We predicted that Brk transgene expression in the mammary gland may increase p38 MAPK activity, perhaps leading to prolonged luminal epithelial cell survival and delayed involution. reference 2 FFPE sections from involuting mammary glands were processed for phospho p38 MAPK IHC, digitally analyzed and assigned H scores, data represent the degree of phospho p38 MAPK positivity and staining intensity. Notably, at both Days 4 and 6 of involution, we detected an approximately three fold increase in Inhibitors,Modulators,Libraries the H scores of glands from Brk transgenic mice relative to glands from same day wild type mice. Both the number of cells staining for phospho p38 and the intensity of staining were increased. These data suggest that Brk can drive p38 MAPK signaling in vivo, activated p38 MAPK may act as a pro survival signal in this setting, leading to delayed mammary gland involution.

Brk dependent signaling and pro survival are linked events in vitro To further link Brk expression to activation of p38 MAPK in the mouse model, we introduced Inhibitors,Modulators,Libraries flag tagged Brk or the Flag vector control into Sik null HC11 murine mammary epithelial cells by transient transfection and treated cells with the breast lactogen, prolactin. p38 MAPK is abundantly expressed in these cells. Following a time course of prolactin treatment, Western blotting revealed robust phosphory lation of p38 in both Flag vector control and Brk Flag transfected cells. However, in HC11 cells expressing Brk Flag, both the intensity and kinetics of p38 phos phorylation were increased relative to vector control cells.

Notably, transient expression of Flag Brk in HC11 cells also induced increased basal p38 MAPK phosphorylation. As a control for intact prolactin signaling and Brk dependent actions, we also blotted Inhibitors,Modulators,Libraries for phos phorylated STAT5, a known substrate of activated Brk. Similar to the published results of Weaver and Silva Inhibitors,Modulators,Libraries in breast cancer cells, we also observed Brk mediated regulation of p STAT5 Inhibitors,Modulators,Libraries in vitro, but only in prolactin treated cells. Phospho selleck chemicals Tubacin STAT3 was not appreciably altered by these treatments. To further link Brk overexpression to activation of p38 MAPK, we made use of our previously engineered human mammary epithelial cell lines stably expressing either a vector control or wild type Brk. Again, Brk expression alone resulted in increased basal phosphorylation of p38 in untreated cells, EGF induced activation of p38 was also slightly increased upon Brk expression relative to vector controls. Finally, EGF dependent activation of p38 MAPK was effectively blocked upon knock down of endogenous Brk in Brk positive breast cancer cells. Recent in vitro studies have demonstrated that Brk promotes anchorage independent survival.