Multiple, functionally distinct proteins are seen dramatically altered in their expression in the resis tant cell sellekchem line. Importantly, ER regulated proteins such as cathepsin D and trefoil factor1 were down regulated, suggesting that suppression of ER signaling pathways is characteristic of tamoxifen resistance Inhibitors,Modulators,Libraries in vitro. Down regulation of cathepsin D and TFF1 PS2 has also been reported in antihormone treated breast cancer cells. Several of the up regulated proteins are involved in the compensatory mechanisms for survival and prolif eration in response to the anti estrogen challenge. For example, up regulation of TROP2 suggests increased survival signaling by activating ERK1 2 mediated cell cycle progression.

Overexpression of the antiapop totic protein, CLU in the tamoxifen resistant cells sug gests that it plays a role in counteracting the growth inhibition effects of tamoxifen. Another group of differentially expressed proteins Inhibitors,Modulators,Libraries are associated with increased cancer cell motility and inva siveness, Inhibitors,Modulators,Libraries which include EphA2, BCAS1, S100 protein family members, Rho family members, Ral A, Rab family members, Cdc42, MARCKS, Ezrin, Galectins 1 and 3 among others. These proteins are generally up regulated and appear to regulate the cytoskeleton dynamics of the resistant cells leading to a more motile and aggressive phenotype. To determine if the observed proteomic changes are due to acquired tamoxifen resistance or other changes including passaging the MCF 7 cells for 12 months, a three way quantitative proteomic control experiment was performed in which an early passage 13, mid passage 25, and late passage 50 MCF 7 control cells are compared.

A total of 635 proteins were compared for their relative abundances by the fold change ratios with statistical assessment. These data confirm that there are no significant proteomic altera tions within the Inhibitors,Modulators,Libraries MCF Inhibitors,Modulators,Libraries 7 control cells after a prolonged period of culture that are comparable to those occurring in the MCF 7 TamR cells. The relatively small fold changes in some protein expressions are not associated with the consistent, statistically significant changes occurring in the MCF 7 TamR resulting from develop ment of resistance to tamoxifen. Overall, these data demonstrate that the progressive culturing of cells over a year in tamoxifen results in changes that are distinct from matched parental cells grown under normal cul ture medium conditions. Proteomic differential expressions are consistent with those at the transcriptional 2nd el To investigate whether the changes observed in protein expression are a result of transcriptional regulation, we performed quantitative before real time PCR of 20 differentially expressed proteins.