Abstract P6-04-24: Overexpression of protein kinase C alpha differentially activates transcription factors in T47D breast cancer cells in the presence of 17β-estradiol both in the 2D and 3D environments.

Abstract

Abstract Background: Our lab has previously shown that overexpression of protein kinase C alpha (PKCα) results in a hormone independent, tamoxifen resistant phenotype in the T47D:A18 breast cancer cell line. Moreover, 17β-estradiol (E2) inhibits colony formation of T47D/PKCα in 3D Matrigel and tumor growth in vivo but not in the 2D environment (Zhang et al, Mol. Cancer Res, 2009). Differential transcriptional activation may account for the phenotypic changes observed in T47D/PKCα cells. In this study, we sought to identify transcription factors (TF) differentially activated in T47D:A18/neo versus T47D:A18/PKCα grown in 3D Matrigel in the presence and absence of E2, and compare these TFs with those activated in the 2D environment using a high-throughput screening (HTS) technology. Methods: A system for rapid, non-invasive, large scale, dynamic quantification of TF activity was developed (Weiss et al, PLoS ONE, 2010). Reporter constructs were created containing a TF binding site that preceeds a basal promoter to produce firefly luciferase (Fluc) using a lentiviral backbone. The control construct contained only the basal promoter and was used for normalization. T47D/PKCα and T47D/neo cells were infected with lentiviral reporter constructs. Cells were seeded either on top of Matrigel or without Matrigel in 384 well plates in E2-free media. After 3 days, cells were treated with either E2 (10–9M) or vehicle (0.1% ethanol). Fluc activity was assessed by bioluminescence imaging at 24, 48 and 72 hours following treatment using Xenogen IVIS Spectrum imaging system. Results: Initial findings showed differential transcriptional activities between the parental T47D/neo and T47D/PKCα cell lines at the basal level (without E2 treatment) in the 3D environment. Specifically, TFs involved in tumorigenesis (such as ETS1, STAT5) or the EMT process (such as SNAI1) were more highly activated in T47D/PKCα cells. Furthermore, E2 has a modulating effect on the activities of TFs in the two cell lines over time; observed both in the 2D and 3D environments. In particular, some TFs, such as Oct4, ETS1, and Stat5; were modulated (either induced or suppressed) by E2 only in T47D/PKCα cells. Conclusion: These findings suggest that overexpression of PKCα alters TF activity in T47D breast cancer cells both basally and in response to E2. This HTS technology allowed us to reveal the dynamic regulation of TF activation in PKCα overexpressing T47D breast cancer cells. We are now beginning to understand the mechanism whereby PKCα may mediate differential response to E2 dependent on the microenvironment. This HTS approach is likely to lead to the identification of new therapeutic targets. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-24.