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. 2015 Sep 29;6(29):27023-36.
doi: 10.18632/oncotarget.4809.

Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes

Jessamy C Tiffen  1   2   3 , Dilini Gunatilake  1   2   3 , Stuart J Gallagher  1   2   3 , Kavitha Gowrishankar  1   2   3 , Anja Heinemann  1   2   3 , Carleen Cullinane  4 , Ken Dutton-Regester  5 , Gulietta M Pupo  6 , Dario Strbenac  7 , Jean Y Yang  7 , Jason Madore  8   3 , Graham J Mann  6   7   3 , Nicholas K Hayward  5 , Grant A McArthur  4   9 , Fabian V Filipp  10 , Peter Hersey  1   2   3
Affiliations

Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes

Jessamy C Tiffen et al. Oncotarget. .

Abstract

The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma.

Keywords: EZH2; H3K27me3; epigenetics; melanoma; targeted therapy.

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Conflict of interest statement

CONFLICTS OF INTEREST

GAM receives research support from Pfizer, Millennium, Novartis and uncompensated consulting Roche-Genentech, GSK, Amgen, Novartis, BMS, Merck. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1. EZH2 and H3K27me3 are overexpressed in both mutant and WT melanoma cell lines but Y646 mutants are more sensitive to EZH2 inhibition
Western blot of endogenous EZH2 and the downstream H3K27me3 mark in a panel of melanoma cell lines. EZH2Y646 mutants are highlighted in red, human epithelial melanocytes (HEM) and human dermal fibroblasts (HDF) were used as untransformed, normal cells. β-actin was used as a loading control for total cell lysates whereas total histone 3 (H3) was used for purified histones A. Dose dependent reduction of H3K27me3 in IGR1Y646 mutant cells treated with GSK126 for 48 hr compared to vehicle treated control (DMSO) B. IC50 values of GSK126 following 72 hr of treatment using a cell titer glow (CTG) viability assay C.
Figure 2
Figure 2. GSK126 inhibits proliferation and causes G2/M cell cycle arrest in EZH2 mutant and WT melanoma
Dose dependent effects on proliferation over time in EZH2 mutant A. wild-type B. and normal cells C. using incucyte. Growth is expressed as a percentage of images acquired at time zero (T0). Histograms representing DNA content following 72 hr of GSK126 treatment in IGR1 cells D–F. Phases of the cell cycle in different cell types are shown in G. treated with either 5 μM (+) or 10 μM (++) GSK126. EZH2Y646 mutants are highlighted in red.
Figure 3
Figure 3. GSK126 induces apoptosis in a caspase independent manner, involves mitochondrial depolarization and AIFM1 release
Cells were treated with different doses of GSK126 for 72 hr and apoptosis (combined early and late) was measured by annexin V staining with PI exclusion via flow cytometry A. GSK126 mediated apoptosis could not be prevented by the addition of a pan caspase inhibitor (Z-VAD-FMK) that could prevent TRAIL induced cell death, used as a control B. Mitochondrial depolarization was observed 48 hr after drug treatment, indicated by JC1 staining and flow cytometry C. Knockdown of apoptosis inducing factor (AIFM1) using two different siRNAs D. was able to protect cells from GSK126 mediated apoptosis in both EZH2 mutant E. and wild-type F. cells. Cell lines were screened for apoptotic proteins following 48 hrs of drug treatment using 7.5 μM GSK126 G. EZH2Y646 mutants are indicated in red.
Figure 4
Figure 4. Knockdown of EZH2 phenocopies GSK126 mediated inhibition in melanoma
EZH2 protein was depleted using two different lentiviral vectors expressing shRNAs A. Following 96 hr of puromycin selection, proliferation was tracked over time using Incucyte in EZH2 mutant B. wild-type C. or normal cells D. Day 8 proliferation values in cell panel with EZH2 knockdown are depicted in E. normalized to control values at 100%. Effects on cell cycle are shown in F. and apoptosis was measured using annexin V/PI staining G. EZH2Y646 mutants are highlighted in red.
Figure 5
Figure 5. GSK126 causes cell growth inhibition via de-repression of tumor suppressor genes
Microarray analysis of the top 30 most differentially expressed genes following 48 hr of 7.5 μM GSK126 treatment. Two sensitive (apoptosis) EZH2Y646 mutant cell lines were selected (IGR1 and MM386) along with a sensitive WT (KMJR138) and an insensitive cell line (MEL-RMU) performed in duplicate. A. The top candidates with known tumor suppressor gene functions were validated by RT-qPCR in IGR1 B. and MM386 C. mutant cells. Western blot of cell lines treated for 48 hr with 7.5 μM GSK126 showing the effect on NDRG1_pT346 protein D. ChIP-qPCR (n = 2) in MM386 cells showing enrichment of H3K27me3 upstream of the transcription start site (primers (A–C) but not downstream (primer D) of the ATF3 gene promotor. CCND2 was used as a positive control and CDC6 as a negative control E. Survival analysis (Cox proportional hazard model) of reverse phase protein array (RPPA) data from 31 TCGA melanoma patients divided into high (>0.2963) or low (<0.2963) NDRG1_pT346 protein expression F. Survival analysis (Kaplan-Meier) of microarray data from 34 stage III melanoma patients [55] divided into high (upper 75th percentile) or low (lower 25th percentile) ATF3 mRNA expression G. Mutants are indicated in red.
Figure 6
Figure 6. GSK126 inhibits the growth of melanoma spheroids growing in 3D culture
The growth of 3D spheroid cultures embedded in collagen was assessed after 72 hr of drug treatment, all images were acquired at 4 × magnification with scale bars representing 2 mm A. Spheroids were dissociated with collagenase treatment and cell death measured by PI staining B. or the spheroid area was measured using ImageJ software, encompassing the entire spheroid to the outside edge of live cells C. Mutants are indicated in red.
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References

    1. Baylin SB. DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol. 2005;2:S4–11. - PubMed
    1. Shen H, Laird PW. Interplay between the cancer genome and epigenome. Cell. 2013;153:38–55. Epub 2013/04/02. - PMC - PubMed
    1. Mills AA. Throwing the cancer switch: reciprocal roles of polycomb and trithorax proteins. Nat Rev Cancer. 2010;10:669–82. - PMC - PubMed
    1. Margueron R, Reinberg D. The Polycomb complex PRC2 and its mark in life. Nature. 2011;469:343–9. - PMC - PubMed
    1. Takeshima H, Wakabayashi M, Hattori N, Yamashita S, Ushijima T. Identification of co-existence of DNA methylation and H3K27me3 specifically in cancer cells as a promising target for epigenetic therapy. Carcinogenesis. 2014;4 - PubMed

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