Epigenetic silencing of EYA2 in pancreatic adenocarcinomas promotes tumor growth

Abstract

To identify potentially important genes dysregulated in pancreatic cancer, we analyzed genome-wide transcriptional analysis of pancreatic cancers and normal pancreatic duct samples and identified the transcriptional coactivator, EYA2 (Drosophila Eyes Absent Homologue-2) as silenced in the majority of pancreatic cancers. We investigated the role of epigenetic mechanisms of EYA2 gene silencing in pancreatic cancers, performed in vitro and in vivo proliferation and migration assays to assess the effect of EYA2 silencing on tumor cell growth and metastasis formation, and expression analysis to identify genes transcriptionally regulated by EYA2. We found loss of tumoral Eya2 expression in 63% of pancreatic cancers (120/189 cases). Silencing of EYA2 expression in pancreatic cancer cell lines correlated with promoter methylation and histone deacetylation and was reversible with DNA methyltransferase and HDAC inhibitors. EYA2 knockdown in pancreatic cancer cell lines increased cell proliferation. Compared to parental pancreatic cancer cells, pancreatic cancers stably-expressing EYA2 grew more slowly and had fewer metastases in orthotopic models. The transcriptional changes after stable expression of EYA2 in pancreatic cancer cells included induction of genes in the TGFbeta pathway. Epigenetic silencing of EYA2 is a common event in pancreatic cancers and stable expression EYA2 limits the growth and metastases of pancreatic adenocarcinoma.

Figure 1. (A) EYA2 expression by real-time PCR in normal HPDE cells and nine pancreatic cancer cell lines

(B-E) Representative figures of immunohistochemical staining of EYA2 with tissue microarrays. (B) Transition from normal ductal epithelium (arrows) to PanIN1. PanIN1 shows a decreased EYA2 expression. (C-E) Pancreatic ductal adenocarcinomas show a decrease or complete loss of EYA2 expression compared to entrapped normal ductal or acinar cells (arrows). Magnification: 10X.

Figure 2

(A) Cells were treated with either 5-Aza-2'-deoxycytidine (5-Aza, 5 μM, 72hrs), trichostatin A (TSA, 0.3 μM, 24hrs) or DMSO prior to RNA extraction and analysis of EYA2 expression by real-time PCR. β-actin was used as an internal control for gene expression. (B) Chromosomal localization of EYA2 gene as represented by the UCSC Genome Browser. The GC percent in 5-base windows is shown for this region. The CpG island spanning the first exon (thick horizontal blue line) and the first part of the first intron (thin horizontal arrowed blue line) is represented in green. Horizontal red bar represents the region studied by bisulfite sequencing (BS). Purple and green arrows represent the primers used for MSP studies (M: Methylated, U: Unmethylated). (C) Representative bisulfite sequencing and MSP in normal pancreatic tissues (upper panels) and in primary pancreatic adenocarcinoma tissues (lower panels). (D) Chromatin immunoprecipitation was performed with specific antibodies against acetylated histone H3 (AcH3) and methylated K27 of histone H3 (mK27H3) in the Human Pancreatic Ductal Epithelial cell line HPDE and four pancreatic cancer cell lines harboring different levels of EYA2 expression (Panc215, Panc1, Panc2.8 and MiaPaca2). Enrichment of each histone modification at EYA2 promoter was assessed by qPCR and reported to the control (Normal rabbit IgG).

Figure 3

(A) Bar graph indicating the level of EYA2 mRNA expression in positive controls (normal HPDE cells and Panc215 cancer cells), non-transfected cancer cell lines (Panc2.5 and Panc3.014, parental), cells transfected with an empty vector (Panc2.5 and Panc3.014, control) and cells transfected with an EYA2 expressing vector (Panc2.5 EYA2 #1 and #2 and Panc3.014 #1, #2 and #3). (B) Confocal microscopy showing EYA2 localization and expression in Panc2.5 and Panc3.014 EYA4 expressing and control clones. (C) Proliferation curves of Panc2.5 and Panc3.014 control cells (blue curves) compared to EYA2 expressing stable transfectants (red curves) and EYA2 non-expressing stable transfectant (grey curve). 1×10⁵ cells were plated in 24-well plates and viable cells were then counted after three and six days. (D) Panc215 cancer cells were transfected with either a non-targeting siRNA or a siRNA targeting EYA2 24hrs after seeding. Cell proliferation was assessed by measuring absorbance at 490 nm after incubation of the cells with Cell Titer 96® Aqueous One Solution Reagent (Promega). *, P < 0.05; **; P < 0.01; ***. P < 0.0001. EYA2 specific knockdown was assessed by qRT-PCR. (E) Migration assay. Wound density was assessed every two hours for 72h using the Incucyte Live-Cell Imaging System

Figure 4

(A-D) Panc2.5 and Panc3.014 control and EYA2-overexpressing cells were used to perform subcutaneous xenografts in Nude mice. For each stable clone, five mice were xenografted and data represent Mean ± SD. *, P < 0.05; **; P < 0.01; ***. P < 0.0001. Tumor size is significantly smaller in mice xenografted with EYA2-overexpressing cells than in control cells. (E-F) Tumours obtained from subcutaneous xenografts of Panc2.5 control and EYA2-overexpressing cells were used to perform orthotopic xenografts in eight additional Nude mice. (E) Tumour weight 77 days after orthotopic implantation. (F) Total number of macroscopically observed metastases in mice xenografted with EYA2-overexpressing cells compared to control cells. The primary tumour in the pancreas was excluded from counting and does not appear in the table.

Figure 5

(A) Summary of differentially expressed genes in Panc2.5 and Panc3.014 EYA2-overexpressing clones compared to control clones. Statistical analysis of gene expression array data as well as Gene Ontology classification were performed with Partek Genomic Suite 6.4 software. (B) Quantitative RT-PCR showing the expression profile of genes from the TGFbeta pathway in EYA2-overexpressing clones compared to control clones and untransfected cells (Parental) in Panc2.5 (green bars) and Panc3.014 (blue bars) cells. Bands were quantified by densitometry.