Ovarian Cancer Stem Cell–Like Side Populations Are Enriched Following Chemotherapy and Overexpress EZH2

The majority of ovarian cancer patients present with advanced disease. Although up to 80% respond well to surgery and platinum-based chemotherapy, tumor recurrence is a common event (1). Following relapse, treatment with platinum can elicit a further response; however, the duration of response tends to decrease with each round of therapy, as patients develop disease with increased chemoresistance. Subpopulations of tumor cells with stem cell–like properties have been proposed to sustain the growth of tumor cells and the inherent drug resistance of these stem cell–like cells could lead to tumor recurrence following chemotherapy (1, 2). Support for the existence of ovarian tumor stem cells comes from studies examining both tumor biopsies and ascites (3–8). Thus, ovarian tumors contain cells that are capable of prolonged growth in an anchorage-independent manner as spheroids and are tumorigenic when injected at low cell numbers into mice (7). Subpopulations of cells capable of forming tumors in xenogeneic mice have been isolated from ascites of ovarian cancer patients (8).

One widely used method for isolating putative cancer stem cells is based on ABC transporter–mediated efflux of the Hoechst 33342 dye to isolate a side population (SP). This dye-excluding SP phenotype has been shown to be enriched with cancer stem–like cells in a variety of tumors (8–14). Furthermore, SP cells from ovarian cancer ascites can form tumors more readily in mice than non-SP cells following 5 days in culture (8). The presence of SP cells with increased in vitro drug resistance has been shown in mouse and human ovarian cancer cell lines (5, 8); although, the relevance to clinical acquired drug resistance is still to be established.

Polycomb group proteins have been shown to be required for the maintenance of embryonic stem cells and could therefore also have a role in maintaining tumor stem/sustaining cells (15–17). Indeed, the key component of polycomb repressive complex (PRC2) EZH2, a specific histone 3 Lys27 (H3K27) methyltransferase, is essential for stem cell maintenance in glioblastoma (18). EZH2 plays a critical role in tumorigenesis and cancer progression through epigenetic gene silencing and chromatin remodeling (19, 20). There is increasing evidence that overexpression of the EZH2 gene occurs in a variety of human malignancies including oral, esophageal, gastric, colon, hepatocellular, bladder, breast, prostate, and endometrial cancers (21–23). Putative oncogenic and tumor-suppressive roles for EZH2 have been suggested (24, 25). Elevated expression of EZH2 has been shown to be associated with advanced stages of ovarian cancer and independently associated with short overall survival of ovarian cancer patients (26). Furthermore, EZH2 knockdown in ovarian cell lines leads to reduced cell growth/proliferation and inhibited cell migration and/or invasion in vitro (26), as well as resensitization of drug-resistant ovarian cancer cells to cisplatin (27). However, these studies have not examined EZH2 in an ovarian tumor stem cell population derived from patients during clinical acquired resistance.

We have address whether SP cells can be isolated from ascites from ovarian cancer patients and whether the size of the SP subpopulation changes during chemotherapy. Given the observed role of EZH2 in platinum resistance of ovarian cell lines (27), we have examined whether EZH2 is overexpressed in these tumor-derived SP cells and whether inhibition of EZH2 may have the potential to inhibit growth of drug-resistant ovarian tumor stem cells.

Ascitic fluid was obtained from ovarian cancer patients requiring therapeutic paracentesis following informed consent for this study, approved by the Royal Marsden Hospital Ethics Review Committee and the Hammersmith Hospital Ethics Review Committee. Within 24 hours of receipt, ascites were centrifuged at 400 × g for 15 minutes to concentrate the cellular content. The concentrated fraction was overlaid onto lymphocyte preparation medium (PAA Laboratories) and centrifuged for 30 minutes at 400 × g without braking. Cells at the interface, enriched for tumor cells and lymphocytes, were collected and washed in RPMI 1640 (Invitrogen) + 10% FBS (PAA Laboratories) then centrifuged for 10 minutes at 400 × g. Contaminating red blood cells were lysed using a RBC Lysis kit (Miltenyi Biotec Ltd.) according to the manufacturer's instructions.

IGROV1 (28), PEO1, PEO4, PEA1, PEA2, PEO14, PEO23 (29), and OSEC2 (30) were obtained from Ovarian Cancer Action Research Centre, Imperial College, London, UK. All cell lines were maintained in RPMI 1640 + 10% FBS. IOSE21 were obtained from Prof. Frances Balkwill, Institute of Cancer, Centre for Cancer and Inflammation, Barts, and The London School of Medicine and Dentistry, London. Cell lines were shown to be identical to those received on the basis of DNA methylation pattern analyzed within 1 month of use and the methylation patterns showed close similarity between samples from the same patient, otherwise no further authentication was carried out.

Purified cell populations were plated and treated 24 hours later with cisplatin (Sigma-Aldrich), carboplatin (Sigma-Aldrich), and paclitaxel (Sigma-Aldrich) for 24 hours. The medium was replaced and the cells were allowed to recover for 48 hours then survival was assessed by MTT assay or the change in SP was measured by flow cytometry following cell enumeration by hemocytometer.

Hoechst 33342 staining of cells (22) in combination with immunophenotyping. Cells were resuspended at 10⁶/mL RPMI 1640 + 2% FBS, 10 mmol/L HEPES (Sigma-Aldrich), and 5 μg/mL Hoechst 33342 then incubated at 37°C with rotation, optimal incubation times were determined for each cell line. Cells were washed in 4 volumes of analysis buffer consisting of ice-cold HBSS containing 2% FBS and 10 mmol/L HEPES. Cells were analyzed and sorted using a BD FACS VantageSE DiVa equipped with 2 Coherent Innova 90 lasers, one with visible optics tuned to 488 nm and set to 200 mW and one with UV optics emitting multiline UV 333.6 to 363.8 nm set to 50 mW. Sheath fluid was sterile PBS (pH 7.2; in-house) and cells were sorted with sheath pressure set at 12 psi (0.82 bar). On reanalysis of sorted populations, the purity generally exceeded 90%. To confirm the presence of an SP and define the gate for cell sorting, verapamil was added to control samples at a final concentration of 20 μmol/L.

For primary ascites cells, CD45-FITC (Fluoroscein isothiocyanate; Clone HI30; BD Biosciences) and/or CD326-APC (EpCAM, Clone HEA-125, Miltenyi Biotech) was added to the cells for 30 minutes on ice and the excess removed by washing, appropriate matched isotype controls were also used to identify nonspecific labeling. Cells were resuspended in HBSS (Invitrogen) containing 2% FBS and 2 μg/mL propidium iodide (Sigma-Aldrich) then analyzed and sorted as described above. Data were analyzed using FCS Express Professional version 3 (De Novo Software).

All procedures were approved by the Institute of Cancer Research Ethics committee and all work performed in accordance with UK Home Office regulations under the Animals (Scientific Procedures) Act 1986 and UKCCR guidelines for animal experimentation (30). SP and non-SP from IGROV1 cells were sorted as described above and resuspended in ice-cold 50% Matrigel (BD biosciences) in RPMI 1640 at 50 μL per xenograft. Between 50 and 250 cells were injected into the mammary fat pad of 10-week-old NOD/SCID mice (Charles River) and allowed to grow for up to 12 weeks. Unsorted cells were grafted as a positive control for cell viability and graft take. Similarly, 48 hours post-siRNA treatment, IGROV1 cells were harvested using TrypLE and counted, 50 μL grafts were prepared and transplanted as described above, using 250 cells per graft. Tumor size was monitored by calliper measurements across 2 diameters (d) at regular intervals to calculate tumor volume (cm³) = 4/3π [(d₁ + d₂)/4]³.

Cells were plated at a range of densities in 6-well ultralow adhesion tissue culture plates in 2 mL RPMI 1640) + 10% FBS and transferred to an incubator with supplementary feeding with same media and serum every 3 to 4 days. Spheroids were allowed to form for 18 days and then counted. In some cases, spheroids were transferred to adherent plates. The adhered spheroids were fixed in 3.7% formalin in PBS then stained with 1% rhodamine B in water. Images of each well were captured then analyzed by Image-Pro Analyser 6.3 to quantitate colony size and frequency.

Optimal plating densities were determined to allow proliferation for up to 96 hours for each cell line. Protocols were optimized as recommended by the manufacturer's instructions. siRNAs were delivered using HiPerfect (Qiagen) within 16 hours of plating cells. All experiments consisted of untreated controls, mock controls, control siRNA (Allstars scrambled siRNA, Qiagen), and positive control siRNA (MAPK, Qiagen).

Total cellular ribonucleic acid (TRNA) was extracted (RNeasy Minikit Plus, Qiagen) and quantified by measurement of optical density at OD_λ260. Up to 2 μg of RNA was reverse transcribed and first-strand cDNA synthesized in 40 μL using the Superscript II reverse transcriptase kit (Invitrogen) according to manufacturer's instructions. Samples were amplified using TaqMan gene expression assays (Applied Biosystems) and a Step One PCR machine (Applied Biosystems). TaqMan probe and primers (see manufacturer's website) were used: ABCB1 (assay ID Hs01067802_m1), ABCG2 (assay ID Hs01053790_m1), GAPDH (part no 4326317E), EZH1 (assay ID Hs00157470_ml), and EZH2 (assay ID Hs01016789_ml).

Cells were resuspended in lysis buffer [PBS containing 0.5% Triton X-100 (v/v), 1× complete protease inhibitor cocktail; Roche] and left to lyse for 10 minutes at 4°C. Nuclei were then precipitated and resuspended in 0.2N HCl overnight at 4°C. Nuclear debris were pelleted and the supernatant recovered. Protein concentration was calculated by Bradford assay (Bio-Rad). The lysate was separated by 8% to 16% SDS-PAGE and transferred to nitrocellulose membranes. Rabbit polyclonal antibodies anti-trimethyl-histone-H3 (Lys27, 07-449; Millipore) was used for immunoblotting.

Gene expression in 3 independently sorted SP and non-SP from IGROV1 cells exponentially growing under standard tissue culture conditions was determined by Affymetrix array using the HG-U133plus2 GeneChip array following amplification labeling. Labeling and hybridizations were carried out at the Paterson Institute Microarray Facility (Manchester, UK) following standard protocols. Full methods are available at http://bioinformatics.picr.man.ac.uk/mbcf.

The raw expression data of IGROV1 SP and non-SP in Affymetrix HG-U133plus2 microarrays were normalized as previously described (31) excluding the probes with low signal intensities (average expression across all the samples <25th quantile of the whole data set). The data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE25191 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25191). Expression values of multiple probes targeting the same gene were averaged, resulting in a total of 20,323 unique genes in Gene Set Analysis (GSA; ref. 32) to assess the differential expression between 3 pairs of SP and non-SP cells in 15 predefined characteristic signatures that embryonic stem cells have. We computed the gene scores (t-statistic z_i) for all the genes in one gene set S. The larger absolute value of the average of the positive and of the negative parts of each z_i in S is called “maxmean statistic”. The significance of the “maxmean statistic” in each gene set was determined by false discovery rate (FDR), estimated on the basis of row randomization and label permutation. FDR less than 5% was used to determine the enrichment of upregulation/downregulation of those signatures in SP cells. This analysis was done in R (version 2.8.1) using GSA package (33).

All further analyses were performed using the statistical analysis package GraphPad Prism v5 (GraphPad Software Inc.). The significant level was set at 2-sided P < 0.05. All figures are shown with means and standard errors unless otherwise stated.

ABC transporter–mediated efflux of the Hoechst 33342 dye to isolate a dye-excludings SP has been shown to be enriched for cells with cancer stem–like properties in a variety of tumors (8-14). SP cells from ascites of ovarian cancer patients have been shown to form tumors more readily in mice than non-SP cells (8). Using differential Hoechst 33342 staining of cells to identify SP of the IGROV1 ovarian tumor cell line, we observe significantly increased tumorigenicity (Repeated measures ANOVA: P < 0.05) of SP compared with non-SP cells when injecting 50 or 100 cells into the mammary fat pad of NOD/SCID mice (Fig. 1A). In addition to the differences in average tumor growth, SP cells initiated tumors more frequently (8/14 injections) than non-SP (1/12) injections (Fisher's exact test: P = 0.0145). Furthermore, SP cells have a greater ability to grow in an anchorage-independent manner as spheroids compared with non-SP (Fig. 1B). The differences in growth properties of SP and non-SP cells could be due to toxicity of Hoechst 33342 and its increase accumulation in non-SP cells. Arguing against this interpretation, we observe non-SP IGROV1 cells to have in fact significantly increased plating efficiency compared with SP cells in standard adherent two-dimensional tissue culture assays (Fig. 1C) and the enhanced growth of SP was only observed in anchorage-independent spheroid growth conditions (Fig. 1B). Asymmetric division is a key feature of stem cells (34). To examine whether isolated SP cells could repopulate SP and non-SP cell populations, multiple SP IGROV1 populations were isolated by FACS (fluorescence-activated cell sorter) and continuously cultured for up to 10 passages. The number of SP cells in the SP selected cultures declined rapidly on passaging with an increase in non-SP cells (Fig. 1D). The mean purity of SP cells following initial separation was 93.3%. It could be argues that a higher proliferative capacity of the contaminating non-SP could lead to the rapid re-emergence of the non-SP. However, the SP became stable as a minority of the total population and the cell culture retained SP and non-SP equivalents to the proportions present in the original cell line. Furthermore, whereas the growth potential of the SP is slightly reduced compared with the non-SP (Fig. 1C), this is unlikely to explain the rapid emergence of the non-SP cells.

GSA (32) was used to interrogate gene expression profiling of SP compared with non-SP from IGROV1 cells for 15 published embryonic stem cell related signatures (Supplementary Table 1). Four signatures with P < 10⁻⁶ and FDR greater than 10⁻⁴ were significantly upregulated in SP compared with non-SP: Oct4-regulated genes, NOS signature (Nanog/Oct4/Sox2-regulated genes), E2F6-associated expression pattern, and PRC2. Consistent with upregulation of PRC2 genes, the PRC2 target genes were significantly repressed in the SP (P < 10⁻⁶ and FDR < 10⁻⁴). The individual GSA scores for the PRC2 target genes are shown in Supplementary Table 4. Thus, on the basis of cell phenotype and overrepresentation of stem cell gene expression patterns, SP cells isolated from the IGROV1 human ovarian tumor cell line have tumor stem cell–like properties.

We assessed the relative proportion of SP cells present in tumors from ovarian cancer patients. We measured the proportion of SP cells present in EpCAM-positive, CD45-negative tumor cells in patient ascites collected either at initial presentation or at relapse after platinum-based chemotherapy (Table 1). Ascites samples collected from relapsed patients show a significant (P = 0.013) increase in proportion of SP cells compared with chemonaive patients (Fig. 2A). Patients were categorized, postascitic drainage, as having either disease control (partial response or stable disease) or progressive disease to subsequent chemotherapy (Table 1). Those with progressive disease tended to have increased SP (mean = 1.8 ± 0.9) compared with those with disease control (mean = 0.42 ± 0.24, P = 0.055, unpaired Student's t test with Welch's correction).

Sequential ascites samples taken from 3 patients during treatment show progressive increase in the SP (Table 1). We have also examined the proportion of SP present in matched cell lines derived from patients' tumors at diagnosis and at platinum-resistant relapse (Table 2). These lines have previously been shown to acquire in vivo increased resistance to carboplatin during chemotherapy (29) and represent isogenic models of clinical drug resistance. We observe an increase in the percent SP in all three of the lines that have acquired platinum resistance compared with the matched chemosensitive tumor line. Two immortalized cell lines generated from normal surface ovarian epithelial cells did not contain a detectable SP.

The increase in SP observed with development of chemoresistance following carboplatin-based chemotherapy argues that SP cells may have a selective survival or growth advantage during chemotherapy. Resistance of tumor stem cells to chemotherapeutic drugs has previously been described in a variety of tumor types including ovarian cancer SP (8, 35). Indeed, we observe increased resistance of IGROV1 SP compared with non-SP to platinum (cisplatin and carboplatin) and to paclitaxel (Fig. 3A). However, it could be argued that this differential drug sensitivity is influenced by differential levels of Hoechst dye remaining in the SP and non-SP cells. Therefore, we examined whether exposure of IGROV1 cells to physiologically relevant levels of carboplatin (in the absence of Hoechst dye) could select for an increased proportion of SP cells. As shown in Figure 3B, SP cells remain relatively unaffected by the carboplatin treatment, whereas there is a significant decrease in viability of the non-SP, consistent with the effects of carboplatin on the viability of the bulk population of cells. The net effect will be an enrichment or selection for the SP cell population during platinum exposure.

Differential expression of ABC transporters has been widely observed in SP from a variety of tumor types; although, in the majority of studies, overexpression of ABCG2 is observed (8, 35). Real-time (RT) PCR quantification of ABCB1 and ABCG2 mRNA showed that ABCB1 was overexpressed in SP cells in comparison with non-SP cells from 4 of 5 ovarian cancer cell lines and in primary patient ascites (n = 10), whereas ABCG2 was below the level of quantification by RT-PCR, except for PEA2, from which ABCB1 was absent (Table 3). Carboplatin is not a substrate for P-glycoprotein (36) and therefore overexpression of ABCB1 is unlikely to be the explanation of the increased resistance to carboplatin or selection of SP cells observed. Because ABCC5 and ABCC6 have been implicated in cisplatin resistance (37, 38), we specifically examined by qRT-PCR (quantitative RT-PCR) their levels in SP and non-SP from patient ascites; but, in the case of ABCC5, no significant difference was observed (1.22 SP to non-SP ratio, n = 10) and for ABCC6, the levels were undetectable in SP and non-SP (n = 10). Paclitaxel is a substrate for ABCB1 and therefore overexpression of ABCB1 in this subpopulation could affect the likelihood of relapse following taxane containing chemotherapy; however, we do not observe increased resistance of the SP cells compared with non-SP for paclitaxel sensitivity (Fig. 3A).

PRC2 contains the histone methyltransferase EZH2, which, together with EED and SUZ12, trimethylates histone H3 on Lys27 (H3K27me3) and is associated with a repressive chromatin state (20). EZH2 is overexpressed in many cancers and levels of EZH2 correlate with poor prognosis including ovarian cancer (21–23, 26). We observe increased expression of EZH2 in 7 of 10 SP compared with non-SP isolated from ascites in patients with recurrent ovarian cancer (Table 4), with up to 14-fold increase expression in SP compared with non-SP in patient samples. The increased EZH2 expression observed in SP from patient ascites is variable and may represent differences in purity of the FACS-sorted SP, although there is no obvious correlation with level of ABCB1 (Table 4). Previous studies have suggested that differential levels of Hoechst dye can affect gene expression in SP cells (39). We observed no effect of Hoechst dye on EZH2 levels or ABCB1 levels (data not shown).

Propagation of the H3K27me3 mark during cell division accounts for the maintenance and somatic inheritance of repressive chromatin domains (40). Therefore, inhibition of EZH2 should reduce H3K27 methylation levels, leading to gene reactivation. To examine whether EZH2 has a direct role in maintaining an SP, we performed siRNA knockdown of EZH2 in 3 independent ovarian tumor cell lines. EZH2 knockdown consistently reduced EZH2 mRNA by greater than 70% and produces a dramatic and prolonged reduction in the levels of H3K27 trimethylation in cells (Fig. 4A).

Because EZH1 also mediates methylation on histone H3 Lys27 and complements EZH2 in maintaining stem cell identity and executing pluripotency (41), we performed double knockdowns of EZH2 and EZH1, as well as EZH2 alone, in case of potential redundancy of functional effects on tumor stem cells. EZH2 knockdown alone, or when combined with EZH1 knockdown, consistently reduced the SP in independent ovarian tumor cell lines (Fig. 4B and C). SP compared with non-SP cells have a greater ability to grow in anchorage-independent culture and form large (>32 cell) spheroids (Fig. 1B). Loss of anchorage-independent growth following knockdown of EZH2 alone or combined with EZH2 in IGROV1, PEO14, and PEO23 is observed (data for IGROV1 shown in Fig. 4D). As well as for the pool of 4 siRNAs for EZH2 shown in Figure 4D, similar reduction in SP and spheroid growth was observed for all 4 siRNAs when tested individually. Following siRNA knockdown of EZH1 + EZH2, IGROV1 or PEO23 cells were injected into a NOD/SCID mouse model and tumor growth measured. Although knockdown will be transitory and not maintained throughout the xenograft experiment, reduced expression of EZH2 and reduced H3K27me is observed 48 to 96 hours following SiRNA treatment, the important time frame for tumor take. Despite the transitory nature of the SiRNA knockdown, a persistent reduction in tumor volume in the EZH2 + EZH1 knockdowns compared with the controls was observed (Fig. 4E and F).

In support of the existence of tumor stem cell–like cells in ovarian tumors, subpopulations of tumor-initiating or -sustaining cells have been identified which grow more readily in an anchorage-independent manner and as tumors in xenogeneic mice (3–8). These subpopulations can be shown to overexpress stem cell–associated markers and to be more resistant to drugs used in the treatment of ovarian cancer (7, 8). One of the methods that has been successfully used to isolate such putative tumor stem cells is differential Hoechst dye uptake (8–13). Using this approach, we have identified a subpopulation of cells from ovarian cell lines that have a more aggressive phenotype, as defined by anchorage-independent growth and tumor formation in NOD/SCID mice, and are resistant to carboplatin and paclitaxel. Consistent with the in vitro observation of a survival advantage of SP following exposure to carboplatin and hence potential drug selection, we observe increased SP in tumor cells isolated from patient ascites following chemotherapy and at relapse compared with chemonaive patients.

SP isolated from ovarian tumor cell lines overexpress a NOS cell signature, frequently associated with embryonic stem cells (17). Furthermore, we show that SP isolated from IGROV1 SP overexpress PRC2 genes, while consistent with this PRC2 repressed targets are underexpressed. PRC2 contains the histone methyltransferase EZH2, which, together with EED and SUZ12, trimethylates histone H3 on Lys27 (H3K27me3) and is associated with a repressive chromatin state (20). Many genes in cancer, including tumor suppressor genes, are epigenetically silenced by mechanisms associated with H3K27me3 which can be independent of DNA methylation (42). Because H3K27me3 is somatically inherited during cell division (40), this argues that it truly is an epigenetic mark that is maintained and as such is a key target for reversing aberrant epigenetic silencing. Consistent with the data presented in our current study in ovarian cancer, EZH2 has been shown to be essential for glioblastoma cancer stem cell maintenance (18). EZH2 is overexpressed in many cancers and levels of EZH2 correlate with poor prognosis in various cancers including ovarian cancer (21–23, 26).

Our data suggest that EZH2 has a key role in maintenance of the drug-resistant SP subpopulation in ovarian tumor cells. We have shown that EZH2 is overexpressed in SP derived from ovarian cancer ascites at relapse. EZH2 knockdown in ovarian cell lines has been shown to lead to reduced cell growth/proliferation, as well as cell migration and/or invasion in vitro (26), whereas overexpression of EZH2 has been associated with acquired cisplatin resistance (27). Furthermore, loss of H3K27 trimethylation has been shown to result in resensitization of ovarian cancer cells to cisplatin (43); although, lower H3K27 trimethylation levels were associated with shorter overall survival. (44). However, these previous studies have not examined EZH2 in an ovarian tumor stem cell population derived from patients during clinical acquired resistance, and our present data strongly support the clinical relevance of EZH2 in this important subpopulation of cells selected for during chemotherapy. This drug-resistant subpopulation will be important to eradicate if we aim to improve the treatment of ovarian cancer. Catalytic site inhibitors of histone methyltransferase (45), have been reported, as has indirect pharmacologic inhibition of PRC2 (46), but so far no specific EZH2 inhibitor has been described. Development of specific inhibitors of EZH2 will have potential as drugs that target this key subpopulation of drug-resistant tumor-sustaining cells.

No potential conflicts of interest were disclosed.

With thanks to all those involved in tissue and patient data collection, including Debbie Tandy, Nicole Martin, Nona Rama, Amy Ford, Michelle Everard, Claudia Hayford, Tim Crook, and Matthew Ng as well as the entire gynacological clinical trials team at Hammersmith Hospital headed by Professor Hani Gabra.

This work was supported by Ovarian Cancer Action (S. Rizzo, P. Mellor, R. Brown) and Cancer Research UK (W. Dai, J. Hersey A. Santos-Silva, R. Brown) grants (C536/A6689), Cancer Research UK Experimental Cancer Medicine Centre, The Biomedical Research Council of the Agency for Science, Technology and Research, Singapore (L. Luk), and Leukaemia Research Fund (I. Titley) and Institute of Cancer Research (I. Titley, D.L. Hudson, R. Brown, S.B. Kaye).

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