Journal: Oncogene

Article Title: TMPRSS2-ERG Fusion, a Common Genomic Alteration in Prostate Cancer Activates C-MYC and Abrogates Prostate Epithelial Differentiation

doi: 10.1038/onc.2008.183

Figure Lengend Snippet: C-MYC and prostate differentiation are targeted by ERG in prostate cancer cells. VCaP cells transfected with ERG si or NT were analyzed by Affymetrix HG U133 Plus 2.0 high-density oligonucleotide human genome arrays, QRT-PCR, Chromatin Immunoprecipitation and by Western blots. (a) Functional co-citation based network was obtained from overlapping genes within the comparative gene expression analyses of ERG expressing prostate tumors (ERG+ Tumor Gene Expression, left side color codes) and ERG si treated VCaP cells (ERG si Gene Expression, VCaP, right side color codes) by the BiblioSphere Software (Genomatix GmbH, Munich, Germany). Red and yellow colors mark upregulation, shades of blue indicate downregulation. For the human tumor gene expression analysis microarray data set was selected from well-differentiated tumors of seven patients with 19-38 folds ERG overexpression (Shaheduzzaman et al., 2007). For assessing gene expression changes in VCaP cells by microarray cells were transfected by ERG si, and were analyzed 48 h post-transfection. The central node of combined ERG si and ERG+ tumor pathways highlights common changes in C-MYC (MYC) and PSA (KLK3) nodes. On the insert (upper left) inhibition of ERG protein expression in VCaP cells in response to various doses of ERG si 48 h post-transfection is shown. (b) MYC expression in response to ERG si inhibition was measured by QRT-PCR and Western blots. Reduced recruitment of ERG to the MYC P2 promoter downstream ETS element was assessed at 48 h post-transfection by ChIP assay by using anti-ERG (ERG) antibody (Ab). IgG and Input was used as controls. (c) PSA mRNA and protein expression were measured by QRT-PCR and Western blot assays respectively. Increased AR binding to the PSA enhancer (ARE) and decreased ERG recruitment to the overlapping ETS cognate element was measured by ChIP assay 48 h after transfection. On the right panel VCaP cells at 9 days after treatment with control NT (upper photographs) and ERG si (lower photographs) were immunostained for CK8/18 (extreme left), PSA (second column from left) and DNA (third column from left) and the separate images merged (extreme right column). The scale bar is 25 micron. (d) SLC45A3 (Prostein) expression in ERG si transfected VCaP cells was measured by Western blots (upper left). Recruitment of AR and ERG to the SLC45A3 promoter upstream ARE and ETS elements was assessed by ChIP assay 48 h post-transfection (lower left). On the table matrix representation of TMPRSS2-ERG expression and SLC45A3 (SLC) immunostaining in prostate tumors of 26 patients (CN=case number) is shown. Sections of whole mounted radical prostatectomy specimens were assessed by immunohistochemistry with anti- SLC45A3 (SLC) antibody. Strong (solid) or weak (grey) staining was correlated with the presence (solid) or absence (hollow) of TMPRSS2-ERG gene fusion transcripts in the same tumors. (e) VCaP cells were transfected with 50 nM of NT or ERG si or MYC si or the combination of 25 nM of ERG si and 25 nM of MYC si. Cell morphology at day 8 is shown. Cell lysates were prepared and assayed by immunoblots with anti-C-MYC or PSA or SLC45A3 (prostein) antibodies. Tubulin was used as the control. ERG-MYC correlation analysis was performed by assessing quantitative gene expression data of C-MYC and ERG or C-MYC and PCA3 from 37 laser capture microdissected human tumors. R and P-values are shown in the table.

Article Snippet: VCaP cells transfected with ERG si or NT were analyzed by Affymetrix HG U133 Plus 2.0 high-density oligonucleotide human genome arrays, QRT-PCR, Chromatin Immunoprecipitation and by Western blots. (a) Functional co-citation based network was obtained from overlapping genes within the comparative gene expression analyses of ERG expressing prostate tumors (ERG+ Tumor Gene Expression, left side color codes) and ERG si treated VCaP cells (ERG si Gene Expression, VCaP, right side color codes) by the BiblioSphere Software (Genomatix GmbH, Munich, Germany).

Techniques: Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Western Blot, Functional Assay, Gene Expression, Expressing, Software, Microarray, Over Expression, Inhibition, Binding Assay, Control, Immunostaining, Immunohistochemistry, Staining