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Aberrant DNA methylation is deeply involved in the development and progression of human breast cancers, but its inducers and molecular mechanisms are still unclear. To reveal such inducers and clarify the molecular mechanisms, animal models are indispensable. Here, to identify genes silenced by promoter DNA methylation in rat mammary carcinomas, we took a combined approach of methylated DNA immunoprecipitation (MeDIP)–CpG island (CGI) microarray analysis and expression microarray analysis after treatment with epigenetic drugs. MeDIP-CGI microarray revealed that among 5031 genes with promoter CGI, 465 were methylated in a carcinoma cell line induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but not in normal mammary epithelial cells. By treatment of the cell line with 5-aza-2′-deoxycytidine and trichostatin A, 29 of the 465 genes were shown to be re-expressed. In primary mammary carcinomas, five (Angptl4, Coro1a, RGD1304982, Tmem37 and Ndn) of the 29 genes were methylated in one or more of 25 samples. Quantitative expression analysis revealed that Angptl4 had high expression in normal mammary glands, but low expression in primary carcinomas. Also in humans, ANGPTL4 was unmethylated and expressed in normal mammary epithelial cells, but was methylated in 11 of 91 (12%) primary breast cancers. This is the first study to identify genes aberrantly methylated in rat mammary carcinomas, and Angptl4 is a novel methylation-silenced gene both in rat and human mammary carcinomas. The combination of the MeDIP-CGI microarray analysis and expression microarray analysis after treatment with epigenetic drugs was effective in reducing the number of methylated genes that are not methylation silenced. (Cancer Sci 2011; 102: 1337–1343)
Aberrant epigenetic modifications, such as aberrant DNA methylation and histone modifications, are deeply involved in the development and progression of human cancers.(1–3) In human breast cancers, tumor-suppressor genes, such as RASSF1A, BRCA1, CDKN2A and PTEN, are silenced by aberrant methylation of promoter CpG islands (CGI).(4) Aberrant methylation could be detected not only in cancers but also in non-cancerous breast tissues, suggesting that an epigenetic field defect is formed in breast cancer patients.(5) Despite the deep involvement of aberrant DNA methylation, limited information is available on the factors that induce aberrant methylation during mammary carcinogenesis. Among the limited information, exposure to estrogen or a nonsteroidal estrogen, bisphenol A, was reported to change the methylation status of mammary epithelial progenitor cells aberrantly in vitro.(6,7) However, inducers and induction mechanisms of aberrant methylation in vivo are still almost unknown. To address these issues, animal models are indispensable.
Rat models are useful for the study of mammary carcinogenesis in terms of several features. Mammary carcinomas can be reproducibly induced by a wide range of a selection of chemicals, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP),(8,9) 7,12-dimethylbenz[a]anthracene (DMBA)(10) and N-nitroso-N-methylurea,(11) and also by radiation,(12) and their characteristics have been well established. The induced mammary carcinomas predominantly originated from mammary ducts similar to the majority of human breast cancers.(13) As an animal model, we can use animals with a homogeneous genetic background and make any intervention to clearly analyze the effects of specific factors, such as overexposure to estrogen and intake of a high fat diet, on a phenotype. However, to analyze inducers of aberrant methylation and its mechanisms, we need genes silenced by aberrant methylation in rat mammary carcinomas, which are not known yet.
In the present study, we aimed to identify methylation-silenced genes in rat primary mammary carcinomas. To this end, we applied two genome-wide methylation analyses, methylated DNA immunoprecipitation (MeDIP)–CGI microarray analysis and expression microarray analysis after treatment with epigenetic drugs.(14)
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- Materials and Methods
- Disclosure Statement
- Supporting Information
In the present study, genes with aberrant methylation of promoter CGI in rat primary mammary carcinomas were identified for the first time, and Angptl4 was demonstrated as a novel methylation-silenced gene both in rat and human mammary carcinomas. The combination of the MeDIP-CGI microarray analysis and expression microarray analysis after epigenetic treatment was effective in reducing the number of methylated genes that were not methylation silenced.
ANGPTL4 is a secreted protein of the angiopoietin-like family, involved in lipid metabolism,(26) and upregulated by hypoxia.(27) It is known to be silenced in human gastric cancers(28) and in human melanoma cell lines.(29) The role of ANGPTL4 in mammary carcinomas remains controversial. Padua et al.,(30) reported that overexpression of ANGPTL4 mediated lung metastasis of estrogen receptor-negative breast cancer cells. In contrast, Foreman et al. and Girroir et al.(31,32) reported that Angptl4 was upregulated by PPARβ/∂ activation, and inhibited growth of human and mouse mammary carcinoma cell lines. Here we found that Angptl4 was expressed in rat mammary glands and human mammary epithelial cells, and methylation silenced in rat mammary carcinomas and human breast cancers. Together with Foreman et al. and Girroir et al. findings, Angptl4 was suggested to be a tumor-suppressor gene.
The other four genes (Coro1a, Tmem37, RGD1304982 and Ndn) were likely to have been methylated as a consequence of rat mammary carcinogenesis because they were not expressed in normal mammary glands. However, several interesting features have been reported about Ndn and RGD1304982. Human NDN is reported to suppress the growth of osteosarcoma cells, have anti-angiogenic effects both in vitro and in vivo, and interact with tumor suppressor p53.(33,34) A putative quinone oxidoreductase-like protein 2, encoded by RGD1304982, has homology to human NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2), which are known to stabilize p53.(35) The possibility remains that Ndn and RGD1304982 expression is induced in response to cellular stresses in normal mammary glands, and that these two genes function as tumor suppressors.
Two (Tmem37 and Ndn) of the five genes aberrantly methylated in mammary carcinomas were slightly methylated in normal-appearing mammary glands of old rats (56–69 weeks old) (Fig. 3A), which did not contain cancerous tissues. This aberrant methylation was not observed in mammary glands of young rats (8 weeks old) (data not shown), suggesting that these two genes are methylated in an age-dependent manner. It is known that age-dependent methylation can be accelerated by inflammation in the colon,(36) and there is a possibility that these genes can be used as efficient markers to assess the effects of possible inducers of aberrant methylation during mammary carcinogenesis.
Four rat primary carcinomas and two human breast cancer cell lines (T-47D and Hs578T) did not express ANGPTL4, although their DNA was unmethylated (Figs 4A, 5A). It is frequently observed that a gene methylation silenced in some cancer cell lines is not expressed in other cell lines by mechanisms other than CGI methylation, such as loss of transcription factors or signal dysregulation.(37) In the case of ANGPTL4, its expression is known to be stimulated by TGFß signaling,(30) which is disrupted in various kinds of cancers. Angptl4 was not expressed even in RMEC (Fig. 4A), suggesting that under in vitro culture conditions factors required for ANGPTL4 expression are lacking.
Methodologically, we integrated the MeDIP-CGI microarray data and those by expression microarray analysis after epigenetic treatment to identify methylation-silenced genes. The integration effectively reduced the number of candidate genes from 465 (by the MeDIP-CGI microarray analysis) and 1705 (by the expression microarray analysis) after epigenetic treatment) to 29 genes. The MeDIP-CGI microarray can isolate methylated CGI, but these are not always located in genomic regions important for gene silencing.(19) The expression microarray analysis after epigenetic treatment, also known as chemical genomic screening(14,38) or pharmacological unmasking,(39) is a simple and cost-effective method in identifying methylation-silenced genes using cell lines.(14,29,38) However, genes isolated by this method are known to contain many genes that were induced by the actions of 5-aza-dC other than DNA demethylation, such as activation of the p53 pathway.(40) The approach taken here is a more effective way to identify methylation-silenced genes by reducing the number of methylated genes that are not methylation silenced.
A rat CGI microarray was custom designed in the present study. Its use successfully led to identification of a novel methylation-silenced gene not only in rat but also in human mammary carcinomas. Rats are widely used in the fields of biomedical research, such as cancers, toxicology, physiology and cardiovascular diseases, and a large amount of data using rat models has been accumulated.(41,42) The rat CGI microarray can be used for various epigenetic research in rats.
In conclusion, this is the first study that identified genes aberrantly methylated in rat mammary carcinomas. Angptl4 is a novel methylation-silenced gene both in rat and human mammary carcinomas.