treated with do ycycline in drinking water. Within three weeks, all the 31 mice injected with control cells gave rise to tumors with a mean diameter of considering 8 mm. In contrast, 38% of mice injected with p130Cas silenced cells did not give rise to detectable tumors and the remaining 45 mice developed small tumors, with a mean diameter of 2 mm. Interestingly, p130Cas silencing was sufficient to halt tumor growth in mice that have already developed tumors with a diameter of 3 to 4 mm. Indeed, by adding do ycycline to drinking water two weeks after cell injection, p130Cas silenced tumors regressed, becoming undetectable by palpation within two to three weeks, while control tumors contin ued to grow. Consistently, after do ycycline withdrawal p130Cas silenced tumors resumed growing.

These data strengthen the in vivo rele vance of p130Cas as a major regulator of the tumorigenic properties of mesenchymal breast cancer cells. We have previously shown that intranipple injection of p130Cas siRNAs in the mammary gland of Balb c NeuT mice sig nificantly decreases the number of cancer lesions com pared to glands injected with control siRNAs, with a significant downregulation of proliferative and survival pathways. Overall these data indicate that tight modula tion of p130Cas levels can affect in vivo tumor properties of distinct breast cancer subtypes, implying the compel ling need of studying its transcriptional regulation in nor mal mammary epithelial cells and in tumors in the near future.

Hemato ylin and eosin staining of tumor sections showed that tumors derived from p130Cas silenced cells consisted of cells with an epithelial like shape, while the control tumors presented elongated, mesenchymal cells. Moreover, immunohistochemis try analysis indicated that tumors from p130Cas silenced cells were characterized by decreased vascularization and proliferation, and increased apoptosis. Western blot analysis of p130Cas silenced tumors showed a significant in vivo p130Cas silencing together with Co 2 downregulation, compromised activation of c Src and JNK kinases and decreased e pression of Cyclin D1. A parallel downregu lation of Snail, Cilengitide Slug and Twist e pression was also detected, indicating that p130Cas silencing compromises tumor growth through inhibition of cell signaling controlling cell cycle progres sion and the acquirement of epithelial like features.

In parallel, syngeneic mice were subcutaneously injected with 105 Co 2 silenced or control A17 cells and treated with do ycycline in drinking water. As shown in Figure 3D, while mice injected with control cells gave rise to tumors with a mean diameter of 10 mm within si weeks, mice injected with Co 2 silenced cells give rise to barely detectable Bosutinib cost tumors. Taken together these data show that p130Cas Co 2 a is controls in vivo survival and proliferative pathways of mesenchymal breast can cer cells and silencing of either p130Cas or Co 2 is suf ficient for switching cells to an epithelial state leading to impaired