Epigenetic reprogramming by tumor-derived EZH2 gain-of-function mutations promotes aggressive 3D cell morphologies and enhances melanoma tumor growth

Abstract

In addition to genetic alterations, cancer cells are characterized by myriad epigenetic changes. EZH2 is a histone methyltransferase that is over-expressed and mutated in cancer. The EZH2 gain-of-function (GOF) mutations first identified in lymphomas have recently been reported in melanoma (~2%) but remain uncharacterized. We expressed multiple EZH2 GOF mutations in the A375 metastatic skin melanoma cell line and observed both increased H3K27me3 and dramatic changes in 3D culture morphology. In these cells, prominent morphological changes were accompanied by a decrease in cell contractility and an increase in collective cell migration. At the molecular level, we observed significant alteration of the axonal guidance pathway, a pathway intricately involved in the regulation of cell shape and motility. Furthermore, the aggressive 3D morphology of EZH2 GOF-expressing melanoma cells (both endogenous and ectopic) was attenuated by EZH2 catalytic inhibition. Finally, A375 cells expressing exogenous EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. This study not only demonstrates the first functional characterization of EZH2 GOF mutants in non-hematopoietic cells, but also provides a rationale for EZH2 catalytic inhibition in melanoma.

Figure 1. EZH2 GOF mutants greatly increase global H3K27me3 levels and cause 3D-culture phenotypes

(A) H3K27me2/3 and Total H3 levels were probed by western blot from cell lines that contain either EZH2 WT or EZH2 GOF: 1.A431, 2.A375, 3.IGR1, 4.OCI-LY19, 5.KARPAS-422, 6.WSU-DL-CL2. (B) Western blot analysis following ectopic expression of EZH2 WT, GOF or LOF mutants in BEAS-2B epithelial cells or A375 melanoma cells. In lanes with ectopic EZH2, the upper band represents the ectopic EZH2 due to the presence of a V5 tag. (C) 4x and 10x images of A375 cells (expressing empty vector, EZH2 WT, EZH2 Y641F, EZH2 Y641N and EZH2 Y726D (LOF)) grown on top of a thick layer of ECM are shown.

Figure 2. EZH2 GOF catalytic activity inhibits cell contractility and is necessary for the maintenance of 3D-branching morphology

(A) Western blot analysis of A375 stable cells cultured on top of ECM (4 days) or tissue-culture treated plastic. pMLC is a marker of cell contractility and ROCK activity. (B) A375 Vector, EZH2 Y641F or EZH2 Y641N cells were seeded on top of ECM and simultaneously treated with 10 μM Y-27632 (ROCK inhibitor). Western blot analysis (top) was performed 4-days post-seeding/treatment. Images (10x, bottom) were captured 2 days post-seeding/treatment. (C and D) A375 stable cell lines were treated with DMSO or 1 μM GSK126 for 7 days in 2D culture and re-plated (and re-treated) on top of ECM and imaged (C, 10x) or lysed for western blot analysis (D) 4 days post-seeding.

Figure 3. Catalytic inhibition of endogenous EZH2 Y641N (GOF) alters 3D-morphology of IGR1 melanoma cells

(A) IGR1 cells were treated for 6 days with GSK126 at the indicated concentrations. Cells were counted or used to make lysates for western blot. Top panel: Western blot analysis of H3K27me3 levels following GSK126 treatment. Bottom panel: quantification of cell number (mean −/+ STDEV). (B) IGR1 cells were treated with DMSO, 1 μM or 2.5 μM GSK126 for 7 days in 2D culture and then re-plated (and re-treated) on top of ECM. Images (4x, 10x) were captured 6 days post-seeding.

Figure 4. RNA-seq analysis of A375 stable cells reveals a correlation between EZH2 catalytic activity and global gene expression patterns

(A) Hierarchical clustering analysis of mRNA expression levels of A375 stable cell lines, including A375 Y641F + GSK126 (left column), is displayed. Red color indicates high gene expression values and blue color indicates low gene expression values (standardized values). (B) A table containing the results from Gene Set Enrichment Analysis is displayed. Left column: A375 Y641F versus vector control cells. Right column: GSK126-treated A375 Y641F versus A375 Y641F.

Figure 5. EZH2 GOF mutants regulate axonal-guidance genes

(A) Ingenuity Pathway Analysis was performed from RNA-seq data of A375 stable cells (left panel) as well as GSK126-treated A375 Y641F and IGR1 cells (right panel). Pathways that were commonly regulated with high statistical significance (p-value < = 0.05) by all EZH2 GOF mutants or GSK126-treatment are shown. Greater –LOG(p-value) values indicates greater statistical significance of pathway enrichment. (B) qPCR was used to validate gene expression changes observed by RNA-seq (mean −/+ STDEV). (C) A model of EZH2 GOF-dependent pathway regulation leading to observed 3D-phenotypes.

Figure 6. A375 cells expressing EZH2 GOF mutants (but not WT or LOF mutant) display increased tumor volume in mouse xenograft models

A375 cells were subcutaneously implanted into nude mice and tumor volume was recorded over a period of 17 days. Data points represent mean tumor volume at each time-point. A two-tailed t-test performed for mean volumes at day 17 was used to calculate significance values. (A) Xenograft growth curves for Vector (control, black), EZH2 WT (red) and EZH2 Y726D (blue) are displayed (left panel). Xenograft growth curves for Vector (control, black), EZH2 Y641F (red), EZH2 Y641N (green), EZH2 A677G (blue) are displayed (right panel). (B) A graph depicting the ratio of mean intensity of H3K27me3 to H3K27me2 for each group is shown.