RESUMO
Atopic dermatitis (AD) is a highly heritable and common inflammatory skin condition affecting children and adults worldwide. Multi-ancestry approaches to AD genetic association studies are poised to boost power to detect genetic signal and identify ancestry-specific loci contributing to AD risk. Here, we present a multi-ancestry GWAS meta-analysis of twelve AD cohorts from five ancestral populations totaling 56,146 cases and 602,280 controls. We report 101 genomic loci associated with AD, including 15 loci that have not been previously associated with AD or eczema. Fine-mapping, QTL colocalization, and cell-type enrichment analyses identified genes and cell types implicated in AD pathophysiology. Functional analyses in keratinocytes provide evidence for genes that could play a role in AD through epidermal barrier function. Our study provides new insights into the etiology of AD by harnessing multiple genetic and functional approaches to unveil the mechanisms by which AD-associated variants impact genes and cell types.
RESUMO
N-linked glycosylation is a common post-translational modification that has various effects on multiple types of proteins. The extent to which an N-linked glycoprotein is modified and the identity of glycans species involved is of great interest to the biopharmaceutical industry, since glycosylation can impact the efficacy and safety of therapeutic monoclonal antibodies (mAbs). mAbs lacking core fucose, for example, display enhanced clinical efficacy through increased antibody-dependent cellular cytotoxicity. We performed a genome-wide CRISPR knockout screen in Chinese hamster ovary (CHO) cells, the workhorse cell culture system for industrial production of mAbs, aimed at identifying novel regulators of protein fucosylation. Using a lectin binding assay, we identified 224 gene perturbations that significantly alter protein fucosylation, including well-known glycosylation genes. This functional genomics framework could readily be extended and applied to study the genetic pathways involved in regulation of other glycoforms. We hope this resource will provide useful guidance toward the development of next generation CHO cell lines and mAb therapeutics.
Assuntos
Anticorpos Monoclonais , Genômica , Cricetinae , Animais , Cricetulus , Glicosilação , Células CHO , Anticorpos Monoclonais/genéticaRESUMO
The function of enhancer RNAs (eRNAs) in transcriptional regulation remains obscure. By analyzing the genome-wide nascent transcript profiles in breast cancer cells, we identify a special group of eRNAs that are essential for estrogen-induced transcriptional repression. Using eRNAs of TM4SF1 and EFEMP1 as the paradigms, we find that these RNA molecules not only stabilize promoter-enhancer interactions but also recruit liganded estrogen receptor α (ERα) to particular enhancer regions, facilitate the formation of a functional transcriptional complex, and cause gene silencing. Interestingly, ERα is shown to directly bind with eRNAs by its DNA-binding domain. These eRNAs help with the formation of a specific ERα-centered transcriptional complex and promote the association of the histone demethylase KDM2A, which dismisses RNA polymerase II from designated enhancers and suppresses the transcription of target genes. Our work demonstrates a complete mechanism underlying the action of eRNAs in modulating and refining the locus-specific transcriptional program.
Assuntos
Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , RNA/metabolismo , Linhagem Celular , Regulação para Baixo/genética , Receptor alfa de Estrogênio/química , Proteínas F-Box/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Modelos Biológicos , Fases de Leitura Aberta/genética , Ligação Proteica , Domínios Proteicos , RNA Polimerase II/metabolismo , Transcrição GênicaRESUMO
Interleukin-17A (IL17A) plays a critical role in the development of numerous autoimmune diseases, including psoriasis. The clinical success of IL17A neutralizing biologics in psoriasis has underlined its importance as a drug discovery target. While many studies have focused on the differentiation and trafficking of IL17A producing T-helper 17 cells, less is known about IL17A-initiated signaling events in stromal and parenchymal cells leading to psoriatic phenotypes. We sought to discover signaling nodes downstream of IL17A contributing to disease pathogenesis. Using IL17A and tumor necrosis factor α (TNF) to stimulate primary human epidermal keratinocytes, we employed two different phenotypic screening approaches. First, a library of â¼22000 annotated compounds was screened for reduced secretion of the pro-inflammatory chemokine IL8. Second, a library of 729 kinases was screened in a pooled format by utilizing CRISPR-Cas9 and monitoring IL8 intracellular staining. The highest-ranking novel hits identified in both screens were the bromodomain and extra-terminal domain (BET) family proteins and bromodomain-containing protein 2 (BRD2), respectively. Comparison of BRD2, BRD3, and BRD4 silencing with siRNA and CRISPR confirmed that BRD2 was responsible for mediating IL8 production. Pan-BRD inhibitors and BRD2 knockout also reduced IL17A/TNF-mediated CXC motif chemokines 1/2/6 (CXCL1/2/6) and granulocyte colony stimulating factor (G-CSF) production. In RNA-Seq analysis, 438 IL17A/TNF dependent genes were reduced in BRD2-deficient primary keratinocytes. KEGG pathway analysis of these genes showed enrichment in TNF signaling and rheumatoid arthritis relevant genes. Moreover, a number of genes important for keratinocyte homeostasis and cornification were dysregulated in BRD2-deficient keratinocytes. In IL17A/TNF/IL22 stimulated three-dimensional organotypic raft cultures, pan-BRD inhibition reduced inflammatory factor production but elicited aberrant cornification, consistent with RNA-Seq analysis. These studies highlight a novel role for BRDs and BRD2 in particular in IL17A-mediated inflammatory signaling.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Inflamação/metabolismo , Interleucina-17/metabolismo , Queratinócitos/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/metabolismo , Fatores de Transcrição/metabolismo , Diferenciação Celular , Células Cultivadas , Técnicas de Silenciamento de Genes , Homeostase , Humanos , Queratinócitos/citologia , RNA Interferente Pequeno/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
MicroRNAs (miRNAs) play important roles in cancer formation and progression by suppressing the production of key functional proteins at the post-transcriptional level in a sequence-specific manner. While differential expression of miRNAs is widely observed in cancers including prostate cancer (PCa), how these miRNAs are transcriptionally regulated is largely unknown. MiRNA-221 and miRNA-222 (miR-221/-222) are well-established oncogenes and overexpressed in breast, liver, pancreas, and lung cancer, but their expression and biological functions in PCa remain controversial. Both up and down regulation have been observed in patient samples. Specifically, studies have demonstrated miR-221/-222 function as oncogenes, and promote PCa cell proliferation and the development of castration-resistant prostate cancer (CRPC). However, the expression level of miR-221/-222 is downregulated in several miRNA expression profiling studies. In this study, we demonstrate miR-221/-222 are androgen receptor (AR)-repressed genes and reside in a long primary transcript (pri-miRNA). Derepression of miR-221/-222 after androgen deprivation therapy (ADT) may enhance PCa cell proliferation potential through promoting G1/S phase transition. This function is likely transient but important in the development of CRPC. Downregulation of miR-221/-222 subsequently occurs once AR activity is restored through AR overexpression in CRPC. Our findings shed light on the complexity of transcriptional regulation of miRNAs in PCa and suggest context-dependent targeting of oncogenic miRNAs.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Regulação para Baixo , Expressão Gênica , Xenoenxertos , Histonas/metabolismo , Humanos , Masculino , Camundongos , Interferência de RNARESUMO
Among androgen-regulated genes, soluble guanylyl cyclase α1 (sGCα1) is significant in promoting the survival and growth of prostate cancer cells and does so independent of nitric oxide (NO) signaling. Peptides were designed targeting sGCα1 to block its pro-cancer functions and one peptide is discussed here. Peptide B-8R killed both androgen-dependent and androgen-independent prostate cancer cells that expressed sGCα1, but not cells that do not express this gene. Peptide B-8R induced apoptosis of prostate cancer cells. Importantly, Peptide B-8R does not affect nor its cytotoxicity depend on NO signaling, despite the fact that it associates with sGCα1, which dimerizes with sGCß1 to form the sGC enzyme. Just as with a previously studied Peptide A-8R, Peptide B-8R induced elevated levels of reactive oxygen species (ROS) in prostate cancer cells, but using a ROS-sequestering agent showed that ROS was not responsible the cytotoxic activity of Peptide B-8R. Interestingly, Peptide B-8R induced elevated levels of p53 and phosphorylated p38, but neither of these changes is the cause of the peptide's cytotoxicity. Additional drugs were used to alter levels of iron levels in cells and these studies showed that Peptide B-8R activity does not depend on Ferroptosis. Thus, future work will be directed at defining the mechanism of cytotoxic action of Peptide B-8R against prostate cancer cells.
Assuntos
Encefalinas/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Precursores de Proteínas/administração & dosagem , Guanilil Ciclase Solúvel/genética , Androgênios/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Encefalinas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Óxido Nítrico/metabolismo , Proteína Oncogênica pp60(v-src)/genética , Fragmentos de Peptídeos/genética , Neoplasias da Próstata/patologia , Precursores de Proteínas/genética , Espécies Reativas de Oxigênio/metabolismo , Guanilil Ciclase Solúvel/antagonistas & inibidoresRESUMO
Whole-exome sequencing of metastatic castration-resistant prostate cancer (mCRPC) reveals that 5% to 7% of tumors harbor promyelocytic leukemia zinc finger (PLZF) protein homozygous deletions. PLZF is a canonical androgen-regulated putative tumor suppressor gene whose expression is inhibited by androgen deprivation therapy (ADT). Here, we demonstrate that knockdown of PLZF expression promotes a CRPC and enzalutamide-resistant phenotype in prostate cancer cells. Reintroduction of PLZF expression is sufficient to reverse androgen-independent growth mediated by PLZF depletion. PLZF loss enhances CRPC tumor growth in a xenograft model. Bioinformatic analysis of the PLZF cistrome shows that PLZF negatively regulates multiple pathways, including the MAPK pathway. Accordingly, our data support an oncogenic program activated by ADT. This acquired mechanism together with the finding of genetic loss in CRPC implicates PLZF inactivation as a mechanism promoting ADT resistance and the CRPC phenotype.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias de Próstata Resistentes à Castração/genética , Animais , Antineoplásicos Hormonais/farmacologia , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Masculino , Camundongos Nus , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The androgen receptor (AR) is a key factor that regulates the behavior and fate of prostate cancer cells. The AR-regulated network is activated when AR binds enhancer elements and modulates specific enhancer-promoter looping. Kallikrein-related peptidase 3 (KLK3), which codes for prostate-specific antigen (PSA), is a well-known AR-regulated gene and its upstream enhancers produce bidirectional enhancer RNAs (eRNAs), termed KLK3e. Here, we demonstrate that KLK3e facilitates the spatial interaction of the KLK3 enhancer and the KLK2 promoter and enhances long-distance KLK2 transcriptional activation. KLK3e carries the core enhancer element derived from the androgen response element III (ARE III), which is required for the interaction of AR and Mediator 1 (Med1). Furthermore, we show that KLK3e processes RNA-dependent enhancer activity depending on the integrity of core enhancer elements. The transcription of KLK3e was detectable and its expression is significantly correlated with KLK3 (R(2) = 0.6213, P < 5 × 10(-11)) and KLK2 (R(2) = 0.5893, P < 5 × 10(-10)) in human prostate tissues. Interestingly, RNAi silencing of KLK3e resulted in a modest negative effect on prostate cancer cell proliferation. Accordingly, we report that an androgen-induced eRNA scaffolds the AR-associated protein complex that modulates chromosomal architecture and selectively enhances AR-dependent gene expression.
Assuntos
Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Inativação Gênica , Humanos , Calicreínas/metabolismo , Masculino , Subunidade 1 do Complexo Mediador/metabolismo , Regiões Promotoras Genéticas , Próstata/metabolismo , Antígeno Prostático Específico/metabolismo , Interferência de RNA , Sequências Reguladoras de Ácido Nucleico , Calicreínas Teciduais/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
The Zinc Finger (ZNF) 280B protein was identified as an unexpected target of an shRNA designed for sGCα1. Further analysis showed that these two proteins are connected in another way, with 280B up-regulation of sGCα1 expression. Knock-down and over-expression experiments showed that 280B serves pro-growth and pro-survival functions in prostate cancer. Surprisingly however, these pro-cancer functions of 280B are not mediated by sGCα1, which itself has similar functions in prostate cancer, but by down-regulated p53. The p53 protein is a second target of 280B in prostate cancer, but unlike sGCα1, p53 is down-regulated by 280B. 280B induces p53 nuclear export, leading to subsequent proteasomal degradation. The protein responsible for p53 regulation by 280B is Mdm2, the E3 ubiquitin ligase that promotes p53 degradation by inducing its nuclear export. We show here that 280B up-regulates expression of Mdm2 in prostate cancer cells, and this regulation is via the Mdm2 promoter. To demonstrate an in vivo relevance to this interaction, expression studies show that 280B protein levels are up-regulated in prostate cancer and these levels correspond to reduced levels of p53. Thus, by enhancing the expression of Mdm2, the uncharacterized 280B protein provides a novel mechanism of p53 suppression in prostate cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Guanilato Ciclase/genética , Neoplasias da Próstata/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/fisiologia , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Expressão Gênica , Técnicas de Silenciamento de Genes , Guanilato Ciclase/metabolismo , Humanos , Masculino , Estabilidade Proteica , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Guanilil Ciclase Solúvel , Proteína Supressora de Tumor p53/metabolismoRESUMO
Among the many identified androgen-regulated genes, sGCα1 (soluble guanylyl cyclase α1) appears to play a pivotal role in mediating the pro-cancer effects of androgens and androgen receptor. The classical role for sGCα1 is to heterodimerize with the sGCß1 subunit, forming sGC, the enzyme that mediates nitric oxide signaling by catalyzing the synthesis of cyclic guanosine monophosphate. Our published data show that sGCα1 can drive prostate cancer cell proliferation independent of hormone and provide cancer cells a pro-survival function, via a novel mechanism for p53 inhibition, both of which are independent of sGCß1, NO, and cGMP. All of these properties make sGCα1 an important novel target for prostate cancer therapy. Thus, peptides were designed targeting sGCα1 with the aim of disrupting this protein's pro-cancer activities. One peptide (A-8R) was determined to be strongly cytotoxic to prostate cancer cells, rapidly inducing apoptosis. Cytotoxicity was observed in both hormone-dependent and, significantly, hormone-refractory prostate cancer cells, opening the possibility that this peptide can be used to treat the usually lethal castration-resistant prostate cancer. In mouse xenograft studies, Peptide A-8R was able to stop tumor growth of not only hormone-dependent cells, but most importantly from hormone-independent cells. In addition, the mechanism of Peptide A cytotoxicity is generation of reactive oxygen species, which recently have been recognized as a major mode of action of important cancer drugs. Thus, this paper provides strong evidence that targeting an important AR-regulated gene is a new paradigm for effective prostate cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Peptídeos/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Guanilato Ciclase/metabolismo , Humanos , Masculino , Camundongos , Orquiectomia , Peptídeos/química , Peptídeos/toxicidade , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Guanilil Ciclase Solúvel , Carga Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Recent studies show that microRNAs (miRNAs), small non-coding RNAs that negatively regulate gene expression, may have potential for monitoring cancer status. We investigated circulating miRNAs in prostate cancer that may be associated with the progression of hormone-sensitive primary tumors to metastatic castration resistant prostate cancer (CRPC) after androgen deprivation therapy. METHODS: Using genome-wide expression profiling by TaqMan Human MicroRNA Arrays (Applied Biosystems) and/or quantitative real-time polymerase chain reaction, we compared the expression levels of miRNAs in serum samples from 28 patients of low-risk localized disease, 30 of high-risk localized disease and 26 of metastatic CRPC. RESULTS: We demonstrated that serum samples from patients of low risk, localized prostate cancer and metastatic CRPC patients exhibit distinct circulating miRNA signatures. MiR-375, miR-378*, and miR-141 were significantly over-expressed in serum from CRPC patients compared with serum from low-risk localized patients, while miR-409-3p was significantly under-expressed. In prostate primary tumor samples, miR-375 and miR-141 also had significantly higher expression levels compared with those in normal prostate tissue. CONCLUSIONS: Circulating miRNAs, particularly miR-375, miR-141, miR-378*, and miR-409-3p, are differentially expressed in serum samples from prostate cancer patients. In the search for improved minimally invasive methods to follow cancer pathogenesis, the correlation of disease status with the expression patterns of circulating miRNAs may indicate the potential importance of circulating miRNAs as prognostic markers for prostate cancer progression.
Assuntos
Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/sangue , Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , MicroRNAs/sangue , Neoplasias da Próstata/sangue , Idoso , Biomarcadores Tumorais/genética , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Orquiectomia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Fatores de RiscoRESUMO
Our laboratory has previously identified soluble guanylyl cyclase α1 (sGCα1) as a novel androgen-regulated gene essential for prostate cancer cell proliferation. sGCα1 expression is highly elevated in prostate tumors, contrasting with the low expression of sGCß1, with which sGCα1 dimerizes to mediate nitric oxide (NO) signaling. In studying its mechanism of action, we have discovered that sGCα1 can inhibit the transcriptional activity of p53 in prostate cancer cells independent of either classical mediators of NO signaling or the guanylyl cyclase activity of sGCα1. Interestingly, sGCα1 inhibition of p53-regulated gene expression was gene specific, targeting genes involved in apoptosis/cell survival. Consistent with this, overexpression of sGCα1 makes prostate cancer cells more resistant to etoposide, a chemotherapeutic and apoptosis-inducing drug. Immunoprecipitation and immunocytochemistry assays show a physical and direct interaction between sGCα1 and p53 in prostate cancer cells. Interestingly, sGCα1 induces p53 cytoplasmic sequestration, representing a new mechanism of p53 inactivation in prostate cancer. Analysis of prostate tumors has shown a direct expression correlation between sGCα1 and p53. Collectively, these data suggest that sGCα1 regulation of p53 activity is important in prostate cancer biology and may represent an important mechanism of p53 down-regulation in those prostate cancers that express significant levels of p53.
Assuntos
Citoplasma/enzimologia , Regulação Neoplásica da Expressão Gênica , Guanilato Ciclase/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Citoplasma/metabolismo , Regulação para Baixo , Genes Reporter , Guanilato Ciclase/genética , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Luciferases/biossíntese , Luciferases/genética , Masculino , Óxido Nítrico/metabolismo , Neoplasias da Próstata , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/genética , Elementos de Resposta , Transdução de Sinais , Guanilil Ciclase Solúvel , Survivina , Transcrição Gênica , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: We have previously identified seven miRs-miR-221, -222, -23b, -27b, -15a, -16-1, and -203, that are differentially expressed in the hormone sensitive LNCaP cell line and the hormone resistant LNCaP-abl cell line and hypothesized that these miRs may characterize certain subtypes of human castration resistant prostate cancer (CRPC). METHODS: Functional studies in cell culture systems have been performed to determine the effect of alternated expression level on cellular response to androgen treatment. To determine the clinical relevance of the expression patterns of these miRs, we compared the expression levels of these seven miRs in normal prostate tissues from 86 individuals, prostate tumor tissues from 34 individuals with localized hormone naïve disease, and bone-derived metastatic CRPC tissues from 17 individuals. RESULTS: The altered expression of miR-221/-222 (as previously described) or miR-203 affected the cellular response to androgen treatment, suggesting their potential involvement in the transition to CRPC. However, the expression of miR-23b, -27b, -15a, and -16-1 did not have a significant influence in the cellular response to androgen treatment, suggesting that these miRs may not play a causative role in the CRPC phenotype. Comparison of the expression levels of these miRs in tissue samples revealed that strikingly, â¼90% of the analyzed metastatic CRPC tumors could be characterized by the increased miR-221/-222 expression and the down-regulated miR-23b/-27b expression. CONCLUSIONS: This finding suggests that altered miR-221/-222 and miR-23b/-27b expression may be associated with the CRPC process.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Masculino , MicroRNAs/genética , Orquiectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
Cellular changes that affect the androgen receptor (AR) can cause prostate cancer to transition from androgen dependent to androgen independent, which is usually lethal. One common change in prostate tumors is overexpression of the AR, which has been shown to lead to androgen-independent growth of prostate cancer cells. This led us to hypothesize that expression of a hyperactive AR would be sufficient for androgen-independent growth of prostate cancer cells. To test this hypothesis, stable lune cancer prostate (LNCaP) cell lines were generated, which express a virion phosphoprotein (VP)16-AR hybrid protein that contains full-length AR fused to the strong viral transcriptional activation domain VP16. This fusion protein elicited as much as a 20-fold stronger transcriptional activity than the natural AR. Stable expression of VP16-AR in LNCaP cells yielded androgen-independent cell proliferation, while under the same growth conditions the parental LNCaP cells exhibited only androgen-dependent growth. These results show that expression of a hyperactive AR is sufficient for androgen-independent growth of prostate cancer cells. To study the molecular basis of this enhanced growth, we measured the expression of soluble guanylyl cyclase-alpha1 (sGCalpha1), a subunit of the sGC, an androgen-regulated gene that has been shown to be involved in prostate cancer cell growth. Interestingly, the expression of sGCalpha1 is androgen independent in VP16-AR-expressing cells, in contrast to its androgen-induced expression in control LNCaP cells. RNA(I)-dependent inhibition of sGCalpha1 expression resulted in significantly reduced proliferation of VP16-AR cells, implicating an important role for sGCalpha1 in the androgen-independent growth of these cells.
Assuntos
Androgênios/fisiologia , Proliferação de Células , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Masculino , Receptores Androgênicos/fisiologia , Transcrição GênicaRESUMO
The multiple transcriptional roles of c-Jun are shown in a novel cross-talk between the androgen receptor (AR) and its new target gene, Ets variant gene 1 (ETV1). In this report, we show that c-Jun can mediate AR induction of ETV1 expression independent of c-Jun transactivation function. Interestingly, c-Jun can transactivate the cloned ETV1 promoter also in the absence of ligand-activated AR, suggesting two mechanisms by which c-Jun can induce ETV1 expression. In addition, both wild-type c-Jun and a transactivation-deficient mutant can enhance the transcriptional activity of ETV1, as measured by both reporter gene assay and endogenous expression of matrix metalloproteinase genes, well-known targets of Ets proteins. Overexpression of the c-Jun mutant protein also led to increased prostate cancer cell invasion. Immunoprecipitation and immunocytochemistry experiments showed copurification and colocalization of c-Jun with AR or ETV1, suggesting that c-Jun acts on AR or ETV1 via a physical association. Collectively, these results, together with a parallel overexpression of ETV1, c-Jun, and AR in prostate tumors, imply that c-Jun plays a pivotal role in the pathway that connects ligand-activated AR to elevated ETV1 expression, leading to enhanced expression of matrix metalloproteinases and prostate cancer cell invasion.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores Androgênicos/metabolismo , Fatores de Transcrição/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metaloproteinases da Matriz/genética , Análise em Microsséries , Invasividade Neoplásica , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Ligação Proteica , Transporte Proteico , Ativação Transcricional/genética , Células Tumorais CultivadasRESUMO
Androgens and the androgen receptor (AR) act in cells by modulating gene expression. Through gene microarray studies, we have identified Ets Variant Gene 1 (ETV1) as a novel androgen-regulated gene. Our data demonstrate that ETV1 mRNA and protein are up-regulated in response to ligand-activated AR in androgen-dependent LNCaP cells, but there is no detectable ETV1 expression in normal prostate cells. The ETV1 promoter is induced by androgens and recruits the AR in the context of chromatin. ETV1-regulated endogenous matrix metalloproteinase genes can be induced by ligand-activated AR. In contrast to the hormone-induced expression in androgen-dependent LNCaP cells, ETV1 expression in androgen-independent LNCaP cells is high and unresponsive to androgen. This androgen-independent ETV1 expression contrasts with the hormone-dependent expression observed for TMPRSS2 in these androgen-independent prostate cancer cells. ETV1 is overexpressed in prostate cancer independent of the TMPRSS2:ETV1 translocation. Disruption of ETV1 expression in both androgen-dependent and androgen-independent prostate cancer cells significantly compromises the invasion capacity of these cells, suggesting an important role for ETV1 in prostate cancer metastasis. Collectively, these results demonstrate that ETV1 expression transitions from androgen-induced to androgen-independent as prostate cancer cells switch from hormone-dependent to hormone-refractory and suggest that this transition may be in part responsible for the elevated levels of ETV1 observed in prostate tumors. Additionally, our data provide an indirect mechanism of AR regulation of gene expression, via the transactivation of the transcription factor ETV1.