Prior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKCalpha)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCalpha preclinical model is tamoxifen resistant, hormone independent, yet is inhibited by 17beta-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCalpha tumor regression requires extranuclear ERalpha and interaction with the extracellular matrix.

T47D:A18/PKCalpha cells were grown in vitro using two-dimensional (2D) cell culture, three dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ERalpha) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERalpha with caveolin-1.

We report that although T47D:A18/PKCalpha cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERalpha rapidly translocates to extranuclear sites during T47D:A18/PKCalpha tumor regression in response to both raloxifene and E2, whereas ERalpha is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ERalpha to the nucleus. T47D:A18/neo tumors that do not overexpress PKCalpha maintain ERalpha in the nucleus during tamoxifen-mediated regression. An association between ERalpha and caveolin-1 increases in tumors regressing in response to E2.

Extranuclear ERalpha plays a role in the regression of PKCalpha overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERalpha in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCalpha expressing tumors encountered in the clinic.