Src kinase is one of the im portant molecules of the signalosome complex which plays a critical role in E2 mediated nongenomic signaling. It has been reported in the literature that Her 2 upregulates and activates PKC through src kinase in Her 2 mediated things cancer cell invasion. Longo et al. has shown that a PKC src kinase ER interaction is critical in the modulation of estrogen responsiveness and the dif ferentiation process in osteoblasts. However, we were unable to detect a physical interaction between PKC and ER, Her2 or src in our tumor model. We detected a physical interaction between ER and caveolin 1 by co IP. These results suggest that caveolin 1 may be responsible for transporting ER to the plasma membrane during E2 induced tumor re gression.

Palmitoylation of ER is known to be necessary for the physical association with caveolin 1 and in particu lar palmitoylation of the E domain of ER at C447 along with nine flanking amino acids are required for association with caveolin 1. The ER caveolin 1 complex in turn facilitates the translocation of the caveolae rafts to the plasma membrane. Caveolin 1 serves Inhibitors,Modulators,Libraries as a scaffold pro tein at the membrane in the recruitment of signaling mole cules to form a signalosome complex that can include ER. Taken together these results suggest that perhaps PKC is capable of modifying the interaction of ER and caveolin 1, potentially Inhibitors,Modulators,Libraries at the membrane via the proposed signalosome to effect tumor regression. It is interesting to note that ER caveolin 1 complex formation correlates with durable tumor regression produced with E2, but not with transient tumor regression as observed with RAL, nor with proliferating T47D,A18 PKC tumors.

Although ER translocation to extranuclear sites Inhibitors,Modulators,Libraries does occur in Matrigel in response to E2, colony regression is not initiated perhaps because a com ponent in the tumor microenvironment is also required to initiate the regression signal. As shown in Figures 3C D, E2 induced tumor regression occurs rapidly and tumors are gone within 2 3 weeks. Matrigel results Inhibitors,Modulators,Libraries reveal that the translocation Inhibitors,Modulators,Libraries of ER may be an early event as ER was seen in the membrane and cytoplasm in some colonies at 24 h further illustrating a rapid response to E2 treat ment. Our results regarding ER translocation in the Matrigel environment compared with in vivo tumors highlight the importance of the ECM in triggering tumor regression.

Brefeldin A protein transport Since we and others have reported that PKC expres sion can be a predictive marker of TAM resistance our T47D,A18 PKC model suggests that detection of extranuclear ER can be used to monitor therapeutic re sponse in TAM resistant, PKC expressing breast can cers. Unfortunately, extranuclear ER is not currently measured clinically and although pathologists may ob serve such staining, it is not reported. A recent report by Welsh et al. with the purpose of testing a panel of ER specific antibodies to detect non nuclear ER in clinical specimens found the average incidence to be only 1. 5%.