Genetic and epigenetic analysis of putative breast cancer stem cell models
© Balic et al.; licensee BioMed Central Ltd. 2013
Received: 25 April 2013
Accepted: 22 July 2013
Published: 24 July 2013
Cancer stem cell model hypothesizes existence of a small proportion of tumor cells capable of sustaining tumor formation, self-renewal and differentiation. In breast cancer, these cells were found to be associated with CD44+CD24-low and ALDH+ phenotype. Our study was performed to evaluate the suitability of current approaches for breast cancer stem cell analyses to evaluate heterogeneity of breast cancer cells through their extensive genetic and epigenetic characterization.
Breast cancer cell lines MCF7 and SUM159 were cultured in adherent conditions and as mammospheres. Flow cytometry sorting for CD44, CD24 and ALDH was performed. Sorted and unsorted populations, mammospheres and adherent cell cultures were subjected to DNA profiling by array CGH and methylation profiling by Epitect Methyl qPCR array. Methylation status of selected genes was further evaluated by pyrosequencing. Functional impact of methylation was evaluated by mRNA analysis for selected genes.
Array CGH did not reveal any genomic differences. In contrast, putative breast cancer stem cells showed altered methylation levels of several genes compared to parental tumor cells.
Our results underpin the hypothesis that epigenetic mechanisms seem to play a major role in the regulation of CSCs. However, it is also clear that more efficient methods for CSC enrichment are needed. This work underscores requirement of additional approaches to reveal heterogeneity within breast cancer.
Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women . Despite combined treatment strategies and advances in treatment, metastatic breast cancer remains currently incurable. One of the possible reasons for therapeutic failure is the existence of tumor cell heterogeneity and presence of cancer stem cells (CSCs) . There are several indicators of intratumoral heterogeneity, including recognized prognostic and predictive markers and candidate biomarkers [3, 4]. Among the clinically established biomarkers are estrogen and progesterone receptor, and human epidermal growth factor receptor 2 (Her2-neu) . An emerging cellular candidate biomarker is the presence of breast CSCs [6, 7]. Increasing experimental evidence supports the cancer stem cell model , which is in favor of only a small proportion of cells with the capability of sustaining tumor formation and growth, self-renewal and differentiation. In breast cancer, CSCs have been identified as CD44+CD24-/low or aldehyde dehydrogenase positive (ALDH+) [9, 10]. Several approaches have been used to enrich and study breast CSCs, including flow cytometry sorting and their capability of forming mammospheres [9, 11]. Tumor sphere culture has been shown to represent a surrogate in vitro model to study CSCs [12, 13]. Identification of distinct properties and molecular biomarkers for breast CSCs may help to identify novel therapeutic targets and thereby allow development of more efficient therapeutic strategies .
We aimed to evaluate molecular heterogeneity of breast cancer cell lines with an emphasis on breast CSCs. For unsorted breast cancer cells and flow-sorted putative stem versus non-stem cells, DNA profiles were generated by array comparative genomic hybridization (aCGH) and methylation analyses of selected candidate genes were done by pyrosequencing. Functional impact of methylation was evaluated by mRNA analysis for selected genes.
Breast cancer cell lines
Breast cancer cell lines MCF7 and SUM159 were used for all experiments. SUM159 were obtained from Asterand (Detroit, MI, USA), and MCF7 from American Type Culture Collection (Manassas, VA, USA). Cells were cultured according to suppliers’ recommendations, harvested at 90% confluence and prepared for further analyses.
The culture of mammospheres (MMO) was performed according to previous publications . Briefly, cells were grown in serum-free Mammary Epithelial Basal medium MEBM (Lonza, Basel, Switzerland), supplemented with 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), 5 μg/mL insulin (both from Peprotech, New York, USA), and 20 μl/ml B27 supplement (Invitrogen, Leek, Netherlands). After the first passage, the mammospheres were filtered through a 40-μm Nylon Cell Strainer (BD, Falcon) to obtain purer spheres for further culture. The cells were then dissociated with TrypLE (Gibco/Invitrogen, Paisley, Renfrewshire, UK), following incubation at 37°C for 4 min. The cells were washed with two volumes of PBS (phosphate buffer saline) to inactivate the enzyme, resuspended in MEBM containing supplements and seeded for generation of secondary spheres.
For all sorting experiments cells were dissociated with Accutase (PAA Laboratories GmbH, Pasching, Austria) for 5 min at 37°C. When using the Aldefluor protocol , dissociated cells were suspended in Aldefluor assay buffer to a concentration of 106 cells/ml. When performing anti-CD44 and anti-CD24 flow-sorting , 106 cells were suspended in 100 μl of PBS/2% fetal calf serum (FCS). After 20 min of incubation on ice with blocking buffer consisting of horse serum diluted 1:20 in 6% bovine serum albumin/PBS solution, aliquots of antibodies (CD44 APC and CD24 FITC), each at a dilution of 1:40 in a final concentration of 0.08 μg/ml and 5 μg/ml, respectively, were added and staining was adapted from the previously published protocol . Briefly, after centrifugation the pellet was dissolved in 100 μl PBS/FCS and 2.5 μl of the antibodies (CD24-FITC and CD44-APC) were mixed with the cells and incubated for 20 min on ice. Then, cells were washed with 500 μl PBS/FCS and centrifuged again. Before sorting, cells were resuspended in 100 μl PBS/FCS, filtered and rinsed with 100 μl PBS/FCS. All fluorochrome-labeled monoclonal antibodies were acquired from BD Bioscience and pretitered to determine their optimal dilutions before use. Cells without staining and isotype controls, also from BD Bioscience, were integrated as controls in all experiments.
Cells with high ALDH activity in MCF7 and SUM159 cells were isolated using the Aldefluor Kit (StemCell Technologies, Durham, NC, USA) according to the manufacturer’s instructions and as previously published [10, 15]. Cells isolated with Aldefluor Kit were used for genetic and epigenetic analysis. Flow cytometry sorting was performed on the Aria fluorescence activating cell sorter (FACS) and acquired data were analyzed using the Diva software (BD Bioscience).
Genomic DNA from cultured cells was extracted using the Gentra Puregene Blood Kit (Qiagen) according to the manufacturer's protocol. DNA was dissolved in a final volume of 100 μL buffer and quantified spectrophotometrically using a BioPhotometer (Eppendorf, Hamburg, Germany).
Evaluation of genomic profiles
Whole genome amplification (WGA)
DNA from sorted SUM159 subpopulations (ALDH+, ALDH-, CD44+CD24-, CD44+ CD24+) and manually picked SUM159 and MCF7 mammospheres were amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, MO, USA) following the instructions of the manufacturer. To prevent any loss, cells were sorted directly into tubes where cell lysis and WGA were performed. Briefly, the volume was adjusted with water to 9 μl. After cell lysis and Proteinase K digest the DNA was fragmented and libraries were prepared. These products were used as templates for the amplification reaction which was performed in a thermal cycler (95°C for 3 min, 25 cycles of 94°C for 30 seconds and 65°C for 5 min, hold 4°C) by adding 7.5 μL of 10 × Amplification Master Mix, 48.5 μL of nuclease-free water and 5 μL WGA DNA polymerase. Amplified samples were purified using GenElute PCR Clean-up Kit (Sigma-Aldrich, St. Louis, MO, USA) and quantified by measuring absorbance on a NanoDrop Spectrophotometer (Thermo Scientific, MA; USA). The quality of the amplification was evaluated using a multiplex PCR .
Samples with significantly lower than the expected average DNA concentration of 250 ng/μl after WGA, or samples that showed only one band in multiplex PCR were excluded from further analyses.
Array CGH (aCGH)
Array CGH was carried out using a genome-wide oligonucleotide microarray platform (Human genome CGH 60 K microarray kit, Agilent Technologies, Santa Clara, CA, USA), following instructions of the manufacturer, and employing commercially available male reference DNA (Promega, Madison, WI, USA). Briefly, 500 ng DNA from SUM159 and MCF7 was digested with restriction endonucleases AluI/RsaI at 37°C for two hours, followed by an enzyme inactivating step at 65°C for 20 min. A smear between 2000 and 100 bp on a 1% agarose gel indicated successful digestion. Due to the previous fragmentation during the WGA this step was omitted for amplified samples (e.g. DNA of cultured MMO of both cell lines and sorted SUM159).
Samples were then labeled with the Bioprime array CGH genomic labeling system (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Five hundred ng each of the test DNA and the reference DNA were differentially labeled with dCTP-Cy5 or dCTP-Cy3 (GE Healthcare, Milwaukee, WI, USA). Unincorporated nucleotides were removed using the Amicon KD30 Kit (Millipore, Billerica, MA, USA). The probes were quantified before hybridization by determining the absorbance at 260 nm (DNA), 550 nm (cyanine 3), and 650 nm (cyanine 5) using NanoDrop spectrophotometer and denatured by incubation at 95°C for 2 min followed by cooling to room temperature. After annealing for 30 min at 37°C, array hybridization was carried out at 65°C for 24 hours with about 200 ng probes/array in Agilent HI-RPM hybridization buffer. Slides were scanned using a microarray scanner (#G2505B; Agilent Technologies, Santa Clara, CA, USA), and images processed using Feature Extraction and DNA Workbench 5.0.14 (Agilent Technologies). In addition, data normalization and calculation of ratio values was performed using the Feature Extraction software 9.1 from Agilent Technologies. Evaluation of data was done based on the previously published algorithm in R (http://www.r-project.org) . The algorithm calculates ratio values with different window sizes that differ significantly from the mean of the ratio profile and are therefore considered as over- or under-represented graphically indicated in a single green or red bar for gained or lost regions, respectively. Furthermore, the algorithm generates a table with all localizations of significant calls, which allows detailed mapping of each copy number variation (CNV).
Evaluation of epigenetic profiles
Epitect methyl qPCR arrays
To identify relevant gene promoter methylation we used EpiTect Methyl qPCR Arrays (SA Biosciences, Qiagen, Hilden, Germany) as a screening method. We analyzed 96 cancer-related genes from four pathways including stem cell transcription factors, homeobox genes, wnt signaling, and epithelial to mesenchymal transition (EMT). Assays were performed according to the manufacturer’s instructions. Results were displayed as percentage of unmethylated and methylated fraction.
For methylation analysis by pyrosequencing, one μg of genomic DNA was subjected to bisulfite conversion with the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. The purified bisulfite converted samples were eluted in 40 μl volume and stored at −20°C for further analysis.
To quantify the percentage of methylated cytosine in individual CpG sites, bisulfite-converted DNA was analyzed using pyrosequencing (Pyromark® Q24, Qiagen) as previously described . Custom Pyromark® CpG assays were used for following genes: HOXB4, HDAC1, FOXA2, CTBP1, LEF1, SMAD2, DSC2 and HIF1A. For genes HOXD3 and WIF1 assays were designed using the Pyromark Assay Design Software Version 2. Following primers were used: HOXD3-fw-5′-AGTTAAAGGTTATTTTAAAGGTTTAT-3′, HOXD3-Bio-rev-5′-CCTCTTACATCTACCCTATACAATT-3′, HOXD3-Pyro 5′-GGTTATTTTAAAGGTTTATGG-3′ and WIF1-fw-5′-GGGGGAGTTGTGGGGTTTTT-3′, WIF1-Bio-rev-5′-CCCAAAAATCTCTAAATACCCTTCTC-3′, WIF1-Pyro-5′-TGTGGGGTTTTTTAGGGGG-3′.
Real-time quantitative PCR (qPCR)
Total RNA was extracted from parental adherent cells, MMO or sorted cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s recommendation. RNA was quantified and assessed for purity by UV spectrophotometry. One microgram of total RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions.
qPCR was performed using LightCycler 480 (Roche). Reactions were performed in a total volume of 20 μl, comprising 1x SYBR Green I Master Mix (Roche), 20 ng cDNA and 25 μM of each primer (final concentration). All qPCR reactions were performed in duplicate and quantification cycle values were averaged. Gene expression was calculated by the comparative Ct method  and relative mRNA expression was presented as fold-differences over that in the parental monolayer cells. Hydroxymethylbilane synthase (HMBS) expression was used as internal control.
Following RT-qPCR primers were chosen from the public database (http://www.rtprimerdb.org) [20, 21], gene (RTPrimerDB-ID): HOXD3 (4603), HIF1A (7974), RGS2 (7770), CTBP1 (4471), LEF1 (8496), HDAC1 (442) and HMBS (4). The program Primer3 (NCBI, Primer-BLAST, http://www.ncbi.nlm.nih.gov/tools/primer-blast) was used to design WIF1 primer sequences (WIF1-fw-5′-GGAAAATGTATTTGCCCTCCA-3′ and WIF1-rv-5′-CCAATGCATTTACCTCCATTTC-3′).
Quantitative data are represented as the mean ± SD. For comparison of means, we used Student’s t-test and ANOVA with Tukey multiple comparison as a post hoc analysis. Statistical significance was defined as p < 0.05. GraphPad Prism software version 6 (GraphPad Software, La Jolla, California, USA) was used for statistical analysis.
Putative stem cell phenotypes in breast cancer cell lines
These results indicate that SUM159 cells have an enriched stem cell phenotype.
Mammosphere formation assay
Genomic profiles of putative breast CSCs
We employed aCGH to analyze 10 manually picked spheres each from MMO cultures of SUM159 and MCF7 and compared them with cells grown in adherent culture.
Methylation analysis of putative breast CSCs
Generally, Epitect Methyl qPCR Arrays revealed less detectable methylation differences in SUM159 cell line. Applying bisulfite pyrosequencing, four selected candidate genes showed higher methylation levels between MMO and parental cells, with HOXD3 being the only gene with significantly higher levels (Figure 10C). Regarding gene expression, WIF1, HDAC1 and HOXD3 mRNA levels were significantly higher in SUM159 MMO cells than in parental cells (Figure 10D). Hence, there was no inverse correlation between HOXD3 methylation and gene expression.
To test whether culturing tumor cells in non-adherent condition could affect methylation of the genes analyzed, we also subjected sorted subpopulations of SUM159 cells (ALDH+, ALDH-, CD44+CD24-, CD44+CD24+) to gene methylation analysis. WIF1 and HDAC1 showed similar levels of gene methylations in all sorted subpopulations and MMO cells, but lower levels for parental SUM159 cells. In contrast, HOXD3 was similarly methylated at lower levels in all sorted populations, but higher methylation levels were found in MMO and parental cells. HOXB4, on the other hand, was similarly higher methylated in sorted populations, but less methylated in MMO and parental cells. These results are summarized in Figure 10E. mRNA expression levels of WIF1, HDAC1 and HOXD3 were different in distinct sorted populations compared to MMO and parental SUM159 cells (Figure 10F).
This is the first study to analyze both genetic and epigenetic alterations in putative breast cancer stem cell models derived from established breast cancer cell lines. At the genomic level we did not find any copy number variations between putative breast cancer stem cells (i.e. mammospheres or sorted subpopulations) and the corresponding parental tumor cells. In contrast, putative breast cancer stem cells showed altered methylation levels of several genes compared to parental tumor cells.
Definition and analysis of CSCs remains a challenge. Among the approaches to enrich and study the cancer stem cell biology are in vitro cultures and flow cytometry sorting based on defined putative phenotypes of CSCs for different diseases [9, 10, 25, 26]. We focused our analyses on evaluation of genetic and epigenetic profiles in an attempt to associate the heterogeneity with the presence of putative CSCs within breast cancer cell lines. Parental cell lines and MMO originating from the parental cell line and distinct populations defined by putative breast cancer stem cell phenotypes were compared.
Two biologically distinct breast cancer cell lines were used: MCF7 represents luminal breast epithelial tumor cells, and SUM159 represents triple negative breast epithelial tumor cells. The phenotypes we used to enrich for breast CSCs were established earlier by Al Hajj et al.  and Ginestier et al. , and are commonly used to address breast CSCs. Ability of these two cell lines to form MMO was previously demonstrated by us and others [15, 22, 27]. The proportions of putative breast CSC subpopulations we observed in both cell lines were comparable with published results [22, 28, 29].
This is the first comparison of aCGH profiles between parental cells and MMO for both cell lines, and indicates that there is no difference in genomic aberrations across the analyzed samples. One explanation for the similar genotype could be the fact that CSCs give rise to all progenitor cells and therefore both share the same genetic profile. However, studies suggest that cancers may contain multiple CSC clones, arising through clonal evolution in CSCs . Such CSC clones are more likely to be found in primary tumors rather than in cell lines. In addition, although CSCs are enriched in these models, not all cells within the mammosphere population represent cells with stem cell properties. As a result, the extent of dilution of the CSC signature, both genetically and epigenetically, is unknown.
Genomic profile of MCF7 cells was comparable with results published for adherent cell cultures [23, 24]. We did not find matching data for SUM159. As expected, owing to their very distinct biology, MCF7 and SUM159 had substantially different genomic profiles.
Proposed plasticity and dynamic property of cancer cells, often associated with the ability to undergo EMT and reverse and to switch stem cell markers off and back on, suggest an underlying epigenetic mechanism [30–32]. These properties determine metastatic potential of tumors , and may be the reason why most disseminated tumor cells in bone marrow have the putative breast CSC phenotype, as we have shown previously . Epigenetic regulation of common CSC genes has recently been demonstrated in triple negative breast cancer . Furthermore, in a recent paper, Park et al. have demonstrated distinct methylation pattern across defined intrinsic molecular breast cancer subtypes and their correlation with described breast CSC markers in archival primary breast cancer tissues . In concordance to our findings in breast cancer cell lines, primary cancer tissues of triple negative breast cancer had higher frequency of ALDH+ and CD44+CD24- cells. Therefore, our further analyses focused on analyzing methylation profiles of putative breast CSCs in vitro. Genes screened by methylation arrays were chosen according to the potentially important molecular pathways involved in stem cell biology, including stem cell transcription factors, homeobox genes, wnt signaling and EMT. Selected differentially methylated genes were further evaluated by bisulfite pyrosequencing.
Bisulfite pyrosequencing revealed differences in methylation levels between parental and MMO cells and also between sorted populations and MMO cells. Differences in sorted subpopulations may reflect not only the heterogeneity in CSCs, but also different ability to enrich for CSC. As already stated, enrichment factor for CSCs is unknown for both approaches. In case of the SUM159 cell line, the majority of cells were associated with CD44+CD24- CSC phenotype. These cells are more aggressive, basal like, with mesenchymal characteristics, and with most of the cells being similar, detection of differentially expressed markers is less likely.
Several groups have used a similar approach in attempt to associate intratumoral heterogeneity with the presence of CSCs. Clearly, the main goal of such an approach is to understand the biological mechanism and finally identify biomarkers of CSCs to design better treatment strategies. One of the first studies was published by Hernandez-Vargas et al. , revealing JAK-STAT pathway activation in putative breast CSCs defined as CD44+CD24- MCF7 cells. Hypomethylation of genes in JAK- STAT pathway was demonstrated further in MMO. The authors provided not only a potential definition of biomarkers associated with CSCs, but also an evidence of underlying epigenetic regulation of CSC properties. In a similar analysis in hepatocellular carcinoma, researchers identified epigenetic regulation of wnt signaling as a potential biomarker of early detection and therapeutic targeting . Epigenetic mechanisms and the role of miRNA regulation of CSCs have also been demonstrated [39, 40] providing a new insight into biology and potential definition of novel therapeutic targets. All these analyses are promising and require further evaluation.
Finally, our mRNA analyses suggest that although some genes showed an inverse correlation between methylation and gene expression, other mechanisms than DNA promoter methylation may be responsible for regulating gene expression. In our study, methylation seemed to play a role only in the regulation of WIF1 and LEF1 in MCF7. One reason for the lacking correlation might be heterogeneous promoter methylation and that at least in part our methylation assay did not detect the regulating region. Also, further mechanisms might be involved in epigenetic silencing of proteins, including histone modifications, long noncoding RNAs and miRNAs . These mechanisms are incompletely understood and are emerging study topics. However, their evaluation was out of the scope of the present study.
Our results, along with others, clearly underpin the hypothesis that epigenetic mechanisms play a major role in the regulation of CSCs, however, more efficient methods for CSC enrichment are needed. Other mechanisms of epigenetic regulation have also to be taken into account. Moreover, these analyses should be extended to patient samples, in order to validate results, and possibly find higher heterogeneity than possible and present in cell lines.
In conclusion, stem cell hypothesis has to be further challenged; we need additional markers and more efficient approaches and models in order to better define the biology of stem cells and identify biomarkers for early diagnosis and treatment with higher precision.
Cancer stem cells
Array comparative genomic hybridization
Whole genome amplification
This work was supported by funds of the Oesterreichische Nationalbank (Anniversary Fund, project number 14001).
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