BCSCs (breast cancer stem cells) have been shown to be resistant to chemotherapy. However, the mechanisms underlying BCSC-mediated chemoresistance remain poorly understood. The Hh (Hedgehog) pathway is important in the stemness maintenance of CSCs. Nonetheless, it is unknown whether the Hh pathway is involved in BCSC-mediated chemoresistance. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain BCSC-enriched MCF-7 MS (MCF-7 mammosphere) cells. We showed that MCF-7 MS cells are sensitive to salinomycin, but not paclitaxel, distinct from parent MCF-7 cells. The expression of the critical components of Hh pathway, i.e. PTCH (Patched), SMO (Smoothened), Gli1 and Gli2, was significantly up-regulated in MCF-7 MS cells; salinomycin, but not paclitaxel, treatment caused a remarkable decrease in expression of those genes in MCF-7 MS cells, but not in MCF-7 cells. Salinomycin, but not paclitaxel, increased apoptosis, decreased the migration capacity of MCF-7 MS cells, accompanied by a decreased expression of c-Myc, Bcl-2 and Snail, the target genes of the Hh pathway. The salinomycin-induced cytotoxic effect could be blocked by Shh (Sonic Hedgehog)-mediated Hh signalling activation. Inhibition of the Hh pathway by cyclopamine could sensitize MCF-7 MS cells to paclitaxel. In addition, salinomycin, but not paclitaxel, significantly reduced the tumour growth, accompanied by decreased expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Furthermore, the expression of SMO and Gli1 was positively correlated with the expression of CD44+/CD24, and the expression of SMO and Gli1 in CD44+/CD24 tissues was associated with a significantly shorter OS (overall survival) and DFS (disease-free survival) in breast cancer patients receiving chemotherapy.

CLINICAL PERSPECTIVES

  • Despite BCSCs (breast cancer stem cells) being resistant to chemotherapy, the mechanisms underlying BCSC-mediated chemoresistance remain unclear. The aim of the present study was to investigate whether the Hh pathway was involved in BCSC-mediated chemoresistance.

  • The findings show that Hh signalling activation contributed to BCSC-mediated chemoresistance in cultured breast cancer MCF-7 MS cells, in xenograft mice and in human breast cancer patients.

  • The present study provides new insights into the molecular mechanisms underlying the BCSC-mediated drug response, and Hh signalling inhibition in combination with conventional chemotherapy is expected to improve therapeutic outcomes in breast cancer patients.

INTRODUCTION

Studies have shown that a rare subpopulation of cancer cells called CSCs (cancer stem cells) exhibit stem-cell-like properties such as self-renewal, multilineage differentiation potential, strong migration capability, chemotherapeutic resistance and high tumorigenicity [15]. CSCs have been thought to play an important role in tumour regrowth [6]. Targeting chemotherapy-resistant CSCs represents a new direction in cancer treatment, but the mechanisms underlying CSC-mediated chemoresistance remain unclear.

The Hh (Hedgehog) signalling pathway plays a crucial role in proliferation and self-renewal of normal stem cells during embryonic development [7,8]. Three mammalian Hh ligands have been identified, including Shh (Sonic Hedgehog), Dhh (Desert Hedgehog) and Ihh (Indian Hedgehog). The Hh signalling pathway is activated by binding of these ligands to the PTCH (Patched) receptor and subsequently alleviating inhibition of SMO (Smoothened). Activation of SMO results in subsequent regulation of the expression of Gli transcription factors that are responsible for cancer cell proliferation, apoptosis and invasion [7,9]. The aberrant activation of the Hh signalling pathway in many tumours such as non-small-cell lung cancer, ovarian cancer and pancreatic cancer has been reported [1014]. In addition, the Hh pathway has been found to be activated in breast cancer [15]. Tanaka et al. [16] reported that the Hh signalling pathway was important in maintaining the highly tumorigenic populations of breast cancer cells. It has been reported that inhibition of Hh signalling by SMO antagonists (cyclopamine and LDE 225) reverses taxane resistance in ovarian cancer [17]. Inhibition of Hh signalling has been shown to reduce multidrug-resistance in prostate cancer cell lines [18]. However, it remains to be determined whether the Hh pathway is involved in the regulation of chemotherapeutic response in BCSCs (breast cancer stem cells).

Cell adhesion molecules CD44 and CD24 are the two main surface markers on breast cancer cells. The CD44+/CD24−/low phenotype is often used as a BCSC marker [5]. However, because BCSCs are rare, identification and isolation of these BCSCs have been a major challenge. Studies have shown that CD44+/CD24−/low cells with stem-cell-like properties are enriched in mammospheres obtained by culturing breast cancer MCF-7 cells in suspension in serum-free medium [19,20].

Paclitaxel, a microtubule-stabilizing drug, has been widely used for the treatment of various types of cancers, including breast cancer [21]. Nevertheless, although paclitaxel can kill most of the bulk tumour cells, BCSCs are resistant to paclitaxel and thereby contribute to cancer recurrence [22]. Salinomycin, a carboxylic polyether ionophore, was identified as a highly effective inhibitor of BCSCs through a high-throughput screening [21]. However, the molecular mechanisms underlying the drug-resistance/sensitivity of BCSCs to paclitaxel/salinomycin treatment remain unclear.

In the present study, we provide both in vitro and in vivo data to show that the Hh pathway contributes to the resistance of BCSC-enriched MCF-7 MS (MCF-7 mammosphere) cells to paclitaxel and their sensitivity to salinomycin. We also show that the expression of the Hh signalling components SMO and Gli1 was closely correlated with the presence of CD44+/CD24 of BCSCs and the poor prognosis in breast cancer patients receiving chemotherapy. We demonstrate that the Hh signalling pathway mediates chemoresistance of BCSCs and contributes to the outcome in breast cancer patients. The present study provides new insights into the mechanisms underlying BCSC-mediated chemoresistance.

MATERIALS AND METHODS

Cell culture

The human breast cancer MCF-7 cell line was purchased from the A.T.C.C. (Manassas, VA, U.S.A.). The cells were maintained in DMEM (Dulbecco's modified Eagle's medium) (Invitrogen) containing 10% (v/v) FBS (HyClone), 100 units/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37°C. The cell line was actively passaged for less than 6 months from the time that it was received from the A.T.C.C., and UKCCCR (United Kingdom Co-ordinating Committee on Cancer Research) guidelines were followed [23].

Mammosphere generation and formation assay

Mammospheres were cultured as reported previously by Ponti et al. [19]. Briefly, MCF-7 cells (5×104 cells/ml) were cultured in suspension in serum-free DMEM/Ham's F12 (Gibco), supplemented with 2% B27 (Invitrogen), 20 ng/ml EGF (Peprotech) and 10 ng/ml bFGF (basic fibroblast growth factor) (Peprotech). Cells were grown under these conditions as non-adherent spherical clusters of cells, named MCF-7 MS cells.

To test the effect of various drugs on mammosphere formation, single MCF-7 MS cells were thoroughly suspended and plated in six-well ultra-low-adherent plates (Corning) at 105 cells/well in 4 ml of mammosphere formation medium described above. After 24 h, cells were treated with paclitaxel (Sigma), salinomycin (Sigma), Shh (R&D Systems), cyclopamine (Merck) or DMSO as a control for 48 h. Cells were then collected, digested into single cells and plated in six-well ultra-low-adherent plates with 2000 cells/well in mammosphere formation medium (2 ml). After culture for 8 days, the number of the mammospheres/2000 cells was counted under an inverted microscope (Nikon TE2000-U).

Flow cytometric analysis

Flow cytometry was performed to determine the expression of CD44 and CD24 in MCF-7 and MCF-7 MS cells. The cells were suspended at a density of 106 cells/ml in PBS and incubated with FITC-conjugated antibodies against CD44 (1:20, BD Pharmingen) and PE (phycoerythrin)-conjugated antibodies against CD24 (1:10 dilution, BD Pharmingen) for 30 min at 4°C in the dark. Single cell suspensions were analysed by flow cytometry, using a FACSCalibur instrument (Becton-Dickinson).

For apoptosis analysis, cells were resuspended in 250 μl of annexin V binding buffer at a density of 106 cells/ml. The suspension (100 μl) was incubated in the dark at room temperature for 15 min with a solution of annexin V–FITC (2.5 μg/ml) and PI (propidium iodide) (5 μg/ml). Cells were analysed for apoptosis by flow cytometry, using a FACSCalibur instrument.

Soft agar colony formation assay

MCF-7 and MCF-7 MS cells (103 cells/ml) were suspended in 0.6% agar with culture medium (1:1), and layered on preformed 1.2% agar with culture medium (1:1) base layer. Culture medium was added on the top agar layer every 3–4 days. After incubation for 3 weeks at 37°C, the number of colonies per well was determined from eight different random fields under an inverted microscope (Nikon TE2000-U).

CCK-8/WST-8 assay

Cell viability was measured using the CCK-8 (Cell Counting Kit-8) (Dojindo). MCF-7 or MCF-7 MS cells (8000 cells/well) were seeded into 96-well ultra-low-adherent plates. To determine the IC50 value of salinomycin or paclitaxel, cells were treated with salinomycin (10–6000 nM) or paclitaxel (0.1–60 nM) for 48 h. To investigate the effect of Shh on salinomycin-induced inhibition or cyclopamine on paclitaxel-induced inhibition on MCF-7 MS proliferation, MCF-7 MS cells were treated with salinomycin (100 nM), Shh (3 μg/ml), salinomycin (100 nM)+Shh (3 μg/ml), paclitaxel (10 nM), cyclopamine (5 μM), paclitaxel (10 nM)+cyclopamine (5 μM) or DMSO for 48 h. After the treatment, the cells in each well were incubated with 10 μl of WST-8 (water-soluble tetrazolium salt 8) (Dojindo) at 37°C for 4 h. The absorbance was then measured at 450 nm using an Anthos 2010 microplate reader (Anthos Labtec Instruments).

Transwell migration assays

Transwell migration assays were carried out using 24-well Transwell migration chambers (Corning) with 8-μm-pore-size polyethylene membranes. The cells were placed in the upper chamber of each insert. MCF-7 cells were starved overnight. The upper chambers were plated with MCF-7 cells in 0.5 ml of serum-free DMEM or MCF-7 MS cells in 0.5 ml of serum-free DMEM/Ham's F12 at a density of 4×105 cells/ml. The lower chambers were filled with 0.5 ml of cell culture medium containing 10% (v/v) FBS. Cells were allowed to migrate towards the lower chamber for 24 h at 37°C. The chambers were then fixed with methanol for 30 min and stained with 0.1% Crystal Violet (Sigma) for 30 min. Cells that did not migrate to the lower chamber were removed with a cotton swab. The number of cells migrating through the membrane was counted under a light microscope (×200 magnification, five random fields per well) and analysed using ImageJ software (NIH). Each experiment was repeated three times.

Western blot analysis

Western blot analysis was conducted as described previously [24]. The primary antibodies were anti-OCT4 (octamer-binding transcription factor 4) (1:1000 dilution; Cell Signaling Technology #2750), anti-keratin 18 (1:2000 dilution; Cell Signaling Technology, #4548), anti-E-cadherin (epithelial cadherin) (1:1000 dilution; Cell Signaling Technology, #4065), anti-vimentin (1:1000 dilution; Cell Signaling Technology, #5741), anti-PTCH (1:1000 dilution; Abcam, ab39266), anti-SMO (1:1000 dilution; Abcam, ab72130), anti-Gli1 (1:500 dilution; Abcam, ab92611), anti-Gli2 (1:800 dilution; Abcam, ab26056), anti-c-Myc (1:1000 dilution; Cell Signaling Technology, #9402), anti-Bcl-2 (1:1000 dilution; Cell Signaling Technology, #2872), anti-Snail (1:300 dilution; Abcam, ab17732), anti-ERα (oestrogen receptor α) (1:500 dilution; Santa Cruz Biotechnology, sc-542), anti-Wnt1 (1:500 dilution; Abcam, ab15251), anti-p-LRP6 (low-density lipoprotein receptor-related protein 6) (1:1000 dilution; Cell Signaling Technology, #2568), anti-β-catenin (1:1000 di-lution; Proteintech, 51067-2AP), anti-p-β-catenin (1:1000 dilu-tion; Cell Signaling Technology, #9561), anti-Axin2 (1:1000 dilution; Cell Signaling Technology, #2151) and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:6000 dilution; Santa Cruz Biotechnology, sc-20357). Protein expression was analysed quantitatively using Scion Image Software.

Immunofluorescence

MCF-7 cells and MCF-7 MS cells were treated with paclitaxel (2 nM), salinomycin (100 nM) or DMSO as a control for 48 h. After treatment, MCF-7 MS cells were collected and fixed in 4% (w/v) paraformaldehyde for 30 min, and then the spheres were embedded in paraffin wax and cut into 4 μm sections using the method of Thurber et al. [25]. Cells were permeabilized with 0.5% Triton X-100 (Sigma) for 10 min, rinsed in PBS and blocked with normal goat serum for 1 h at room temperature. Cells were then incubated overnight at 4°C with primary antibodies against PTCH (1:200 dilution), SMO (1:100 dilution), Gli1 (1:100 dilution) and Gli2 (1:100 dilution). Immunoreactivity was detected by incubation with FITC-conjugated secondary antibodies (1:300 dilution; Invitrogen) for 1 h at room temperature. Nuclei were counterstained with DAPI for 15 min. Fluorescence was detected using confocal laser-scanning microscopy (FV1000S-SIM/IX81, Olympus). For MCF-7 cells, cells were seeded on to the glass coverslips, fixed in 4% (w/v) paraformaldehyde and stained as above.

In vivo xenograft experiments

To study the tumorigenic ability of MCF-7 and MCF-7 MS cells, equal numbers (2×106) of MCF-7 MS cells or MCF-7 cells were suspended in 200 μl of PBS and Matrigel™ (1:1) (BD Biosciences) and subcutaneously injected into the right flank of athymic nude 6–8-week-old female BALB/c mice (n=5 per group). At 3 days before inoculating cells, mice received a 0.72 mg subcutaneous injection of β-oestradiol (Sigma). Tumour volume was measured and calculated as described previously [24]. Mice were killed 44 days after the initial injection of the cells, and xenograft tumours were weighed and harvested for Western blot analysis and histological examination.

To study the effects of salinomycin and paclitaxel on the xenograft tumours, 2×106 MCF-7 MS cells were subcutaneously inoculated into the nude mice as above. At 8 days after inoculation, when the average tumour volume reached approximately 100–200 mm3, the transplanted nude mice were randomly divided into control, salinomycin-treated and paclitaxel-treated groups (n=5 per group). Mice in the three groups were intraperitoneally injected with DMSO, salinomycin (5 mg/kg) or paclitaxel (5 mg/kg) once every other day for 20 days. The tumour volume was measured and calculated. Mice were killed 4 weeks after the initial injection, and tumours were weighed and harvested. All mice were bred in pathogen-free conditions at the Animal Center of China Medical University. All animal studies were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Patients

Breast cancer tissues were obtained from 290 breast cancer patients, who underwent surgery at the Tumor Hospital of Liaoning Province, China from 2006 to 2008. The study has been approved by the Institutional Review Board of China Medical University, and all subjects gave their written informed consent before their inclusion in the study.

Immunohistochemistry

Immunohistochemistry staining was carried out as described previously [26]. Sections obtained from paraffin-embedded tumour tissues from xenograft mice or breast cancer patients were incubated with primary antibodies against PTCH (1:100 dilution), SMO (1:50 dilution), Gli1 (1:100 dilution) and Gli2 (1:200 dilution). To detect the expression of CD44 and CD24 in breast cancer patients, double-immunostaining with antibodies against CD44 (clone 156-3C11, 1:800 dilution; Thermo) and CD24 (clone SN3b, 1:400 dilution; Thermo) was performed according to the manufacturer's instructions as described previously by Lee et al. [27]. CD24 was detected with Permanent Red and CD44 with DAB (3′-diaminobenzidine) (Sigma).

Images from each section were captured by a Digital Sight digital camera under a Nikon Eclipse 80i microscope (at ×200 magnification). The immunoreactivity was evaluated by two independent investigators, blinded to the patients’ clinicopathological characteristics, according to the percentage of stained cells and the intensity of the immunoreactivity [12,28]. The intensity of immunoreactivity was scored as follows: 0 (none), 1 (weak), 2 (moderate) and 3 (strong). The percentage of stained cells was scored as follows: 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%) and 4 (>75%). The final immunoreactive score was determined by multiplying the intensity score with the score for the percentage of positively stained cells. The ROC (receiver operating characteristic) curve analysis with respect to OS (overall survival) was used to determine the cut point of the final score, as described by Kim et al. [29]. According to the optimal sensitivity and specificity of the ROC curve by DFS (disease-free survival), 2.5, 2.5, 3.5 and 3.5 were defined as the optimal cut points for SMO, Gli1, CD44 and CD24 respectively. Since the final immunoreactive scores were integers, the negative and positive immunoreactivity were defined by a final score of <3 and ≥3 for SMO and Gli1, and <4 and ≥4 for CD44 and CD24 respectively.

Statistical analysis

Data were analysed using the SPSS statistics 16.0 software package. Quantitative data are presented as means±S.D. for at least three experiments. Student's t test was used to compare the differences between two groups. One-way ANOVA was used to compare the differences among three or more groups. Pearson's χ2 test and Mann–Whitney U analysis were used to assess the relationship between the expression of SMO or Gli1 and CD44+/CD24 expression in breast cancer tissues. Survival curves were estimated by the Kaplan–Meier method and evaluated by a log-rank test. P<0.05 was considered statistically significant.

RESULTS

BCSC-like MCF-7 MS cells exhibit a strong capacity for self-renewal, migration and tumorigenesis

It is known that BCSCs have a CD44+/CD24 phenotype and exhibit strong tumorigenic potential [5]. We first examined the presence of CD44 and CD24 in MCF-7 MS cells using flow cytometry. The proportion of CD44+/CD24 cells was 2.41±1.09% in parental MCF-7 cells, and was increased to 87.89±5.21% in MCF-7 MS after seven or eight passages (Figure 1A). Furthermore, Western blot analysis showed that the expression of OCT4 (a stem cell marker) was significantly higher, whereas the expression of keratin 18 (a differentiation marker) was significantly lower in MCF-7 MS cells compared with MCF-7 cells (Figure 1B). In addition, we found that MCF-7 MS cells formed significantly more colonies than MCF-7 cells using a soft agar colony formation assay (Figure 1C). These data suggest that MCF-7 MS cells exhibited BCSC-like self-renewal capacity. Furthermore, we found that the number of MCF-7 MS cells that migrated into the lower Transwell chamber was significantly higher than that of MCF-7 cells, using Transwell migration assays (Figure 1D), and the expression of the epithelial marker E-cadherin was significantly lower, whereas the expression of the mesenchymal marker vimentin was significantly higher in MCF-7 MS cells than in MCF-7 cells determined using Western blot analysis (Figure 1E). These data suggest that MCF-7 MS cells exhibited increased migration capacity.

MCF-7 MS cells possess BCSC-like properties

Figure 1
MCF-7 MS cells possess BCSC-like properties

(A) Flow cytometry showing the percentage of CD44+/CD24 cells in MCF-7 and MCF-7 MS cells. (B) Western blot analysis showing the expression level of OCT4 and keratin 18. GAPDH was used as a loading control. The expression level of OCT4 or keratin 18 was normalized to that of GAPDH. The OCT4 or keratin 18/GAPDH ratio in MCF-7 cells was set as 1. (C) MCF-7 cells and MCF-7 MS were grown in soft agar as described in the Materials and methods section. The colony numbers were counted manually under a microscope after culture for 7 days. Scale bar, 50 μm. (D) Transwell migration assays showing the enhanced ability of migration in MCF-7 MS cells. Cells that migrated into the lower Transwell chambers were counted. Scale bar, 20 μm. The number of MCF-7 cells migrating to the lower chambers was set as 1. (E) Western blot analysis showing the expression level of E-cadherin and vimentin. GAPDH was used as a loading control. The E-cadherin or vimentin/GAPDH ratio in MCF-7 cells was set as 1. (F) Equal numbers (2×106) of MCF-7 and MCF-7 MS cells were subcutaneously inoculated into the female BALB/c athymic nude mice (n=5). Tumour volumes were calculated as described in the Materials and methods section. (G) The mass of tumours induced by inoculation of MCF-7 and MCF-7 MS cells after mice were killed 44 days after cell inoculation (n=5). Results are means±S.D. *P<0.05, **P<0.01 compared with MCF-7 cells.

Figure 1
MCF-7 MS cells possess BCSC-like properties

(A) Flow cytometry showing the percentage of CD44+/CD24 cells in MCF-7 and MCF-7 MS cells. (B) Western blot analysis showing the expression level of OCT4 and keratin 18. GAPDH was used as a loading control. The expression level of OCT4 or keratin 18 was normalized to that of GAPDH. The OCT4 or keratin 18/GAPDH ratio in MCF-7 cells was set as 1. (C) MCF-7 cells and MCF-7 MS were grown in soft agar as described in the Materials and methods section. The colony numbers were counted manually under a microscope after culture for 7 days. Scale bar, 50 μm. (D) Transwell migration assays showing the enhanced ability of migration in MCF-7 MS cells. Cells that migrated into the lower Transwell chambers were counted. Scale bar, 20 μm. The number of MCF-7 cells migrating to the lower chambers was set as 1. (E) Western blot analysis showing the expression level of E-cadherin and vimentin. GAPDH was used as a loading control. The E-cadherin or vimentin/GAPDH ratio in MCF-7 cells was set as 1. (F) Equal numbers (2×106) of MCF-7 and MCF-7 MS cells were subcutaneously inoculated into the female BALB/c athymic nude mice (n=5). Tumour volumes were calculated as described in the Materials and methods section. (G) The mass of tumours induced by inoculation of MCF-7 and MCF-7 MS cells after mice were killed 44 days after cell inoculation (n=5). Results are means±S.D. *P<0.05, **P<0.01 compared with MCF-7 cells.

Furthermore, we observed that equal numbers of MCF-7 MS cells generated tumours earlier and faster in xenografted mice than MCF-7 cells (Figure 1F). At 44-days after tumour cell inoculation, the average mass of MCF-7 MS xenograft tumours (1.75±0.24 g) was significantly higher than that of MCF-7 xenograft tumours (0.59±0.11 g) (Figure 1G). In addition, we measured the expression of CD44 and CD24 in the MCF-7 and MCF7-MS xenograft tumours using immunohistochemistry. The percentage of CD44- and CD24-positive expression was higher in MCF-7 xenograft tumours, whereas the percentage of CD44-positive expression was higher and the percentage of CD24-positive expression was lower in MCF-7 MS xenograft tumours (Supplementary Figure S1). Taken together, these data show that MCF-7 MS cells obtained from serum-free suspension culture express the BCSC-specific CD44+CD24 marker and possess BCSC-like properties.

MCF-7 MS cells are sensitive to salinomycin, but resistant to paclitaxel

It has been reported that BCSCs are resistant to paclitaxel and sensitive to salinomycin [21]. To investigate whether the BCSC-like MCF-7 MS cells also exhibited differential sensitivity to paclitaxel and salinomycin, we first assessed the sensitivity of MCF-7 and MCF-7 MS cells to paclitaxel and salinomycin using CCK-8 assays, and found that paclitaxel inhibited cell viability of MCF-7 cells in a dose-dependent manner with an IC50 value of 2.04 nM (Supplementary Figure S2A). At a concentration of 10 nM, paclitaxel inhibited cell viability of MCF-7 cells by approximately 90%, but only inhibited cell viability of MCF-7 MS cells by approximately 30%, and lower concentrations of paclitaxel (0.1–1 nM) even promoted the proliferation of MCF-7 MS cells, suggesting that MCF-7 MS cells exhibited resistance to paclitaxel. In contrast, MCF-7 MS cells exhibited a greater sensitivity to salinomycin compared with MCF-7 cells (Supplementary Figure S2B). Salinomycin inhibited cell viability of MCF-7 MS cells in a dose-dependent manner with an IC50 value of 106 nM. At a concentration of 1000 nM, salinomycin inhibited cell viability of MCF-7 MS cells by approximately 90%, but only inhibited cell viability of MCF-7 cells by approximately 10%. In addition, salinomycin (30, 100 and 300 nM) significantly inhibited mammosphere formation in MCF-7 MS cells with approximately 80% inhibition at a concentration of 300 nM, whereas paclitaxel (0.1 and 1 nM) promoted mammosphere formation and 10 nM paclitaxel only slightly inhibited mammosphere formation of MCF-7 MS cells with approximately 15% inhibition (Supplementary Figure S2C).

Hh signalling activation contributes to the drug response of MCF-7 MS cells

Previous studies have shown that the Hh signalling pathway is aberrantly activated in breast cancer and plays an important role in maintaining the highly tumorigenic populations of breast cancer cells including the CD44+/CD24−/low subpopulation, and that inhibition of this pathway reverses drug-resistance of ovarian and prostate cancers [1518]. We therefore speculated that the Hh signalling pathway may play a role in the response of MCF-7 MS cells to different drugs. To test this hypothesis, we first compared the expression levels of the main components of the Hh pathway in MCF-7 and MCF-7 MS cells, including PTCH, SMO, Gli1 and Gli2. Western blot analysis showed that the expression levels of PTCH, SMO, Gli1 and Gli2 were significantly higher in MCF-7 MS cells than in MCF-7 cells (Figure 2A). Furthermore, we found that the expression levels of PTCH, SMO, Gli1 and Gli2 were also remarkably higher in MCF-7 MS xenograft tumours than in MCF-7 xenograft tumours as determined using immunohistochemistry and Western blotting (Figure 2B and 2C). These findings suggest that the Hh pathway was highly activated in MCF-7 MS cells.

Hh pathway is activated in the MCF-7 MS cells in vitro and in vivo

Figure 2
Hh pathway is activated in the MCF-7 MS cells in vitro and in vivo

(A) Western blot analysis showing the expression levels of PTCH, SMO, Gli1 and Gli2 in MCF-7 and MCF-7 MS cells. (B) Representative immunohistochemical staining for the expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours induced by MCF-7 or MCF-7 MS cells. Scale bar, 10 μm. (C) Western blot analysis showing the expression levels of PTCH, SMO, Gli1 and Gli2 in the xenograft tumours induced by MCF-7 and MCF-7 MS cells. GAPDH was used as a loading control. The expression level of PTCH, SMO, Gli1 and Gli2 was normalized to that of GAPDH. The expression levels of these proteins in MCF-7 cells or MCF-7-inudced tumours were set as 1. Results are means±S.D. from there independent experiments. *P<0.05 compared with MCF-7.

Figure 2
Hh pathway is activated in the MCF-7 MS cells in vitro and in vivo

(A) Western blot analysis showing the expression levels of PTCH, SMO, Gli1 and Gli2 in MCF-7 and MCF-7 MS cells. (B) Representative immunohistochemical staining for the expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours induced by MCF-7 or MCF-7 MS cells. Scale bar, 10 μm. (C) Western blot analysis showing the expression levels of PTCH, SMO, Gli1 and Gli2 in the xenograft tumours induced by MCF-7 and MCF-7 MS cells. GAPDH was used as a loading control. The expression level of PTCH, SMO, Gli1 and Gli2 was normalized to that of GAPDH. The expression levels of these proteins in MCF-7 cells or MCF-7-inudced tumours were set as 1. Results are means±S.D. from there independent experiments. *P<0.05 compared with MCF-7.

We then assessed the expression of PTCH, SMO, Gli1 and Gli2 in MCF-7 and MCF-7 MS cells pre-treated with paclitaxel (2 nM) and salinomycin (100 nM) for 48 h. Paclitaxel treatment did not significantly alter the expression of PTCH, SMO, Gli1 or Gli2 in either MCF-7 or MCF-7 MS cells. Salinomycin treatment significantly inhibited the expression of PTCH, SMO, Gli1 and Gli2 to 44.8±4.56%, 49.4±5.10%, 50.5±5.41% and 52.4±3.56% of the control in MCF-7 MS cells, but not in MCF-7 cells (Figures 3A and 3B). Consistent with the above Western blot data, immunofluorescence staining also showed that paclitaxel and salinomycin did not change the expression of PTCH, SMO, Gli1 or Gli2 in MCF-7 cells (Figure 3C), whereas salinomycin, but not paclitaxel, inhibited their expression in MCF-7 MS cells (Figure 3D).

The Hh signalling pathway is involved in the drug-sensitivity of MCF-7 MS cells

Figure 3
The Hh signalling pathway is involved in the drug-sensitivity of MCF-7 MS cells

Western blot analysis showing the expression of PTCH, SMO, Gli1 and Gli2 in (A) MCF-7 and (B) MCF-7 MS cells pre-treated with 2 nM paclitaxel, 100 nM salinomycin or DMSO as a control for 48 h. GAPDH was used as a loading control. The expression level of PTCH, SMO, Gli1 and Gli2 was normalized to that of GAPDH. The expression levels of these proteins in the control MCF-7 cells or MCF-7 MS cells were set as 100%. Results are means±S.D. from there independent experiments. **P<0.01 compared with control. Immunofluorescence showing the expression of PTCH, SMO, Gli1 and Gli2 in (C) MCF-7 and (D) MCF-7 MS cells pre-treated with 2 nM paclitaxel, 100 nM salinomycin or DMSO as a control for 48 h. Scale bars, 10 μm.

Figure 3
The Hh signalling pathway is involved in the drug-sensitivity of MCF-7 MS cells

Western blot analysis showing the expression of PTCH, SMO, Gli1 and Gli2 in (A) MCF-7 and (B) MCF-7 MS cells pre-treated with 2 nM paclitaxel, 100 nM salinomycin or DMSO as a control for 48 h. GAPDH was used as a loading control. The expression level of PTCH, SMO, Gli1 and Gli2 was normalized to that of GAPDH. The expression levels of these proteins in the control MCF-7 cells or MCF-7 MS cells were set as 100%. Results are means±S.D. from there independent experiments. **P<0.01 compared with control. Immunofluorescence showing the expression of PTCH, SMO, Gli1 and Gli2 in (C) MCF-7 and (D) MCF-7 MS cells pre-treated with 2 nM paclitaxel, 100 nM salinomycin or DMSO as a control for 48 h. Scale bars, 10 μm.

We also measured the protein expression of Hh pathway target genes, including c-Myc, Bcl-2 and Snail in MCF-7 MS cells pre-treated with paclitaxel (2 nM) and salinomycin (100 nM) for 48 h. Paclitaxel treatment did not alter the expression of c-Myc, Bcl-2, and Snail in MCF-7 MS cells. Salinomycin treatment significantly inhibited their expression in MCF-7 MS cells (Supplementary Figure S3A). Furthermore, compared with the control, we found that salinomycin, but not paclitaxel, treatment significantly increased the percentages of early apoptotic MCF-7 MS cells using flow cytometry (Supplementary Figure S3B) and significantly decreased the number of MCF-7 MS cells that migrated into the lower chambers in the Transwell migration assay (Supplementary Figure S3C).

Furthermore, salinomycin-induced inhibition of the expression of PTCH, SMO, Gli1 and Gli2 in MCF-7 MS cells could be partially blocked by Shh (3 μg/ml), an activator of the Hh signalling pathway (Figure 4A). In addition, Shh treatment could partially reverse the salinomycin-induced decrease in cell viability and mammosphere formation of MCF-7 MS cells (Figures 4B and 4C). Conversely, cyclopamine (5 μM), an inhibitor of the Hh signalling pathway, could promote inhibition of the expression of PTCH, SMO, Gli1 and Gli2 in MCF-7 MS cells treated with paclitaxel (Figure 4D). Additionally, cyclopamine treatment could enhance the paclitaxel-induced decrease in cell viability and mammosphere formation of MCF-7 MS cells (Figures 4E and 4F).

Hh signalling activation affects the drug-sensitivity of MCF-7 MS

Figure 4
Hh signalling activation affects the drug-sensitivity of MCF-7 MS

Western blot analysis showing the expression of PTCH, SMO, Gli1 and Gli2 in MCF-7 MS cells treated with (A) Shh (3 μg/ml), salinomycin (100 nM) or Shh (3 μg/ml)+salinomycin (100 nM), or (D) cyclopamine (5 μM), paclitaxel (10 nM), cyclopamine (5 μM)+paclitaxel (10 nM) or DMSO as a control for 48 h. GAPDH was used as a loading control. The expression level of PTCH, SMO, Gli1 and Gli2 was normalized to that of GAPDH. The expression levels of these proteins in control cells were set as 100%. The cell viability of MCF-7 MS cells was measured after treatment with (B) Shh (3 μg/ml), salinomycin (100 nM) or Shh (3 μg/ml)+salinomycin (100 nM), or (E) cyclopamine (5 μM), paclitaxel (10 nM), cyclopamine (5 μM)+paclitaxel (10 nM) or DMSO as a control for 48 h using the CCK-8 assay. The mammosphere formation of MCF-7 MS cells was measured after treatment with (C) Shh (3 μg/ml), salinomycin (100 nM), Shh (3 μg/ml)+salinomycin (100 nM), or (F) cyclopamine (5 μM), paclitaxel (10 nM), cyclopamine (5 μM)+paclitaxel (10 nM) or DMSO as a control for 48 h. Results means±S.D. from there independent experiments. *P<0.05, **P<0.01 compared with control. #P<0.05, ##P<0.01 compared with salinomycin or paclitaxel alone. Scale bars, 100 μm.

Figure 4
Hh signalling activation affects the drug-sensitivity of MCF-7 MS

Western blot analysis showing the expression of PTCH, SMO, Gli1 and Gli2 in MCF-7 MS cells treated with (A) Shh (3 μg/ml), salinomycin (100 nM) or Shh (3 μg/ml)+salinomycin (100 nM), or (D) cyclopamine (5 μM), paclitaxel (10 nM), cyclopamine (5 μM)+paclitaxel (10 nM) or DMSO as a control for 48 h. GAPDH was used as a loading control. The expression level of PTCH, SMO, Gli1 and Gli2 was normalized to that of GAPDH. The expression levels of these proteins in control cells were set as 100%. The cell viability of MCF-7 MS cells was measured after treatment with (B) Shh (3 μg/ml), salinomycin (100 nM) or Shh (3 μg/ml)+salinomycin (100 nM), or (E) cyclopamine (5 μM), paclitaxel (10 nM), cyclopamine (5 μM)+paclitaxel (10 nM) or DMSO as a control for 48 h using the CCK-8 assay. The mammosphere formation of MCF-7 MS cells was measured after treatment with (C) Shh (3 μg/ml), salinomycin (100 nM), Shh (3 μg/ml)+salinomycin (100 nM), or (F) cyclopamine (5 μM), paclitaxel (10 nM), cyclopamine (5 μM)+paclitaxel (10 nM) or DMSO as a control for 48 h. Results means±S.D. from there independent experiments. *P<0.05, **P<0.01 compared with control. #P<0.05, ##P<0.01 compared with salinomycin or paclitaxel alone. Scale bars, 100 μm.

Hh signalling activation is associated with the drug response of breast cancer tumours induced by MCF-7 MS cells in mice

We then investigated whether Hh signalling activation was also involved in the drug response of breast cancer tumours in mice. As shown in Figure 5(A), salinomycin significantly reduced the volume of breast cancer xenograft tumours induced by MCF-7 MS cells in mice compared with paclitaxel, which only slightly reduced the tumour volume. After killing, the tumour mass was significantly lower in the salinomycin-treated group (0.68±0.21 g) compared with the control (1.21±0.31 g) and paclitaxel-treated groups (1.04±0.27 g) (Figure 5B). Accordingly, salinomycin, but not paclitaxel, treatment reduced the expression levels of PTCH, SMO, Gli1 and Gli2 in xenograft tumours (Figures 5C and 5D). These data suggest that the Hh signalling pathway may mediate the drug response of xenograft tumours.

Hh signalling activation is associated with the drug-sensitivity of MCF-7 MS cell-induced tumours in mice

Figure 5
Hh signalling activation is associated with the drug-sensitivity of MCF-7 MS cell-induced tumours in mice

(A) The time course of the volume of MCF-7 MS cell-induced tumours in mice treated with salinomycin (5 mg/kg), paclitaxel (5 mg/kg) or DMSO as a control 8 days after inoculation. (B) The tumour mass in mice treated with salinomycin (5 mg/kg), paclitaxel (5 mg/kg) or DMSO as a control when mice were killed 4 weeks after cell inoculation. (C) Representative immunohistochemical staining for the expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Scale bar, 10 μm. (D) Western blot analysis showing the expression of PTCH, SMO, Gli1 and Gli2 in the xenograft tumours. GAPDH was used as a loading control. The expression level of PTCH, SMO, Gli1 and Gli2 was normalized to that of GAPDH. The ratios of the proteins to GAPDH in the control tumours were set as 100%. Results are means±S.D. from three independent experiments. *P<0.05, **P<0.01 compared with control.

Figure 5
Hh signalling activation is associated with the drug-sensitivity of MCF-7 MS cell-induced tumours in mice

(A) The time course of the volume of MCF-7 MS cell-induced tumours in mice treated with salinomycin (5 mg/kg), paclitaxel (5 mg/kg) or DMSO as a control 8 days after inoculation. (B) The tumour mass in mice treated with salinomycin (5 mg/kg), paclitaxel (5 mg/kg) or DMSO as a control when mice were killed 4 weeks after cell inoculation. (C) Representative immunohistochemical staining for the expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Scale bar, 10 μm. (D) Western blot analysis showing the expression of PTCH, SMO, Gli1 and Gli2 in the xenograft tumours. GAPDH was used as a loading control. The expression level of PTCH, SMO, Gli1 and Gli2 was normalized to that of GAPDH. The ratios of the proteins to GAPDH in the control tumours were set as 100%. Results are means±S.D. from three independent experiments. *P<0.05, **P<0.01 compared with control.

The expression of SMO and Gli1 is positively associated with the presence of CD44+/CD24 in human breast cancer tissues

To investigate further the role of the Hh signalling pathway in BCSCs, we measured the expression of SMO, Gli1, CD44 and CD24 in human breast cancer samples from 290 patients, using immunohistochemistry according to the cut point of the final score from ROC curve analysis with respect to OS as shown in Supplementary Figure S4(A). Immunoreactivity for SMO and Gli1 was observed in 62.4% (181/290) and 54.8% (159/290) of breast cancer patients respectively (Supplementary Figure S4B). Immunoreactivity for the BCSC marker CD44+/CD24 was observed in 39.0% (113/290) of breast cancer patients (Supplementary Figure S4C). We next analysed the correlation between the expression of SMO and Gli1 and the presence of CD44+/CD24 in 290 human breast cancer patients using Pearson's χ2 test. Of the 113 CD44+/CD24 samples, 83 (73.5%) and 73 (64.6%) samples were positive for SMO and Gli1 respectively. The expression of SMO and Gli1 was significantly positively correlated (P<0.01) with the presence of CD44+/CD24 (Supplementary Figure S4D). As expected, the percentage of SMO-positive and Gli1-postive samples was significantly higher in CD44+/CD24 samples compared with non-CD44+/CD24 samples using Mann–Whitney U analysis (P<0.01, Supplementary Figure S4E). These data suggest that Hh signalling activation is closely associated with the stemness of breast cancer cells.

The expression of SMO or Gli1 in CD44+/CD24 tissues is associated with the poor outcome in breast cancer patients

Of the 290 breast cancer patients, 207 patients received chemotherapy, including 156 patients on the CEF (cyclophosphamide/epirubicin/fluorouracil) regimen, 25 patients on the CAF (cyclophosphamide/adriamycin/fluorouracil) regimen, and 26 patients on the CET [cyclophosphamide/epirubicin/taxol (i.e. paclitaxel)] regimen. We evaluated the correlation of the expression of SMO and Gli1 with OS or DFS in breast cancer patients with chemotherapy, using Kaplan–Meier analysis. The expression of SMO and Gli1 was associated with a significantly shorter OS and DFS in breast cancer patients (P<0.05 or P<0.01; Figure 6A), suggesting that Hh signalling activation was associated with the poor therapeutic response in breast cancer patients who received chemotherapy. Notably, the correlation of the expression of SMO and Gli1 with poor OS and DFS was more significant in the subgroup of chemotherapy-treated breast cancer patients whose tumour tissues are CD44+/CD24 (n=113; Figure 6B) than in the whole patient set (Figure 6A); in contrast, no significant correlation was observed in the subgroup of the patients whose tumour tissues are not CD44+/CD24 (results not shown). Together, our data suggests that Hh signalling activation may be responsible for BCSC-induced therapeutic resistance and poor outcomes in breast cancer patients.

The expression of SMO and Gli1 in CD44+/CD24 tissues is associated with the poor therapeutic response in breast cancer patients

Figure 6
The expression of SMO and Gli1 in CD44+/CD24 tissues is associated with the poor therapeutic response in breast cancer patients

Kaplan–Meier survival curves showed the expression of SMO or Gli1 alone (A) and in combination with CD44+/CD24 (B) was associated with a shorter overall survival and disease-free survival in breast cancer patients with chemotherapy.

Figure 6
The expression of SMO and Gli1 in CD44+/CD24 tissues is associated with the poor therapeutic response in breast cancer patients

Kaplan–Meier survival curves showed the expression of SMO or Gli1 alone (A) and in combination with CD44+/CD24 (B) was associated with a shorter overall survival and disease-free survival in breast cancer patients with chemotherapy.

DISCUSSION

BCSCs have been thought to be responsible for tumour chemoresistance, recurrence and metastasis [3,5,30]. However, the mechanisms underlying BCSC-mediated chemoresistance remain unclear. It has been reported that enhanced antioxidative and anti-apoptotic capacities contribute to chemoresistance in BCSCs [1,4]. In addition, multidrug-resistance in BCSCs has been attributed to increased drug efflux as a result of overexpression of members of ABC (ATP-binding cassette) transporters [31,32]. Sims-Mourtada et al. [18] reported that Hh signalling activation induced chemoresistance by increasing drug efflux in an ABC transporter-dependent manner in prostate cancer cell lines. In the present study, we obtained CD44+/CD24 cell-enriched MCF-7 mammospheres by culturing MCF-7 cells in suspension in serum-free medium as reported previously by Ponti et al. [19]. The MCF-7 MS cells exhibited BCSC-like characteristics such as high expression of the stem cell marker OCT4, low expression of the differentiation marker keratin 18, strong colony-forming ability, increased migration capability and strong tumorigenicity in vivo. We also compared the expression of ERα in MCF-7 and MCF-7 MS cells, as well as their tumours using Western blotting, and no significant difference was identified (Supplementary Figure S5), suggesting that no variant appeared, as reported by Cariati et al. [20]. In addition, we found that MCF-7 MS cells were sensitive to salinomycin and resistant to paclitaxel. The lower concentrations of paclitaxel (0.1–1 nM) promoted the proliferation of MCF-7 MS cells and the higher concentrations of paclitaxel could not achieve the same inhibitory effects on MCF-7 MS cells as on MCF-7 cells as determined by cell viability analysis and mammosphere formation assay, which was consistent with the findings by Oak et al. [33]. These data suggest that MCF-7 MS cells exhibited BCSC-like pharmacological properties as reported previously by Gupta et al. [21].

It is well known that the Hh signalling pathway is crucial for regulating embryonic development through control of stem cells [34], and maintaining stemness of CSCs [3537]. However, the contribution of the Hh signalling pathway to the drug resistance/sensitivity of BCSCs is largely unknown. In the present study, we showed that the major components of the Hh signalling pathway, including PTCH, SMO, Gli1 and Gli2, were expressed at a significantly higher level in MCF-7 MS cells and MCF-7 MS cell-induced tumours than in parental MCF-7 cells and MCF-7 cell-induced tumours, suggesting that the Hh signalling pathway is highly activated in BCSC-like MCF-7 MS cells. The role of the Hh signalling pathway in the drug sensitivity of MCF-7 cells has been reported by Ramaswamy et al. [38] showing that the mRNA expression of SMO and Gli1 was significantly higher in tamoxifen-resistant MCF-7 cells than in their parental MCF-7 cells, but they did not study the contribution of the Hh pathway to the BCSCs. Hh signalling activation results in an increase in the expression of many downstream target genes including c-Myc, Bcl-2 and Snail, which regulate cell proliferation, apoptosis and migration [3941]. In the present study, we then showed that salinomycin, but not paclitaxel, inhibited cell survival, increased the percentages of early apoptotic cells, decreased the migration capacity of MCF-7 MS cells, accompanied by a decreased expression of PTCH, SMO, Gli1 and Gli2, as well as c-Myc, Bcl-2 and Snail, suggesting that the Hh signalling pathway may contribute to the drug response of MCF-7 MS cells.

We confirmed further the role of the Hh signalling pathway in the drug response of MCF-7 MS cells through manipulating the activation of Hh signalling by using an Hh signalling activator Shh and a SMO inhibitor cyclopamine. Shh activated the Hh signalling pathway and largely blocked salinomycin-induced inhibition of the expression of PTCH, SMO, Gli1 and Gli2, as well as inhibiting salinomycin-induced cytotoxicity and reversing salinomycin-induced inhibition of mammosphere formation in MCF-7 MS cells. Conversely, cyclopamine partially inhibited the Hh signalling pathway and enhanced paclitaxel-induced cytotoxicity and inhibition of mammosphere formation in MCF-7 MS cells. Consistent with our in vitro data, we also found that salinomycin treatment significantly reduced the tumour volume and mass compared with paclitaxel, accompanied with decreased expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours, suggesting that salinomycin inhibits tumour growth likely also through inhibiting the Hh signalling pathway. In contrast, paclitaxel only slightly reduced the tumour volume and mass with no changes in the expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. The mild paclitaxel-mediated inhibition of tumour growth may be due to its inhibition of cancer cells that were differentiated from MCF-7 MS cells in vivo. Gupta et al. [21] reported that paclitaxel-treated mice exhibited a greater reduction in tumour growth in mice transplanted with SUM150 human breast cancer cells than in the present study using MCF-7 MS cells. Different breast cancer cells with different differentiation capacities probably contribute to the different response to paclitaxel between the two studies.

Wnt signalling pathway is another important pathway regulating the stemness of CSCs. We also found higher expression of Wnt1, p-LRP6 and β-catenin, the main components of the Wnt pathway, and higher expression of Axin2, one of the major targets of Wnt pathway, but lower expression of p-β-catenin, representing the degradable form of β-catenin, in MCF-7 MS cells compared with MCF-7 (Supplementary Figure S6A). Lu et al. [42] reported that salinomycin inhibited Wnt signalling and selectively induced apoptosis in chronic lymphocytic leukaemia cells. We observed that salinomycin, but not paclitaxel, could inhibit the expression of Wnt1, p-LRP6, β-catenin and Axin2 and increased the expression of p-β-catenin in MCF-7 MS cells (Supplementary Figure S6C), but the inhibitory effect of salinomycin on the Wnt pathway was weaker than that on the Hh pathway. Neither paclitaxel nor salinomycin significantly altered the expression of these proteins in MCF-7 cells (Supplementary Figure S6B). In addition, the rescue of the salinomycin effect by Wnt3a, an activator of the Wnt pathway, was found to be modest through examining the expression of the components of the Wnt pathway, the cell viability and mammosphere formation in the salinomycin-treated MCF-7 MS cells (Supplementary Figure S7). Taken together, our findings suggest that the Hh signalling pathway rather than the Wnt pathway plays an essential role in the drug response of BCSCs and inhibition of this pathway is likely to be important for killing BCSCs. Given the potential important role of the Hh signalling pathway in drug resistance of other types of cancers [17,18,43], inhibition of this pathway may be a potential therapeutic strategy for reducing chemoresistance in various types of cancers.

We investigated further the role of the Hh signalling pathway in breast cancer patients receiving chemotherapy. We found overexpression of SMO and Gli1 in breast cancer patients, suggesting that the Hh signalling pathway was activated in human breast cancer. Consistent with our findings, Kubo et al. [15] found overexpression of Shh, PTCH and Gli1 in 52 human breast cancer samples [15]. In addition, we found that the expression of SMO and Gli1 was positively correlated with the expression of CD44+/CD24, and the percentage of SMO-positive and Gli1-postive samples was significantly higher in CD44+/CD24 samples than in non-CD44+/CD24 samples. These findings suggest that the Hh signalling pathway is activated in human BCSCs.

Currently, the most common chemotherapy regimens include anthracycline-based therapy such as AC (adriamycin/cyclophosphamide), CEF and CAF regimens and taxol (i.e. paclitaxel)-based therapy such as CET, CAT (cyclophosphamide/adriamycin/taxol) and AT (adriamycin/taxol) regimens [44]. In the present study, we analysed the correlation between the expression of SMO and Gli1 and therapeutic response in 207 breast cancer patients receiving CEF, CAF and CET chemotherapy regimens. We found that the expression of SMO and Gli1 was associated with a shorter OS and DFS in breast cancer patients. In particular, the correlation of the expression of SMO with poor OS and DFS was more significant in the subgroup of chemotherapy-treated breast cancer patients whose tumour tissues are CD44+/CD24, whereas no significant correlation was observed in the subgroup of the patients whose tumour tissues are not CD44+/CD24. Together, our data suggest that Hh signalling activation may mediate BCSC-mediated chemoresistance and thereby result in poor therapeutic response in breast cancer patients who received chemotherapy.

In summary, we showed that Hh signalling activation contributed to BCSC-mediated chemoresistance in cultured breast cancer MCF-7 MS cells, in xenograft mice and in human breast cancer patients. Our studies provide new insights into the molecular mechanisms underlying the BCSC-mediated drug response. Moreover, our findings suggest that the Hh signalling pathway may represent an important target for reversing BCSC-mediated chemoresistance, and Hh signalling inhibition in combination with conventional chemotherapy is expected to improve therapeutic outcomes in breast cancer patients.

AUTHOR CONTRIBUTION

Minjie Wei and Miao He designed the experiments. Yingzi Fu, Yuanyuan Yan, Qian Jiang, Weifan Yao, Haishan Zhao and Miao He performed the experiments. Yingzi Fu, Yuanyuan Yan, Huizhe Wu, Zhaojin Yu and Miao He analysed the data. Feng Jin, Xiaoyi Mi, Enhua Wang, Liwu Fu and Zeshi Cui provided technical and material support. Miao He, Minjie Wei, Lin Zhao, Qinghuan Xiao and Jianjun Chen wrote and reviewed the paper.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant numbers 81373427 and 81102472], Program for Liaoning Innovative Research Team in University (LNIRT), China [grant number LT2014016], the open project of State Key Laboratory of Oncology in South China [grant number HN2014-03] and the S&T Projects in Shenyang, China [grant number F12-148-9-00].

Abbreviations

     
  • ABC

    ATP-binding cassette

  •  
  • BCSC

    breast cancer stem cell

  •  
  • CCK-8

    Cell Counting Kit-8

  •  
  • CAF

    cyclophosphamide/adriamycin/fluorouracil

  •  
  • CEF

    cyclophosphamide/epirubicin/fluorouracil

  •  
  • CET

    cyclophosphamide/epirubicin/taxol

  •  
  • CSC

    cancer stem cell

  •  
  • DFS

    disease-free survival

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • E-cadherin

    epithelial cadherin

  •  
  • ERα

    oestrogen receptor α

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • Hh

    Hedgehog

  •  
  • LRP6

    low-density lipoprotein receptor-related protein 6

  •  
  • MCF-7 MS

    MCF-7 mammosphere

  •  
  • OCT4

    octamer-binding transcription factor 4

  •  
  • OS

    overall survival

  •  
  • PTCH

    Patched

  •  
  • ROC

    receiver operating characteristic

  •  
  • Shh

    Sonic Hedgehog

  •  
  • SMO

    Smoothened

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Supplementary data