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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

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)

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Rat primary tissue samples and carcinoma cell lines.  PhIP-induced mammary adenocarcinomas were obtained from female (F344 × SD)F1 rats at the age of 56–69 weeks who were administrated 10 doses of PhIP (75 mg/kg) at the age of 6 weeks and a high-fat diet (23.5% corn oil).(15) DMBA-induced mammary adenocarcinomas were obtained from female (F344 × SD)F1 rats at the age of 25–32 weeks who were administrated a single dose of DMBA (50 mg/kg) at the age of 7 weeks.(16) Normal mammary glands were collected from 8-week-old untreated female (F344 × SD)F1 rats and 56–69-week-old female (F344 × SD)F1 rats without administration of PhIP by the gland isolation technique for mammary ducts.(17) Primary-cultured epithelial cells were maintained as rat mammary epithelial cells (RMEC).(17) Two carcinoma cell lines, PhIP7-4 and PhIP12-1, were established from two mammary carcinomas induced by PhIP, as previously reported.(18) Two carcinoma cell lines, DMBA334 and DMBA397, were established from the DMBA-induced mammary carcinomas described above.

Human breast cancer cell lines and tissue samples.  MCF-7, T47-D, SK-BR-3, MDA-MB-231, MDA-MB-468, ZR-75-1, BT-474, Hs578T and mammary epithelial cell line (MCF10A) were purchased from the American Type Culture Collection (Rockville, MD, USA). Human mammary epithelial cells (HMEC) were purchased from Cambrex (East Rutherford, NJ, USA). Human breast cancers (= 91) and adjacent non-cancerous tissues (= 21), which were located at least 3 cm apart from cancers, were obtained from the surgical specimens of patients who underwent mastectomy. Human samples were obtained with informed consent and the analysis was approved by the institutional review boards.

5-Aza-2′-deoxycytidine and trichostatin A treatments.  PhIP7-4 cells were seeded at a density of 2 × 105 cells/10 cm plate on day 0, exposed to freshly prepared 1 or 5 μM 5-aza-2′-deoxycytidine (5-aza-dC; Sigma, St Louis, MO, USA) for 24 h on days 1 and 2, treated with 100 or 300 nM trichostatin A (TSA; Sigma) for 24 h on day 3, and then harvested on day 4. BT-474 and MDA-MB-231 cells were seeded at a density of 5 × 105 cells/6 cm plate and 6 × 105 cells/10 cm plate, respectively, on day 0, and treated with 5-aza-dC (1 or 5 μM for BT474; 0.5 or 1 μM for MDA-MB-231) for 24 h on days 1 and 3, and harvested on day 4. The doses of 5-aza-dC (and TSA) were adjusted so that the growth of treated cells is suppressed to 40–80% of non-treated cells.

MeDIP-CGI microarray analysis.  As described previously,(19,20) 5 μg of sonicated DNA was immunoprecipitated with 6 μg antibody against 5-methylcytidine (Diagnode, Liége, Belgium), and the precipitated DNA and input DNA were labeled with Cy5 and Cy3, respectively. The labeled probes were hybridized to a rat CGI oligonucleotide microarray (Agilent Technologies, Santa Clara, CA, USA) that contained 93 024 probes in or within 95 bp of 13 026 CGI with an average probe spacing of 100 bp. The microarray was scanned with an Agilent G2565BA microarray scanner (Agilent Technologies), and the scanned data were processed using Feature Extraction Ver.9.1 (Agilent Technologies) and Agilent G4477AA ChIP Analytics 1.3 software (Agilent Technologies). A signal of probes was converted into a “Me value”, which represented the methylation level as a value from 0–0.3 (unmethylated) to 0.6–1 (methylated).(19) For the rat CGI microarray, the formula for the Me value was optimized as follows: inline image

Definition of promoter CGI, and hierarchical clustering analysis.  A CGI was defined as an assembly of probes within intervals <500 bp. A promoter CGI was defined as a CGI within a nucleosome-free region, which was defined as a region between a transcription start site (TSS) and its 200 bp upstream.(21) A TSS was determined using UCSC rn4 (Baylor Build 3.4, November 2004). According to these definitions, 5031 assemblies were defined as promoter CGI. The methylation level of a CGI was assessed by an average Me value of the probes located within the CGI, and cut-off values of 0.6 and 0.3 were used for methylated and unmethylated CGI, respectively. Hierarchical clustering of promoter CGI by the Euclidean distance of their Me value was performed using MultiExperimental Viewer v4.1 software (Dana-Farber Cancer Institute, Boston, MA, USA).

Oligonucleotide expression microarray analysis.  Total RNA was isolated using ISOGEN (NIPPON GENE, Tokyo, Japan). Oligonucleotide microarray analysis was performed using a GeneChip Rat Genome 230 2.0 Array (Affymetrix, Santa Clara, CA, USA) with 54 000 probe sets, and 30 000 transcripts from 30 000 genes. From 7 μg of total RNA, double-stranded cDNA was synthesized and biotin-labeled cRNA was prepared using a BioArray High-Yield ENA transcript labeling kit (Enzo, Farmingdale, NY, USA). Twenty micrograms of labeled cRNA were fragmented and hybridized to the GeneChip oligonucleotide microarray. The scanned data were processed using GeneChip operating software and normalized so that the average of all genes on a GeneChip would be 500. A P-value for differential expression (change P-value) was calculated for each probe by an algorithm based on the Wilcoxon Signed-Rank test.

Methylation-specific PCR (MSP) and bisulfite sequencing.  Sodium bisulfite treatment was performed as described previously using 1 μg of DNA digested with BamHI (Toyobo, Tokyo, Japan),(22) and suspended in 40 μL of Tris-EDTA (TE) buffer. Fully methylated DNA was prepared by methylating genomic DNA using SssI-methylase (New England Biolabs, Beverly, MA, USA). Fully unmethylated DNA was prepared by amplifying genomic DNA with ø29 DNA polymerase (GenomiPhi DNA Amplification kit; GE Healthcare UK, Buckinghamshire, UK).

Conventional MSP for screening purposes was conducted with primers specific to methylated DNA up to 39 cycles (Table S1), and samples with no PCR products amplified from methylated DNA were determined to be unmethylated samples.(22) Quantitative MSP (qMSP) was performed by real-time PCR using SYBR Green I and primers specific to methylated DNA at a locus and to a B2 repeat sequence, regardless of its methylation statuses for rat genes, or to unmethylated DNA for a human gene (Table S1). The number of DNA molecules was calculated as previously described.(23) The methylation level of a rat gene was calculated as the methylation percentage obtained as: ([number of DNA molecules methylated at a target CGI in a sample]/[number of B2 repeat in the sample])/([number of DNA molecules methylated at the target CGI in a SssI-treated DNA]/[number of B2 repeat in the SssI-treated DNA]) × 100; and that of a human gene as: (number of DNA molecules methylated)/([number of DNA molecules methylated] + [number of DNA molecules unmethylated]) × 100. The methylation level in a cancer sample was considered as aberrant when it was two or more times higher than the highest methylation level in normal samples.

For bisulfite sequencing, sodium bisulfite-treated DNA was amplified with primers common to methylated and unmethylated DNA sequences (Table S1). The PCR product was cloned into a pGEM-T Easy Vector (Promega, Madison, WI, USA), and 10 clones were sequenced using an ABI PRISM 310 sequencer (Applied Biosystems, Foster City, CA, USA).

Quantitative reverse transcription-PCR (qRT-PCR).  DNase-treated total RNA (1 μg) was reverse-transcribed with a random hexamer (Invitrogen, Carlsbad, CA, USA) and Superscript III reverse transcriptase (Invitrogen). Quantitative PCR was carried out by real-time PCR using SYBR Green I and primers specific to genes. The primer sequences and PCR conditions are shown in Table S1. The amplification curve of a sample was compared with those of standard DNA samples with known copy numbers to obtain a copy number in the sample, as in qMSP. The number of target cDNA molecules was normalized to those of rat Ppia or human GAPDH cDNA molecules.(24)

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Isolation of genes methylation-silenced in a rat mammary carcinoma by integration of MeDIP-CGI microarray and expression microarray.  To isolate CGI methylated in rat mammary carcinomas and identify a carcinoma with the largest number of aberrantly methylated CGI, we analyzed three primary carcinomas (PhIP7-4#1, DMBA334#1 and DMBA397#1), three carcinoma cell lines established from these primary samples, RMEC and a pool of four normal mammary glands by MeDIP-CGI microarray. Among the 5031 promoter CGI, 1745 were methylated in at least one carcinoma but not in any of the normal mammary glands. Hierarchical clustering analysis using the 1745 promoter CGI identified a group of promoter CGI methylated both in cell lines and primary carcinomas (Group A) and a group methylated mainly in carcinoma cell lines (Group B) (Fig. 1A). The number of methylated promoter CGI was the largest in the PhP7-4 cell line among the three cell lines, and 84% and 78% of the promoter CGI methylated in the DMBA334 and DMBA397 cell lines, respectively, overlapped with those in the PhIP7-4 cell line. Thus, we selected the PhIP7-4 cell line for further analysis, and isolated 465 promoter CGI methylated in this cell line but not in RMEC (Fig. 1B).

image

Figure 1.  Selection procedures of genes with methylated promoter CpG islands (CGI) in a rat mammary carcinoma cell line and its primary mammary carcinoma. (A) DNA methylation statuses of promoter CGI revealed by methylated DNA immunoprecipitation (MeDIP)–CGI microarray analysis. Hierarchical clustering of promoter CGI was performed using Me values of 1745 promoter CGI methylated in at least one carcinoma and not methylated in normal mammary glands. The Me values are represented by colors from blue (0.0) to yellow (1.0). (B) Selection procedure of genes with methylated promoter CGI in a rat mammary carcinoma cell line and then in its primary carcinoma. The number of methylated promoter CGI and that of genes upregulated by treatment of the PhIP7-4 cell line with 5-aza-2′-deoxycytidine (5-aza-dC) and trichostatin A (TSA) are shown. After integration of both sets of data, 29 candidate genes silenced in the PhIP7-4 cell line were obtained, and 15 genes were shown to be methylated also in its primary carcinoma (PhIP7-4#1). RMEC, rat mammary epithelial cells.

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To select candidates for methylation-silenced genes, expression microarray analyses were conducted using the PhIP7-4 cell line before and after treatment with 5 μM 5-aza-dC and 100 nM TSA. The expression microarray data showed that 1705 genes were upregulated twofold or more by the treatment, with P-values < 0.002 (Fig. 1B). By integrating the expression microarray data and the MeDIP-CGI microarray data, 29 genes were found to be methylated in the PhIP7-4 cell line and re-expressed by the epigenetic treatment. Based on the MeDIP-CGI microarray data, 14 genes methylated only in the cell line were excluded, and 15 genes methylated also in its primary tumor (PhIP7-4#1) were selected for further analysis (Fig. 1B).

Isolation of six genes methylated in rat mammary carcinoma cell lines.  The data of MeDIP-microarray analysis was confirmed by conventional MSP of the PhIP7-4 cell line, and 11 of the 15 genes were methylated. Seven (Angptl4, Coro1a, Tmem37, RGD1304982, Scin, Ndn and Sts) of the 11 genes were not methylated in RMEC, while the remaining four were methylated (data not shown). The methylation levels of the seven genes were further quantified by qMSP in the RMEC and four carcinoma cell lines (the three cell lines used for the initial microarray analysis and PhIP12-1) (Fig. 2). Six genes (Angptl4, Coro1a, Tmem37, RGD1304982, Ndn and Sts) were highly methylated in one or more carcinoma cell lines (Fig. 2), supporting that aberrant methylation of these genes was present in epithelial cancer cells. In contrast, Scin had methylation levels <10% in all the carcinoma cell lines and the RMEC (data not shown).

image

Figure 2.  Methylation levels of the six genes (Angptl4, Coro1a, Tmem37, RGD1304982, Ndn and Sts) in rat mammary carcinoma cell lines. Methylation levels of the seven genes selected by the data of methylated DNA immunoprecipitation–CpG islands (MeDIP-CGI) microarray data and expression microarray data were analyzed by quantitative methylation-specific PCR using rat mammary epithelial cells (RMEC) and the four mammary carcinoma cell lines (PhIP7-4, PhIP12-1, DMBA334 and DMBA397). Data of the six genes with high methylation levels in at least one carcinoma cell line are shown.

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Identification of five genes methylated in rat primary mammary carcinomas.  Methylation levels of the six genes in primary carcinomas were quantified using 13 PhIP-induced primary mammary carcinomas, 12 DMBA-induced primary mammary carcinomas and seven normal mammary glands from age-matched control rats by qMSP (Fig. 3A). Two genes (Angptl4 and Coro1a) were barely methylated in normal mammary glands, and were aberrantly methylated in three or more PhIP- and DMBA-induced carcinomas, showing carcinoma-specific aberrant methylation. RGD1304982 was also barely methylated in normal mammary glands, and only one PhIP-induced carcinoma had aberrant methylation. Two genes (Tmem37 and Ndn) had background methylation in normal mammary glands. Tmem37 was aberrantly methylated in three of the 13 PhIP-induced and one of the 12 DMBA-induced carcinomas, and Ndn was aberrantly methylated only in two PhIP-induced carcinomas. The remaining one gene, Sts, was highly methylated in normal mammary glands, and carcinoma-specific methylation could not be confirmed.

image

Figure 3.  Methylation of six genes in primary rat mammary carcinomas. (A) Methylation levels analyzed by quantitative methylation-specific PCR (qMSP) of seven normal mammary glands (Normal), and 13 PhIP-induced (PhIP) or 12 DMBA-induced (DMBA) primary mammary carcinomas. A carcinoma sample was considered to have aberrant methylation when its methylation level was two or more times higher than the highest methylation level in normal samples. (B) Presence of dense methylation in the PhIP7-4 cell line and a primary carcinoma, PhIP2-2#2. The methylation status of individual CpG sites (vertical lines, shown at the top) in the upstream region of Angptl4 was determined by bisulfite sequencing. Closed circles, methylated CpG islands (CGI) sites; open circles, unmethylated CpG sites; closed arrows, location of the primers for MSP.

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The presence of dense methylation of the promoter CGI of Angptl4 was analyzed by bisulfite sequencing of the PhIP7-4 cell line and a primary carcinoma (methylation level of 30% by qMSP) (Fig. 3B). Densely methylated DNA molecules were present in these two samples while they were absent in the RMEC and normal mammary glands (Fig. 3B).

Identification of Angptl4 as a gene methylation silenced in rat mammary carcinomas with abundant expression in normal mammary glands.  Expression in normal mammary glands was examined by qRT-PCR. Angptl4 was highly expressed (Fig. 4A), but the other four genes (Coro1a, Tmem37, RGD1304982 and Ndn) were not expressed (data not shown). Angptl4 was not expressed in two carcinoma cell lines and primary carcinomas, and unexpectedly also not in RMEC (Fig. 4A). Re-expression after the treatment of PhIP7-4 with 5-aza-dC and TSA was confirmed by qRT-PCR (Fig. 4B), supporting methylation silencing of Angptl4.

image

Figure 4.  Expression analysis of rat Angptl4. (A) Angptl4 expression in rat mammary epithelial cells (RMEC), normal mammary glands, carcinoma cell lines (PhIP7-4 and PhIP12-1) and 10 PhIP-induced primary carcinomas analyzed by qRT-PCR. Angptl4 expression was lost in the cell lines, and decreased in all the carcinomas. (B) Re-expression of Angptl4 after epigenetic treatment of PhIP7-4 cells with 5-aza-2′-deoxycytidine (5-aza-dC) and trichostatin A (TSA). Angptl4 re-expression was confirmed.

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Methylation silencing of ANGPTL4 in human breast cancer samples.  Finally, we investigated methylation silencing of human ANGPTL4, which has a promoter CGI, as does rat Angptl4, in human breast cancers. ANGPTL4 was expressed in two kinds of mammary epithelial cells (HMEC and MCF10A) that had unmethylated DNA (Fig. 5A). Among eight breast cancer cell lines, ANGPTL4 was highly methylated in three cell lines (MDA-MB-231, MDA-MB-468 and BT-474), and had little expression in these cell lines (Fig. 5A). ANGPTL4 was re-expressed by 5-aza-dC treatment of BT-474 and MDA-MB-231, showing that ANGPTL4 was also silenced by methylation in human breast cancer cell lines (Fig. 5B).

image

Figure 5.  Methylation silencing of human ANGPTL4. (A) Expression levels of human ANGPTL4 in normal mammary epithelial cells and cancer cell lines by qRT-PCR, along with its methylation levels by qMSP. ANGPTL4 expression was very low in the three cell lines (MDA-MB-231, MDA-MB-468 and BT-474) with high methylation levels. (B) Re-expression of ANGPTL4 after treatment of breast cancer cell lines (BT-474 and MDA-MB-231) with 5-aza-2′-deoxycytidine (5-aza-dC).

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Methylation of ANGPTL4 was analyzed in 91 primary human breast cancers and non-cancerous breast tissues of 21 cancer patients by qMSP. Using a cut-off value of 10%,(25) methylation was detected in 11 of the 91 breast cancers (12%), while little methylation was detected in the non-cancerous tissues (Fig. 6). The methylation status in cancer was not associated with any clinicopathological characteristics, including age, clinical stage, tumor size, estrogen receptor status, progesterone receptor status, HER2 expression and recurrence (data not shown). These data showed that methylation silencing of Angptl4 is commonly present in mammary carcinomas of both rats and humans.

image

Figure 6.  Methylation levels of human ANGPTL4 obtained by quantitative methylation-specific PCR (qMSP) of primary human tissue samples. Methylation was barely observed in non-cancerous tissues, but high methylation levels were observed in breast cancer tissues. Using a threshold of 10%, 11 cancers showed aberrant methylation.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. 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.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

N.H. is the recipient of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research. This study was supported by a Grant-in-Aid for the Third-Term Comprehensive Cancer Control Strategy from the Ministry of Health, Labour and Welfare, Japan, and by the A3 Foresight Program from the Japan Society for the Promotion of Science.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Table S1. Primers for MSP, RT-PCR and bisulfite sequencing.

FilenameFormatSizeDescription
CAS_1955_sm_st1.xls37KSupporting info item

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