January 13, 2011;
Epigenetic reprogramming of breast cancer cells with oocyte extracts.
BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.
regulation of gene expression, epigenetic
Disease Ontology terms:
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Figure 1. Expression of tumour suppressor genes after reprogramming in oocyte, egg and embryonic stem cell extracts Expression of RARB, CST6, CCND2, GAS2 and CDKN2A after 6 hours reprogramming analysed by Q-PCR. Data are shown as fold increase compared to the calibrator sample (UN: untreated cells). Relative quantification to the expression of ACTIN (ACTB) was performed for each gene. Study of CDKN2A expression of was only performed in HCC1954 since this gene is deleted in MCF-7 cells.* indicates P <0.05 for treated groups different from UN.
Figure 2. DNA methylation analysis of tumour suppressor genes by bisulfite sequencing. Bisulfite sequencing of RARB, CST6, CCND2 (MCF-7 cells), and CDKN2A (HCC1954 cells) gene promoters after 6 hours reprogramming. Schematics indicate the position of analysed CpG islands in promoter regions. A minimum of 10 clones were analysed for each gene and average loss of methylation was calculated for each reprogramming treatment. Black circles indicate metylated CGs, white circles indicate unmethylated CGs. Reprogramming in AOE produced the highest levels of demethylation (P <0.05; a = AOE, XOE, ESCE vs UN; b = AOE vs XOE; c = AOE vs ESCE).
Figure 3. Reprogramming of histone marks by oocyte extracts. Analysis of RARB, CDKN2A and GAS2 gene promoters by ChIP. Data are presented as fold enrichment to input chromatin and indicate reprogramming of histone repressive (H3K9me3, H3K9me2, H3K27me3) and active (H3K4me3, H3K9Ac) marks by different extracts after 6 hours of treatment. * indicates P <0.05 for treated groups different from UN.
Figure 4. Epigenetic reprogramming stability of tumour suppressor genes in AOE. Reprogramming of the tumour suppressor gene RARB persists 6 days after treatment of MCF-7 and HC1954 cells with AOE. (A) Promoter activity in HMEC, MCF-7 and HCC1954 cells with or without RA treatment measured by luciferase assay (* indicates P <0.05 for RA treated cells different from the untreated group). (B) RARB promoter in retinoic acid resistant HCC1954 cells can respond to RA after reprogramming (P <0.05; a = AOE and AOE+RA vs UN; b = AOE + RA vs AOE). (C) RARB expression by Q-PCR. * indicates P <0.05 of treated cells compared to UN. (D) DNA demethylation is maintained in HCC1954 cells after 6 days of treatment as shown by bisulfite sequencing (similar results were obtained with MCF-7 cells).
Figure 5. Effect of epigenetic reprogramming on malignant cell phenotype. Cancer cell growth after reprogramming with AOE. (A) Proliferation of MCF-7 cells after 1, 3 and 6 days of culture in adherent conditions as measured by MTT assay. (B) Growth of MCF-7 cells in anchorage-independent conditions. The top panels show cultures stained with crystal violet and representative fields of view of the same cultures in soft agar. The bottom panel show quantification of colony number as counted under a stereomicrosope. Bar = 100 μm. * indicates P <0.05.
Figure 6. Effect of epigenetic reprogramming on in vivo tumorigenicity. Macroscopic and microscopic analyses of tumour xenografts. (A) Macroscopic appearance of untreated (UN) and AOE-treated tumour xenografts (AOE) and relative tumour growth curves (* indicates P <0.05). (B) Eosin-Haematoxylin staining of untreated tumour sections (black arrows: mitotic figures; Bar = 50 μm). (C) Number of mitotic figures for two independent tumours in untreated (UN) and AOE-treated xenografts (AOE) (* indicates P <0.05). (D) Interstitial stroma present in AOE-treated tumours stained for collagen (blue staining; Bar = 50 μm).
Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm.