ABSTRACT: Prostate cancer cell lines that express ERG acquire a neuron-like phenotype. The human prostate tumor cell line LNCap was transfected with lenti-ERG or control lenti-vector. Gene expression profiling was performed to establish the ERG-associated phenotype.
Project description:Androgen receptor (AR) is a transcription factor that plays a central role in the growth and development of the normal prostate and its malignant transformation. More recently, a majority of prostate cancers have been shown to harbor recurrent gene fusions of the androgen-regulated gene, TMPRSS2, to the oncogenic ETS transcription factor ERG. Here we employed chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-Seq) to explore the genome-wide localization of these transcription factors in human prostate cancer cell lines as well as tissues. Unexpectedly, transcriptional networks emanating from AR and ERG were found to be highly overlapping. Furthermore, AR was found to regulate known 5’ fusion partners in prostate cancer including TMPRSS2, as well as negatively regulating its own expression. While induced by androgen through fusion to TMPRSS2, ERG itself was shown to inhibit AR expression and positively regulate the genomic locus of wild-type ERG, thus revealing multiple levels of molecular cross-talk between AR and ERG. Importantly, androgen-sensitive prostate cancer cells in which ERG is overexpressed are able to proliferate and invade in the absence of androgen. Thus, we dissected the intertwined genomic landscape of two master transcriptional regulators of prostate cancer and suggest a role for ERG in maintaining transcriptional networks necessary for androgen-independent prostate cancer growth. These studies may suggest that future therapies against prostate cancer should target both AR and ERG, rather than AR alone, in order to achieve maximum effectiveness. ChIP_Seq examination of histone modifications and key transcription factors in LNCaP and VCaP prostate cancer cell lines in un-treated, vehicle treated or 10nM R1881 treated conditions. LNCaP ChIP-Seq experiments include samples GSM353609-GSM353618, GSM353625-GSM353628, GSM353633-GSM353635, GSM353641-GSM353644, and GSM353648. VCaP ChIP-Seq experiments include samples GSM353601-GSM353608, GSM353619-GSM353624, GSM353629-GSM353632, and GSM353645-GSM353647. In addition, we performed re-ChIP of AR and ERG in VCaP cells (GSM356767), and examined the effect of ERG knockdown on AR and ERG binding (samples GSM353636-GSM353639). To study ectopic ERG binding we performed ERG ChIP-Seq in stable RWPE+ERG or control cells (samples GSM353649-GSM353650). AR ChIP-Seq was also done in the AR-positive but ETS fusion-negative 22RV1 cells (GSM353640). To further study transcription factor binding and chromatin state we performed ChIP-Seq of AR, ERG, H3K4me3, H3K9me3, H3K27me3 and RNA Pol II in a metastatic prostate tumor tissue (samples GSM353651-GSM353656). To couple the ChIP-Seq experiments with gene expression, we have also done Illumian SAGE-tag profiling in LNCaP cells following androgen treatment for 0, 24 and 48hrs. These DGE experiments correspond to samples GSM353657-GSM353659.
Project description:The ERG gene belongs to the ETS family of transcription factors and has been found involved in atypical chromosomal rearrangements in several cancers. To gain insight into the oncogenic activity of ERG, we compared the gene expression profile of NIH-3T3 cells stably expressing the coding regions of the three main ERG oncogenic fusions: TMPRSS2/ERG (tERG), EWS/ERG and FUS/ERG,. We found that all the three ERG fusions significantly up-regulate PIM-1 expression in the NIH-3T3 cell line. PIM-1 is a serine/threonine kinase frequently over-expressed in cancers of haematological and epithelial origin. We show here that tERG expression induces PIM-1 in the non-malignant prostate cell line RWPE-1, strengthening the relation between tERG and PIM-1 up-regulation in the initial stages of prostate carcinogenesis. Silencing of tERG reversed PIM-1 induction. A significant association between ERG and PIM-1 expression in clinical prostate carcinoma specimens was found, suggesting that such a mechanism may be relevant in vivo. Chromatin Immunoprecipitation experiments showed that tERG directly binds to PIM-1 promoter in the RWPE-1 prostate cell line, suggesting that tERG could be a direct regulator of PIM-1 expression. The up-regulation of PIM-1 induced by tERG over-expression significantly modified CyclinB1 levels and increased the percentage of aneuploid cells in the RWPE-1 cell line after 24hrs of taxane-based treatment. Here we provide the first evidence for an ERG-mediated PIM-1 up-regulation in prostate cells in vitro and in vivo, suggesting a direct effect of ERG transcriptional activity in the alteration of genetic stability. NIH-3T3 cells stably expressing the coding regions of the three main ERG oncogenic fusions: TMPRSS2/ERG (tERG), EWS/ERG and FUS/ERG together with the empty vector where profiled in triplicate. Quality control using NUSE and RLE plots identified one array as problematic (R540_TMP-ERG_P1) which was removed.
Project description:Chromosomal rearrangements involving ETS factors, ERG and ETV1, occur frequently in prostate cancer. How these factors contribute to tumorigenesis and whether they play similar in vivo roles remain elusive. We show that ERG and ETV1 control a common transcriptional network but in an opposing fashion. In mice with ERG or ETV1 targeted to the endogenous Tmprss2 locus, either factors cooperated with Pten-loss, leading to localized cancer, but only ETV1 supported development of advanced adenocarcinoma, likely through enhancement of androgen receptor signaling and steroid biosynthesis. Indeed, ETV1 expression promotes autonomous testosterone production, which may contribute to tumor progression to castration-resistant prostate cancer. Patient data confirmed association of ETV1 expression with aggressive disease. We conclude that despite many shared targets, ERG and ETV1 contribute differently to prostate tumor biology. Hence, prostate cancers with these fusions should be considered as distinct subtypes for patient stratification and therapy. Genomic targets of ERG and ETV1 transcription factors were identified by antibody-mediated and biotin-mediated ChIP-chip in human VCaP and LNCaP cells, respectively.
Project description:Fusion proteins involving the ETS factor ERG have been associated with multiple cancers such as Ewing's sarcoma and prostate cancer. In acute myeloid leukemias harboring t(16;21) another ERG fusion protein is expressed, FUS-ERG. Here, we found that this FUS-ERG oncofusion protein acts in the context of a heptad of proteins (ERG, FLI1, GATA2, LYL1, LNMO2, RUNX1 and TAL1) central to proper expression of genes involved in maintaining a stem cell hematopoietic phenotype. Moreover, in t(16;21) FUS-ERG co-occupies genomic regions bound by the nuclear receptor heterodimer RXR-RARA inhibiting target gene expression and interfering with hematopoietic differentiation. All-Trans Retinoic Acid treatment of t(16;21) cells as well as FUS-ERG knock down alleviate the myeloid differentiation block. Together, the results suggest that FUS-ERG acts as a transcriptional repressor of the retinoic acid signaling pathway. Cell lines were used for RNA extraction for RNA-seq and ChIP experiments for ChIP-seq.
Project description:Recently, a frequent chromosomal aberration fusing Androgen regulated TMPRSS2 promoter and the ERG gene (T/ERG) was discovered in prostate cancer. Several studies demonstrated cooperation between the T/ERG and other defective pathways in cancer progression however, the biological mechanism by which the T/ERG operates is yet to be determined. Using immortalized prostate epithelial cells (EP) model we were able to show that EP with the combination of androgen receptor(AR) and T/ERG(EP-AR T/ERG cell line) demonstrate an Epithelial to Mesenchymal Transition (EMT) manifested by a mesenchyme-like morphological appearance and behavior. To further elucidate the mechanism by which T/ERG executes the EMT program at large, we took a genome-wide approach and conducted micro-array based comparison between EP-AR and EP-AR T/ERG cells Four biological replicate EP-AR and EP-AR T/ERG cell lines were infected in two different infections, two of each were used for expression analysis.
Project description:VCaP cells expressing inducible shRNAs for either ERG or a non-targeting control were treated with Doxycycline for 1, 3, 7 and 10 days prior to collection This experiment is designed to see which genes and pathways are modulated by ERG knockdown VCaP cells stably expressing a Doxycycline (dox)-inducible control nontargeting shRNA (Pak4) or an ERG shRNA (2217) were exposed to 100ng/ml Dox for the noted days.
Project description:Chromosomal translocations juxtaposing the androgen-responsive TMPRSS2 promoter with the ETS-family transcription factor ERG result in aberrant ERG up-regulation in approximately 50% of prostate cancers. Studies to date have demonstrated important roles of ERG in inducing oncogenic properties of prostate cancer. Its molecular mechanisms of action, however, are yet to be fully understood. To address these questions, we generated engineered LNCaP cells with ERG overexpression followed by LEF1 knockdown as well as control cell lines. To further investigate the role of LEF1 in ERG fusion positive samples, we also knockdown ERG in VCaP cell line. We performed microarray analysis on LNCaP cells with ERG overexpression followed by LEF1 knockdown using siRNA. We also knockdown endogenous ERG in fusion-positive cell line VCaP.
Project description:A transgenic TMPRSS2:ERG mouse model was engineered in FVB background and compared to its wildtype counterpart in the absence of any treatment This experiment is designed to look at ERG-dependent changes in phenotype and gene expression A loxP-GFP-loxP-hERG exon 4-11 cassette was inserted into a BAC clone containing the TMPRSS2 locus using a recombineering kit. This modified BAC was used for pronuclear injection and generation of germline-transmitting mice. One line expressing high GFP was used for pronuclear injection of Cre protein and one sub-line that transmitted the TMPRSS2:ERG transgene into the germline was subsequently bred to homozygosity.
Project description:ERG overexpression was conducted in stably transfected K562 cell line with a tet-on inducible plasmid habouring ERG3. Prolonged induction of ERG (8 days) produced spindle cell shape changes whereas non-induced cells retained the round morphology. In oder to determine the genes responsible for inducing cell shape changes, a genome wide transcriptional screen was conducted. Three clones that potently induced ERG overexpression (8 days) with the corresponding non-induced cells were hybridized.
Project description:The goal of this project was to analyze the global gene expression profiles of RWPE1 and VCAP cells following transfection of GFP, GFP-ERG at 48 and 72hrs time points and stable ERG shRNA, scramble shRNA, respectively. Overall design: RWPE1 cells were transfected with GFP or GFP-ERG. VCAP cells were transfected with ERG lenti-shRNA or scramble shRNA. Transfections were performed in duplicate. Total cellular RNA was isolated with Trizol and quality analysed by the bioanalyser kit.