RETRACTED ARTICLE: Extranuclear ERα is associated with regression of T47D PKCα-overexpressing, tamoxifen-resistant breast cancer

Abstract

BackgroundPrior 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 (PKCα)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCα preclinical model is tamoxifen-resistant, hormone-independent, yet is inhibited by 17β-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCα tumor regression requires extranuclear ERα and interaction with the extracellular matrix.MethodsT47D:A18/PKCα 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 (ERα) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERα with caveolin-1.ResultsWe report that although T47D:A18/PKCα cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERα rapidly translocates to extranuclear sites during T47D:A18/PKCα tumor regression in response to both raloxifene and E2, whereas ERα 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 ERα to the nucleus. T47D:A18/neo tumors that do not overexpress PKCα maintain ERα in the nucleus during tamoxifen-mediated regression. An association between ERα and caveolin-1 increases in tumors regressing in response to E2.ConclusionsExtranuclear ERα plays a role in the regression of PKCα-overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERα in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCα-expressing tumors encountered in the clinic.

Novel Selective Estrogen Mimics for the Treatment of Tamoxifen-Resistant Breast Cancer

“…In an effort to find a potential alternative to E2, and to expand on positive data obtained in vivo with RAL (11), two benzothiophene SEMs, BTC and TTC-352 (Figure 2A and 2B), were selected from a library of compounds and screened by DNA growth assay in 2D.…”

“…However, RAL did not yield complete tumor regression and importantly was without activity in 3D cultures (11). In contrast to RAL, the SEMs, BTC and TTC-352, are ER agonists in 2D cultures (Figure 2) and are able to inhibit both T47D:A18/PKCα and T47D:A18-TAM1 colony formation in 3D (Figures 3B and 3C).…”

“…E2 was administered via silastic capsules (1.0 cm) implanted subcutaneously between the scapulae, producing a mean serum E2 level of 379.5 pg/mL (11, 15). BTC and TTC-352 were administered p.o.…”

“…Tumor sections (4 μm) were cut from paraffin blocks and prepared for IF staining by deparaffinization and rehydration as previously described (11). Briefly, antigen retrieval was performed by incubating slides in Tris-EDTA (pH = 9.0) buffer.…”

“…Since treatment with E2, DES and TAM are now all associated with side effects including increased risk of thromboembolic disorders and unwanted agonist-driven uterine growth, we sought an alternative treatment strategy that would have therapeutic efficacy in the TAM-resistant setting. We have previously reported that TAM-resistant T47D:A18/PKCα tumors regress upon treatment with both E2 and the benzothiophene SERM raloxifene (RAL), although the effects of RAL did not persist after treatment withdrawal (11). RAL has a favorable antiestrogenic profile in the uterus and has proven safety over 15 years of clinical use in postmenopausal osteoporosis and breast cancer chemoprevention.…”