LNCaP and C42B LSD1 knockdown microarray gene expression data and C42B androgen (DHT) stimulation microarray gene expression data
ABSTRACT: LSD1 (also known as KDM1A) is a histone demethylase and a key regulator of gene expression in embryonic stem cells and cancer.1,2 LSD1 was initially identified as a transcriptional repressor via its demethylation of active histone H3 marks (di-methyl lysine 4 [2MK4]).1 In prostate cancer, specifically, LSD1 also co-localizes with the AR and demethylates repressive 2MK9 histone marks from androgen-responsive AR target genes, facilitating androgen-mediated induction of AR-regulated gene expression and androgen-induced proliferation in androgen-dependent cancers.3,4 Recently, it was shown that treatment with high doses of androgens (e.g.10-fold higher doses than those required for induction of expression of androgen-activated genes such as PSA) recruits LSD1 and AR to an enhancer within the AR; this AR and LSD1 recruitment represses AR transcription.5 Thus, LSD1 appears to play a role in mediating both the proliferative and repressive phases of the biphasic androgen dose-response curve. For these reasons, we hypothesized that LSD1 might be important for maintenance of AR signaling in castration-resistant prostate cancer (CRPC) tumors. However, in this report, we describe a distinct role of LSD1 as a driver of proliferation and survival of prostate cancer cells, including CRPC cells, irrespective of androgens or even AR expression. Specifically, LSD1 activates expression of cell cycle, mitosis, and embryonic stem cell maintenance pathways that are enriched in lethal prostate cancers – pathways not activated by androgens. Finally, we observe that treatment with a new LSD1 inhibitor potently and specifically suppresses LSD1 function and suppresses CRPC growth and survival in vitro and in vivo. Our data place LSD1 as a key driver of androgen-independent survival in lethal prostate cancers and demonstrate the potential of LSD1-directed therapies in the near-term. The enclosed files are from microarrays experiments after suppressing LSD1 with RNAi or stimulating cells with the androgen agonist dihydrotestosterone (DHT).
Project description:The androgen receptor (AR) is a ligand-inducible transcription factor that mediates androgen action in target tissues. Upon ligand binding, the AR binds to thousands of genomic loci and activates a cell-type specific gene program. Prostate cancer growth and progression depend on androgen-induced AR signalling. Treatment of advanced prostate cancer through medical or surgical castration leads to initial response and durable remission, but resistance inevitably develops. In castration-resistant prostate cancer (CRPC), AR activity remains critical for tumor growth despite androgen deprivation. While previous studies have focused on ligand-dependent AR signalling, in this study we explore AR function under the androgen-deprived conditions characteristic of CRPC. Our data demonstrate that the AR persistently occupies a distinct set of genomic loci after androgen deprivation in CRPC. These androgen-independent AR occupied regions have constitutively open chromatin structures that lack the canonical androgen response element and are independent of FoxA1, a transcription factor involved in ligand-dependent AR targeting. Many AR binding events occur at proximal promoters, which can act as enhancers to augment transcriptional activities of other promoters through DNA looping. We further show that androgen-independent AR binding directs a distinct gene expression program in CRPC, which is necessary for the growth of CRPC after androgen withdrawal. LNCaP, C4-2B, or 22RV1 cells were cultured in hormone-free media for 3 days and then treated with ethanol vehicle or DHT (10nM) for 4h or 16h prior to ChIP-seq or RNA-seq assays. For siRNA transfection, cells were transfected with AR siRNA or control siRNA for 3 days prior to RNA-seq assays.
Project description:The spliced variant forms of androgen receptor (AR-Vs) have been identified recently in castration-resistant prostate cancer (CRPC) cell lines and clinical samples. Here we identified the cistrome and transcriptome landscape of AR-Vs in CRPC cell lines and determine the clinical significance of AR variants regulated gene.The AR variants binding sites can be identified in 22Rv1 cell line in the absence of androgen. Knocking down full-length AR (AR-FL) doesn't affect AR-Vs binding sites in genome-wide. A set of genes were identified to be regulated uniquely by AR-Vs, but not by AR-FL in androgen-depleted condition. Integrated analysis showed that some genes may be modulated by AR-Vs directly. Unsupervised clustering analysis demonstrated that AR variants gene signature can separate not only the benign and malignant prostate tissue, but also the localized prostate cancer and metastatic CRPC specimens. Some genes modulated uniquely by AR variants were also identified to correlate with the Gleason Pattern of prostate cancer and PSA failure. We conclude that AR spliced variants bind to DNA independent of full-length AR, and can modulate a unique set of genes which is not regulated by full-length AR in the absence of androgen. AR variants gene signature correlate with CRPC and prostate cnacer disease progress. Androgen receptor (AR) binding sites in human prostate cancer 22Rv1 cell lines were studied using ChIP-seq. ChIP enriched and input DNA were sequenced using Illumina HiSeq 2000.
Project description:Androgen receptor (AR) is reactivated in castration resistant prostate cancer (CRPC) through mechanisms including marked increases in AR gene expression. We identify an enhancer in the AR second intron contributing to increased AR expression at low androgen levels in CRPC. Moreover, at increased androgen levels the AR binds this site and represses AR gene expression through recruitment of lysine specific demethylase 1 (LSD1) and H3K4me1,2 demethylation. AR similarly represses expression of multiple genes mediating androgen synthesis, DNA synthesis and proliferation, while stimulating genes mediating lipid and protein biosynthesis. Androgen levels in CRPC appear adequate to stimulate AR activity on enhancer elements, but not on suppressor elements, resulting in increased expression of AR and AR repressed genes that contribute to cellular proliferation. Custom Agilent 44K whole human genome expression oligonucleotide microarrays were used to profile pre-castrated androgen dependent, 4d-post-castrated, and relapsed castration resistant VCaP xenograft tumors in 3 mice. Total RNA was isolated and amplified prior to hybridization against a common reference pool of prostate tumor cell lines.
Project description:Prostate cancer is the most commonly diagnosed and second-most lethal cancer among men in the United States. The vast majority of prostate cancer deaths are due to castration-resistant prostate cancer (CRPC) – the lethal form of the disease that has progressed despite therapies that interfere with activation of androgen receptor (AR) signaling. One emergent resistance mechanism to medical castration is synthesis of intratumoral androgens that activate the AR. This insight led to the development of the AR antagonist enzalutamide. However, resistance to enzalutamide invariably develops, and disease progression is nearly universal. One mechanism of resistance to enzalutamide is an F877L mutation in the AR ligand-binding domain that can convert enzalutamide to an agonist of AR activity. However, mechanisms that contribute to the agonist switch had not been fully clarified, and there were no therapies to block AR F877L. Using cell line models of castration-resistant prostate cancer (CRPC), we determined that cellular androgen content influences enzalutamide agonism of mutant F877L AR. Further, enzalutamide treatment of AR F877L-expressing cell lines recapitulated the effects of androgen activation of F877L AR or wild-type AR. Because the BET bromodomain inhibitor JQ-1 was previously shown to block androgen activation of wild-type AR, we tested JQ-1 in AR F877L-expressing CRPC models. We determined that JQ-1 suppressed androgen or enzalutamide activation of mutant F877L AR and suppressed growth of mutant F877L AR CRPC tumors in vivo, demonstrating a new strategy to treat tumors harboring this mutation. RNA-seq profiles of prostate cancer cell lines to understand gene expression associated with enzalutamide treatment
Project description:LSD1 (also known as KDM1A) is a histone demethylase and a key regulator of gene expression in embryonic stem cells and cancer.1,2 LSD1 was initially identified as a transcriptional repressor via its demethylation of active histone H3 marks (di-methyl lysine 4 [2MK4]).1 In prostate cancer, specifically, LSD1 also co-localizes with the AR and demethylates repressive 2MK9 histone marks from androgen-responsive AR target genes, facilitating androgen-mediated induction of AR-regulated gene expression and androgen-induced proliferation in androgen-dependent cancers. We report here that the LSD1 protein is universally upregulated in human CRPC and promotes survival of CRPC cell lines. This effect is explained in part by LSD1-induced activation of cell cycle and embryonic stem cell gene sets—gene sets enriched in transcriptomal studies of lethal human tumors. Importantly, despite the fact that many of these genes are direct LSD1 targets, we did not observe histone methylation changes at the LSD1-bound regions, demonstrating non-canonical histone demethylation-independent mechanisms of gene regulation. This ChIP-seq dataset included H3K4me2 and H3K9me2 ChIP-seq data for siRNA target against LSD1 and non-targeting control, as well as SP2509 inhibition of LSD1 and mock treatment 4 conditions: siRNA against LSD1, siRNA against luciferase (non-targeting control); SP2509 inhibition of LSD1, mock treatment. There are 2 replicates per condition.
Project description:Castration resistant prostate cancer (CRPC) is a lethal disease1-4. Aberrant activation of the androgen receptor (AR) becomes a central mechanism contributing to the resistance of endocrine therapies2,3. Here we demonstrate that non-coding RNAs transcribed from the AR bound-enhancers RNAs (AR-eRNAs) are upregulated in human CRPC cells in vitro, xenografts in vivo and patient tissues. Expression of a subset of genes with elevated AR-eRNAs, including TLE1 and HTR3A, is inversely correlated with biochemical recurrence-free survival of CRPC patients. We identify aan HIV-1 TAR-like (TAR-L) motif in AR-eRNAs of AR target genes including KLK3 (or PSA) and TMPRSS2. The TAR-L motif is important for these eRNAs to bind to CYCLIN T1 of the positive transcription elongation factor b (P-TEFb) complex. Knockdown of PSA eRNA diminishes RNA polymerase II (Pol II) serine-2 (Ser-2) phosphorylation at the PSA promoter. The TAR-L motif in KLK3 eRNA is crucial for effective transcription of PSA mRNA. Together, wWe demonstrate a P-TEFb activation function of eRNA and reveal aberrant eRNA expression as a functional indicator of AR abnormality in CRPC. Our results also suggest that eRNAs as amay be a potential target for CRPC therapy. Total RNA sequencing of two prostate cancer cells. Data were generated by deep sequencing, in diplicate, using Illumina HiSeq 2000.
Project description:The development and progression of castrate resistant prostate cancer (CRPC), a lethal disease, is thought to be driven by multiple events. A hallmark of CRPC is the ability to evade the cytotoxic effects of anti-androgen therapy. Importantly, persistent androgen receptor (AR) signalling is thought to play a principal role in maintaining CRPC. The precise molecular alterations driving this condition, however, are not clearly understood. Our previous studies identified specific metabolic alterations associated with localized prostate cancer (PCa) and CRPC, implicating metabolic re-programming in disease progression. Building on these findings, using a novel network-based integromics approach, here we show distinct alterations in the Hexosamine Biosynthetic Pathway (HBP) to be critical for sustaining the castrate resistant state. We found expression of the HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) was regulated by androgens and elevated in androgen dependent (AD) PCa while relatively diminished in CRPC possessing either full length AR (AR-FL) or the spliced V7 variant (AR-V7). Genetic loss of function experiments for GNPNAT1 in CRPC-like cells led to increased proliferation and aggressiveness, both, in vitro and in vivo. This was mediated by specific cell cycle genes regulated by the PI3K-AKT pathway activating either AR in cells with AR-FL or SP1-ChREBP (carbohydrate response element binding protein) in cells containing AR-V7. Strikingly, addition of HBP metabolite UDP-N-acetylglucosamine (UDP) to CRPC-like cells reduced the expression of cell cycle genes and attenuated tumor cell proliferation, both in vitro and in vivo. Furthermore, addition of UDP sensitized CRPC-like cells, inclusive of those possessing AR-V7, to enzalutamide, demonstrating the therapeutic value of targeting altered metabolic pathways in lethal PCa. We anticipate that our findings will motivate the development of novel metabolic therapeutic strategies that complement existing treatments for men with lethal prostate cancer We used microarray analysis to determine key molecular alterations associated with inhibition of HBP pathway in CRPC by knocking down GNPNAT1 transcript level using lentiviral particle bearing shRNA in 22Rv1 and LNCaP-ABL cells GNPNAT1 expression was knockdown in two independent prostate cancer cells, 22Rv1 and LNCaP-ABL
Project description:Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. The development and progression to CRPC following androgen ablation therapy is predominantly driven by unregulated androgen receptor (AR) signaling. Despite the success of recently approved therapies targeting AR signaling, such as abiraterone and second-generation anti-androgens MDV3100 (enzalutamide), durable responses are limited, presumably due to acquired resistance. Recently, JQ1 and I-BET, two selective small molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit antiproliferative effects in a range of malignancies. Here we show that AR signaling-competent CRPC cell lines are preferentially sensitive to BET bromodomain inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ1. Like the direct AR antagonist, MDV3100, JQ1 disrupted AR recruitment to target gene loci. In contrast to MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR mediated gene transcription including induction of TMPRSS2-ERG and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer. Examination of ASH2L genome-wide binding in prostate cancer cells after AR stimulation.
Project description:Enzalutamide (formerly MDV3100 and available commercially as Xtandi), a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration-resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: (1) binding of androgens to AR, (2) AR nuclear translocation, and (3) association of AR with DNA. Here we used Affymetrix human genome microarray technology to investigate the global programme of gene expression of LNCaP cells in response to enzalutamide alone and in the context of DHT-stimulated androgen receptor gene expression. LNCaP cells were grown in RPMI 1640 supplemented with 5% hormone depleted FBS and treated with vehicle (control sample) , DHT (100 nM), enzalutamide (1 or 10 µM) or DHT (100 nM) plus enzalutamide (1 or 10 µM)for 16 hours for RNA extraction and hybridization. Each condition was done in triplicate.
Project description:Androgen receptor (AR) is required for castration resistant prostate cancer (CRPC) progression, but the function and disease relevance of AR-bound enhancers remain poorly understood. Here, we identify a group of AR-regulated enhancer RNAs (e.g. PSA eRNA) that are upregulated in CRPC cells, patient-derived xenografts (PDX) and patient tissues. PSA eRNA binds to CYCLIN T1, activates P-TEFb and promotes in cis and trans gene transcription by increasing serine-2 phosphorylation of RNA polymerase II (Pol II-Ser2p). To avoid the total Pol II changing by PSA eRNA. We measured the total Pol II using N20 and 8WG16 antibodies with or without PSA eRNA knocking down. To avoid the AR binding changes by PSA eRNA, we also measured the AR binding using AR N20 antibodies with or without PSA eRNA knocking down. Androgen receptor (AR) binding sites in human prostate cancer cell lines, C4-2, were studied using ChIP-seq. Total Pol II Ser-2p and AR binding sites in human prostate cancer cell lines C4-2 with or without PSA eRNA knockdown, were studied using ChIP-seq. ChIP enriched DNA were sequenced using Illumina HiSeq 2500and input DNA were sequenced using Illumina HiSeq 2000.