606-treated tumors overlap significantly with genes increased during lactation (Rudolph et al., 2007
) and repressed in mouse transgenic tumors caused by c-Myc, SV40 T antigen and ETV6-NTRK3 (Bild et al., 2006
; Klein et al., 2005
; Li et al., 2007
) and human breast cancer cell lines (Klein et al., 2005
). These results suggest that the SKI-606 induced differentiation reflects the reversal of a common transcriptional response to oncogene transformation. The induction of differentiation by SKI-606 may be related to the function of Src in ER signaling (Ishizawar and Parsons, 2004
). Cross talk between ER and Src is well documented and may include effects on the nuclear localization of ER. Src null mice have defects in mammary gland development and ER signaling (Kim et al., 2005b
). While we found no differences in the cellular distribution of ER in both tumor cells or in stromal cells of mice treated with SKI-606, alterations in ER activity may still contribute to the diverse differentiation responses observed.
Differentiation of SKI-606-treated PyMT tumors was associated with changes in vascular organization, although not vascular density. Src is involved in the production of VEGF (Mukhopadhyay et al., 1995
) and Src is activated by VEGF receptors (Eliceiri et al., 1999
). Significantly, the observed changes in vascular organization after SKI-606 treatment are consistent with normalization of the more permeable and tortuous vessels found in these tumors (Cheung et al., 1997
). SKI-606 treatment reduces tumor cell extravasation (Weis et al., 2004
) at least in part by strengthening VE-cadherin (cadherin 5) and beta-catenin interaction. SKI-606 was also effective in models for ischemic stroke where VEGF-induced vascular leakage mediated by Src activation is believed to play a dominant role in disease etiology. These observations suggest that Src inhibition by SKI-606 contributes to tumor vascular normalization. While SKI-606 was originally developed as a Src inhibitor, subsequent studies indicated that it targets multiple kinases including Abl kinases (Remsing Rix et al., 2009
). At the RNA level, Lck, Egfr and Csk are expressed at detectable levels in PyMT tumors and are reported to have SKI-606 IC50
of 100 nM or less (Remsing Rix et al. 2009
). However, the observed biological responses to SKI-606 treatment are consistent with existing knowledge of the roles of Src in angiogenesis, tumor cell survival, proliferation and motility (Finn, 2008
). Src inhibition alone may be responsible for a large part of the observed biological effect on these tumors.
Apoptosis outside of obviously necrotic areas was not increased significantly in tumors treated for either 18 hours (data not shown) or two weeks. Thus, the response of tumor cells to SKI-606 appears to be decreased proliferation but not elevated cell death. Within tumor cells, Src binds to and mediates the oncogenic signaling of Polyoma middle T antigen (Courtneidge and Smith, 1983
). While the presence of Src is required for PyMT-induced tumors to form in the mammary gland (Guy et al., 1994
), deleting Src is not equivalent to inhibiting its kinase activity, as demonstrated by partial restoration of osteoclast function by expression of catalytically inactive Src in Src-deficient osteoclasts (Schwartzberg et al., 1997
). SKI-606 treatment for 4 weeks resembles the result of genetically inactivating Src. This is consistent with Src inhibition being an important mediator of the effects of SKI-606.
PyMT activates both Src and PI3 kinase pathways, much like the combination of ErbB2 and ErbB3 signaling. Src is also activated by ErbB2 receptor signaling (Muthuswamy et al., 1994
), directly associates with the same receptor (Kim et al., 2005a
) and facilitates the heterodimerization of ErbB2 and ErbB3 (Ishizawar et al., 2007
). Thus, SKI-606 might be expected to have similar effects on ErbB2-driven mammary tumors as those observed here for PyMT-driven tumors.
Ezh2 is a core component of the Polycomb Repressive Complex 2 (PRC2), that mediates gene repression by methylating lysine 27 on histone H3 and recruiting histone deacetylase (Bracken and Helin, 2009
). PRC2 may maintain pluripotency by silencing developmental regulators that drive differentiation (Lee et al., 2006
). Poorly differentiated human tumors show preferential repression of PRC2-regulated genes and this expression pattern is associated with poor clinical outcome. There is a strong correlation between elevated Ezh2 levels and poorly differentiated breast carcinomas (Kleer et al., 2003
). Deregulated expression of Ezh2 and its downstream target genes may play a critical role in maintaining the arrested differentiation phenotype observed in aggressive tumors.
Treatment of MMTV-PyMT tumors with SKI-606 resulted in a rapid reduction in Ezh2 protein, and a modest increase in E-cadherin mRNA, as might be expected from the transcriptional inhibition of the E-cadherin promoter by Ezh2, (Cao et al., 2008
). However, E-cadherin protein and RNA are expressed within PyMT tumor cells suggesting that EZH2 is not sufficient to silence E-cadherin in this system. SKI-606 may alter Ezh2 levels indirectly through miR-101; a group of microRNAs that have been shown to negatively regulate Ezh2 levels (Varambally et al., 2008
). The relatively rapid inhibition of Ezh2 expression following SKI-606 treatment is consistent with a functional and early role of Ezh2 rather than only a reflection of the later differentiated state of treated tumors. This is not likely due only to an anti-proliferative effect because a single treatment with SKI-606 for 18 hours did not result in a significant decrease in PCNA labeling index (not shown), even though Ezh2 protein was clearly decreased. Over-expression of Ezh2 in the mouse mammary epithelium leads to intraductal hyperplasia and delayed involution (Li et al., 2009
). Similarly, MMTV-driven expression of activated Src causes mammary hyperplasia and arrested lobuloalveolar development (Webster et al., 1995
), suggesting that both proteins may cooperate to restrict the cell fates and maintain the undifferentiated cellular phenotypes that are common in aggressive breast cancers. The restriction of MMTV-PyMT tumors by SKI-606 induced differentiation may be mediated by both tumor and host cell responses. The restriction of mammary tumor progression by induced differentiation is a relatively rare example that stimulates consideration of differentiation therapy. It will be of great interest to determine the link between Src inhibition and Ezh2 expression in developing breast cancer.