Estrogen promotes stemness and invasiveness of ER-positive breast cancer cells through Gli1 activation

We examined the endogenous mRNA expression of breast cancer stem cell markers ALDH1, Gli1 and ER in several human breast cancer cell lines using real-time polymerase chain reaction (PCR; Figure 1A–C). Our data showed that ALDH1 and Gli1 mRNAs were detectable in all cell lines. We then used linear correlation analysis to evaluate the relationship among Gli1, ALDH1 and ER expression levels. We found that ER expression positively correlated with Gli1 and ALDH1 (Figure 1D & E). Next, we examined the expression of the ER protein using western blotting and immunofluorescence assays in MCF-7, HCC1428, MDA-MB-231 and BT549 cells. As shown in Figure 2A–C, ER expression was higher in MCF-7 and HCC1428 cells and barely detectable in MDA-MB-231 and BT549 cells.

Because ER expression was correlated with Gli1, we then asked whether estrogen could influence Shh pathway activation in breast cancer cells. MCF-7, HCC1428, MDA-MB-231 and BT549 cells were incubated with 10 nM estrogen (E2) with or without 1 μM 4-hydroxy tamoxifen (4OHT) for 4 days, after which Shh and Gli1 protein and mRNA expression were measured. In ER-positive MCF-7 and HCC1428 cells, Gli1 expression was significantly increased in estrogen-treated cells compared with that in control (ETOH-treated) cells. Additionally, 4OHT inhibited estrogen-induced expression of Gli1 (Figure 3A, B & Additional file1: Figure S1A). However, E2 failed to significantly increase Gli1 expression in ER-negative MDA-MB-231 and BT549 cells (Figure 3C, D & Additional file1: Figure S1B). Shh expression was not affected in any of the four cell lines tested. Our results indicated that estrogen activated the Shh/Gli1 pathway only in ER-positive breast cancer cells through noncanonical Shh signaling.

To elucidate the mechanism by which E2 activated the Shh/Gli1 pathway, we tested cyclopamine, a canonical inhibitor of Smo, in the Shh signaling pathway. Cyclopamine plus E2 were incubated with MCF-7 cells for 4 days. We then analyzed and compared Gli1 protein and mRNA expression levels in ETOH and E2-treated cells. Cyclopamine did not inhibit estrogen-induced activation of Gli1 (Figure 3E & F).

We also treated breast cancer cells with the Shh ligand to examine the effect of Shh on Gli1 and Ptch1 mRNA expression. Addition of various concentrations of Shh to these cells for 24 h increased both Gli1 and Ptch1 mRNA expression levels relative to untreated cells (Additional file2: Figure S2). These results indicated that Gli1 activation was not mediated by canonical Shh signaling. Given that E2 modulated Gli1 transcription, quantitative chromatin immunoprecipitation (qChIP) assays were performed in ETOH and E2-treated MCF7 cells to determine the mechanism of this E2 effect. We found increased ER protein binding to the Gli1 promoter (area #1), as well as to the Gli1 gene body (area #3) in E2-treated MCF7 cells compared with ETOH-treated control cells (Figure 3H). The occupancy of IgG at the Gli1 gene promoter was not changed by E2 treatment (Figure 3I). These results indicated that E2 induced transcriptional activation of Gli1, probably through enriching ER occupancy at the Gli1 gene promoter and gene body.

To determine whether Gli1 mediated E2-induced stemness and invasiveness in breast cancer cell lines, MCF-7 and HCC1428 cells were transfected with pSingle vectors carrying short hairpin RNA (shRNA) targeting Gli1. Cells were grown in the absence (shVEC transfected cells) or presence (shGli1-1 and shGli1-2 transfected cells) of Dox for 4 days. They were then harvested and analyzed using western blotting. As shown in Figure 4A and Additional file1: Figure S1C, Gli1 protein levels in shGli1-1 and shGli1-2 cells were significantly reduced when compared with those of the control (shVEC).

Cells were then transfected with shVEC, shGli1-1 or shGli1-2 (the latter two representing Gli1-knockdown cells) and treated with E2 for 4 days, following which the CD44⁺/CD24^–/low cell population was observed using flow cytometry. We found that treatment of shVEC cells with E2 induced a statistically significant expansion of CD44⁺/CD24^–/low stem-like cells. In Gli1-knockdown cells, E2 failed to significantly increase the proportion of CD44⁺/CD24^–/low cells (Figure 4B, C and Additional file1: Figure S1D & E). The parallel expression patterns of Gli1 and CSC markers indicated that Gli1 may regulate the expansion of CSCs in estrogen-treated ER-positive breast cancer cells (Figure 4A–C & Additional file1: Figure S1C–E).

Next, shVEC and Gli1-knockdown cells were cultured at very low densities (1 cell/μL) in 96-well plates containing serum-free medium with or without E2 for seven days. In the presence of E2, shVEC cells produced more and larger spheres compared with control cells (p < 0.01; Figure 4D–F). In Gli1-knockdown cells, E2 failed to significantly increase the number and size of these spheres (p < 0.01; Figure 4D–F). The significant inhibition in Gli1-knockdown cells of the estrogen-mediated increase in sphere formation suggested that Gli1 was indeed involved in the regulation of estrogen-induced self-renewal of breast CSCs.

The proliferative potential of these breast cells under the same conditions were also assessed using a colony formation assay. We observed that shVEC cells showed a significant increase in the number and size of colonies in the presence of E2. However, E2-induced proliferation was not observed in Gli1-knockdown cells (Figure 5A–C). Flow cytometric analyses of apoptosis and cell cycle progression further confirmed the role of Gli1 in E2-induced ER-positive breast cancer cell survival and proliferation (Additional file3: Figure S3).

To further investigate the mechanism through which Gli1 regulated CSCs, MCF7 cells were treated with E2 (0–10 nM) for 4 days. Gli1, Shh, ALDH1, Nanog, SOX2 and Bmi-1 expression levels were measured using real-time PCR and western blotting. As shown in Figure 6A and B, Gli1, ALDH1, Nanog, SOX2 and Bmi-1 expression levels all gradually increased with increasing E2 concentrations, whereas Shh was virtually unchanged. The expression of CSC markers was also measured following transfection of MCF-7 cells with shVEC or Gli1-knockdown constructs and subsequent treatment with E2. As expected, ALDH1, Nanog, SOX2 and Bmi-1 expression levels were significantly enhanced in shVEC cells after E2 treatment, but remained unchanged in Gli1-knockdown cells following E2 treatment (Figure 6C & D). Because E2 simultaneously induced CSC-related gene expression and Gli1 activation without affecting Shh expression, these results implied that E2 induced the expansion of breast CSCs through a noncanonical ligand-independent Shh/Gli signaling pathway.

Recent data has indicated that cancer cells often undergo EMT during cancer metastasis, which results in a mesenchymal fibroblast-like morphology, reduced intercellular adhesion, increased motility and increased invasive and migratory properties[25]. EMT-type tumor cells are closely associated with tumor recurrence and therapeutic resistance and display several characteristics of CSCs[26]. Firstly, we investigated whether estrogen-treated breast cancer cells exhibited Gli1-dependent changes in cell motility using a wound healing assay in MCF-7 and HCC1428 cells. An area devoid of cells was created at time 0 h by scraping the monolayer of shVEC-transfected and Gli1-knockdown cells, followed by incubation of the cells with ETOH or E2 for 48 h. Compared with Gli1-knockdown cells, estrogen-treated shVEC-transfected MCF-7 and HCC1428 cells showed a significantly higher rate of migration (p < 0.01), and the leading edges along the scraped areas had almost coalesced 48 h after scraping (Figure 7A, B and Additional file4: Figure S4A & B). A Matrigel invasion assay was also performed. The results showed that the number of E2-treated shVEC-transfected cells that migrated across both the Matrigel and the insert were 3.23 and 1.97 times higher than those of ETOH-treated MCF-7 and HCC1428 cells, respectively (Figure 7C, D and Additional file4: Figure S4C & D). In Gli1-knockdown cells, E2 failed to alter the number of migrated or invaded cells. These data indicated that Gli1 played a significant role in mediating the invasiveness of ER-positive breast cancer cells.

Next, we examined the impact of Gli1 knockdown on estrogen-induced EMT. As shown in Figure 8A, MCF-7 cells transfected with shGli1-1 and shGli1-2 exhibited an epithelial phenotype in the presence of E2, similar to that displayed by MCF-7 cells in the absence of E2, regardless of whether they were transfected with shGli1-1, shGli1-2 or shVEC. In contrast, MCF-7 cells transfected with shVEC displayed a spindle-shaped morphology after E2 stimulation. This morphological transformation was consistent with the reduction in E-cadherin expression and increase in vimentin expression in estrogen-treated shVEC-transfected cells (Figure 8B & C). In Gli1-knockdown cells, Gli1 knockdown partly reversed E2-induced expression of E-cadherin and vimentin, as shown using real-time PCR and western blot analysis (Figure 8B & C). This observation was consistent with the notion that Gli1 may be responsible for the estrogen-induced EMT in ER-positive breast cancer cells.

To determine whether there were any correlations among ER, Gli1 and the CSC marker ALDH1 in breast cancer specimens, we used a tissue microarray containing 100 breast cancer samples and 10 adjacent normal breast tissue or adenosis samples to analyze the expression of ER, Gli1 and ALDH1 using immunohistochemical staining. Gli1 and ALDH1 protein expression levels were low or undetectable in the 10 normal samples (data not shown) but comparatively high in breast tumor tissues (Figure 9A). Moreover, ER expression was positively correlated with Gli1 and ALDH1 expression levels (Figure 9B).

Lipofectamine 2000 transfection reagent and TRIzol LS Reagent were purchased from Invitrogen (Grand Island, NY, USA). The DAB substrate kit for peroxidase was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Antibodies against Gli1, ERα, E-cadherin, Vimentin, Nanog, Bmi-1, SOX2, Smo and β-actin were from Cell Signaling Technology (Danvers, MA, USA). Anti-ALDH antibodies were from BD (Franklin Lakes, NJ, USA). Anti-Shh antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Amino-terminal Shh (ShhN) peptide was from R&D Systems (Minneapolis, MN, USA). Unless otherwise noted, all other chemicals were from Sigma (St. Louis, MO, USA).

All the human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCC1428, MDA-MB-231, BT549, HCC1937, HCC1569, HCC70, HCC1500, HS578T, SK-BR-3, AU565 and ZR-75-1 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MCF-7, T47D, BT474, BT483, MDA-MB-468, MDA-MB-453, MDA-MB-435 and MDA-MB-361 cells were grown in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. MCF-10A cells were cultured in DMEM/F12 supplemented with 20 ng/mL epidermal growth factor, 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, 5% heat-inactivated HS, 100 IU/mL penicillin and 100 μg streptomycin. All the cell lines were grown at 37°C in an atmosphere containing 5% CO₂ and 95% air.

To knock down Gli1 expression, shRNA targeting Gli1 expressed in the pSingle vector was prepared as described previously[37]. Cells were grown in culture dishes until they reached 75% confluence, at which point they were transfected for 24 h with pSingle-shRNA specific to Gli1 using the Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. The tight on/off regulation of the pSingle vector system and coordinate inactivation of the target gene was mediated using doxycycline (Dox). Expression of the shRNA in the absence of induction was extremely low and prevented unwanted suppression of the target gene. When Dox was added to the culture medium, transcriptional suppression was relieved, permitting the shRNA to be transcribed. After transfection, cells were trypsinized, collected and subjected to various experiments.

Cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl [pH 7.4], 1 mM EDTA, 1 mM EGTA, 2 mM NaF, 1 mM sodium orthovanadate, 10 μg/mL leupeptin, 10 μg/mL pepstatin, 10 μg/mL aprotinin, 10 μg/mL E 64 and 1 mM Pefabloc; EMD). Protein concentrations were determined using an Enhanced BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China) and then boiled for 5 min. Protein samples (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). Membranes were rinsed in Tris-buffered saline containing Tween 20 (TBST), blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature, and incubated with the primary antibody at 4°C overnight. The membranes were then rinsed and incubated in peroxidase-conjugated secondary antibodies for 1 h at room temperature. After washing, proteins were detected using enhanced chemiluminescence (ECL) (Millipore Corporation). Membranes were stripped and reprobed with anti-β-actin mouse monoclonal antibodies to confirm equal loading of samples.

Cell lines were plated on culture slides (Costar, Manassas, VA, USA). After 4 days, cells were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS, and permeabilized using 0.5% Triton X-100. Cells were then blocked for 30 min in 10% BSA (Sigma Aldrich, St. Louis, MO, USA) in PBS and then incubated with primary monoclonal antibodies in 10% BSA overnight at 4°C. After three washes in PBS, slides were incubated for 1 h in the dark with FITC-conjugated secondary goat anti-mouse or goat anti-rabbit antibodies (Invitrogen). After three additional washes, slides were stained with 4-,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich) for 5 min to visualize the nuclei and examined using a Carl Zeiss confocal imaging system (LSM 780; Carl Zeiss, Jena, Germany).

Anti-CD44-APC and anti-CD24-PE antibodies used for FACS analysis were obtained from Biolegend (San Diego, CA, USA). Briefly, for each cell line, 1 × 10⁶ cells were aliquoted into two tubes; tube 1 was stained with IgG isotype controls for APC and PE, and tube 2 was stained with anti-CD44-APC and anti-CD24-PE antibodies. Cells were incubated with the appropriate antibodies for 30 min on ice and then washed with PBS. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences); each sample required 10,000 cells for analysis.

Aliquots of 1 × 10⁵ cells were collected using trypsinization and treated with 50 μg/mL DNase-free RNase and 20 μg/mL propidium iodide (PI) following the manufacturer’s instructions. Cells were analyzed using an FC500 instrument (Beckman Coulter, Brea, CA) with MultiCycle for Windows software (Beckman Coulter) for detailed cell cycle status.

Apoptosis was determined using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, cells were detached and resuspended in 100 μL binding buffer containing FITC-Annexin V and PI. After incubation for 15 min at room temperature in the dark, cells were analyzed using an FC500 instrument (Beckman Coulter). Annexin V-positive cells were classified as apoptotic.

Mammosphere culture was performed as described by Dontu et al. with slight modifications[38]. Single-cell suspensions were plated in ultralow attachment 96-well plates (Costar) at different densities of viable cells. Cells were grown in serum-free mammary epithelial growth medium (MEGM), supplemented with 1:50 B27 (Invitrogen), 20 ng/mL epithelial growth factor (EGF), 20 ng/mL basic fibroblast growth factor (bFGF; BD) and 10 μg/mL heparin (Sigma). The number of spheroids was counted after 7–10 days. For in vitro propagation, primary spheres were collected, dissociated into single-cell suspensions and plated in ultralow attachment 96-well plates. The secondary number of spheroids was counted 14 days after plating.

Cells were seeded in triplicate at 500 cells/6-cm dish in complete medium. After 3 weeks of growth, cells were fixed and stained with crystal violet (0.1% w/v in 20 nM 4-morpholinepropanesulfonic acid; Sigma), and visible colonies were counted according to the number of cells in each colony. All experiments were repeated at least three times. Plating efficiency was determined as the number of colonies formed divided by the total number of cells plated.

Cells were seeded in 6-cm culture dishes, and cell monolayers were wounded by scratching with sterile plastic 200-μL micropipette tips and photographed using phase-contrast microscopy immediately following and 48 h after wounding. Migration assays were independently performed in triplicate. The migration distance of each cell was measured after the photographs were converted to Photoshop files.

Invasion of cells was measured in Matrigel (BD)-coated transwell inserts (6.5 mm, Costar) containing polycarbonate filters with 8-μm pores, as detailed previously[39]. Inserts were coated with 50 μL of 1 mg/mL Matrigel matrix according to the manufacturer’s recommendations. A total of 2 × 10⁵ cells in 200 μL serum-free medium were plated in the upper chamber, and 600 μL of medium containing 10% FBS was added to the lower chamber. After 24 h incubation, top cells (noninvasive) were removed, and bottom cells (invasive) were counted. Cells that invaded to the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. For each membrane, five random fields were counted at 10× magnification. Data were presented as the mean ± SD from three independent experiments performed in triplicate.

Tissue microarrays of breast samples (BC081116a) were from Alenabio (Xian, China). The manufacturer provided clinical and pathological information. Immunostaining was performed using the avidin-biotin-peroxidase complex method (UltrasensitiveTM, MaiXin, Fuzhou, China). Sections were deparaffinized in xylene, rehydrated in a graded series of alcohols and then boiled in 0.01 M citrate buffer (pH 6.0) for 2 min in an autoclave. Hydrogen peroxide (0.3%) was applied to block endogenous peroxide activity, and sections were incubated with normal goat serum to reduce nonspecific binding. Tissue sections were incubated with rabbit polyclonal anti-Gli1 antibodies (1:100 dilution), mouse monoclonal anti-ER antibodies (1:50 dilution) or mouse monoclonal anti-ALDH1 antibodies (1:100 dilution). Staining for these antibodies was performed at room temperature for 2 h. Biotinylated goat anti-mouse serum IgG was used as a secondary antibody. After washing, sections were incubated with streptavidin-biotin conjugated with horseradish peroxidase and the peroxidase reaction was developed with 3,30-diaminobenzidine tetrahydrochloride. Two independent, blinded investigators examined the slides randomly. Five views were examined per slide and 100 cells were observed per view at 400× magnification.