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Epigenetic repression of phosphatidylethanolamine N-methyltransferase (PEMT) in BRCA1-mutated breast cancer.

Li D, Bi FF, Chen NN, Cao JM, Sun WP, Zhou YM, Cao C, Li CY, Yang Q - Oncotarget (2014)

Bottom Line: However, the epigenetic mechanism regulating PEMT transcription remains largely unknown.Mechanistically, hypermethylated -132 site-mediated loss of active histone marks H3K9ac and increase of repressive histone marks H3K9me enrichment synergistically inhibited PEMT transcription.Clinicopathological data indicated that a hypermethylated -132 site was associated with histological grade (P = 0.031) and estrogen receptor status (P = 0.004); univariate survival and multivariate analyses demonstrated that lymph node metastasis was an independent and reliable prognostic factor for BRCA1-mutated breast cancer patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, China.

ABSTRACT
Phosphatidylethanolamine N-methyltransferase (PEMT) plays a critical role in breast cancer progression. However, the epigenetic mechanism regulating PEMT transcription remains largely unknown. Here, we show that the first promoter-specific transcript 1 is the major PEMT mRNA species, and methylation of the -132 site is a key regulatory element for the PEMT gene in BRCA1-mutated breast cancer. Mechanistically, hypermethylated -132 site-mediated loss of active histone marks H3K9ac and increase of repressive histone marks H3K9me enrichment synergistically inhibited PEMT transcription. Clinicopathological data indicated that a hypermethylated -132 site was associated with histological grade (P = 0.031) and estrogen receptor status (P = 0.004); univariate survival and multivariate analyses demonstrated that lymph node metastasis was an independent and reliable prognostic factor for BRCA1-mutated breast cancer patients. Our findings imply that genetic (e.g., BRCA1 mutation) and epigenetic mechanisms (e.g., DNA methylation and histone modifications) are jointly involved in the malignant progression of PEMT-related breast cancer.

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H3K9ac and H3K9me-mediated transcriptional regulation of PEMT in BRCA1-mutated breast cancer cellsAi, RT-PCR showing GCN5, PCAF, HLCS, and EHMT-1 levels before and after knockdown by shRNAs, and normalized to β-actin expression. Aii, the results from three independent experiments are represented as mean ± SD. Bi, EdU labeling showing the proliferation of GCN5-, PCAF-, HLCS-, and EHMT-1-silenced in primary BRCA1-mutated breast cancer cells (each group, n = 68). Blue, Hoechst 33342 labeling of cell nuclei; red, EdU labeling of nuclei of proliferative cells. Bii, the EdU incorporation rate was expressed as the ratio of EdU-positive cells to total Hoechst 33342-positive cells. Ci, analysis of histone modification H3K9ac enrichment around the -132 site after the deletion of GCN5 and PCAF in primary BRCA1-mutated breast cancer cells (each group, n = 68). Cii, analysis of histone modification H3K9me enrichment around the -132 site after the deletion of HLCS and EHMT-1 in primary BRCA1-mutated breast cancer cells (each group, n = 68). D, PEMT transcript 1 levels after the deletion of H3K9ac and H3K9me around the -132 site in primary BRCA1-mutated breast cancer cells (each group, n = 68). Bar graphs show mean ± SD. * P < 0.05 vs. control.
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Figure 5: H3K9ac and H3K9me-mediated transcriptional regulation of PEMT in BRCA1-mutated breast cancer cellsAi, RT-PCR showing GCN5, PCAF, HLCS, and EHMT-1 levels before and after knockdown by shRNAs, and normalized to β-actin expression. Aii, the results from three independent experiments are represented as mean ± SD. Bi, EdU labeling showing the proliferation of GCN5-, PCAF-, HLCS-, and EHMT-1-silenced in primary BRCA1-mutated breast cancer cells (each group, n = 68). Blue, Hoechst 33342 labeling of cell nuclei; red, EdU labeling of nuclei of proliferative cells. Bii, the EdU incorporation rate was expressed as the ratio of EdU-positive cells to total Hoechst 33342-positive cells. Ci, analysis of histone modification H3K9ac enrichment around the -132 site after the deletion of GCN5 and PCAF in primary BRCA1-mutated breast cancer cells (each group, n = 68). Cii, analysis of histone modification H3K9me enrichment around the -132 site after the deletion of HLCS and EHMT-1 in primary BRCA1-mutated breast cancer cells (each group, n = 68). D, PEMT transcript 1 levels after the deletion of H3K9ac and H3K9me around the -132 site in primary BRCA1-mutated breast cancer cells (each group, n = 68). Bar graphs show mean ± SD. * P < 0.05 vs. control.

Mentions: We observed that the knockdown of GCN5, PCAF, HLCS, and EHMT-1 (Fig. 5A) had no detectable effect on cell morphology and proliferation (Fig. 5B). Combined knockdown of GCN5 and PCAF, or HLCS and EHMT-1 specifically induced a decrease in H3K9ac or H3K9me enrichment around the -132 site, respectively (Figs. 5Ci and Cii). Notably, after the deletion of H3K9ac or H3K9me, the transcription of PEMT was significantly down-regulated or up-regulated, respectively (Fig. 5D). The phenomenon was further confirmed by other specific siRNAs (Supplementary Fig. S4).


Epigenetic repression of phosphatidylethanolamine N-methyltransferase (PEMT) in BRCA1-mutated breast cancer.

Li D, Bi FF, Chen NN, Cao JM, Sun WP, Zhou YM, Cao C, Li CY, Yang Q - Oncotarget (2014)

H3K9ac and H3K9me-mediated transcriptional regulation of PEMT in BRCA1-mutated breast cancer cellsAi, RT-PCR showing GCN5, PCAF, HLCS, and EHMT-1 levels before and after knockdown by shRNAs, and normalized to β-actin expression. Aii, the results from three independent experiments are represented as mean ± SD. Bi, EdU labeling showing the proliferation of GCN5-, PCAF-, HLCS-, and EHMT-1-silenced in primary BRCA1-mutated breast cancer cells (each group, n = 68). Blue, Hoechst 33342 labeling of cell nuclei; red, EdU labeling of nuclei of proliferative cells. Bii, the EdU incorporation rate was expressed as the ratio of EdU-positive cells to total Hoechst 33342-positive cells. Ci, analysis of histone modification H3K9ac enrichment around the -132 site after the deletion of GCN5 and PCAF in primary BRCA1-mutated breast cancer cells (each group, n = 68). Cii, analysis of histone modification H3K9me enrichment around the -132 site after the deletion of HLCS and EHMT-1 in primary BRCA1-mutated breast cancer cells (each group, n = 68). D, PEMT transcript 1 levels after the deletion of H3K9ac and H3K9me around the -132 site in primary BRCA1-mutated breast cancer cells (each group, n = 68). Bar graphs show mean ± SD. * P < 0.05 vs. control.
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Figure 5: H3K9ac and H3K9me-mediated transcriptional regulation of PEMT in BRCA1-mutated breast cancer cellsAi, RT-PCR showing GCN5, PCAF, HLCS, and EHMT-1 levels before and after knockdown by shRNAs, and normalized to β-actin expression. Aii, the results from three independent experiments are represented as mean ± SD. Bi, EdU labeling showing the proliferation of GCN5-, PCAF-, HLCS-, and EHMT-1-silenced in primary BRCA1-mutated breast cancer cells (each group, n = 68). Blue, Hoechst 33342 labeling of cell nuclei; red, EdU labeling of nuclei of proliferative cells. Bii, the EdU incorporation rate was expressed as the ratio of EdU-positive cells to total Hoechst 33342-positive cells. Ci, analysis of histone modification H3K9ac enrichment around the -132 site after the deletion of GCN5 and PCAF in primary BRCA1-mutated breast cancer cells (each group, n = 68). Cii, analysis of histone modification H3K9me enrichment around the -132 site after the deletion of HLCS and EHMT-1 in primary BRCA1-mutated breast cancer cells (each group, n = 68). D, PEMT transcript 1 levels after the deletion of H3K9ac and H3K9me around the -132 site in primary BRCA1-mutated breast cancer cells (each group, n = 68). Bar graphs show mean ± SD. * P < 0.05 vs. control.
Mentions: We observed that the knockdown of GCN5, PCAF, HLCS, and EHMT-1 (Fig. 5A) had no detectable effect on cell morphology and proliferation (Fig. 5B). Combined knockdown of GCN5 and PCAF, or HLCS and EHMT-1 specifically induced a decrease in H3K9ac or H3K9me enrichment around the -132 site, respectively (Figs. 5Ci and Cii). Notably, after the deletion of H3K9ac or H3K9me, the transcription of PEMT was significantly down-regulated or up-regulated, respectively (Fig. 5D). The phenomenon was further confirmed by other specific siRNAs (Supplementary Fig. S4).

Bottom Line: However, the epigenetic mechanism regulating PEMT transcription remains largely unknown.Mechanistically, hypermethylated -132 site-mediated loss of active histone marks H3K9ac and increase of repressive histone marks H3K9me enrichment synergistically inhibited PEMT transcription.Clinicopathological data indicated that a hypermethylated -132 site was associated with histological grade (P = 0.031) and estrogen receptor status (P = 0.004); univariate survival and multivariate analyses demonstrated that lymph node metastasis was an independent and reliable prognostic factor for BRCA1-mutated breast cancer patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, China.

ABSTRACT
Phosphatidylethanolamine N-methyltransferase (PEMT) plays a critical role in breast cancer progression. However, the epigenetic mechanism regulating PEMT transcription remains largely unknown. Here, we show that the first promoter-specific transcript 1 is the major PEMT mRNA species, and methylation of the -132 site is a key regulatory element for the PEMT gene in BRCA1-mutated breast cancer. Mechanistically, hypermethylated -132 site-mediated loss of active histone marks H3K9ac and increase of repressive histone marks H3K9me enrichment synergistically inhibited PEMT transcription. Clinicopathological data indicated that a hypermethylated -132 site was associated with histological grade (P = 0.031) and estrogen receptor status (P = 0.004); univariate survival and multivariate analyses demonstrated that lymph node metastasis was an independent and reliable prognostic factor for BRCA1-mutated breast cancer patients. Our findings imply that genetic (e.g., BRCA1 mutation) and epigenetic mechanisms (e.g., DNA methylation and histone modifications) are jointly involved in the malignant progression of PEMT-related breast cancer.

Show MeSH
Related in: MedlinePlus