RESUMO
Androgen receptor splice variant 7 (AR-V7) is crucial for prostate cancer progression and therapeutic resistance. We show that, independent of ligand, AR-V7 binds both androgen-responsive elements (AREs) and non-canonical sites distinct from full-length AR (AR-FL) targets. Consequently, AR-V7 not only recapitulates AR-FL's partial functions but also regulates an additional gene expression program uniquely via binding to gene promoters rather than ARE enhancers. AR-V7 binding and AR-V7-mediated activation at these unique targets do not require FOXA1 but rely on ZFX and BRD4. Knockdown of ZFX or select unique targets of AR-V7/ZFX, or BRD4 inhibition, suppresses growth of castration-resistant prostate cancer cells. We also define an AR-V7 direct target gene signature that correlates with AR-V7 expression in primary tumors, differentiates metastatic prostate cancer from normal, and predicts poor prognosis. Thus, AR-V7 has both ARE/FOXA1 canonical and ZFX-directed non-canonical regulatory functions in the evolution of anti-androgen therapeutic resistance, providing information to guide effective therapeutic strategies.
Assuntos
Processamento Alternativo/genética , Carcinogênese/genética , Fatores de Transcrição Kruppel-Like/genética , Oncogenes/genética , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genéticaRESUMO
BACKGROUND AND AIMS: Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. APPROACH AND RESULTS: Fah-/- , Rag2-/- , and Il2rg-/- knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. CONCLUSIONS: LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.
Assuntos
Benzoatos/farmacocinética , Benzilaminas/farmacocinética , Colesterol/metabolismo , Hepatócitos/metabolismo , Lipoproteínas/metabolismo , Receptores X do Fígado/agonistas , Fígado/metabolismo , Animais , Hepatócitos/transplante , Humanos , Fígado/cirurgia , Masculino , Camundongos , Camundongos KnockoutRESUMO
The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1's effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1's inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT: The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.
Assuntos
Anticolesterolemiantes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Colesterol/biossíntese , Receptor Constitutivo de Androstano/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Ácidos Tricarboxílicos/farmacologia , Animais , Linhagem Celular , Citocromo P-450 CYP2B6/biossíntese , Citocromo P-450 CYP2D6/biossíntese , Citocromo P-450 CYP2D6/genética , Farneseno Álcool/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Pravastatina/farmacologia , RatosRESUMO
Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials. The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome. Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human-murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes in vivo. As predicted from preclinical and clinical studies, rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly-approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Xenoenxertos/metabolismo , Fígado/metabolismo , Transdução Genética/métodos , Transgenes/genética , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Ensaios Clínicos como Assunto , Dependovirus/isolamento & purificação , Modelos Animais de Doenças , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/transplante , Humanos , Fígado/citologia , Fígado/patologia , Masculino , Camundongos , Especificidade da EspécieRESUMO
BACKGROUND & AIMS: A major limitation in the field of liver transplantation is the shortage of transplantable organs. Chimeric animals carrying human tissue have the potential to solve this problem. However, currently available chimeric organs retain a high level of xenogeneic cells, and the transplantation of impure organs needs to be tested. METHODS: We created chimeric livers by injecting Lewis rat hepatocytes into C57Bl/6Fah-/-Rag2-/-Il2rg-/- mice, and further transplanted them into newly weaned Lewis rats (45⯱â¯3â¯g) with or without suboptimal immunosuppression (tacrolimus 0.6â¯mg/kg/day for 56 or 112â¯days). Control donors included wild-type C57Bl/6 mice (xenogeneic) and Lewis rats (syngeneic). RESULTS: Without immunosuppression, recipients of chimeric livers experienced acute rejection, and died within 8 to 11â¯days. With immunosuppression, they all survived for >112â¯days with normal weight gain compared to syngeneic controls, while all xenogeneic controls died within 98â¯days due to rejection with Banff scores >6 (pâ¯=â¯0.0014). The chimeric grafts underwent post-transplant remodelling, growing by 670% on average. Rat hepatocytes fully replaced mouse hepatocytes starting from day 56 (absence of detectable mouse serum albumin, histological clearance of mouse hepatocytes). In addition, rat albumin levels reached those of syngeneic recipients. Four months after transplantation of chimeric livers, we observed the development of diffuse mature rat bile ducts through transdifferentiation of hepatocytes (up to 72% of cholangiocytes), and patchy areas of portal endothelium originating from the host (seen in one out of five recipients). CONCLUSIONS: Taken together, these data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant recipient-oriented graft remodelling. Validation in a large animal model is still needed. LAY SUMMARY: Chimeric animals are composed of cells from different species. Chimeric animals carrying human tissue have the potential to increase the availability of transplantable organs. We transplanted rat-to-mouse liver grafts into newly weaned rats. The chimeric grafts underwent post-transplant remodelling with rat hepatocytes replacing all mouse hepatocytes within 56â¯days. In addition, we observed the post-transplant development of diffuse mature rat bile ducts through the transformation of hepatocytes, and patchy areas of portal endothelium originating from the host. These data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant graft remodelling.
Assuntos
Transplante de Fígado/métodos , Transplante Heterólogo/métodos , Animais , Quimera , Feminino , Rejeição de Enxerto , Hepatócitos/transplante , Imunossupressores/uso terapêutico , Transplante de Fígado/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Transplante Heterólogo/efeitos adversosRESUMO
Melanoma antigen-A11 (MAGE-A11) is an X-linked and primate-specific steroid hormone receptor transcriptional coregulator and proto-oncogenic protein whose increased expression promotes the growth of prostate cancer. The MAGEA11 gene is expressed at low levels in normal human testis, ovary, and endometrium, and at highest levels in castration-resistant prostate cancer. Annotated genome predictions throughout the surviving primate lineage show that MAGEA11 acquired three 5' coding exons unique within the MAGEA subfamily during the evolution of New World monkeys (NWM), Old World monkeys (OWM), and apes. MAGE-A11 in all primates has a conserved FXXIF coactivator-binding motif that suggests interaction with p160 coactivators contributed to its early evolution as a transcriptional coregulator. An ancestral form of MAGE-A11 in the more distantly related lemur has significant amino acid sequence identity with human MAGE-A11, but lacks coregulator activity based on the absence of the three 5' coding exons that include a nuclear localization signal (NLS). NWM MAGE-A11 has greater amino acid sequence identity than lemur to human MAGE-A11, but inframe premature stop codons suggest that MAGEA11 is a pseudogene in NWM. MAGE-A11 in OWM and apes has nearly identical 5' coding exon amino acid sequence and conserved interaction sites for p300 acetyltransferase and cyclin A. We conclude that the evolution of MAGEA11 within the lineage leading to OWM and apes resulted in steroid hormone receptor transcriptional coregulator activity through the acquisition of three 5' coding exons that include a NLS sequence and nonsynonymous substitutions required to interact with cell cycle regulatory proteins and transcription factors.
Assuntos
Antígenos de Neoplasias/genética , Evolução Molecular , Antígenos Específicos de Melanoma/genética , Proteínas de Neoplasias/genética , Filogenia , Primatas/genética , Sequência de Aminoácidos , Animais , Éxons , HumanosRESUMO
BACKGROUND: High affinity androgen binding to the androgen receptor (AR) activates genes required for male sex differentiation and promotes the development and progression of prostate cancer. Human AR transcriptional activity involves interactions with coregulatory proteins that include primate-specific melanoma antigen-A11 (MAGE-A11), a coactivator that increases AR transcriptional activity during prostate cancer progression to castration-resistant/recurrent prostate cancer (CRPC). METHODS: Microarray analysis and quantitative RT-PCR were performed to identify androgen-regulated MAGE-A11-dependent genes in LAPC-4 prostate cancer cells after lentivirus shRNA knockdown of MAGE-A11. Chromatin immunoprecipitation was used to assess androgen-dependent AR recruitment, and immunocytochemistry to localize an androgen-dependent protein in prostate cancer cells and tissue and in the CWR22 human prostate cancer xenograft. RESULTS: Microarray analysis of androgen-treated LAPC-4 prostate cancer cells indicated follistatin-like 1 (FSTL1) is up-regulated by MAGE-A11. Androgen-dependent up-regulation of FSTL1 was inhibited in LAPC-4 cells by lentivirus shRNA knockdown of AR or MAGE-A11. Chromatin immunoprecipitation demonstrated AR recruitment to intron 10 of the FSTL1 gene that contains a classical consensus androgen response element. Increased levels of FSTL1 protein in LAPC-4 cells correlated with higher levels of MAGE-A11 relative to other prostate cancer cells. FSTL1 mRNA levels increased in CRPC and castration-recurrent CWR22 xenografts in association with predominantly nuclear FSTL1. Increased nuclear localization of FSTL1 in prostate cancer was suggested by predominantly cytoplasmic FSTL1 in benign prostate epithelial cells and predominantly nuclear FSTL1 in epithelial cells in CRPC tissue and the castration-recurrent CWR22 xenograft. AR expression studies showed nuclear colocalization of AR and endogenous FSTL1 in response to androgen. CONCLUSION: AR and MAGE-A11 cooperate in the up-regulation of FSTL1 to promote growth and progression of CRPC. Prostate 77:505-516, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Antígenos de Neoplasias/biossíntese , Proteínas Relacionadas à Folistatina/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/biossíntese , Regulação para Cima/fisiologia , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Masculino , Análise em Microsséries/métodos , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
X-linked primate-specific melanoma antigen-A11 (MAGE-A11) is a human androgen receptor (AR) coactivator and proto-oncogene expressed at low levels in normal human reproductive tract tissues and at higher levels in castration-resistant prostate cancer where it is required for androgen-dependent cell growth. In this report, we show that MAGE-A11 is targeted for degradation by human p14-ARF, a tumor suppressor expressed from an alternative reading frame of the p16 cyclin-dependent kinase inhibitor INK4a/ARF gene. MAGE-A11 degradation by the proteasome was mediated by an interaction with p14-ARF and was independent of lysine ubiquitination. A dose-dependent inverse relationship between MAGE-A11 and p14-ARF correlated with p14-ARF inhibition of the MAGE-A11-induced increase in androgen-dependent AR transcriptional activity and constitutive activity of a splice variant-like AR. Reciprocal stabilization between MAGE-A11 and AR did not protect against degradation promoted by p14-ARF. p14-ARF prevented MAGE-A11 interaction with the E2F1 oncoprotein and inhibited the MAGE-A11-induced increase in E2F1 transcriptional activity. Post-translational down-regulation of MAGE-A11 promoted by p14-ARF was independent of HDM2, the human homologue of mouse double minute 2, an E3 ubiquitin ligase inhibited by p14-ARF. However, MAGE-A11 had a stabilizing effect on HDM2 in the absence or presence of p14-ARF and cooperated with HDM2 to increase E2F1 transcriptional activity in the absence of p14-ARF. We conclude that degradation of MAGE-A11 promoted by the human p14-ARF tumor suppressor contributes to low levels of MAGE-A11 in nontransformed cells and that higher levels of MAGE-A11 associated with low p14-ARF increase AR and E2F1 transcriptional activity and promote the development of castration-resistant prostate cancer.
Assuntos
Antígenos de Neoplasias/metabolismo , Regulação para Baixo , Proteínas de Neoplasias/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Fator de Transcrição E2F1/metabolismo , Humanos , Masculino , Fenótipo , Neoplasias de Próstata Resistentes à Castração/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proto-Oncogene Mas , Transcrição GênicaRESUMO
BACKGROUND: The previously established CWR-R1 cell line has been used as an in vitro model representing castration-recurrent prostate cancer. Microscopic observation of subconfluent cells demonstrated two distinct cellular morphologies: polygonal closely aggregated epithelial cells surrounded by bipolar fibroblastic cells with long processes. This study sought to establish and characterize a fibroblast-free derivative of the CWR-R1 cell line. METHODS: The CWR-R1ca cell line was established from CWR-R1 cells by removing fibroblasts using multiple cycles of short-term trypsinization, cloning, and pooling single-cell colonies. Authentication of fibroblast-free CWR-R1ca cells was demonstrated by analyzing the expression of cytodifferentiation and prostate-associated markers, DNA and cytogenetic profiling, and growth pattern in the absence or presence of androgen. RESULTS: CWR-R1ca is an androgen-sensitive cell line that expresses the androgen receptor (AR) and its splice variant 7 and the luminal epithelia markers, CK-8, CK-18, and c-Met. CWR-R1fb fibroblasts isolated from CWR-R1 cells express AR, hepatocyte growth factor-α, and mouse ß-actin but not AR-V7 or epithelial markers. Cytogenetic analysis of CWR-R1ca cells revealed a hyperdiploid male with numerical gains in chromosomes 1, 7, 8, 10, 11, and 12, deletion of one chromosome 2 allele, structural abnormalities that include der(1)t(1:4), der(4)t(2:4), der(10)t(4:10), and an unbalanced reciprocal translocation between chromosome 6 and 14. DNA-profiling revealed that CWR-R1ca cells had significant short-tandem repeat marker homology with CWR22Pc and CWR22Rv1 cell lines, which indicated lineage derivation from CWR22 prostate cancer xenografts. CWR-R1ca cells were responsive to the growth stimulatory effects of dihydrotestosterone (DHT) in the femtomolar range. CONCLUSION: This study establishes CWR-R1ca cells as a fibroblast-free derivative of the castration-recurrent CWR-R1 cell line. Prostate 76:1067-1077, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Fibroblastos/citologia , Neoplasias de Próstata Resistentes à Castração/patologia , Actinas/análise , Animais , Biomarcadores/análise , Linhagem Celular Tumoral , Separação Celular/métodos , Aberrações Cromossômicas , Deleção Cromossômica , Impressões Digitais de DNA , Expressão Gênica , Fator de Crescimento de Hepatócito/análise , Xenoenxertos , Humanos , Cariotipagem , Masculino , Camundongos , Células NIH 3T3 , Transplante de Neoplasias , Próstata/química , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Isoformas de Proteínas/análise , Receptores Androgênicos/análise , Receptores Androgênicos/genéticaRESUMO
Melanoma antigen-A11 (MAGE-A11) is a low-abundance, primate-specific steroid receptor coregulator in normal tissues of the human reproductive tract that is expressed at higher levels in prostate cancer. Increased expression of MAGE-A11 enhances androgen receptor transcriptional activity and promotes prostate cancer cell growth. Further investigation into the mechanisms of MAGE-A11 function in prostate cancer demonstrated interactions with the retinoblastoma-related protein p107 and Rb tumor suppressor but no interaction with p130 of the Rb family. MAGE-A11 interaction with p107 was associated with transcriptional repression in cells with low MAGE-A11 and transcriptional activation in cells with higher MAGE-A11. Selective interaction of MAGE-A11 with retinoblastoma family members suggested the regulation of E2F transcription factors. MAGE-A11 stabilized p107 by inhibition of ubiquitination and linked p107 to hypophosphorylated E2F1 in association with the stabilization and activation of E2F1. The androgen receptor and MAGE-A11 modulated endogenous expression of the E2F1-regulated cyclin-dependent kinase inhibitor p27(Kip1). The ability of MAGE-A11 to increase E2F1 transcriptional activity was similar to the activity of adenovirus early oncoprotein E1A and depended on MAGE-A11 interactions with p107 and p300. The immunoreactivity of p107 and MAGE-A11 was greater in advanced prostate cancer than in benign prostate, and knockdown with small inhibitory RNA showed that p107 is a transcriptional activator in prostate cancer cells. These results suggest that MAGE-A11 is a proto-oncogene whose increased expression in prostate cancer reverses retinoblastoma-related protein p107 from a transcriptional repressor to a transcriptional activator of the androgen receptor and E2F1.
Assuntos
Antígenos de Neoplasias/metabolismo , Fator de Transcrição E2F1/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Proteína p107 Retinoblastoma-Like/metabolismo , Transcrição Gênica , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animais , Antígenos de Neoplasias/genética , Células COS , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Fator de Transcrição E2F1/genética , Células HeLa , Humanos , Masculino , Proteínas de Neoplasias/genética , Fosforilação/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Estabilidade Proteica , Proto-Oncogene Mas , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Proteína p107 Retinoblastoma-Like/genéticaRESUMO
Prostate cancer growth and progression depend on androgen receptor (AR) signaling through transcriptional mechanisms that require interactions with coregulatory proteins, one of which is the primate-specific steroid receptor coregulator melanoma antigen-A11 (MAGE-A11). In this report, we provide evidence how increased expression of MAGE-A11 during prostate cancer progression enhances AR signaling and prostate cancer growth. MAGE-A11 protein levels were highest in castration-recurrent prostate cancer. The cyclic AMP-induced increase in androgen-dependent and androgen-independent AR transcriptional activity correlated with an increase in MAGE-A11 and was inhibited by silencing MAGE-A11 expression. MAGE-A11 mediated synergistic AR transcriptional activity in LAPC-4 prostate cancer cells. The ability of MAGE-A11 to rescue transcriptional activity of complementary inactive AR mutants and promote coimmunoprecipitation between unlike forms of AR suggests that MAGE-A11 links transcriptionally active AR dimers. A model for the AR·MAGE-A11 multidimeric complex is proposed in which one AR FXXLF motif of the AR dimer engages in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction, whereas the second FXXLF motif region of the AR dimer interacts with dimeric MAGE-A11. The AR·MAGE-A11 multidimeric complex accounts for the dual functions of the AR FXXLF motif in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction and binding MAGE-A11 and for synergy between reported AR splice variants and full-length AR. We conclude that the increased expression of MAGE-A11 in castration-recurrent prostate cancer, which is enhanced by cyclic AMP signaling, increases AR-dependent growth of prostate cancer by MAGE-A11 forming a molecular bridge between transcriptionally active AR dimers.
Assuntos
Antígenos de Neoplasias/genética , Melanoma/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Ativação Transcricional , Motivos de Aminoácidos , Animais , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Células COS , Chlorocebus aethiops , AMP Cíclico/metabolismo , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Neoplasias/metabolismo , Plasmídeos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Multimerização Proteica , Receptores Androgênicos/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Androgênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores Androgênicos/genética , Antagonistas de Receptores de Andrógenos/metabolismo , Androgênios/metabolismo , Anilidas/farmacologia , Ligação Competitiva , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Indóis/metabolismo , Indóis/farmacologia , Luciferases/genética , Luciferases/metabolismo , Masculino , Metribolona/metabolismo , Metribolona/farmacologia , Microscopia de Fluorescência , Nitrilas/farmacologia , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Bibliotecas de Moléculas Pequenas , Compostos de Tosil/farmacologiaRESUMO
Naturally occurring germ line mutations in the X-linked human androgen receptor (AR) gene cause incomplete masculinization of the external genitalia by disrupting AR function in males with androgen insensitivity syndrome. Almost all AR missense mutations that cause androgen insensitivity syndrome are located in the highly structured DNA and ligand binding domains. In this report we investigate the functional defect associated with an AR exon 1 missense mutation, R405S, that caused partial androgen insensitivity. The 46,XX heterozygous maternal carrier had a wild-type Arg-405 CGC allele but transmitted an AGC mutant allele coding for Ser-405. At birth, the 46,XY proband had a bifid scrotum, hypospadias, and micropenis consistent with clinical stage 3 partial androgen insensitivity. Androgen-dependent transcriptional activity of AR-R405S expressed in CV1 cells was less than wild-type AR and refractory in androgen-dependent AR NH(2)- and carboxyl interaction transcription assays that depend on the coregulator effects of melanoma antigen-A11. This mutation created a Ser-405 phosphorylation site evident by the gel migration of an AR-R405S NH(2)-terminal fragment as a double band that converted to the wild-type single band after treatment with λ-phosphatase. Detrimental effects of the R405S mutation were related to the proximity of the AR WXXLF motif (433)WHTLF(437) required for melanoma antigen-A11 and p300 to stimulate transcriptional activity associated with the AR NH(2)- and carboxyl-terminal interaction. We conclude that the coregulator effects of melanoma antigen-A11 on the AR NH(2)- and carboxyl-terminal interaction amplify the androgen-dependent transcriptional response to p300 required for normal human male sex development in utero.
Assuntos
Síndrome de Resistência a Andrógenos/genética , Antígenos de Neoplasias/metabolismo , Éxons/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Ativação Transcricional/genética , Motivos de Aminoácidos , Síndrome de Resistência a Andrógenos/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína p300 Associada a E1A/metabolismo , Feminino , Humanos , Masculino , Fenótipo , Fosforilação/genética , Receptores Androgênicos/genéticaRESUMO
Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH(2)-terminal (110)LLXXVLXXLL(119) motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium.
Assuntos
Antígenos de Neoplasias/metabolismo , Endométrio/metabolismo , Proteínas de Neoplasias/metabolismo , Gravidez/fisiologia , Receptores de Progesterona/metabolismo , Proteínas de Ligação a Tacrolimo/biossíntese , Ativação Transcricional/fisiologia , Adolescente , Adulto , Motivos de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação para Baixo/fisiologia , Implantação do Embrião/fisiologia , Endométrio/citologia , Feminino , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Multimerização Proteica/fisiologia , Estrutura Terciária de Proteína , Receptores de Progesterona/genética , Elementos de Resposta/fisiologia , Proteínas de Ligação a Tacrolimo/genética , Regulação para Cima/fisiologia , Proteínas ras/biossíntese , Proteínas ras/genéticaRESUMO
Murine hepatocytes become polyploid and then undergo ploidy reversal and become aneuploid in a dynamic process called the ploidy conveyor. Although polyploidization occurs in some types of human cells, the degree of aneuploidy in human hepatocytes is not known. We isolated hepatocytes derived from healthy human liver samples and determined chromosome number and identity using traditional karyotyping and fluorescence in situ hybridization. Similar to murine hepatocytes, human hepatocytes are highly aneuploid. Moreover, imaging studies revealed multipolar spindles and chromosome segregation defects in dividing human hepatocytes. Aneuploidy therefore does not necessarily predispose liver cells to transformation but might promote genetic diversity among hepatocytes.
Assuntos
Aneuploidia , Cromossomos Humanos , Variação Genética , Hepatócitos/patologia , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Feminino , Hepatócitos/transplante , Humanos , Hidrolases/deficiência , Hidrolases/genética , Hibridização in Situ Fluorescente , Lactente , Cariotipagem , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Quimeras de Transplante , Adulto JovemRESUMO
The synchronization of circadian clock depends on a central pacemaker located in the suprachiasmatic nuclei. However, the potential feedback of peripheral signals on the central clock remains poorly characterized. To explore whether peripheral organ circadian clocks may affect the central pacemaker, we used a chimeric model in which mouse hepatocytes were replaced by human hepatocytes. Liver humanization led to reprogrammed diurnal gene expression and advanced the phase of the liver circadian clock that extended to muscle and the entire rhythmic physiology. Similar to clock-deficient mice, liver-humanized mice shifted their rhythmic physiology more rapidly to the light phase under day feeding. Our results indicate that hepatocyte clocks can affect the central pacemaker and offer potential perspectives to apprehend pathologies associated with altered circadian physiology.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Humanos , Camundongos , Animais , Ritmo Circadiano/genética , Fígado/metabolismo , Hepatócitos , Relógios Circadianos/genética , Núcleo Supraquiasmático/metabolismoRESUMO
Male sex development and growth occur in response to high affinity androgen binding to the androgen receptor (AR). In contrast to complete amino acid sequence conservation in the AR DNA and ligand binding domains among mammals, a primate-specific difference in the AR NH(2)-terminal region that regulates the NH(2)- and carboxyl-terminal (N/C) interaction enables direct binding to melanoma antigen-A11 (MAGE-11), an AR coregulator that is also primate-specific. Human, mouse, and rat AR share the same NH(2)-terminal (23)FQNLF(27) sequence that mediates the androgen-dependent N/C interaction. However, the mouse and rat AR FXXLF motif is flanked by Ala(33) that evolved to Val(33) in primates. Human AR Val(33) was required to interact directly with MAGE-11 and for the inhibitory effect of the AR N/C interaction on activation function 2 that was relieved by MAGE-11. The functional importance of MAGE-11 was indicated by decreased human AR regulation of an androgen-dependent endogenous gene using lentivirus short hairpin RNAs and by the greater transcriptional strength of human compared with mouse AR. MAGE-11 increased progesterone and glucocorticoid receptor activity independently of binding an FXXLF motif by interacting with p300 and p160 coactivators. We conclude that the coevolution of the AR NH(2)-terminal sequence and MAGE-11 expression among primates provides increased regulatory control over activation domain dominance. Primate-specific expression of MAGE-11 results in greater steroid receptor transcriptional activity through direct interactions with the human AR FXXLF motif region and indirectly through steroid receptor-associated p300 and p160 coactivators.
Assuntos
Antígenos de Neoplasias/metabolismo , Evolução Molecular , Regulação da Expressão Gênica/fisiologia , Proteínas de Neoplasias/metabolismo , Receptores Androgênicos/metabolismo , Transcrição Gênica/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Masculino , Camundongos , Proteínas de Neoplasias/genética , Estrutura Terciária de Proteína , Ratos , Receptores Androgênicos/genética , Especificidade da Espécie , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Androgen receptor (AR)-mediated gene regulation involves interactions with coregulatory proteins that include the melanoma antigen gene protein-A11 (MAGE-11). To understand the functional significance of sequence similarity between MAGE-11 and the adenovirus early protein E1A, we determined whether MAGE-11 contributes to AR transcriptional activity through an interaction with p300, a potent and ubiquitous transcriptional regulator. Here, we report that MAGE-11 interacts with the NH(2)-terminal region of p300 through the MAGE-11 MXXIF motif (185)MXXIF(189), with transcriptional activity depending on the MAGE-11 F-box and MAPK phosphorylation. The MAGE-11- and p300-dependent increase in AR transactivation required the NH(2)-terminal regions of AR and p300, p300 acetyltransferase activity, and the AR FXXLF motif (23)FQNLF(27) interaction with MAGE-11. MAGE-11 linked AR to p300 and the p160 coactivator, transcriptional intermediary protein 2 (TIF2). The p300 NH(2)-terminal FXXLF motif (33)FGSLF(37) was required for transcriptional activation by TIF2. Increased expression of p300 decreased the ubiquitinylation of MAGE-11 and transiently increased endogenous MAGE-11 levels. Autoacetylation of p300 and decreased acetylation of TIF2 were evident in the MAGE-11, p300, and TIF2 complex. The studies suggest that MAGE-11 links NH(2)-terminal domains of AR and p300 to promote transcriptional synergy through a cadre of FXXLF-related interacting motifs.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteína p300 Associada a E1A/metabolismo , Proteínas de Neoplasias/metabolismo , Motivos de Aminoácidos , Androgênios/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Modelos Biológicos , Fosforilação , Receptores Androgênicos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Polyglutamine expansion within the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA) and is associated with misfolded and aggregated species of the mutant AR. We showed previously that nuclear localization of the mutant AR was necessary but not sufficient for SBMA. Here we show that an interdomain interaction of the AR that is central to its function within the nucleus is required for AR aggregation and toxicity. Ligands that prevent the interaction between the amino-terminal FXXLF motif and carboxyl-terminal AF-2 domain (N/C interaction) prevented toxicity and AR aggregation in an SBMA cell model and rescued primary SBMA motor neurons from 5α-dihydrotestosterone-induced toxicity. Moreover, genetic mutation of the FXXLF motif prevented AR aggregation and 5α-dihydrotestosterone toxicity. Finally, selective androgen receptor modulators, which prevent the N/C interaction, ameliorated AR aggregation and toxicity while maintaining AR function, highlighting a novel therapeutic strategy to prevent the SBMA phenotype while retaining AR transcriptional function.