Introduction
Cancers are well known to consist of heterogeneous populations of cells that differ in marker expression, proliferation capacity, and tumorigenicity [
1,
2]. The existence of cancer stem cells (CSCs) has been reported in a variety of malignancies, including leukemia [
3], and solid tumors such as brain cancer [
4], breast cancer [
5], and colon cancer [
6]. In breast cancer, CD24
-CD44
+ [
5] or cells with high aldehyde dehydrogenase (ALDH) activity [
7] have been shown to be enriched in breast cancer stem cells (BCSCs). In addition to their tumor-initiating capacity, BCSCs were reported to be radiation resistant [
8] and prone to metastasis [
9,
10]. Eradication of BCSCs is thus a key to curative therapy of breast cancer [
11], and identifying pathways crucial for BCSCs may provide valuable clues for therapeutic targets.
The phosphatidylinositol-3-kinase (PI3K)/Akt (also known as protein kinase B) pathway has been demonstrated to be dysregulated in many types of cancer, including breast cancer [
12], and to be associated with poor prognosis [
13,
14]. In tumors, hyperactivation of the PI3K/Akt pathway may occur by activation of upstream growth factor receptors, overexpression or amplification of Akt, or inactivation of a phosphatase and tensin homolog tumor suppressor [
15]. One of the growth receptors associated with activation of Akt is insulin-like growth factor-1 receptor (IGF-1R), which can turn on the signaling cascade of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway upon stimulation with insulin-like growth factor-1 (IGF-1) [
16]. The expression of IGF-1 in breast cancer tissues [
17] and serum of breast cancer patients [
18] was significantly higher than those in normal healthy individuals. Besides, overexpression and hyperphosphorylation of the IGF-1R in primary breast tumors were reported to correlate with radioresistance and tumor recurrence [
19]. Although the IGF-1/IGF-1R pathway seems to be important in breast cancer, its role in BCSCs remains to be delineated. In this study, we investigated the possibility that IGF-1R signal might play an important role in the tumorigenicity and maintenance of BCSCs.
Methods
Ethics statement
All of the studies involving human participates were fully encoded to protect patient confidentiality and were utilized under a protocol approved by the Institutional Review Board of Human Subjects Research Ethics Committees of Tri-Service General Hospital and by Academia Sinica, Taipei, Taiwan. All patients enrolled in this study have signed an informed consent form to agree to participate in this study and for publication of the results.
All of the animal studies were operated following a protocol approved by the Institutional Animal Care & Utilization Committee of Academia Sinica, Taipei, Taiwan.
Isolation and transplantation of primary tumor cells
Primary breast cancer cells were harvested from tumor tissues as described previously [
20]. All human breast cancer specimens were obtained from patients who had undergone initial surgery at the Tri-Service General Hospital (Taipei, Taiwan). Samples were fully encoded to protect patient confidentiality and were utilized under a protocol approved by the Institutional Review Board of Human Subjects Research Ethics Committees of Tri-Service General Hospital and Academia Sinica, Taipei, Taiwan.
After receiving the specimens, tumor mass was sliced into 1 mm pieces and digested with collagenase/hyalurondiase digestion buffer (StemCell Technologies, Vancouver, BC, Canada) at 37°C for 2 hours. The released tumor cells were collected after filtration with a 40 μm cell strainer (BD Biosciences, San Jose, CA, USA). Before inoculation of primary tumor cells, 8-week-old female NOD/SCID mice (Tzu Chi University, Hualien, Taiwan) received a sublethal dose of gamma irradiation. For initial establishment and serial passages of xenografts, 1×10
6 tumor cells were mixed with 5×10
5 normal human breast fibroblasts/site in 2 mg/ml Matrigel and were subcutaneously injected into mammary fat pads of mice. For CSC frequency determination, a serial dilution of sorted tumor cells was mixed with normal human breast fibroblasts and Matrigel and was injected into mammary fat pads of NOD/SCID mice as described above. The tumor formation was monitored weekly. CSC frequency was calculated by Extreme Limiting Dilution Analysis software [
21].
Fluorescence-activated cell sorting
Anti-CD24-PE, anti-CD44-APC, anti-H2Kd-FITC, and anti-IGF-1R-PE antibody were purchased form BD Biosciences and the ALDEFLUOR assay kit was purchased from StemCell Technologies. Cell labeling with fluorescent-conjugated antibodies or ALDEFLUOR assay was performed according to the manufacturer's recommendations. Sorting of antibody-labeled cells was carried out on a FACSAria cell sorter (BD Biosciences).
Cell culture and reagents
Sorted H2Kd-CD24-CD44+ cells from BC0145 xenograft and H2Kd-ALDH+ cells from BC0244 xenograft were cultured in MEM containing 10% fetal bovine serum and insulin (10 μg/ml) at 37°C with 5% CO2 and designated AS-B145 and AS-B244, respectively. They could be propagated in serial passages, with emergence of phenotypic diversity of ALDH activity as noted in xenografted tumors. These cultured cells served as convenient in vitro cell models for investigating the signaling pathways involved in the maintenance of BCSCs. CB-124005 (Akt inhibitor), PI-103 (PI3K/mTOR inhibitor), rapamycin (mTOR inhibitor), and picropodophyllin (PPP; IGF-1R inhibitor) were purchased from Calbiochem (Billerica, MA, USA), and FPA-124 (Akt inhibitor) was purchased from Tocris Bioscience (Bristol, BS, UK). All of the small-molecule inhibitors were dissolved in dimethylsulfoxide.
Knockdown of IGF-1R expression
Negative control siRNA or IGF-1R-specific siRNA were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and delivered into cells by Metafectene SI transfection reagent (Biontex Laboratories GmbH, Martinsried, Germany) at 100 nM according to the manufacturer's protocol. For
in vivo xenograftment assay, knockdown of IGF-1R was performed by lentivirus-mediated gene silencing. The lentivirus that carry luciferase-specific shRNA (sh-Luc) or IGF-1R-specific shRNA (sh-IGF-1R) were obtained from the National RNAi Core Facility at the Institute of Molecular Biology, (Academia Sinica, Taipei, Taiwan), produced and transduced into cells as described previously [
20].
Fluorescence-activated cell sorting analysis of pAktSer473 and E-cadherin
Tumor cells from primary breast tumor tissue were resuspended in staining buffer (0.2% BSA in PBS containing 0.05% NaN3) containing an antibody against phosphor-Akt (Ser473; BD Biosciences), anti-CD45-PerCP-Cy5.5, anti-CD24-PE, and anti-CD44-APC. Phosphor-AktSer473-expressing cells in BCSCs (CD45-/CD24-/CD44+) and non-BCSCs (other cells in the CD45- population) were further analyzed with FACSCalibur (BD Biosciences) flow cytometer and WinMDI software (The Scripps Research Institute, La Jolla, CA, USA). For determination of E-cadherin expression by fluorescence-activated cell sorting (FACS), cells were harvested by 5 mM ethylenediamine tetraacetic acid treatment, incubated with mouse monoclonal anti-E-cadherin antibody (Santa Cruz Biotechnology), followed by Alexa-488 conjugated secondary antibody (Molecular Probes, Grand Island, NY, USA).
Cells were resuspended in Dulbecco's MEM-F12 medium containing 1% methyl cellulose to avoid cell aggregation, and basic fibroblast growth factor (20 ng/ml; PeproTech, Rocky Hill, NJ, USA), human epidermal growth factor (20 ng/ml; PeproTech), insulin (5 μg/ml), and B27 supplement (at a 50× dilution; GIBCO, Grand Island, NY, USA). Cells were seeded at 1,000 cells/well into ultralow-attachment 96-well plates (Corning Life Sciences, Tewksbury, MA, USA). After 7 days of incubation, the number of mammospheres was counted using bright-field optical microscopy under a 20× objective lens, and data were presented as the sphere number per 1,000 cells.
Western blot analysis
Cells were lysed in RIPA lysis buffer containing NP-40. Twenty-five micrograms of extracted protein was separated using a 4 to 12% gradient NuPAGE (Invitrogen, Grand Island, NY, USA) and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA). The membrane was then incubated with antibodies against Akt, phosphor-Akt (Ser473), mTOR, phosphor-mTOR (Ser2448), GAPDH (Cell Signaling Technology, Danvers, MA, USA), phospho-insulin receptor (Tyr972), insulin receptor (IR; GeneTex Inc., Irvine, CA, USA) phospho-IGF-1R (Tyr1165/1166; Santa Cruz Biotechnology), β-actin (Sigma-Aldrich, St. Louis, MO, USA), and the IGF-1R (R&D Systems, Minneapolis, MN, USA). Alkaline phosphatase-conjugated anti-rabbit or anti-mouse immunoglobulin G (Promega, Madison, WI, USA) was used as the secondary antibody. Fluorescent signals from catalyzed ECF substrate were scanned using a Typhoon9400 Variable Mode Imager (Amersham BioScience, Pittsburgh, PA, USA). The quantifications of band intensities were calculated with ImageJ software (National Institutes of Health, Bethesda, MD, USA) or Bio1D (Vilber Lourmat, Marne-la-Vallée, France).
p-IGF-1RTyr1165/1166 analysis after immunoprecipitation of IGF-1Rβ
Total cell lysates (500 μg) from sorted ALDH- or ALDH+ BC0244 xenograft tumor cells were used for immunoprecipitation analysis. Briefly, 1 μg IGF-1Rβ specific antibody (sc-713; Santa Cruz Biotechnology) was added into cell lysates (500 μg/200 μl Tris-buffered saline) and incubated at 4°C overnight. After adding 10 μl Protein G Mag Sepharose beads (GE Healthcare Life Science, Pittsburgh, PA, USA), the solutions were further incubated for 2 hours at room temperature. The beads were then proper washed and the binding proteins were eluted by 1× SDS-PAGE sample loading dye. The eluted proteins were further separated by 10% SDS-PAGE and blotted with anti-p-IGF-1RTyr1165/1166 and anti-IGF-1Rβ antibodies according the protocol of western blot analysis.
Cell migration assay
Cells were suspended in serum-free culture medium, seeded at in the upper chamber insert of a transwell plate (Corning Life Sciences) and then inserted into 24-well plates with serum-containing medium. After incubation at 37°C for 16 hours, cells that had migrated across the membrane of the insert were stained with crystal violet after removing the cells attached on the inner face of the insert and results were recorded by microscopy.
Immunofluorescence staining of E-cadherin
Cells were fixed with cold methanol followed by 3.7% formaldehyde/PBS. After blocking with 1% BSA/PBS, cells were incubated with an anti-E-cadherin antibody and then further incubated with an Alexa-488-conjugated secondary antibody. Fluorescence signals were captured under an inverted fluorescence microscope (Olympus, Shinjuku-ku, Tokyo, Japan).
Discussion
In this study, we used the reported BCSC markers, CD44/CD24, and ALDH activity to examine the role of IGF-1R in BCSCs. We showed greater phosphorylation of IGF-1R in BCSCs than in non-BCSCs and preferential sensitivity of BCSCs to PPP, a specific inhibitor of the IGF-1R, leading to reduced phosphorylation of Akt
Ser473 and decreased ALDH
+ BCSC populations. In human malignancies, increased circulating IGF-1 was associated with a greater risk of several cancers, including breast cancer [
18]. A crosstalk between IGF-1R and the Wnt pathway has been reported in colon cancer [
31], oligodendroglial cells [
32], and chondrocytes [
33]. The interaction of these two pathways in breast cancer is intriguing and awaits further investigation. Recently, two reports demonstrated the essential role of IGF/IGF-1R signaling in the maintenance of leukemia-initiating cells in T-cell acute lymphoblastic leukemia [
34] or in the transformation of hematopoietic progenitor cells in the mouse model of acute myelogenous leukemia [
35]. The IGF-1R expression of leukemia-initiating cells in T-cell acute lymphoblastic leukemia was maintained by Notch signaling [
34], which also contributed to the maintenance of BCSCs [
36,
37]. Whether the Notch pathway is involved in the IGF-1R signaling in BCSCs remains to be investigated. In solid tumors, chemoresistant colorectal cells displayed a CSC phenotype and became more sensitive to IGF-1R inhibition [
38]. In hepatocellular carcinoma, the IGF-2/IGF-1R signal was shown to be involved in Nanog-mediated self-renewal of hepatic CSCs [
39]. These reports also support the importance of IGF-1R in CSC biology. In breast cancer, activation of the IGF-1R could result in stimulation of proliferation and metastasis through activation of insulin receptor substrate-1 [
40] and insulin receptor substrate-2 [
41]. Furthermore, it has been reported that IGF-1R expression was positively correlated with a shorter disease-free survival in triple negative breast cancer [
42], the particular subtype with the highest rate of recurrence and higher percentage of BCSCs than other breast cancer subtypes [
43]. In a recent report by Jones and colleagues, recurrence of breast cancer was observed in 16% of inducible IGF-1R transgenic mice upon the discontinuation of doxycycline and the recurrence involved IGF-1R-reactivation and IGF-1R-independent mechanisms [
44]. Although the IGF-1R-independent tumors displayed EMT phenotypes, their metastatic potential was much lower than tumors with IGF-1R reactivation [
44].
Moreover, induction of EMT in immortalized human mammary epithelial cells by overexpressing EMT-related transcriptional factors, twist or snail, or treatment with transforming growth factor β1 generated CD44
+ BCSCs [
10]. Recently, Lorenzatti and colleagues found that CCN6, a tumor inhibitory protein, could suppress the expression of EMT transcriptional factor ZEB1 in breast cancer cells through attenuation of IGF-1R signaling [
26]. Along this line, we also showed that inhibition of IGF-1R signaling suppressed the cell migration ability of CD44
+ BCSCs through induction of E-cadherin, the adhesion molecule that blocks the EMT process, as well as suppression of other mesenchymal markers (vimentin, twist and N-cadherin). More importantly, IGF-1R could serve as a novel marker for a particular population of cancer cells with stem/progenitor features within breast cancer since IGF-1R high-expressing human breast cancer cells displayed the capacity for mammosphere formation
in vitro and tumorigenicity
in vivo. Furthermore, mammosphere formation of IGF-1R expressing BCSCs was sensitive to PPP treatment. When comparing the CSC frequency of different populations of breast cancer cells, high expression of IGF-1R seems to be most efficient in enriching BCSCs in a triple negative breast cancer BC0244 xenograft (one out of 909 cells; Figure
1D). For BC0145 xenograft, the identification of cancer stem/progenitors based on IGF-1R expression (one out of 54,785; Figure
1D) was slightly better than ALDH
+ (one out of 93,412; see Table S1 in Additional file
1) but not as good as CD24
-CD44
+ (one out of 6,401; see Table S1 in Additional file
1). These results are consistent with the known heterogeneity in the BCSC-enriched population. It has been shown that overexpression of IGF-1R in MCF7 increased the size of colonies under three-dimensional culture conditions [
45], which corroborated our observation. To the best of our knowledge, this is the first demonstration of IGF-1R as a marker for cancer stem/progenitors in breast cancer.
To further investigate the downstream signaling of IGF-1R, we explored the involvement of the PI3K/Akt/mTOR pathway. The upstream stimuli of the PI3K/Akt/mTOR pathway in mammary stem/progenitor cells have been linked to Wnt/β-catenin signaling. Korkaya and colleagues demonstrated that phosphatase and tensin homolog knockdown increased phosphorylation of Akt, leading to enriched normal and malignant mammary stem/progenitor cells, and that this Akt-driven process was mediated by the Wnt/β-catenin pathway [
46]. They also demonstrated that perifosine, an Akt inhibitor, could suppress both
in vivo tumorigenicity and the
in vitro ALDH
+ population of a breast cancer cell line and two xenografts of primary breast cancer [
46]. In this study, we documented enhanced activation of PI3K/Akt/mTOR in BCSCs of primary human breast cancer and two xenografts of primary tumors, and suppressive effects of the mTOR inhibitor, rapamycin, on their growth
in vitro and
in vivo, as well as mammosphere formation. Herein, we have provided evidence for another mechanism of activation of the PI3K/Akt/mTOR pathway via IGF-1R signaling in BCSCs. The importance of PI3K/Akt/mTOR in BCSCs of clinical samples is consistent with findings from previously reported studies of breast cancer cell lines [
46,
47]. Enhanced phosphorylation of Akt
Ser473 as determined by immunohistochemical staining was reported to correlate with a poor prognosis for breast cancer [
14]. In the present study, it was demonstrated for the first time that pAkt
Ser473 was higher in CD45
-CD24
-CD44
+ BCSCs than the non-BCSCs, in a majority (7/11, 63.6%) of those primary tumors with detectable pAkt and their xenografts in mice. There was no obvious correlation with clinical and histopathological features (see Table S2 in Additional file
1). Although BCSCs are frequently enriched in CD24
-CD44
+ cells, such markers cannot be generalized to all patients, given the inherent heterogeneity of breast cancer. The variation of markers useful for enrichment of BCSCs among patients may thus explain the lack of consistent elevation of pAkt
Ser473 in CD24
-CD44
+ cells in some patients.
Taken together, our findings support the notion that IGF-1R signaling with activation of the downstream PI3K/Akt/mTOR pathway plays an important role in breast cancer progression by controlling both the maintenance of BCSCs and their EMT behavior. These studies also provide an impetus for developing cancer therapy targeting BCSCs by combining inhibitors or mAb against IGF-1R with inhibitors of the PI3K/Akt/mTOR pathway. Although preclinical evidence for the efficacy of several small molecule inhibitors and monoclonal anti-IGF-1R antibodies (figitumumab, robatumumab and R1507) was strong, large-scale clinical trials were halted due to very modest activity [
48]. The failure may be attributed in part to the selection of appropriate target population, and in part to the increased dependency of cancer cells on insulin [
49]. Along this line, targeting IGF-1R and IR simultaneously with OSI-906 and BMS 754807, which are small molecule inhibitors of tyrosine kinase activity of both IGF-1R and IR, is undergoing clinical trials [
48]. In addition, recent preclinical studies have shown that dual inhibition of mTOR with rapamycin and Akt with perifosine prevents mTOR inhibition-initiated Akt activation and significantly enhances antitumor effects in lung cancer [
50] and multiple myeloma [
51]. Also, combination of IGF-1R and mTOR inhibition showed clinical benefits in Ewing's sarcoma [
52]. With a similar strategy, dalotuzumab will be combined with Akt or mTORC1 inhibition [
48]. Thus, co-targeting the PI3K/Akt/mTOR pathway and its upstream signal, IGF-1R may prove to have synergistic anti-tumor effects and is worthy of further investigation.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
W-WC designed and performed the research, analyzed data and wrote the manuscript. R-JL performed the research and analyzed the data. W-YC helped the sorting and animal experiments, C-HF helped the FACS analysis of clinical specimens. AC-YL helped the western blot analysis of IGF-1R/Akt expression. J-CY contributed clinical specimens/analytic tools. JY contributed the manuscript preparation and revision. ALY participated in the research design, data analysis and manuscript preparation. All authors read and approved the final manuscript.