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. 2010 Sep 13;5(9):e12710.
doi: 10.1371/journal.pone.0012710.

Epigenetic Drugs Can Stimulate Metastasis Through Enhanced Expression of the Pro-Metastatic Ezrin Gene

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Free PMC article

Epigenetic Drugs Can Stimulate Metastasis Through Enhanced Expression of the Pro-Metastatic Ezrin Gene

Yanlin Yu et al. PLoS One. .
Free PMC article

Abstract

Ezrin has been reported to be upregulated in many tumors and to participate in metastatic progression. No study has addressed epigenetic modification in the regulation of Ezrin gene expression, the importance of which is unknown. Here, we report that highly metastatic rhabdomyosarcoma (RMS) cells with high levels of Ezrin have elevated acetyl-H3-K9 and tri-methyl-H3-K4 as well as reduced DNA methylation at the Ezrin gene promoter. Conversely, poorly metastatic RMS cells with low levels of Ezrin have reduced acetyl-H3-K9 and elevated methylation. Thus epigenetic covalent modifications to histones within nucleosomes of the Ezrin gene promoter are linked to Ezrin expression, which in fact can be regulated by epigenetic mechanisms. Notably, treatment with histone deacetylase (HDAC) inhibitors or DNA demethylating agents could restore Ezrin expression and stimulate the metastatic potential of poorly metastatic RMS cells characterized by low Ezrin levels. However, the ability of epigenetic drugs to stimulate metastasis in RMS cells was inhibited by expression of an Ezrin-specific shRNA. Our data demonstrate the potential risk associated with clinical application of broadly acting covalent epigenetic modifiers, and highlight the value of combination therapies that include agents specifically targeting potent pro-metastatic genes.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Epigenetic modifications at the Ezrin gene locus are linked to its expression.
(A) Comparative expression and activity of histone H3 tail proteins were analyzed by Western blotting in RMS772 and RMS14 cells. RMS772 cells with low level Ezrin have a higher level of di-methyl-H3K9, but lower levels of actyl-H3K9 and tri-methyl-H3K4; in contrast, RMS14 cells with higher level Ezrin have higher levels of acetyl-H3K9 and tri-methyl-H3K4, but a lower level di-methyl-H3K9. (B) Comparative analysis of histone modifications at the Ezrin gene promoter was performed by chromatin immunoprecipitation (ChIP) in RMS772 cells (purple line) and RMS14 cells (blue line). Six pairs of primers were designed for covering an 8 kb region of the Ezrin gene promoter (a). Enriched chromatin DNA immunoprecipitated with anti-di-methyl-H3K9 (b), anti-acetyl-H3K9 (c) or anti-tri-methyl-H3K4 (d) were amplified by PCR using six pairs of primers. The data were analyzed using Image J software and normalized with input. Purple line represents data from RMS772 cells; blue line represents data from RMS14 cells. (C) The status of DNA methylation within the Ezrin gene promoter was analyzed by methylation-specific PCR using specific methylated Ezrin promoter primers and specific unmethylated Ezrin promoter primers. M, methylated promoter; U, unmethylated promoter.
Figure 2
Figure 2. HDAC inhibitor (TSA) and DNA demethylating agent (5-Aza) reactivate Ezrin gene expression.
The expression of Ezrin was analyzed by RT-PCR after treatment with 300 nM TSA or 1 µM 5-Aza in RMS772 and RMS14 cells. (A) and (B) Ezrin expression in different time courses after treatment with TSA (A) or 5-Aza (B) in RMS772 and RMS14 cells. M, DNA molecular marker; 18s rRNA, internal controls.
Figure 3
Figure 3. Epigenetic agents regulate metastatic potential in pretreated cultured RMS772 cells.
(A) Gross pulmonary metastases from pretreated cultured RMS772 cell with either 300 nM TSA (24 or 48 hours) or 1 µM 5-Aza (48 hours) in cell culture. After pretreated with epigenetic agents, 2×105 cells were injected into athymic nude mice by tail vein. (B) Ezrin expression after treatment with TSA and 5-Aza for 48 hours in RMS772 cells with stably transfected shRNA Ezrin expression vector (RMS772 shEz) or empty vector (RMS772C). M, DNA molecular marker; 18s rRNA, internal controls. (C) and (D) Gross pulmonary metastases from cells with stably transfected shRNA Ezrin expression vector (+) or empty vector (−) pretreated with either 300 nM TSA (C) or 1 µM 5-Aza (D) for 48 hours in cell culture. After pretreated with epigenetic agents, 5×105 cells were injected into athymic nude mice by tail vein. (E) Cell growth was represented by the number of cells. RMS772 cells with stably transfected shRNA Ezrin expression vector (shEzrin) or empty vector (vector) were treated with 300 nM TSA (red rectangle), 1 µM 5-Aza (green rectangle) or mock DMSO (Blue rectangle) for 48 hours.
Figure 4
Figure 4. Epigenetic agents regulate metastatic potential in preclinical mice model.
(A) and (B) Gross pulmonary metastases from mice treated with TSA (A) or VPA (B). Mice harboring 5×105 RMS772 cells stably transfected with the shRNA Ezrin expression vector (shEzrin) or empty vector (vector) were treated with two different doses of TSA or VPA for four days. (C) and (D) Representative histopathology (H&E staining) of lung sections with metastases from mice treated with TSA (C) or 5-Aza (D). Vector, lung section from mouse inoculated with RMS772 cells with empty vector control; shEzrin, lung section from mouse inoculated with RMS772 transfected with the shRNA Ezrin expressing vector. (E) Ezrin expression as determined by immunohistochemical staining in metastases derived from mice bearing RMS772 cells treated with 2 mg/kg TSA (TSA) or 0.8% VPA (VPA). H&E, hematoxylin and eosin staining; Ezrin, immunohistochemical staining with anti-Ezrin antibody; 20X. (F) Immunoreactivity score of Ezrin expression in metastases from mice treated with 2 mg/kg TSA (TSA) or 0.8% VPA (VPA). Quantitative scores, which were analyzed using ImageScope V10.0 software from Aperio Technololgies, presenting the percentage of cells in strongly positive, positive, weakly positive and negative for immunohistochemical staining with an anti-Ezrin antibody.
Figure 5
Figure 5. Phenotypic effects of epigenetic drugs on RMS14 cells.
RMS14 cells with high-level endogenous Ezrin have high metastatic potential. (A) and (B) Gross pulmonary metastases from RMS14 cells stably transfected with an shRNA Ezrin expression vector (+) or empty vector (−), pretreated with 300 nM TSA (A) or 1 µM 5-Aza (B) for 48 hours in cell culture. (C) Growth rate of RMS14 cells treated with TSA or 5-Aza stably transfected with either shRNA Ezrin expression vector (RMS14 shEz) or empty vector (RMS14C). There was no statistic difference in cell number between RMS14C and RMS14 shEz in the DMSO group, but treatment with either TSA or 5-Aza significantly inhibited cell growth in both RMS14C and RMS14 shEz cells (*p<0.0001). (D) Gross pulmonary metastases from mice treated with TSA or VPA. Mice harboring RMS14 cells stably transfected with either the shRNA Ezrin expression vector (RMS14 shEz) or empty vector (RMS14C) were treated with TSA or VPA for four days. (E) Representative histopathology (H&E staining) of lung sections with metastases from mice bearing RMS14C and RMS14 shEz cells treated with TSA or VPA. (F) Ezrin expression assessed by immunohistochemical staining in metastases derived from mice treated with 2 mg/kg TSA or 0.8% VPA; H&E, hematoxylin and eosin staining; Ezrin, immunohistochemistry staining with anti-Ezrin antibody; IgG, negative control. 20X. (G) Immunoreactivity score of Ezrin expression in metastases from mice treated with 2 mg/kg TSA or 0.8% VPA. Ezrin expression in tissue sections was visualized using an anti-Ezrin antibody, and quantified using ImageScope V10.0 software from Aperio Technololgies; the percentage of cells that were strongly positive, positive, weakly positive or negative for Ezrin is presented.

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