Epigenetic reprogramming of breast cancer cells with oocyte extracts
© Allegrucci et al; licensee BioMed Central Ltd. 2011
Received: 22 February 2010
Accepted: 13 January 2011
Published: 13 January 2011
Breast cancer is a disease characterised by both genetic and epigenetic alterations. Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. In this study we apply the reprogramming capacity of oocytes to cancer cells in order to study breast oncogenesis.
We show that breast cancer cells can be directly reprogrammed by amphibian oocyte extracts. The reprogramming effect, after six hours of treatment, in the absence of DNA replication, includes DNA demethylation and removal of repressive histone marks at the promoters of tumour suppressor genes; also, expression of the silenced genes is re-activated in response to treatment. This activity is specific to oocytes as it is not elicited by extracts from ovulated eggs, and is present at very limited levels in extracts from mouse embryonic stem cells. Epigenetic reprogramming in oocyte extracts results in reduction of cancer cell growth under anchorage independent conditions and a reduction in tumour growth in mouse xenografts.
This study presents a new method to investigate tumour reversion by epigenetic reprogramming. After testing extracts from different sources, we found that axolotl oocyte extracts possess superior reprogramming ability, which reverses epigenetic silencing of tumour suppressor genes and tumorigenicity of breast cancer cells in a mouse xenograft model. Therefore this system can be extremely valuable for dissecting the mechanisms involved in tumour suppressor gene silencing and identifying molecular activities capable of arresting tumour growth. These applications can ultimately shed light on the contribution of epigenetic alterations in breast cancer and advance the development of epigenetic therapies.
Tissue homeostasis depends on tightly regulated mechanisms controlling cell proliferation and differentiation. Expression of proto-oncogenes and tumour suppressor genes controls normal cell function, and misregulation of these genes by both genetic and epigenetic alterations is at the origin of cancer [1, 2]. Genetic changes include deletion, mutation and amplification of genes, whereas epigenetic alterations occur without change in DNA sequence via modification of chromatin organisation, including DNA methylation, histone modifications and expression of non-coding RNAs. The role of epigenetic alterations in tumourigenesis has been recognised in different types of malignancies, including breast cancer .
In the breast, abnormal epigenetic regulation of genes regulating the cell cycle, apoptosis, DNA repair, cell adhesion and signalling leads to tumour formation, its progression, and drug resistance . Epigenetic alterations prevail over genetic abnormalities in initial stages of breast tumour development. For instance, silencing of CDKN2A (p16INK4A), HOXA and PCDH gene clusters by DNA methylation together with over-expression of Polycomb proteins BMI-1, EZH2 and SUZ12 occurs during spontaneous or induced transformation of human mammary epithelial cells [4, 5]. Methylation of several homeobox genes is also observed in ductal carcinoma in situ and stage I breast tumours .
Unlike genetic alterations, epigenetic modifications of the chromatin are reversible and therefore are suitable targets for reversal or attenuation of malignancy. The question of how tumours can be reprogrammed is intriguing, and determining how a cancer cell can be reprogrammed back to a normal cell phenotype is important not only for understanding the molecular pathways of the disease but also for diagnostic and therapeutic intervention .
Embryonic environments that program cell fate during development are able to reverse tumorigenicity . Landmark experiments have shown that teratocarcinoma cells are reprogrammed when injected into a mouse blastocyst resulting in normal tissue derived from tumour cells in chimeric mice . Tumorigenicity of metastatic melanoma cells is also reduced when cells are injected into zebrafish , chicken  and mouse embryos  or when they are cultured on 3D-matrices conditioned with human embryonic stem cells .
Nuclear transfer (NT) experiments have demonstrated that oocytes can fully reset the epigenotype of somatic cells  and this ability has been exploited to re-establish developmental potential in teratocarcinoma, medulloblastoma and melanoma cells to extents that depend on the degree of non-reprogrammable karyotypic abnormalities of the donor tumour cell nucleus [15–17]. Because NT experiments depend on the ability of reprogrammed cells to support embryonic development, with either formation of viable offspring or blastocyst-derived embryonic stem cells as potential outcomes, they are not easily amenable to dissecting the molecular mechanisms involved in tumour reversion. Understandably, NT experiments also do not allow the study of human tumour cells.
An alternative method to reprogram cells is using oocyte extracts as an ex-ovo system . Extracts made from amphibian oocytes are of particular interest, since they are available in large quantities and they possess reprogramming abilities similar to those of mammalian oocytes [19–22]. We have previously shown that amphibian oocyte extracts possess activities able to modify DNA methylation and histone marks, together contributing to the remodelling of somatic cell chromatin [21, 23]. In addition, we have introduced oocytes from axolotls, a urodele (salamander) amphibian, as a source of reprogramming extract based on our previous demonstrations that urodeles are genetically more similar to mammals and the molecular mechanisms governing the early development of urodeles and mammals are conserved [24–28]. In this study we analyse the relative efficiencies of extracts from oocytes of axolotl and Xenopus for their ability to reverse epigenetic alterations within breast cancer cell chromatin. Our results show that axolotl oocyte extracts reprogram cancer cell chromatin with high efficiency, reversing epigenetic silencing and activating expression from tumour suppressor genes whose repression is involved in breast tumorigenesis. In addition, we show long term suppression of tumour growth in vivo by reprogramming with oocyte molecules.
Results and Discussion
Oocyte extracts induce expression of silenced tumour suppressor genes
Expression of tumour suppressor genes in reprogrammed breast cancer cells
MCF-7 in AOE
HCC1954 in AOE
We next compared the reprogramming capacity of these extracts with extracts prepared from embryonic stem cells (ESC), as it has been reported that ESC extracts (ESCE) can also reprogram transcription of somatic cells to pluripotency [20, 23, 30]. Mouse ESCE were used so that we could control for activation of human genes in this mammalian heterologous system; reprogramming was performed at mammalian physiological temperature (37°C). Surprisingly, ESCE only induced expression of GAS2 (Figure 1). Epigenetic silencing of tumour suppressor genes involves diverse mechanisms, including DNA methylation, histone modifications and expression of non coding RNAs . It is therefore possible that activities contained in oocyte and ESC extracts can differentially reprogram these epigenetic marks. The limited reprogramming capacity of ESCE that we demonstrate here is in agreement with a previous report in which changes in tumour suppressor gene expression were not observed when teratocarcinoma cells were used to reprogram somatic cells to pluripotency . Taken together, our results highlight intrinsic differences in the reprogramming ability of oocytes and ESC for tumour suppressor genes, which we believe extend from the natural chromatin remodelling activities found in oocytes .
Intriguingly, AOE was consistently more efficient in re-activating the majority of silenced genes compared with the activities in XOE. It is important in this regard to note that axolotl oocytes were chosen for this study for the specific reason that urodele amphibians reflect the ancestral amphibian state from which mammals evolved, and they are therefore more genetically similar to mammals than are frogs [25–28]. As a consequence, transcription factor compatibility with mammalian target sequences would be expected to be greater, and axolotl oocytes would more closely reflect the epigenetic remodelling activity of mammalian oocytes [25–28], (ADJ, unpublished). Hence, AOE provides a powerful tool to identify mechanisms that mediate the reversal of epigenetic silencing of tumour suppressor genes involved in human cancers.
Demethylation of tumour suppressor gene promoters by oocyte extracts
Sp1 sites in demethylated tumour suppressor gene promoters
Number of demethylated CGs
1-8, 14-18, 25, 31
Number of demethylated CGs containing Sp1 sites
5, 12, 13
9, 10, 22
Putative transcription factors binding sites contained in demethylated CGs
CREB, MYC, USF1, MAX, SRY, MZF-1
USF1, MZF-1, CP2
Previous work demonstrates that amphibian oocytes induce expression of pluripotency genes in somatic cells [22, 23]. As re-expression of these genes in cancer cells may induce an adverse cancer stem cell phenotype, we investigated whether AOE can induce demethylation of OCT-4 and NANOG gene promoters, as well as expression of the respective proteins. After 6 hours of reprogramming and 6 days of culture, we observed no change in DNA methylation at pluripotency gene promoters (Additional file 2: Figure S2A). Consistently, OCT-4 was not expressed after 6 days; however we detected NANOG protein expression in untreated and treated cells, likely as result of cross-reactivity of the NANOG antibody with the protein encoded by the NANOGP8 pseudogene (Additional file 2: Figure S2B). Cancer cells predominantly express NANOG transcripts derived from the NANOGP8 retrogene and because the protein encoded by the pseudogene is almost identical to the native NANOG protein, it can be easily recognised by anti-NANOG antibodies .
Remodelling of histone marks by oocyte extracts
Taken together, our results show very effective epigenetic reprogramming of tumour suppressor gene promoters by oocyte extracts, encompassing DNA demethylation and reversion of histone marks to a more euchromatic state. By comparison, treatment with 5-aza-2'-deoxycytidine can demethylate DNA in the promoters of tumour suppressor genes, but H3K9me3 and H3K27me3, which characteristically mark heterochromatin, are often retained . Most importantly, oocyte extracts induce expression from repressed tumour suppressor genes, and the higher level of expression induced by AOE than XOE correlates with a more robust targeting of demethylating activity in these extracts. Clearly, identifying the molecules that participate in the reprogramming of tumour suppressor gene expression could provide a route to the development of therapeutic strategies for the treatment of breast cancer.
Stability of tumour suppressor gene reprogramming
Reprogramming of tumour suppressor genes and reversal of malignant phenotype
This study describes a new method for investigating reprogramming of silenced tumour suppressor genes using oocyte extracts. Understanding the molecular mechanisms of tumourigenesis has been central to cancer research for decades and reports of tumour reversion are creating exciting avenues to address this important biological and biomedical question. Reprogramming in response to embryonic environments  or by over-expression of embryonic factors [41, 42] can change cancer cell fate over many cell divisions. Reprogramming with oocyte molecules which does not require DNA replication, can directly remodel cancer cell chromatin and induce re-expression of silenced tumour suppressor genes after only 6 hours of treatment. The long-term effects of this treatment are reflected in reduced tumour growth in vivo. This reprogramming system can easily be adapted to understand how oocyte chromatin remodelers and DNA demethylating complexes are targeted to promoter regions of tumour suppressor genes. Since oocyte-mediated demethylation does not occur randomly, this system can be employed to identify the mechanisms that maintain tumour suppressor gene silencing in cancer cells. Although this study focussed on reprogramming of selected tumour suppressor genes, we show that AOE can induce stable epigenetic reprogramming. Future studies will focus on pure populations of reprogrammed cells, isolated by selection in culture, to study their epigenotype at a genome-wide level. In this study we have used cancer cell lines with complex genetic abnormalities. Since NT studies highlight that genetically abnormal cells are resistant to tumour reversion , future studies will test oocyte-mediated reprogramming on cells derived from early stage tumours to elucidate the contribution of epigenetic alterations to the onset of breast oncogenesis.
Our data show that DNA methylation represents a bottleneck to reprogramming since extracts of ESC cannot efficiently reprogram hypermethylated tumour suppressor genes. Defining the relationship between different levels of epigenetic regulation for cancer-related genes is essential for devising epigenetic therapies and this system could be of paramount importance for dissecting this aspect of the problem. Extracts of axolotl oocytes show superior reprogramming capacity and they present several practical advantages, including cell size and availability. We propose that they can be a valuable tool to understand how cells become malignant and to advance the discovery of novel cancer therapies.
Cell culture and permeabilization
All culture reagents were from Invitrogen and chemicals from Sigma unless otherwise indicated. Cells were incubated in a 37°C humidified incubator with 5% C02.
MCF-7 and HCC1954 cell lines were purchased from ATCC and maintained in RPMI medium containing 10% fetal calf serum (FCS), 2 mM glutamine, 1% non-essential amino acids, 1% sodium pyruvate, and 1% penicillin/streptomycin. HMEC cells and HMEC complete medium were from Invitrogen. HMEC were passaged by incubation with 0.25% trypsin for 15 min at 37°C and neutralisation with 0.1% soybean trypsin inhibitor. CGR8 mouse ESC were obtained from ECACC and cultured on gelatin-coated culture dishes in DMEM containing 15% Hyclone stem cell screened FCS, 2 mM glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 1000 U/ml of leukemia inhibitory factor (Millipore) and 0.1 mM beta-mercaptoethanol. NTERA2 cells (kindly donated by Prof. Andrews, University of Sheffield) were cultured in DMEM medium containing 10% FCS, 2 mM glutamine, 1% non-essential amino acids, and 1% penicillin/streptomycin.
Cell permeabilisation was performed as previously reported . Briefly, cell suspensions (2 × 106 cells/ml) were treated with 20 μg/ml digitonin in PB buffer (170 mM potassium gluconate, 5 mM KCl, 2 mM MgCl2, 1 mM KH2PO4, 1 mM EGTA, 20 mM Hepes, supplemented with 3 μg/ml leupeptin, 1 μg/ml aprotinin and 1 μg/ml pepstatin A, pH 7.25, freshly prepared prior to use) for 1-2 min on ice. Cells were washed in cold PB buffer and permeabilisation was assessed by staining with propidium iodide (PI) and 70 kDa FITC-dextran.
Treatment of cells in oocyte, egg and ESC extracts
Axolotl and Xenopus oocyte/egg extracts (AOE, XOE and XEE, respectively) were prepared from mature females as described previously [21, 23]. Mouse ESC extracts (ESCE) were prepared according to Taranger et al., .
Permeabilised cells were added to oocyte/egg and ESC extracts (5,000 cells/μl extract) supplemented with an energy regenerating system (150 μg/ml creatine phosphokinase, 60 mM phosphocreatine, 1 mM ATP) and incubated at 17°C for AOE, 21°C for XOE and XEE, and 37°C for ESCE.
Gene expression analysis
Cells were collected and processed for RNA extraction using Qiagen RNAeasy mini kit with Qiashredder and DNAse treatment. cDNA synthesis was performed with Superscript III reverse transcriptase (Invitrogen). Real time PCR (Q-PCR) was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems). TaqMan Gene expression master mix and TaqMan Gene expression assays were used (assay ID can be found in Additional file 5: Table S1). After validation of the amplification efficiencies, the Relative Quantification method (ΔΔCt) was used to quantify the gene expression levels of each gene relative to ACTB (ACTIN, endogenous control) for each sample. Results are represented as fold increase in expression relative to untreated sample (UN) used as calibrator (mean ± sd, n = 3).
Nuclear proteins were extracted with NucBuster™ Protein Extraction Kit (Calbiochem).
Extracted proteins were loaded into a 12% Acrylamide gel (10 μg/lane), separated by SDS-PAGE electrophoresis and blotted onto a PVDF membrane. Membranes were blocked with 10% skimmed milk and then probed overnight at 4°C with a rabbit anti-NANOG antibody (1:1,000, Peprotech) or goat anti-OCT-4 antibody (1:1,000, Santa Cruz Biotechnology), in the presence of 0.05% Tween 20 and 5% milk. Peroxidase conjugated donkey anti-rabbit (1:10,000; GE Healthcare) or anti-goat (1:10,000; Sigma) antibodies were incubated for 1h at RT. ECL plus kit (Amersham Biosciences) was used to detect chemiluminescence.
Bisulfite genomic sequencing
Genomic DNA was isolated using DNeasy Tissue kit (Qiagen). Bisulfite genomic sequencing was carried out as described previously . Briefly, 1 μg of genomic DNA was used for bisulfite treatment (5 hours, 55°C) and 1 μl of bisulfite converted DNA was used for PCR reactions using 2.5 U of Platinum Taq polymerase (Invitrogen) (primers are listed in Additional file 5: Table S1). Primers spanning CpG island sequences were designed using Methprimer software http://www.urogene.org/methprimer/index1.html. Purified PCR products were either directly sequenced or cloned into pGEM-T easy (Promega), with 10 or more clones of each sample subjected to sequencing.
Chromatin Immunoprecipitation (ChIP)
ChIP experiments were performed using the Magna ChIP A kit (Millipore). One million cells were used with the following antibodies: ChIP grade rabbit polyclonal anti-H3K27me3 (3 μg, Millipore 07-449), ChIP grade rabbit polyclonal anti-H3K4me3 (3 μg, Abcam ab8580), ChIP grade rabbit polyclonal anti-H3K9ac (1.5 μg, Abcam ab4441), ChIP grade rabbit polyclonal anti-H3K9me3 (2 μg, Abcam ab8898), ChIP grade mouse monoclonal anti-H3K9me2 (2 μg, Abcam ab1220), IgG from rabbit serum (4 μg, Sigma I5006). Immunoprecipitated DNA was quantified by Q-PCR using the 7500 Fast Real-Time PCR System (Applied Biosystems) with 5 μl DNA (from a total of 50 μl). TaqMan Gene expression master mix and TaqMan Gene expression custom assays (Applied Biosystem) were used (primers and probes are listed in Additional file 5: Table S1). Data are presented as "Fold enrichment" of precipitated DNA for each histone modification relative to a 1/100 dilution of input chromatin (mean ± sd, n = 3).
HMEC, MCF-7 and HC1954 cells were transfected with Firefly RARB reporter and Renilla luciferase transfection control pRL-TK (Promega) plasmids using Lipofectamine 2000 (Invitrogen). The RARB reporter (containing promoter sequence fragment -522 to +156) was obtained by cloning into pGL3-Basic (Promega) at the Nhe and Xho1 restriction sites in both orientations. The vector with antisense promoter orientation was used as control in transfection experiments. Retinoic acid (RA 1 μM, Sigma) treatment was performed for 24 hours. After normalizing the Firefly values to Renilla, the data are presented as relative luciferase values to the negative control (mean ± sd, n = 3).
Cell proliferation assay
Permeabilised MCF-7 cells were incubated in AOE and after 6 hours plated at a density of 12.5 × 103 cells/cm2 in triplicate. After 1, 3, and 6 days in culture MTT was added at 5 mg/ml and incubated for 3 hours at 37°C. The converted dye was dissolved by treatment with isopropanol containing 0.04N HCl and quantified by measuring the absorbance at 570 nm with background subtraction at 650 nm in a SmartSpec 3000 Spectrophotometer (Bio-Rad Laboratories). Results are presented as mean ± sd, n = 3.
Apoptosis and cell cycle assays
MCF-7 cells treated in AOE were plated at a density of 5 × 103 cells/cm2 in triplicate. After 1, 3, and 6 days in culture cells were trypsinised and fixed with 70% ice-cold ethanol for 30 min at -20°C. Cell were then centrifuged and stained in 50 μg/ml PI solution containing 0.1 mg/ml RNase A and 0.05% Triton X-100 for 30 min. After washing, cells were resuspended in PBS and 50,000 cells analysed with a Beckman Coulter FC-500 flow cytometer.
Soft agar assay
MCF-7 cells (30,000/6 well) were seeded in 0.5 ml of 0.3% noble agar in complete RPMI medium overlaying a 1 ml 0.5% agar in the same medium. After 2 weeks culture cell colonies were stained with crystal violet and colonies ≥ of 100 μm counted under a MZ125 Leica stereomicroscope. For control experiments with non-permeabilised cells, cells were either incubated with AOE for 6 hours and then plated in soft agar or cultured with different amounts of AOE into the agar top layer. In the latter case 10, 50 and 100 μl of AOE were added to the top layer of soft agar together with MCF-7 cells (corresponding to the same, 5-fold and 10-fold higher ratio of cell/extract used with permeabilised cells).
Female MF1 nude mice (Harlan-Olac) were anaesthetised with Ketamine/Medetomidine and MCF-7 cells at 1.5 × 106 cells in a volume of 200 μl Matrigel were injected sub-cutaneously into the left flank. In addition, 0.1 mg 17-beta-estradiol pellet (60-day release; Innovative Research of America, US) implanted subcutaneously into the scruff of each mouse. Tumour dimensions were measured by calliper measurement of length and width three times weekly and the volume calculated [(length2 × width)/2] and clinical condition of the mice were monitored by weekly body weight measurements for the duration of the study (n = 4-6 for each time point). The project was run under Home Office project PPL 40/2962 which was awarded in November 2006 (Watson) following local ethical approval. The study also adhered to the UK Co-ordinating Committee for Cancer Research (UKCCCR) guidelines. At termination, tumours were excised, fixed in formalin and paraffin embedded.
Histology sections (5 μm) were stained with Eosin & Haematoxylin and observed under a Leica DM5000B microscope and Leica Application Suite software.
Mitotic figures were quantified by examination of 10 fields of view at high power magnification (630x) in two independent tumours. Collagen was stained with the Trichrome Stains (Masson, Sigma) according to manufacturer's instructions.
GraphPad InStat3 software was used to perform statistical analysis. Q-PCR and ChIP data were analysed by one-way ANOVA with post Tukey's multiple comparison test with a significance level set at P < 0.05. Bisulfite sequencing data were analysed by χ2 test with a significance level set at P < 0.05. Cell proliferation, soft agar assay and luciferase assay, data were analysed with unpaired Student's t-test (P < 0.05). Tumour growth data were analysed by two-way ANOVA with Bonferroni post-test (P < 0.05). Mitotic figures data were analysed by one-way ANOVA with post Tukey's multiple comparison test with a significance level set at P < 0.05.
We thank Jodie Chatfield and Ceri Allen for valuable technical support. We acknowledge Peter Brown for his assistance with histopathology. We thank Peter Andrews for donating the NTERA2 cells. This work was supported by EvoCell Ltd., Nottingham, UK.
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