Introduction
Breast cancer is the second most common malignancy and cause of cancer death among American women [
1]. Despite advances in early detection, about 30% of patients with early-stage breast cancer have recurrent disease. Although systemic treatment of patients with chemotherapy, hormonal therapy and immunotherapy produce a high response rate initially, progression invariably occurs after a variable time interval [
2]. This may result from a small subpopulation of cells, i.e., cancer 'stem' cells or tumor-initiating cells (TICs) that exhibit a high capacity for self-renewal, and thus contribute to tumor maintenance and metastasis [
3], as well as relapse and drug resistance [
4].
CD133 has been implicated as a TIC marker for malignancies of the lung [
5], liver [
6,
7], colon [
8,
9], brain [
10,
11]. High CD133 expression has been noted in MCF-7 breast cancer cells that are either drug-resistant [
12] or resistant to TRAIL-mediated apoptosis [
13]. CD133
+ breast cancer cells form spheroid under low attachment conditions, and are enriched in stem cell markers, and rapidly form tumors in NOD/SCID mice [
14]. Thus, by these criteria, CD133 may be considered a TIC marker. Another protein associated with breast TICs is Musashi1 (Msi1) [
15‐
17]. Msi1 was first identified as a cell fate determinant for sensory organ development in
Drosophila[
18], and subsequent studies found the mammalian homolog to be a stem cell marker in the brain [
19,
20]. Msi1 is a highly conserved RNA-binding protein [
21] that recognizes a (G/A)U
n = 1-3AGU motif in the 3'-untranslated region of target mRNAs, such as Numb [
22] and p21
CIP1[
23], as well as other genes involved in cell cycle regulation, proliferation and apoptosis [
24]. When expressed in mammary epithelial cells, Msi1 activated a unique gene expression pattern associated with an autocrine pathway involving the growth factor Proliferin1 and inhibition of the secreted Wnt pathway inhibitor, Dickkopf3 [
15,
16]. This led to activation of Notch and Wnt signaling and mammary stem/progenitor cell proliferation.
The role of Msi1 in the etiology and progression of breast cancer is unknown. We hypothesized that Msi1 may promote breast TIC proliferation, and hereby influence survival outcome. Here we show that Msi1 is expressed in approximately two-thirds of primary breast cancers, and is associated with reduced survival. Msi1 was enriched in CD133+ MCF-7 and T47D cells, particularly when grown as spheroid cultures, and Msi1 'knockdown' reduced spheroid colony formation and tumor xenograft growth. These results suggest that Msi1 regulates TIC proliferation and is a negative prognosticator for survival in breast cancer patients.
Discussion
Msi1 is an RNA-binding protein that functions as a translational repressor [
20], and is widely used as a marker of stem cells in embryonic and adult tissues [
18,
21]. Although Msi1 has been reported to be elevated in high grade gliomas [
26] and liver cancer [
27], little is known about its function and prognostic value. Here we report that Msi1 is expressed in a high proportion of primary breast cancers and cell lines, particularly in metastatic disease. The most important prognostic factor influencing the outcome of patients with invasive breast cancer is whether the tumor has spread regionally or systemically [
28], and there is evidence that TICs play an important role in mediating metastasis. Our finding that 84% of breast cancer lymph node metastases expressed Msi1, in comparison to 42% of matched primary tumors, is consistent with the role of TICs in metastatic disease. Since Msi1 predicted shorter survival as well, it may ultimately be a useful prognostic indicator of regional spread of the disease.
In normal breast tissue, Msi1 is co-localized predominantly with myoepithelial cells in the TDLU, where approximately 10% of the cells are Msi1
+. Msi1 has been reported to be present in 0.6% of the total lobular epithelial cells, and enriched in label-retaining cells [
17], which divide asymmetrically as stem cells [
29]. Msi1-overexpressing mammary epithelial cells express stem and progenitor cell markers, where Msi1 co-localizes with CK14
+ myoepithelial cells [
16], a phenotype consistent with the basal cell characteristics of mammary stem cells [
30]. In the present study, Masi1 was functionally active in breast cancer cells, since inhibition of colony expansion and tumor growth by Msi1 KD resulted in increased Notch and reduced p21
CIP1. Numb, a translational target of Msi1, promotes the degradation of intracellular Notch [
22], and hence Msi1 KD would reduce Notch expression (Figures
3 and
5). p21
CIP1, an inhibitor of cyclin-dependent kinases, is a translational target of Msi1 [
23], and hence Msi1 KD would increase its expression and stimulate growth (Figures
3 and
5). These data indicate that Notch and p21
CIP1, among other factors, regulate the TIC capacity of MCF-7 and T47D cells, and are consistent with the growth inhibitory activity of Msi1 KD in medulloblastoma [
31] and colon tumor [
32] cells.
CD44
+/CD24
- and CD133 have been reported as TIC markers for breast cancer [
5‐
11,
13,
14,
36‐
38]. CD44
+/CD24
- cells represented a very small percentage (0.02-0.36%) of MCF-7 and T47D cells [
33] (Additional file
1, Figure S1). In contrast, CD133 was expressed in 1.7-6.2% of the cells, which were also enriched in Msi1. Msi1 was also enriched in spheroid cells, which have been reported to be less sensitive to ionizing radiation than monolayer cells and exhibit reduced senescence [
34,
35]. Mammary dysplasia induced by constitutively active hedgehog signaling also leads to an expansion of murine stem cells as high as 15-33% with an increased proliferative capacity [
36]. Similar findings of an increased tumorigenic capacity have been reported for spheroid breast cancer cells in pleural effusions [
37]. Thus, the capacity to form anchorage-independent spheroid cells reflects the TIC capacity of tumor cells, and is consistent with our tumor xenograft data.
In addition to affecting TIC proliferation, Msi1 regulated the expression of several somatic and ES cell markers, including CD133, Bmi1, Nanog, Oct4, Sox2 and c-Myc (Figure
3). Although, CD133 is TIC marker [
5‐
11,
13,
14,
38], its function in this context is largely unknown. Bmi1 is a polycomb protein that plays a critical role in somatic stem cell self-renewal based on gain-of-function and loss-of-function analyses [
39,
40]. Oct4, Sox2, Nanog and Lin-28 [
41], as well as Oct3/4, Sox2, Klf4, and c-Myc [
42] were shown to induce pluripotent stem cells from human fibroblasts. An ES cell gene signature has also been reported in poorly differentiated breast cancers and other tumors, and correlated with poor survival [
43]. Thus, Msi1 appears to regulate transcriptional pathways required for survival and maintenance of the undifferentiated state.
Previously, Msi1 was found to activate an autocrine process in mammary epithelial cells that resulted in progenitor cell expansion through proliferin-mediated activation of ERK and Wnt and Notch signaling [
15,
16]. These findings are in agreement with the reduction of pERK1/2 and Notch1 expression after Msi1 KD. Wnt and ERK signaling constitute an oncogenic positive feedback loop [
44], and inhibition of these pathways led to the differentiation of ES cells [
16]. It therefore seems likely that activation of Wnt signaling through ERK by Msi1 plays a similar role in the maintenance of TIC proliferation and the undifferentiated state [
15,
16].
There was also a correlation between Msi1 and ErbB2 expression. ErbB2 was reduced in cells after Msi1 KD, suggesting that either ErbB2-expressing cells are only affected or that Msi1 acts downstream of ErbB2 signaling. ErbB2 is notable for its role in the pathogenesis of breast cancer and as a therapeutic target, and is frequently associated with metastatic disease and tumor progression [
45]. Recently, ErbB2 was shown to induce Notch1 activity in breast cancer cells [
46], which is consistent with its ability to drive mammary stem cell proliferation, tumorigenesis and invasion [
47]. Since Msi1 activates the Notch pathway by directly targeting Numb, our findings suggest a common endpoint between the Msi1 and ErbB2 pathways that merits further examination in a larger subset of patients.
Methods
Cell Lines
Human breast cancer cell lines BRC-230, BT20, BT483, BT474, BT549, DU4475, Hs578T, MCF-7, MDA-MB-134VI, MDA-MB-157, MDA-MB-231, MDA-MB-330, MDA-MB-361, MDA-MB-415, MDA-MB-435, MDA-MB-436, MDA-MB-468, SK-BR-3, T47D and ZR-75-1, and immortalized human mammary epithelial cell line MCF-10A were obtained from the Tissue Culture Shared Resource, Lombardi Comprehensive Cancer Center. MCF-7 and T47D cells were maintained in Dulbecco's- modified Eagle's medium (DMEM) (Sigma-Aldrich Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum at 37°C under 5% CO2 in a Sanyo CO2 incubator.
Western Blotting
Cells lysates were prepared on ice [
48], and protein concentration was determined using the Bicinchonic Acid Assay (Pierce, Rockford, IL). Cell lysates (20 μg protein) were separated in Criterion XT Bis-Tris Gels (Bio-Rad), transferred to a polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ) and separated in the Criterion System (Bio-Rad, Hercules, CA). Proteins were detected with the appropriate primary antibody using horseradish peroxidase-linked secondary antibodies, and visualized by chemiluminescence with the West Pico system (Amersham Biosciences, Piscataway, NJ). Primary antibodies included rat anti-Msi1 or anti-biotinylated Msi1 (1:1000) [
49] (14H-1, Dr. Hideyuki Okano, Keio University, Tokyo, Japan), rabbit anti-ErbB2 (1:1000) (Millipore, Danvers, MA), mouse anti-ERα (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-CD133 (1:1000) (Cell Signaling Technology, Danvers, MA), rabbit anti-pERK1/2 (1:1000)(Cell Signaling Technology), mouse anti-β-actin (1:5000) (Sigma-Aldrich Corp), rabbit anti-Notch1(D1E11) (Cell Signaling Technology) and mouse anti-p21
CIP1 (EMD Chemicals, Gibbstown, NJ).
Flow Cytometry
For CD133, CD24 and CD44 detection, MCF-7 and T47D cells were suspended at a concentration of 0.5-1.0 × 106 cells/ml in ice-cold PBS containing 3% fetal bovine serum (PBS/FBS). One μg of CD133 unconjugated primary antibody (Cell Signaling Technology), CD24-biotin or CD44-phyoerythrin(PE)-Cy5 (eBioscience, Inc. San Diego, CA) or control IgG isotype antibody was incubated with 106 cells on ice for 60 min. Cells were washed three times by centrifugation at 400 × g for 5 min, and resuspended in ice-cold PBS/FBS. CD24 was measured with FITC-conjugated streptavidin (eBioscience) (1:100 dilution) after incubation for 45 min. CD133 was measured with an anti-rabbit secondary antibody conjugated with PE after incubation at room temperature for 45 min. Cells were sorted with a Becton Dickinson FACSort system into CD133+ and CD133- cell populations and analyzed with FCS Express V3 software (De Novo Software, Ontario, Canada). Cells were washed twice with PBS and the cell pellet stored at -80°C for RT-PCR.
Msi1 expression was 'knocked down' using an shRNAmir GIPZ lentiviral vector targeting the sequence, 5'-CGT-CCT-GTA-TCA-TAT-GTA-AAT-3' in the 3'-UTR of Msi1 mRNA (Oligo ID # V2HS_280120; Open Biosystems, Huntsville, AL) [
31]. TLA-HEK293T cells (Open Biosystems) were transfected with the Trans-Lentiviral Packaging Mix and pGIPZ transfer vector at 50% confluence using Arrest-In transfection reagent (Open Biosystems) according to the manufacturer's protocol. After incubation for 48-72 hr, the virus-containing supernatant was collected and centrifuged at 3,000 rpm for 20 min at 4°C, mixed 50:50 with fresh cell culture media, and used to transduce MCF-7 and T47D cells. Lentivirus expressing either a non-silencing control shRNA (shRNAmir, Open Biosystems) served as a negative control. Cells were selected for stable integration of the virus by incubation with 2.5 μg/ml puromycin (Sigma-Aldrich Corp.) for 10 days. The efficiency of integration was monitored by green fluorescent protein (
GFP) co-expressed by the lentivirus.
RT-PCR
RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA) using the manufacturer's protocol. One μg RNA was reverse-transcribed in a total volume of 20 μl using the Omniscript RT kit (Qiagen). PCR was performed in triplicate using an ABI-Prism 7700 instrument (Applied Biosystems, Foster City, CA) and SYBR Green I detection (Applied Biosystems) according to the manufacturer's protocol. Amplification using the appropriate primers (Additional file
2, Table S1) was confirmed by ethidium bromide staining of the PCR products in an agarose gel. The expression of each target gene was normalized to the expression of 18S RNA and is presented as the ratio of the target gene to 18S RNA, expressed as 2
-ΔCt, where Ct is the threshold cycle and ΔCt = Ct
Target - Ct
18S as described previously[
16].
Spheroid Cell Culture
Spheroid cell culture was carried out in ultra low attachment plates as described with small modifications [
25]. Briefly, MCF-7 and T47-D cells growing as monolayer cultures were trypsinized with 0.05% trypsin-0.5 mM EDTA (Invitrogen, Carlsbad, CA), washed twice with PBS, counted and seeded into a 6-well plate at a density of 3,000 viable cells/ml or into a 96-well plate at serial dilutions of 125 to 16 cells per 200 μl. Cells were grown in serum-free DMEM medium supplemented with 1× B27 (Invitrogen), 20 ng/ml EGF (Sigma) and 20 ng/ml FGF-2 (Invitrogen) at 37°C under 5% CO
2. Spheroid clusters were counted and collected by gravity or gentle centrifugation (800
g, 10 sec) after 21 days, and dissociated in 0.05% trypsin-0.5 mM EDTA for 10-15 min by gentle pipeting. Cells were filtered through a 40-μm nylon mesh sieve (Falcon), analyzed microscopically for single cellularity and counted. Successive passages were plated at 1,000 cells/ml in 6-well plates and colony size quantitated using MetaMorph software.
Growth Assay
Cells were seeded in 96-well plates at 5,000 cells/well in 200 μl medium, and growth was determined 24 to 72 h later by sulforhodamine B staining [
50]
Tissue Microarrays
Breast cancer tissue microarray slides were obtained from Imgenex (San Diego, CA, Cat# IMH-371 and IMH-364), US Biomax (Rockville, MD, Cat#BR951) and Cybrdi (Xian, China, cat# CC08-01). There were a total of 140 cases of primary breast cancer, 19 metastatic lymph node carcinomas and 24 normal adjacent breast tissues. All patients were female between 20 and 84 years of age, with a median age of 47. Eighty four of 140 cases contained TNM stage data, 78 of 140 cases contained ER, PR and p53 status, and 40 of 110 cases were analyzed for ErbB2 expression (Table
1). After diagnosis, 75 of 140 patients were followed up for 1 to 107 months, with a median follow-up time of 76 months, and 11 were deceased by 60 months.
Immunohistochemistry
Tissue microarray slides were baked at 62°C for 1 hr, deparaffinized in xylene for 15 min, and rehydrated in 100%, 95% and 70% ethanol for 5 min each. Antigen retrieval was achieved by steaming slides for 10 min in 10 mM citrate buffer, pH 6.0. Slides were washed three times in PBS and blocked for 1 hr in a buffer containing 10% goat serum in PBS, and incubated overnight at 4°C with a 1:1,000 dilution of rat anti-Msi1 antibody 14H-1 conjugated with biotin [
49]. Slides were washed three times in PBS and antigen visualized with ABC Vectastain and DAB as substrate (Vector Labs, Burlingame, CA). Slides were counterstained with Harris-modified hematoxylin (Thermo-Fisher, Pittsburgh, PA) and mounted in Permount. The level of Msi1 expression in breast cancer tissue was categorized as negative, low (1~30% positive) and high (>31% positive). Four high power fields (400×) were randomly chosen for counting Msi1
+ cells in each core of the slides. The total cell number counted was ~1000 per core per patient. All slides were reviewed by one pathologist (Linnoila RI, MD) and one well-trained researcher in pathology (Wang XY, MD, PhD) blinded to the patients' clinical information. The agreement between the two researchers for the determinations of all tumor samples was 90.7% with a 0.76 of Cohen's Kappa indicating substantial agreement.
Dual Label Immunofluorescence
Dual label immunofluorescence was performed using methods similar to those described previously [
16]. The primary antibodies used were: mouse monoclonal anti-human smooth muscle actin (SMA) (clone 1A4, Dako North America, Inc. Carpinteria, CA) and rat monoclonal anti-mouse Msi1 antibody 14H-1 [
49]. Antigens were retrieved as described above, and detected sequentially on sections by incubation for 1 hr with the first (Msi1) primary antibody followed by incubation with the appropriate PE-conjugated streptavidin (eBioscience, Inc), and then incubation for 1 hr with the second primary antibody followed by FITC-conjugated secondary antibodies. All incubations were at room temperature and sections were washed in PBS (3 × 5 min) between each step. Sections were mounted in an anti-fading reagent with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). Control slides were included in each analysis in which non-immune serum was substituted for primary antibodies and secondary antibodies individually.
Tumor Xenografts
MCF-7 and T47D cells were grown as spheroid cultures and transduced with either the non-silencing shRNA or Msi1 shRNA as described above. Ten athymic nu/nu female mice of 6 weeks of age were each injected s.c. with a 60-day release pellet containing 0.72 mg 17-β-estradiol. Five animals were then injected s.c. into opposite flanks with either 100,000 MCF-7 cells transduced with a non-silencing shRNA or MCF-7 cells transduced with the Msi1 shRNA mixed with 0.45 mg Matrigel (BD Biosciences, Inc.). The same protocol was used for xenografts of T47D cells except that the inoculum was 10,000 cells. The frequency of engraftment of MCF-7 and T47D cells at these inocula was approximately 80%, which is similar to what is observed for standard xenograft procedures using inocula of ~5 × 106 cells. After 8 weeks, tumor size was imaged for GFP fluorescence using a Maestro imaging system (Cambridge Instruments, Inc.) with 1-3% isofluorane in oxygen as anesthetic.
Statistics
Fisher's exact test was used to analyze the correlation between Msi1 and ErbB2 protein expression in breast cancer cell lines. Fisher's exact two-tailed test was also used to determine the association between Msi1 and the clinic stage, grade and 5-year survival events in breast cancer patients. Student's t test was used to determine the significance of the differences between cell growth, spheroid cell number and sphere diameters in cell lines, as well as tumor size resulting from Msi1 KD. The Kaplan-Meier method, log rank test and Cox regression model were used to evaluate the effects of Msi1 protein expression on the overall survival of patients with breast cancer. All tests were two-sided and determined with SAS software (version 9.1; SAS Institute, Cary, NC, USA). P < 0.05 was considered statistically significant.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
XYW designed research, carried out the molecular and cell studies, analyzed data, wrote the paper, and LP contributed to the design of the shRNA studies, HY contributed to the xenograft studies, RIL contributed to the IHC slide review and analysis of clinical data, JL contributed to the tissue microarray studies, HO contributed to the IHC studies, and RIG contributed to the conception, design, analysis and interpretation of data. All authors read and approved the final manuscript.