Introduction
Patients with estrogen receptor α (ERα)-positive breast cancer are candidates for treatment with endocrine therapies such as the selective estrogen receptor modulator (SERM) tamoxifen (TAM), aromatase inhibitors (AIs) letrozole, anastrozole, or exemestane or the selective estrogen receptor downregulator (SERD), fulvestrant. However, both
de novo and acquired endocrine resistance represent a significant clinical problem. Mechanisms of endocrine resistance include activation of growth factor signaling and downstream pathway activation including phosphatidyl inositol 3-kinase (PI3K) and mitogen activated protein kinase (MAPK) (reviewed in[
1]). Numerous reports from our laboratory and others suggest that activation of protein kinase C (PKC) signaling, specifically PKCα, is associated with endocrine resistance in the clinic[
2‐
4].
We developed and previously described a preclinical TAM-resistant model where PKCα is stably overexpressed in the T47D:A18 breast cancer cell line[
5]. Under two-dimensional (2D) culture conditions, T47D:A18/PKCα cells exhibit both TAM-resistance and hormone-independence characterized by proliferation in the presence and absence of 17β-estradiol (E2). Paradoxically when T47D:A18/PKCα cells are grown
in vivo as xenograft tumors, E2 administration inhibits tumor growth and induces complete tumor regression in established tumors[
6,
7]. Similarly, we previously reported that the MCF-7 TAM tumor model that exhibits the E2-inhibitory phenotype[
8] also overexpresses PKCα[
7]. Previous mechanistic studies in our laboratory determined that E2-induced T47D:A18/PKCα tumor regression is dependent upon ERα, increased Fas/FasL–mediated apoptosis and decreased AKT signaling[
9]. Moreover, we showed that T47D:A18/PKCα cultured in three-dimensional (3D) Matrigel™ partially recapitulated the
in vivo E2-inhibitory effects by inhibiting colony formation. Further, the membrane impermeable E2-BSA conjugate was shown to inhibit T47D:A18/PKCα colony formation in a manner similar to E2, suggesting the potential involvement of a plasma membrane localized ERα[
9].
In addition to genomic signaling by nuclear ERα, examples of nongenomic rapid responses of extranuclear ERα in the presence of E2 are abundant in the literature[
10‐
14]. Extranuclear ERα plays an important role in cell proliferation, cell cycle regulation and blockade of cell death by activating MAPK[
15,
16] and the AKT signaling pathways[
17‐
19] in breast cancer cell lines. There is evidence that extranuclear ERα interacts with several growth factor receptors as a mechanism for endocrine-resistant breast cancer by promoting downstream proliferation and survival signals[
20‐
22].
In the present study we determined that in 2D and 3D cell culture, TAM-resistant T47D:A18/PKCα cells exhibit cross-resistance to raloxifene (RAL). Similar to the paradoxical effects of E2 in this model, RAL induces T47D:A18/PKCα tumor regression. Based on our previous findings showing the dependence of ERα in tumor regression and the involvement of extranuclear ERα in colony inhibition, in this study we determined the subcellular localization of ERα in T47D:A18/PKCα tumors during regression (E2 and RAL) and during proliferation (absence or presence of TAM) using immunofluorescence (IF) confocal microscopy. Interestingly, ERα localizes to the nucleus in tumors proliferating in a hormone-independent manner or in mice treated with TAM, whereas ERα localizes to extranuclear sites in tumors undergoing regression with either E2 or RAL. Withdrawal of RAL treatment results in the resumption of T47D:A18/PKCα tumor growth accompanied by relocalization of ERα back into the nucleus. We further report an association of extranuclear ERα with caveolin-1 suggesting a mechanism whereby ERα may influence growth factor signaling. These findings are in agreement with our previous report that E2-induced tumor regression is accompanied by downregulation of AKT signaling in this model[
9]. To our knowledge this is the first study to report an association of extranuclear ERα with tumor regression, as opposed to the activation of growth factor receptor signaling. With the renewed interest in the use of E2 for treatment of endocrine resistant breast cancer[
23,
24], our model offers a potential inhibitory mechanism involving extranuclear ERα.
Discussion
In this paper we have shown by IF confocal microscopy that ERα translocates from the nucleus to the extranuclear space upon E2 and RAL-induced tumor regression in our T47D:A18/PKCα preclinical TAM-resistant model. This model is clinically relevant as evidenced by the reported success of E2 in the clinic[
23,
24]. We initially associated PKCα expression with TAM resistance[
2], and others further identified PKCα as a marker of endocrine resistance and breast cancer aggressiveness[
3,
4]. Extranuclear ERα was previously reported to play a role in endocrine-resistant breast cancers specifically by interacting with growth factor receptors to activate proliferative and pro-survival signals[
20‐
22]. However we demonstrate here that ERα translocation is associated with tumor regression only in PKCα overexpressing tumors in response to E2 and RAL. Our findings imply that a specific subset of endocrine-resistant breast cancers that express PKCα may be uniquely susceptible to E2 therapy. Although the literature is conflicting regarding the level of PKCα expression in breast cancers compared to the normal breast[
32‐
36], variability in PKCα expression amongst breast cancers and the link to endocrine resistance and tumor aggressiveness is clear. Based on three reports in the literature, the prevalence of PKCα expression in all breast cancers ranges between 28% to as high as 70%[
3,
4,
37]. Even if the lowest estimate of 28% prevalence is the most accurate, this still represents a significant number of patients that may benefit from E2 treatment.
There are numerous reports of nongenomic signaling by estrogen in breast cancer cell lines[
38,
39] and there is evidence that this pathway is upregulated in endocrine resistant breast cancers. Translocation of nuclear ERα to extranuclear sites is reported to be involved in cytoskeletal remodeling, migration and invasion[
40] and recently shown to play an important role in breast cancer cell motility and metastasis[
41]. High expression of the MTA1 protein is reported to sequester ERα in the cytoplasm and activate MAPK signaling[
42], and the same group reported that overexpression of Her-2 causes ERα nuclear to cytoplasmic translocation[
43]. Fan
et al.[
44] showed that long term exposure to TAM causes translocation of ERα from the nucleus to the cytoplasm and enhances the interaction between ERα and EGFR. All of these examples in the literature describe the activation of signaling pathways by extranuclear ERα leading to cancer cell proliferation and survival. However in our study, we present a novel finding that translocation of ERα from the nucleus to extranuclear sites occurs following E2- and RAL-induced T47D:A18/PKCα tumor regression. We previously reported that E2-induced regression is accompanied by apoptosis mediated in part by Fas/FasL and downregulation of the AKT pathway[
9]. An additional novel finding is that TAM and RAL elicit opposite growth effects in our T47D:A18/PKCα tumor model. We hypothesize that PKCα, a cytoplasmic protein that translocates to the plasma membrane when activated[
45], may physically interact with other growth factor receptors and signaling pathways[
46]. A recent publication by Guttierez et al. shows that translocation of ERα to the plasma membrane in response to E2 results in activation of PKCα/ERK 1/2 signaling in anterior pituitary cells, yet PKCα is not responsible for mediating the physical translocation of ERα to the plasma membrane[
47]. Src kinase is one of the important molecules of the signalosome complex which plays a critical role in E2-mediated nongenomic signaling[
48]. It has been reported in the literature that Her-2 upregulates and activates PKCα through src kinase in Her-2 mediated cancer cell invasion[
49]. Longo
et al. has shown that a PKCα-src kinase-ERα interaction is critical in the modulation of estrogen responsiveness and the differentiation process in osteoblasts[
50]. However, we were unable to detect a physical interaction between PKCα and ERα, Her2 or src in our tumor model.
We detected a physical interaction between ERα and caveolin-1 by co-IP (Figures
5A-B). These results suggest that caveolin-1 may be responsible for transporting ERα to the plasma membrane during E2-induced tumor regression. Palmitoylation of ERα is known to be necessary for the physical association with caveolin-1 and in particular palmitoylation of the E domain of ERα at C447 along with nine flanking amino acids are required for association with caveolin-1[
30,
31,
51,
52]. The ERα-caveolin-1 complex in turn facilitates the translocation of the caveolae rafts to the plasma membrane. Caveolin-1 serves as a scaffold protein at the membrane in the recruitment of signaling molecules to form a signalosome complex that can include ERα. Taken together these results suggest that perhaps PKCα is capable of modifying the interaction of ERα and caveolin-1, potentially at the membrane via the proposed signalosome to effect tumor regression. It is interesting to note that ERα/caveolin-1 complex formation correlates with durable tumor regression produced with E2, but not with transient tumor regression as observed with RAL, nor with proliferating T47D:A18/PKCα tumors (NT, TAM, RAL W/D). Although ERα translocation to extranuclear sites does occur in Matrigel™ in response to E2 (Figure
6), colony regression is not initiated perhaps because a component in the tumor microenvironment is also required to initiate the regression signal. As shown in Figures
3C-D, E2-induced tumor regression occurs rapidly and tumors are gone within 2–3 weeks. Matrigel™ results reveal that the translocation of ERα may be an early event as ERα was seen in the membrane and cytoplasm in some colonies at 24 h further illustrating a rapid response to E2 treatment. Our results regarding ERα translocation in the Matrigel™ environment compared with
in vivo tumors highlight the importance of the ECM in triggering tumor regression.
Since we and others have reported that PKCα expression can be a predictive marker of TAM resistance[
2‐
4] our T47D:A18/PKCα model suggests that detection of extranuclear ERα can be used to monitor therapeutic response in TAM-resistant, PKCα-expressing breast cancers. Unfortunately, extranuclear ERα is not currently measured clinically and although pathologists may observe such staining, it is not reported. A recent report by Welsh
et al.[
53] with the purpose of testing a panel of ERα-specific antibodies to detect non-nuclear ERα in clinical specimens found the average incidence to be only 1.5%. In an accompanying commentary, Levin points out that while it is possible that the number of breast tumors that express extranuclear ERα may indeed be small, it is also possible that more sensitive techniques are required to detect the very small ERα pools located outside of the nucleus[
54]. We offer the possibility that extranuclear ERα may be detected more frequently in PKCα-expressing tumors that are regressing possibly indicating a response to treatment. It remains to be seen whether other techniques will be developed that may improve the detection of extranuclear ERα in clinical specimens.
We have previously suggested that PKCα may be used as predictive biomarker for the use of E2 or an E2-like compound to effect tumor regression[
9], and in fact the utility of using E2 was demonstrated[
23]. We report here that not only E2, but RAL is capable of eliciting T47D:A18/PKCα tumor regression, despite the fact that these tumors are TAM-resistant. Further we have shown that following 5 years of TAM treatment, these tumors are still sensitive to RAL-induced tumor regression (Figure
2B). Although RAL may be considered as a potential treatment for patients with PKCα-expressing breast cancers, RAL is not as durable as E2 to elicit complete tumor regression (Figure
3D). Since RAL has poor bioavailability, we are currently testing a series of benzothiophene analogues in our T47D:A18/PKCα preclinical model for improved tumor inhibitory activity.
Methods
Reagents
For in vitro experiments dimethylsulfoxide (DMSO), ethanol, E2, 4-OHT and RAL were obtained from Sigma-Aldrich (St. Louis, MO USA). For in vivo experiments E2 and TAM were obtained from Sigma. RAL (Evista®, Eli Lilly and Company, Indianapolis, IN USA) was purchased from the University of Illinois at Chicago Hospital Pharmacy. Cell culture reagents were obtained from Life Technologies (Carlsbad, CA USA). Tissue cultureware was purchased from Becton-Dickinson (Franklin Lakes, NJ USA). The following antibodies were used: rabbit monoclonal ERα (for tissue and cells, SP1, Lab Vision, Thermo Scientific, Kalamazoo, MI USA), mouse monoclonal ERα (alternative epitope to confirm specificity for tissue, 1D5, N-terminal epitope, Abcam, Cambridge, MA USA), rabbit polyclonal ERα (for colonies, HC20, Santa Cruz Biotechnology, Santa Cruz, CA USA), and mouse monoclonal caveolin-1 (Clone2234, BD Transduction Laboratories, Franklin Lakes, NJ USA). Secondary antibodies included: anti-rabbit Alexa Fluor 488 (Life Technologies, Carlsbad, CA USA), anti-mouse Cy3 (Jackson Immunoresearch Laboratories, West Grove, PA USA) and HRP-cojungated anti-rabbit and anti-mouse (GE Healthcare UK Limited, Buckinghamshire, UK).
Cell culture conditions
T47D:A18/neo and T47D:A18/PKCα[
5] cells were maintained in RPMI 1640 with phenol red supplemented with 10% fetal bovine serum (FBS) and G418 (500 μg/ml) at 37°C, 5% CO
2. Prior to experiments cell lines were placed in phenol red-free RPMI 1640 supplemented with 10% stripped FBS (E2-depleted media) for 3 days and maintained in the same manner for the duration of experiments. Cell lines were tested for Mycoplasm contamination on a regular basis (MycoAlert™ Mycoplasm Detection Kit, Lonza Ltd., Rockland, ME, USA). Cell lines were not authenticated by the authors.
DNA growth assay
Cells were plated at a density of 15,000 cells/well in 24-well plates. Treatment media (vehicle, DMSO [0.1%], E2 [10-9M], 4-OHT [10-7M] or RAL [10-7M]) was added the following day (Day 1) and changed every three days. Growth was determined by incubating cells with Hoechst 33342 cell permeable dye (Life Technologies, Carlsbad, CA USA) for 1 h at 37°C and reading fluorescence at excitation 355 nm/emission 460 nm on a Perkin Elmer Victor3 V (Waltham, MA USA) plate reader.
Treatments (ethanol [0.1%], E2 [10-9M], 4-OHT [10-7M] or RAL [10-7M]) were added to liquefied phenol-red free Matrigel™ matrix (BD Biosciences, Franklin Lakes, NJ USA) and used to coat 6-well plates and solidified at 37°C for 30 min. Cells (5000) were seeded in E2-depleted media containing treatments on top of pre-gelled Matrigel™ and incubated at 37°C with 5% CO2. Treatment media were changed every three days. Colonies were stained with 0.25% crystal violet (Sigma-Aldrich, St. Louis, MO USA) solution for 30 min and then destained with 0.9% saline for 20 min at room temperature. Colony number was determined by counting five 1.0 cm2 areas.
Xenograft tumor establishment
All procedures involving animals were approved by the Animal Care and Use Committee of the University of Illinois at Chicago according to institutional and national guidelines. T47D:A18/neo and T47D:A18/PKCα tumors were established in 4–6 week old ovariectomized athymic nude mice (Harlan Laboratories) as previously described[
7]. LT-TAM tumors were derived by
in vivo serial transplantation in the presence of TAM for 5 years. Where indicated, mice were given the following treatments as previously described: E2 (1.0 cm silastic capsule, s.c.), TAM (1.5 mg/day, p.o.), RAL (0.5 mg/day, p.o.), or RAL (1.5 mg/day, p.o.)[
55]. Tumor cross-sectional area was determined at least weekly and sometimes daily using digital calipers and calculated using the formula: length/2 × width/2 × π. Mice were euthanized by CO
2 inhalation and cervical dislocation. Tumors were immediately excised and either fixed in 10% buffered formalin for paraffin block preparation or snap frozen in liquid nitrogen and stored at −80°C for co-immunoprecipitation and western blot analysis.
Tumor IF confocal microscopy and co-localization analysis
Tumors sections (4 μm) were prepared from paraffin blocks for IF staining by deparaffinization and rehydration. Antigen retrieval was performed by incubating slides in Tris-EDTA (pH = 9.0) buffer at 90°C and allowed to cool at room temperature for 45 min. Slides were blocked with antibody diluent (DAKO, Carpinteria, CA USA) for 20 min followed by primary antibody at 1:100 in antibody diluent for 1 h at room temperature. Slides were incubated with fluorescence-conjugated secondary antibodies at 1:100 in antibody diluent for 45 min at room temperature followed by 4’, 6-diamidino-2-phenylindole (DAPI) (1 μg/mL), DAKO, Carpinteria, CA USA) for 15 min and mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA USA). Confocal microscopy was performed with a Zeiss LSM 510 microscope (Carl Zeiss, Incorporated, North America, Thornwood, NY USA). The objective used was a C-Apochromat 63X with a numerical aperture of 1.2. Image acquisition scaling was X: 0.14 μm and Y: 0.14 μm and stack size was X: 142.86 and Y: 142.86, these two parameters were kept constant across samples. Pinholes and laser intensities were kept constant for each wavelength (green: λ = 488 nm, laser = 15%, pinhole = 228 μm and blue: λ = 405 nm, laser = 5%, pinhole 194 μm) across all samples. Images were modified following acquisition using the Zeiss LSM Image Browser by similarly enlarging images 2X and increasing the brightness and contrast by 10%.
Co-IP and western blot
Tumors were ground into a fine powder in liquid nitrogen and resuspended in cell lysis buffer (20 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, with protease [Sigma, St. Louis, MO] and phosphatase [Calbiochem, Bilerica, MA] inhibitor cocktails) and homogenized using a Polytron hand-held homogenizer (Fisher Scientific, Pittsburgh, PA USA). Protein concentration was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA USA). Equal amounts of total tumor extract (500 μg) were immunoprecipitated by rotating for 2 hr at 4°C with antibody followed by overnight rotation with protein-A Dynabeads (Life Technologies, Carlsbad, CA), at 4°C. Samples were washed and boiled for 10 min then eluted from beads with sample buffer containing 2-mercaptoethanol (Sigma, St. Louis, MO USA). Samples were subjected to 8% SDS-PAGE, followed by western blot with respective primary and secondary antibodies. Proteins were detected by chemiluminescence using a Chemi Doc Gel Documentation System (Bio-Rad Laboratories, Hercules, CA USA).
Cell IF microscopy
Cells were seeded in phenol red-containing media onto Lab-Tek II 4-well chamber slides (Millipore, Billerica, MA) at a density of 3 × 104 cells/well. The following day cells were placed in E2-depleted media for 3 days then given treatment media (DMSO [0.1%], E2 [10-9M], 4-OHT [10-7M] or RAL [10-7M]). For IF, cells were fixed in 100% methanol overnight at −20°C and stained as described above for tissue sections. Cells were imaged using Zeiss Axiovision Observer D1 microscope (Carl Zeiss, LLC, Thornwood, NY USA).
Colony IF microscopy
Colonies were formed by ding cells in Matrigel™ as described above and treated with DMSO (0.1%), E2 (10-9M), 4-OHT (10-7M) or RAL (10-7M). Colonies were extracted from the Matrigel™ by adding ice-cold PBS-EDTA to the rinsed and aspirated wells. Gel was lifted from the bottom of the well with a cell scraper and plates were shaken gently on ice. Colonies were then transferred to a conical tube and shaken on ice for an additional 30 min until Matrigel™ was completely dissolved, collected by centrifugation at 115g for 2 min and pipetted onto a slide. Slides were then fixed in ice cold methanol and stored at −80°C until staining (as described above). Confocal microscopy was performed with a Zeiss LSM 510 microscope. The objective used was a C-Apochromat 63X with a numerical aperture of 1.2. Image acquisition scaling was X: 0.14 μm and Y: 0.14 μm and stack size was X:142.86 and Y: 142.86, these two parameters were kept constant across samples. Pinholes and laser intensities were kept constant for each wavelength (green: λ = 488 nm, laser = 10%, pinhole = 200 μm and blue: λ = 405 nm, laser = 13%, pinhole 92 μm) across all samples. Images were modified following acquisition using the Zeiss LSM Image Browser by similarly enlarging images 2X and increasing the brightness and contrast by 10%.
Statistical analysis
The specific statistical test applied to the data is described in the figure legends. All of the statistics on the data were performed using GraphPad Prism 5.02 Software (La Jolla, CA USA).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BPW and MEM contributed equally to this study and contributed to writing portions of the manuscript. BPW made figures and designed layout. All authors contributed to xenograft experiments, HZ and YZ developed the LTTAM tumor model, HZ performed IF staining and microscopy on cell lines, BPW and MEM performed IF and confocal microscopy on tumor sections and colonies. DAT conceived of the study and wrote the manuscript. All authors read and approved the final manuscript.