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Genome-wide DNA methylation analysis reveals estrogen-mediated epigenetic repression of metallothionein-1 gene cluster in breast cancer.

Jadhav RR, Ye Z, Huang RL, Liu J, Hsu PY, Huang YW, Rangel LB, Lai HC, Roa JC, Kirma NB, Huang TH, Jin VX - Clin Epigenetics (2015)

Bottom Line: Bioinformatics analysis determined seven gene clusters with a significant difference in overall survival (OS) and further revealed a distinct feature that the conservation of a large gene cluster (approximately 70 kb) metallothionein-1 (MT1) among 45 species is much lower than the average of all RefSeq genes.Our data suggests that DNA methylation in large contiguous gene clusters can be potential prognostic markers of breast cancer.Further investigation of these clusters revealed that estrogen mediates epigenetic repression of MT1 cluster in ERα + breast cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, STRF, Room 225, 7703 Floyd Curl Drive, San Antonio, 78229 TX USA.

ABSTRACT

Background: Recent genome-wide analysis has shown that DNA methylation spans long stretches of chromosome regions consisting of clusters of contiguous CpG islands or gene families. Hypermethylation of various gene clusters has been reported in many types of cancer. In this study, we conducted methyl-binding domain capture (MBDCap) sequencing (MBD-seq) analysis on a breast cancer cohort consisting of 77 patients and 10 normal controls, as well as a panel of 38 breast cancer cell lines.

Results: Bioinformatics analysis determined seven gene clusters with a significant difference in overall survival (OS) and further revealed a distinct feature that the conservation of a large gene cluster (approximately 70 kb) metallothionein-1 (MT1) among 45 species is much lower than the average of all RefSeq genes. Furthermore, we found that DNA methylation is an important epigenetic regulator contributing to gene repression of MT1 gene cluster in both ERα positive (ERα+) and ERα negative (ERα-) breast tumors. In silico analysis revealed much lower gene expression of this cluster in The Cancer Genome Atlas (TCGA) cohort for ERα + tumors. To further investigate the role of estrogen, we conducted 17β-estradiol (E2) and demethylating agent 5-aza-2'-deoxycytidine (DAC) treatment in various breast cancer cell types. Cell proliferation and invasion assays suggested MT1F and MT1M may play an anti-oncogenic role in breast cancer.

Conclusions: Our data suggests that DNA methylation in large contiguous gene clusters can be potential prognostic markers of breast cancer. Further investigation of these clusters revealed that estrogen mediates epigenetic repression of MT1 cluster in ERα + breast cancer cell lines. In all, our studies identify thousands of breast tumor hypermethylated regions for the first time, in particular, discovering seven large contiguous hypermethylated gene clusters.

No MeSH data available.


Related in: MedlinePlus

Differential response to E2 suppression and DAC activation of MT1 gene expression is seen in ERα + cell lines but not in ERα − cell lines. (A) Luciferase activity of ERE reporter vector normalized to Renilla reporter activity in luminal and basal breast cancer cell lines. (B) MT1 expression was carried out by RT-qPCR following E2 and/or DAC treatments as described in the Methods section.
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Fig6: Differential response to E2 suppression and DAC activation of MT1 gene expression is seen in ERα + cell lines but not in ERα − cell lines. (A) Luciferase activity of ERE reporter vector normalized to Renilla reporter activity in luminal and basal breast cancer cell lines. (B) MT1 expression was carried out by RT-qPCR following E2 and/or DAC treatments as described in the Methods section.

Mentions: To further investigate whether estrogen mediates epigenetic repression of the cluster in ERα + breast cancer cell lines, we conducted E2 and DAC treatment in various breast cancer cell types. We first used estrogen response element (ERE) luciferase assay to confirm that ERE responds to E2 in ERα + cell lines, MCF7, MDAMB134, and BT474, and not in ERα − cell lines, BT20 and MDA-MB-231 (Figure 6A). We then examined the differential gene expression of eight genes in the cluster before and after the E2 and DAC treatment (Figure 6B). We removed MT1B from this experiment as there was no detectable expression level in the selected cell lines. Surprisingly, for MCF7, a ERα + cell line, the expression level for six genes (MT1A, F, H, M, E, and G) significantly decreased in response to E2 or/and DAC treatment, while no changes were observed for two of them (MT1L and X). Similarly, MDAMB134 which is ERα + cell line but with a slightly lower ERE activity compared to MCF7 showed a marked decrease in expression of five genes (MT1E, F, H, L, and X). However, for BT474, another ERα + cell line, we found that expression levels showed no significant changes for six genes after E2 treatment but significantly increased upon DAC treatment for all eight genes. We also observed that the expression levels for the combination of E2 and DAC treatment are similar to those of E2 treatment. We did not detect any expression changes after E2 or/and DAC treatment in both ERα − cell lines. The distinct expression response of BT474 compared to other ERα + cell lines may be attributed to the altered ER signal transduction pathway which also contributes to its tamoxifen resistance as reported by Wang et al. [34]. Clearly, more experiments are needed to further explore the underlying mechanism; our data nevertheless suggest that estrogen plays an important role in mediating epigenetic repression (mainly DNA hypermethylation) of the MT1 gene cluster in ERα + breast cancer cell lines.Figure 6


Genome-wide DNA methylation analysis reveals estrogen-mediated epigenetic repression of metallothionein-1 gene cluster in breast cancer.

Jadhav RR, Ye Z, Huang RL, Liu J, Hsu PY, Huang YW, Rangel LB, Lai HC, Roa JC, Kirma NB, Huang TH, Jin VX - Clin Epigenetics (2015)

Differential response to E2 suppression and DAC activation of MT1 gene expression is seen in ERα + cell lines but not in ERα − cell lines. (A) Luciferase activity of ERE reporter vector normalized to Renilla reporter activity in luminal and basal breast cancer cell lines. (B) MT1 expression was carried out by RT-qPCR following E2 and/or DAC treatments as described in the Methods section.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4355986&req=5

Fig6: Differential response to E2 suppression and DAC activation of MT1 gene expression is seen in ERα + cell lines but not in ERα − cell lines. (A) Luciferase activity of ERE reporter vector normalized to Renilla reporter activity in luminal and basal breast cancer cell lines. (B) MT1 expression was carried out by RT-qPCR following E2 and/or DAC treatments as described in the Methods section.
Mentions: To further investigate whether estrogen mediates epigenetic repression of the cluster in ERα + breast cancer cell lines, we conducted E2 and DAC treatment in various breast cancer cell types. We first used estrogen response element (ERE) luciferase assay to confirm that ERE responds to E2 in ERα + cell lines, MCF7, MDAMB134, and BT474, and not in ERα − cell lines, BT20 and MDA-MB-231 (Figure 6A). We then examined the differential gene expression of eight genes in the cluster before and after the E2 and DAC treatment (Figure 6B). We removed MT1B from this experiment as there was no detectable expression level in the selected cell lines. Surprisingly, for MCF7, a ERα + cell line, the expression level for six genes (MT1A, F, H, M, E, and G) significantly decreased in response to E2 or/and DAC treatment, while no changes were observed for two of them (MT1L and X). Similarly, MDAMB134 which is ERα + cell line but with a slightly lower ERE activity compared to MCF7 showed a marked decrease in expression of five genes (MT1E, F, H, L, and X). However, for BT474, another ERα + cell line, we found that expression levels showed no significant changes for six genes after E2 treatment but significantly increased upon DAC treatment for all eight genes. We also observed that the expression levels for the combination of E2 and DAC treatment are similar to those of E2 treatment. We did not detect any expression changes after E2 or/and DAC treatment in both ERα − cell lines. The distinct expression response of BT474 compared to other ERα + cell lines may be attributed to the altered ER signal transduction pathway which also contributes to its tamoxifen resistance as reported by Wang et al. [34]. Clearly, more experiments are needed to further explore the underlying mechanism; our data nevertheless suggest that estrogen plays an important role in mediating epigenetic repression (mainly DNA hypermethylation) of the MT1 gene cluster in ERα + breast cancer cell lines.Figure 6

Bottom Line: Bioinformatics analysis determined seven gene clusters with a significant difference in overall survival (OS) and further revealed a distinct feature that the conservation of a large gene cluster (approximately 70 kb) metallothionein-1 (MT1) among 45 species is much lower than the average of all RefSeq genes.Our data suggests that DNA methylation in large contiguous gene clusters can be potential prognostic markers of breast cancer.Further investigation of these clusters revealed that estrogen mediates epigenetic repression of MT1 cluster in ERα + breast cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, STRF, Room 225, 7703 Floyd Curl Drive, San Antonio, 78229 TX USA.

ABSTRACT

Background: Recent genome-wide analysis has shown that DNA methylation spans long stretches of chromosome regions consisting of clusters of contiguous CpG islands or gene families. Hypermethylation of various gene clusters has been reported in many types of cancer. In this study, we conducted methyl-binding domain capture (MBDCap) sequencing (MBD-seq) analysis on a breast cancer cohort consisting of 77 patients and 10 normal controls, as well as a panel of 38 breast cancer cell lines.

Results: Bioinformatics analysis determined seven gene clusters with a significant difference in overall survival (OS) and further revealed a distinct feature that the conservation of a large gene cluster (approximately 70 kb) metallothionein-1 (MT1) among 45 species is much lower than the average of all RefSeq genes. Furthermore, we found that DNA methylation is an important epigenetic regulator contributing to gene repression of MT1 gene cluster in both ERα positive (ERα+) and ERα negative (ERα-) breast tumors. In silico analysis revealed much lower gene expression of this cluster in The Cancer Genome Atlas (TCGA) cohort for ERα + tumors. To further investigate the role of estrogen, we conducted 17β-estradiol (E2) and demethylating agent 5-aza-2'-deoxycytidine (DAC) treatment in various breast cancer cell types. Cell proliferation and invasion assays suggested MT1F and MT1M may play an anti-oncogenic role in breast cancer.

Conclusions: Our data suggests that DNA methylation in large contiguous gene clusters can be potential prognostic markers of breast cancer. Further investigation of these clusters revealed that estrogen mediates epigenetic repression of MT1 cluster in ERα + breast cancer cell lines. In all, our studies identify thousands of breast tumor hypermethylated regions for the first time, in particular, discovering seven large contiguous hypermethylated gene clusters.

No MeSH data available.


Related in: MedlinePlus