Estradiol Induced Regression of T47D:A18/PKCα Tumor Involves Translocation of Estrogen Receptor alpha from the Nucleus to the Cytoplasm.

Abstract

Abstract Background: Resistance to tamoxifen (TAM) is one of the most challenging problems in the treatment of breast cancer. In our laboratory we have developed a PKCα overexpressing T47D:A18 cell line which is TAMresistant and 17β estradiol (E2) independent compared to the E2 dependent T47D:A18/neo cell line (Chisamore et al, 2001). PKCα overexpression is known to be associated with TAM treatment failure in breast cancer (Tonetti et al, 2003). This T47D:A18/PKCα model exhibits E2-induced tumor regression in in vivo or when grown in matrigel (3D matrix) but not on 2D plastic. Recently we have demonstrated that tumor regression involves participation of estrogen receptor alpha (ERα), Fas/FasL and the extracellular matrix (Zhang et al, 2009). Interestingly membrane impermeable E2-BSA conjugate mediates growth inhibition of T47D:A18/PKCα colonies in matrigel similar to E2 suggesting a potential role of extranuclear ERα. On the basis of our preliminary data we investigated whether extranuclear ERα may play a critical role in E2-induced tumor regression.Materials and methods: T47D:A18/PKCα cells were injected into mammary fat pads of 20 ovariectomized athymic mice. Mice were left untreated and tumors were allowed to grow until the mean tumor size reached 0.5 cm2, then 10 mice were implanted with a 1.0 cm E2 capsule and the other 10 continued with no treatment (control). T47D:A18/neo tumors were also established in 10 athymic mice in the presence of E2. Four micrometer thick sections were prepared from the paraffin embedded T47D:A18/PKCα tumor tissues of control and E2-treated mice and T47D:A18/neo tumors. For immunofluorescence staining, alexa fluor 488 and Cy3 fluorescence tagged secondary antibodies were used against ERα and β-actin primary antibodies respectively.Results: Immunofluorescence images of T47D:A18/PKCα tumor sections from untreated control mice showed that the ERα was mainly localized in the nucleus. Interestingly, treatment with E2 resulted in significant translocation of ERα from the nucleus to the cytoplasm and the plasma membrane of tumor cells. Immunofluorescence analysis of tumor sections obtained from E2 treated mice indicated that approximately 5-10% of cells contained ERα localized to the plasma membrane whereas the majority of ERα was localized to the cytosol. However in T47D:A18/neo tumors the majority of ERα was found in the nucleus suggesting the involvement of the ERE mediated classical pathway in tumor growth.Discussion: This is the first time we are reporting that E2-induced T47D:A18/PKCα tumor regression is accompanied by translocation of ERα to extranuclear sites. E2-induced tumor regression observed in our pre-clinical model suggests E2 or an E2-like compound may be a potential treatment option for patients harboring PKCα overexpressing tumors. Furthermore the extranuclear ERα signaling pathway may be an attractive therapeutic target to treat PKCα overexpressing tumors that are refractory to current endocrine therapies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4140.