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

Abstract

Background: 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 (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.

Methods: T47D: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.

Results: We 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.

Conclusions: Extranuclear 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.

Figure 1

T47D:A18/PKCα cells are resistant to 4-OHT and RAL in 2D and 3D cell culture. DNA and Matrigel™ colony formation assays were performed as described in materials and methods. Cells were grown in the presence of vehicle (DMSO, 0.1%), E2 (10^-9M), 4-OHT (10^-7M) or RAL (10^-7M) with media changes every three days. A. T47D:A18/neo cells. B. T47D:A18/PKCα cells. RFU, relative fluorescence units. C. Quantification of T47D:A18/PKCα colonies. Graphs are representative of at least three independent experiments and error bars represent SEM. *P < 0.05 compared to vehicle; by one-way ANOVA followed by Bonferroni’s post-test. D. Photographic representation of T47D:A18/PKCα colonies. Total magnification: 6X.

Figure 2

RAL inhibits TAM-resistant T47D:A18/PKCα xenograft tumors. Xenograft tumors were formed as described in materials and methods. Treatments for TAM and RAL were given by oral gavage 5 days/week. A. T47D:A18/PKCα tumors. Mice (10/group) were given TAM (1.5 mg/day) or RAL (0.5 mg/day or 1.5 mg/day). B. LT-TAM-treated T47D:A18/PKCα tumors. Mice (10/group) received TAM (1.5 mg/day) or RAL (1.5 mg/day). C. T47D:A18/neo tumors. Mice (6/group) were given no treatment, E2 capsule (1.0 cm), TAM (1.5 mg/day) or RAL (1.5 mg/day). The dotted line indicates initiation of TAM or RAL treatment following 8 weeks of E2 treatment. Error bars represent SEM.

Figure 3

Growth of T47D:A18/neo and T47D:A18/PKCα xenograft tumors. Xenograft tumors were formed as described in materials and methods. A. T47D:A18/neo tumors (NT, 15 mice/group and E2, 3 mice/group). B. T47D:A18/PKCα tumors (10 mice/group). C. T47D:A18/PKCα tumors. Tumors were grown to an average size of 0.5 cm². Mice were then randomized into NT, RAL or E2 groups (large arrow, 9 mice/group). Two weeks later RAL treatment was stopped (small arrow). D. T47D:A18/PKCα tumors (5 mice/group). Tumors were grown to an average size of 0.3 cm². Mice were then randomized into NT or E2 groups (arrow).

Figure 4

ERα localizes to extranuclear sites in E2- and RAL-induced T47D:A18/PKCα regressing tumors. A. Tissue sections were immunostained as described in materials and methods. Images are representative photographs of immunostained tumor sections. Sections were costained for ERα (green) and nuclei (blue). Scale bar = 20 μm. All images were acquired and processed using parameters described in materials and methods. PKCα, T47D:A18/PKCα; neo, T47D:A18/neo. B. Quantification of ERα localization in tumor sections. At least three fields from each tumor were counted. T47D:A18/neo is represented by two individual tumors. Bars representing T47D:A18/PKCα tumors show the mean (± SEM) of three individual tumors. ***, P < 0.001 compared to PKCα NT, TAM and RAL W/D by two-way ANOVA. C. Expression of ERα in whole cell tumor lysates. Molecular weights of ERα and β-actin are 67 kDa and 42 kDa, respectively. Values represent β-actin-normalized ERα expression relative to T47D:A18/neo E2-treated tumors.

Figure 5

ERα/caveolin-1 complex formation in response to E2, TAM and RAL treatment in T47D:A18/PKCα tumors. A. Representative western blot of co-IP experiments in T47D:A18/PKCα and T47D:A18/neo tumor extracts as detailed in materials and methods. B. Densitometric quantification of three co-IP experiments from three independent tumors for each group. Error bars represent SEM. *, P < 0.05 compared to all groups determined by one-way ANOVA followed by Bonferroni’s post-test.

Figure 6

E2 induces complete relocalization of ERα in established T47D:A18/PKCα colonies after 10 days. A. T47D:A18/neo colonies (neo) and T47D:A18/PKCα colonies (PKCα) colonies were immunostained for ERα (green) and nuclei (blue). All images were acquired and processed using parameters described in materials and methods. Colonies were grown for 10 days then treated for 10 days with vehicle (EtOH, 0.1%), E2 (10^-9 M), 4-OHT (10^-7 M) or RAL (10^-7M). Scale bar = 20 μm. B. Expression of ERα in whole cell colony lysates. Molecular weights of ERα and β-actin are 67 kDa and 42 kDa, respectively. Values represent β-actin-normalized ERα expression relative to T47D:A18/neo E2-treated colonies.