Project description:Alternative splicing (AS) plays a crucial role in the diversification of gene function and regulation. Consequently, the systematic identification and characterization of temporally regulated splice variants is of critical importance to understanding animal development. We have used high-throughput RNA sequencing and microarray profiling to analyze AS in C. elegans across various stages of development. This analysis identified thousands of novel splicing events, including hundreds of developmentally regulated AS events. To make these data easily accessible and informative, we constructed the C. elegans Splice Browser, a web resource in which researchers can mine AS events of interest and retrieve information about their relative levels and regulation across development. The data presented in this study, along with the Splice Browser, provides the most comprehensive set of annotated splice variants in C. elegans to date, and is therefore expected to faciliate focused, high resolution in vivo functional assays of AS function.
Project description:The androgen receptor (AR) plays a key role in progression to incurable androgen-ablation resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand binding domain (designated as AR3, AR4 and AR5) in hormone insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly upregulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells while specific knock-down of AR3 expression (without altering AR level) in hormone resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct yet essential role in ablation-independent growth through regulating a unique set of genes including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation-independence during PCA progression and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available anti-androgen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA. Total RNA was extracted from CWR-R1 and 22Rv1 cells treated with shAR3-1, shARa and the scrambled shRNA control, respectively. Each of CWR-R1 and 22Rv1 cells treated with shAR3-1 was compared with the scrambled shRNA control. The same experiments were performed for the cells treated with shARa.
Project description:Prostate cell lines from diverse backgrounds are important to addressing disparities in prostate cancer (PCa) incidence and mortality rates among Black men. ACRJ-PC28 was developed from a transrectal needle biopsy and established via inactivation of the CDKN2A locus and simultaneous expression of human telomerase. Characterization assays included growth curve analysis, immunoblots, IHC, 3D cultures, immunofluorescence imaging, confocal microscopy, flow cytometry, WGS and, RNA-Seq. RNA-Seq confirmed the expression of prostate specific genes alpha-methylacyl-CoA racemase (AMACR) and NKX3.1 and Neuroendocrine specific markers, synaptophysin (SYP) and enolase 2 (ENO2) and IHC confirmed the presence of AMACR. WGS confirms the absence of exonic mutations and the presence of intronic variants that appear to not affect function of AR, p53 and pRB. RNA-Seq data revealed numerous TP53 and RB1 mRNA splice variants and the lack of AR mRNA expression. The novel cell line described here should advance research addressing the disparity in prostate cancer among Black men.
Project description:Chromosomal abnormalities that give rise to elevated expression levels of the ETS genes ETV1, ETV4, ETV5, or ERG are prevalent in prostate cancer, but the function of these transcription factors in carcinogenesis is not clear. Previous work implicates ERG, ETV1, and ETV5 as regulators of invasive growth but not transformation in cell lines. Here we show that the PC3 prostate cancer cell line provides a model system to study the over-expression of ETV4. Anchorage independent growth assays and microarray analysis indicate that high ETV4 expression is critical for the transformation phenotype of PC3 cells. However, genes up-regulated upon ETV4 over-expression were very similar to genes up-regulated by ETV1 over-expression in the RWPE-1 normal prostate cell line. Together these data indicate that the ETV4 dependent transformation phenotype observed in PC3 cells is due to the genetic background of the cell line, rather than a distinct characteristic of ETV4. Furthermore, these findings suggest that the function of ETS genes in prostate cancer may differ based on other genetic alterations in a tumor. Two sets of two color experiments. First is PC3 cells expressing one of two independent ETV4 shRNAs versus PC3 cells expressing a control shRNA (luciferase). Second is RWPE-1 cells expressing 3xFlag tagged ETV4 versus RWPE-1 cells with a control (empty) vector.
Project description:Identification and characterization of androgen receptor splice variants preferred bindings that drive prostate cancer progression [RNA-seq]
Project description:Identification and characterization of androgen receptor splice variants preferred bindings that drive prostate cancer progression [ChIP-seq]
Project description:Transcriptional profiling of human PC-3 prostate cancer cells lentivirally infected with non-target control (NTC) short hairpin (sh)RNA comparing with lentivirally shRNA mediated human ETV4 knock-down. Cells were either cultured for 24 hours at 20% oxygen tension or 0.2% oxygen. Goal was to determine (i) genes affected by hypoxia in PC-3 NTC cells and (ii) identification of hypoxically induced genes requiring ETV4 for hypoxic regulation.
Project description:Chromosomal translocations or upregulations involving ETS transcription factor are frequent events in prostate cancer pathogenesis and significantly co-occurrence with p53 or PTEN loss. Caused by the low stabilities of ETS proteins in cytosol, mouse models with aberrant expression of wild type ETS transcription factors had subtle phenotypes and only drive prostate cancer progression in the setting of Pten loss. Here we show that prostate specific aberrant expression of mutated ETV4 (V70P71D72-AAA, ETV4-AAA), which is resistence to COP1 mediated protein degradation, results in more stabilized ETV4 protein in mouse prostate. We found that ETV4-AAA mice develop marked prostatic intraepithelial neoplasia (mPin) and p53-dependent cell senescence within 2 weeks, but without tumor development when aged. Interestingly, ETV4-AAA positive cells reduce dramatically in a PTEN loss background, which means that there is no cooperation between ETV4-AAA and PTEN loss. Aberrant ETV4-AAA expression promotes progression of mPin to prostatic adenocarcinoma in a Tp53 deficiency or haploinsufficiency background. In contrast to PTEN loss induced mouse prostate cancers which loss NKX3.1 expression and resistant to castration therapy, these ETV4-AAA tumor cells well maintain AR and NKX3.1 expression and are sensitive to castration therapy.