RESUMEN
N6-methyladenosine (m6A) of mRNAs modulated by the METTL3-METTL14-WTAP-RBM15 methyltransferase complex and m6A demethylases such as FTO play important roles in regulating mRNA stability, splicing, and translation. Here, we demonstrate that FTO-IT1 long noncoding RNA (lncRNA) was upregulated and positively correlated with poor survival of patients with wild-type p53-expressing prostate cancer (PCa). m6A RIP-seq analysis revealed that FTO-IT1 knockout increased mRNA m6A methylation of a subset of p53 transcriptional target genes (e.g., FAS, TP53INP1, and SESN2) and induced PCa cell cycle arrest and apoptosis. We further showed that FTO-IT1 directly binds RBM15 and inhibits RBM15 binding, m6A methylation, and stability of p53 target mRNAs. Therapeutic depletion of FTO-IT1 restored mRNA m6A level and expression of p53 target genes and inhibited PCa growth in mice. Our study identifies FTO-IT1 lncRNA as a bona fide suppressor of the m6A methyltransferase complex and p53 tumor suppression signaling and nominates FTO-IT1 as a potential therapeutic target of cancer.
Asunto(s)
Neoplasias , ARN Largo no Codificante , Masculino , Ratones , Animales , ARN Largo no Codificante/genética , Proteína p53 Supresora de Tumor/genética , Adenosina/metabolismo , ARN Mensajero/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismoRESUMEN
TMPRSS2-ERG gene fusion occurs in approximately 50% of cases of prostate cancer (PCa), and the fusion product is a key driver of prostate oncogenesis. However, how to leverage cellular signaling to ablate TMPRSS2-ERG oncoprotein for PCa treatment remains elusive. Here, we demonstrate that DNA damage induces proteasomal degradation of wild-type ERG and TMPRSS2-ERG oncoprotein through ERG threonine-187 and tyrosine-190 phosphorylation mediated by GSK3ß and WEE1, respectively. The dual phosphorylation triggers ERG recognition and degradation by the E3 ubiquitin ligase FBW7 in a manner independent of a canonical degron. DNA damage-induced TMPRSS2-ERG degradation was abolished by cancer-associated PTEN deletion or GSK3ß inactivation. Blockade of DNA damage-induced TMPRSS2-ERG oncoprotein degradation causes chemotherapy-resistant growth of fusion-positive PCa cells in culture and in mice. Our findings uncover a previously unrecognized TMPRSS2-ERG protein destruction mechanism and demonstrate that intact PTEN and GSK3ß signaling are essential for effective targeting of ERG protein by genotoxic therapeutics in fusion-positive PCa.
Asunto(s)
Proteínas de Ciclo Celular/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Proteínas de Fusión Oncogénica/genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/genética , Proteínas Tirosina Quinasas/genética , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Quimioterapia , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Xenoinjertos , Humanos , Masculino , Ratones , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
In light of the increasing number of identified cancer-driven gain-of-function (GOF) mutants of p53, it is important to define a common mechanism to systematically target several mutants, rather than developing strategies tailored to inhibit each mutant individually. Here, using RNA immunoprecipitation-sequencing (RIP-seq), we identified the Polycomb-group histone methyltransferase EZH2 as a p53 mRNA-binding protein. EZH2 bound to an internal ribosome entry site (IRES) in the 5'UTR of p53 mRNA and enhanced p53 protein translation in a methyltransferase-independent manner. EZH2 augmented p53 GOF mutant-mediated cancer growth and metastasis by increasing protein levels of mutant p53. EZH2 overexpression was associated with worsened outcome selectively in patients with p53-mutated cancer. Depletion of EZH2 by antisense oligonucleotides inhibited p53 GOF mutant-mediated cancer growth. Our findings reveal a non-methyltransferase function of EZH2 that controls protein translation of p53 GOF mutants, inhibition of which causes synthetic lethality in cancer cells expressing p53 GOF mutants.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Mutación con Ganancia de Función , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/patología , ARN Mensajero/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Proliferación Celular , Proteína Potenciadora del Homólogo Zeste 2/genética , Humanos , Sitios Internos de Entrada al Ribosoma , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Estabilidad Proteica , ARN Mensajero/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SPOP mutations and TMPRSS2-ERG rearrangements occur collectively in up to 65% of human prostate cancers. Although the two events are mutually exclusive, it is unclear whether they are functionally interrelated. Here, we demonstrate that SPOP, functioning as an E3 ubiquitin ligase substrate-binding protein, promotes ubiquitination and proteasome degradation of wild-type ERG by recognizing a degron motif at the N terminus of ERG. Prostate cancer-associated SPOP mutations abrogate the SPOP-mediated degradation function on the ERG oncoprotein. Conversely, the majority of TMPRSS2-ERG fusions encode N-terminal-truncated ERG proteins that are resistant to the SPOP-mediated degradation because of degron impairment. Our findings reveal degradation resistance as a previously uncharacterized mechanism that contributes to elevation of truncated ERG proteins in prostate cancer. They also suggest that overcoming ERG resistance to SPOP-mediated degradation represents a viable strategy for treatment of prostate cancers expressing either mutated SPOP or truncated ERG.
Asunto(s)
Proteínas Nucleares/fisiología , Proteínas de Fusión Oncogénica/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Represoras/fisiología , Transactivadores/fisiología , Secuencia de Aminoácidos , Proliferación Celular , Puntos de Rotura del Cromosoma , Células HEK293 , Humanos , Masculino , Fragmentos de Péptidos/fisiología , Neoplasias de la Próstata/metabolismo , Unión Proteica , Proteolisis , Regulador Transcripcional ERG , UbiquitinaciónRESUMEN
Nuclear FOXO proteins act as tumor suppressors by transcriptionally activating genes involved in apoptosis and cell cycle arrest, and these anticancer functions are inhibited by AKT-induced phosphorylation and cytoplasmic sequestration of FOXOs. We found that, after AKT-mediated phosphorylation at serine 319, FOXO1 binds to IQGAP1, a hub for activation of the MAPK pathway, and impedes IQGAP1-dependent phosphorylation of ERK1/2 (pERK1/2). Conversely, decreased FOXO1 expression increases pERK1/2 in cancer cell lines and correlates with increased pERK1/2 levels in patient specimens and disease progression. Treatment of cancer cells with PI3K inhibitors or taxane causes FOXO1 localization in the nucleus, increased expression of pERK1/2, and drug resistance. These effects are reversed by administering a small FOXO1-derived phospho-mimicking peptide inhibitor in vitro and in mice. Our results show a tumor suppressor role of AKT-phosphorylated FOXO1 in the cytoplasm and suggest that this function of FOXO1 can be harnessed to overcome chemoresistance in cancer.
Asunto(s)
Resistencia a Antineoplásicos , Proteína Forkhead Box O1/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Hidrocarburos Aromáticos con Puentes/farmacología , Línea Celular Tumoral , Proteína Forkhead Box O1/genética , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/genética , Taxoides/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
BACKGROUND: Aberrant activation of the androgen receptor (AR) is a major factor highly relevant to castration-resistant progression of prostate cancer (PCa). FOXO1, a key downstream effector of PTEN, inhibits androgen-independent activation of the AR. However, the underlying mechanism remains elusive. METHODS: The inhibitory effect of FOXO1 on full-length and constitutively active splice variants of the AR was examined by luciferase reporter assays and real-time reverse transcription polymerase chain reaction (RT-qPCR). In vitro protein binding assays and western blot analyses were used to determine the regions in FOXO1 and AR responsible for their interaction. RESULTS: We found that a putative transcription repression domain in the NH2-terminus of FOXO1 is dispensable for FOXO1 inhibition of the AR. In vitro protein binding assays showed that FOXO1 binds to the transcription activation unit 5 (TAU5) motif in the AR NH2-terminal domain (NTD), a region required for recruitment of p160 activators including SRC-1. Ectopic expression of SRC-1 augmented transcriptional activity of some, but not all AR splice variants examined. Forced expression of FOXO1 blocked the effect of SRC-1 on AR variants' transcriptional activity by decreasing the binding of SRC-1 to the AR NTD. Ectopic expression of FOXO1 inhibited expression of endogenous genes activated primarily by alternatively spliced AR variants in human castration-resistant PCa 22Rv1 cells. CONCLUSIONS: FOXO1 binds to the TAU5 motif in the AR NTD and inhibits ligand-independent activation of AR splice variants, suggesting the PTEN/FOXO1 pathway as a potential therapeutic target for inhibition of aberrant AR activation and castration-resistant PCa growth.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal/fisiología , Empalme Alternativo , Línea Celular Tumoral , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Humanos , Masculino , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Próstata/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Androgénicos/genética , Transcripción Genética/fisiología , Activación TranscripcionalRESUMEN
Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and ß-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. ß-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals ß-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.
Asunto(s)
Neoplasias de la Próstata , Regulador Transcripcional ERG , Proteína p53 Supresora de Tumor , Animales , Humanos , Masculino , Ratones , beta Catenina/genética , beta Catenina/metabolismo , Mutación con Ganancia de Función , Proteínas de Fusión Oncogénica/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proto-Oncogenes , Pirimidinas/biosíntesis , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Retinoblastoma (RB) protein can exert tumor suppressor functions even when it becomes phosphorylated. It is thus essential to understand how phosphorylated RB (p-RB) expression and function are regulated. Here, we demonstrated that RING finger domain protein TRIM28 bound and promoted ubiquitination and degradation of CDK4/6-phosphorylated RB protein. SETDB1, a known TRIM28 binding partner, protected p-RB from degradation through the binding of methylated RB by its Tudor domain independent of its methyltransferase activity. SETDB1 was found to be frequently overexpressed due to gene amplification and positively correlated with p-RB in prostate cancer patient specimens. Inhibition of SETDB1 expression using a gene-specific antisense oligonucleotide (ASO) reduced tumor growth but accelerated RB protein degradation, limiting the therapeutic efficacy. However, coadministration of the CDK4/6 inhibitor palbociclib blocked ASO-induced RB degradation and resulted in a much greater cancer-inhibitory effect than each inhibitor alone both in vitro and in vivo. This study identified CDK4/6-dependent, TRIM28-mediated proteasomal degradation as a mechanism of RB inactivation and reveals SETDB1 as a key inhibitor of this process. Our findings suggest that combined targeting of SETDB1 and CDK4/6 represents a viable approach for the treatment of cancers with SETDB1 gene amplification or overexpression. SIGNIFICANCE: The identification of a role for TRIM28 and SETDB1 in regulating CDK4/6-phosphorylated RB stability uncovers a combination strategy using CDK4/6 and SETDB1 inhibition to decrease RB degradation and inhibit cancer growth.
Asunto(s)
Neoplasias , Humanos , Masculino , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Fosforilación , Proteína de Retinoblastoma/genéticaRESUMEN
53BP1 promotes nonhomologous end joining (NHEJ) over homologous recombination (HR) repair by mediating inactivation of DNA end resection. Ubiquitination plays an important role in regulating dissociation of 53BP1 from DNA double-strand breaks (DSBs). However, how this process is regulated remains poorly understood. Here, we demonstrate that TRABID deubiquitinase binds to 53BP1 at endogenous level and regulates 53BP1 retention at DSB sites. TRABID deubiquitinates K29-linked polyubiquitination of 53BP1 mediated by E3 ubiquitin ligase SPOP and prevents 53BP1 dissociation from DSBs, consequently inducing HR defects and chromosomal instability. Prostate cancer cells with TRABID overexpression exhibit a high sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. Our work shows that TRABID facilitates NHEJ repair over HR during DNA repair by inducing prolonged 53BP1 retention at DSB sites, suggesting that TRABID overexpression may predict HR deficiency and the potential therapeutic use of PARP inhibitors in prostate cancer.
Asunto(s)
Inhibidores de Poli(ADP-Ribosa) Polimerasas , Neoplasias de la Próstata , Masculino , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Mutaciones Letales Sintéticas , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Reparación del ADN , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reparación del ADN por Unión de Extremidades , ADN/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismoRESUMEN
BACKGROUND: Androgens control homeostasis of the normal prostate and growth of prostate cancer (PCa) through the androgen receptor (AR) by regulating gene networks involving in cell proliferation, differentiation, and survival. We demonstrated previously that expression of Skp2, a key protein regulating cell entry into the S phase, is inhibited by androgens in an AR-dependent manner (Oncogene, 2004; 23(12): 2161-2176). However, the underlying mechanism of this regulation is unknown. METHODS: Using the LNCaP PCa cell line as a working model, the effect of androgens on the expression of Skp2 was examined by Western and Northern blot analyses. Cell cycle was measured by fluorescence-activated cell sorting (FACS). Gene transfection was performed by electroporation to manipulate the expression levels of proteins studied. RESULTS: At physiological levels androgens markedly repressed Skp2 expression but slightly induced Skp2 expression at subphysiological levels. Androgens modestly decreased the stability of the Skp2 protein. Androgenic repression of Skp2 expression was completely abolished by E1A-mediated inactivation of pocket proteins including RB, p130, and p107. Moreover, ectopic expression of p107 inhibited Skp2 expression, and silencing of p107 partially blocked androgenic repression of Skp2. CONCLUSIONS: Our data indicate that androgens repress Skp2 expression via p107-dependent and -independent pathways in PCa cells. These regulatory mechanisms may be targeted for the development of new therapeutics of androgen-refractory PCa.
Asunto(s)
Ciclo Celular/fisiología , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Proteína p107 Similar a la del Retinoblastoma/metabolismo , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Northern Blotting , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Masculino , Metribolona/farmacología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Congéneres de la Testosterona/farmacologíaRESUMEN
In this study, we identified 3-aminophthalic acid as a new ligand of cereblon (CRBN) E3 ubiquitin ligase and developed a phthalic acid-based O'PROTAC for degradation of the ERG transcription factor. This phthalic acid-based O'PROTAC presented an efficacy in degrading ERG comparable to those displayed by pomalidomide-based ERG O'PROTACs. Moreover, phthalic acid-being more chemically stable and economical than classical immunomodulatory drugs (IMiDs)-represents, as a ligand, a new alternative for the development of PROTACs, especially O'PROTACs.
Asunto(s)
Ácidos Ftálicos/farmacología , Factores de Transcripción/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Estructura Molecular , Ácidos Ftálicos/química , Proteolisis/efectos de los fármacos , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Molecular mechanisms underlying intratumoral androgenesis and aberrant androgen receptor (AR) activation in prostate cancer remain poorly understood. Here we demonstrate that ectopic expression of the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger domain protein (SPOP) stabilizes 17ßHSD4. SPOP bound a functional substrate-binding consensus (SBC) motif 315RATST319 in 17ßHSD4 and promoted nondegradable K27- and K29-linked polyubiquitination of 17ßHSD4. The effect of SPOP was antagonized by serum- and glucocorticoid kinase-3 (SGK3)-mediated phosphorylation of serine 318 (S318) in the SBC and S318 phosphorylation-dependent binding of SKP2 E3 ligase and subsequent K48-linked polyubiquitination and proteasomal degradation of 17ßHSD4. Prostate cancer-associated SPOP mutations impaired the SPOP-17ßHSD4 interaction, caused 17ßHSD4 protein destruction in prostate cancer cells in culture and patient specimens, and increased testosterone production and prostate cancer cell growth in vitro and in mouse models. Thus, we have identified SPOP and SKP2 as two essential E3 ubiquitin ligases that exert opposite effects on 17ßHSD4 protein degradation and intratumoral androgenesis in prostate cancer cells. We further demonstrate that SPOP mutations or SKP2 overexpression contribute to prostate cancer progression by decreasing 17ßHSD4 expression and increasing intratumoral androgen synthesis. SIGNIFICANCE: This study reveals a novel mechanism of aberrant AR activation in SPOP-mutated prostate cancer and uncovers putative biomarkers for effective treatment by AR-targeted therapies.
Asunto(s)
Andrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica , Mutación , Proteínas Nucleares/metabolismo , Proteína-2 Multifuncional Peroxisomal/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismo , Proteínas Represoras/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Humanos , Masculino , Ratones , Ratones SCID , Proteínas Nucleares/genética , Proteína-2 Multifuncional Peroxisomal/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteolisis , Receptores Androgénicos/genética , Proteínas Represoras/genética , Células Tumorales Cultivadas , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DNA-binding proteins, including transcription factors (TFs), play essential roles in various cellular processes and pathogenesis of diseases, deeming to be potential therapeutic targets. However, these proteins are generally considered undruggable as they lack an enzymatic catalytic site or a ligand-binding pocket. Proteolysis-targeting chimera (PROTAC) technology has been developed by engineering a bifunctional molecule chimera to bring a protein of interest (POI) to the proximity of an E3 ubiquitin ligase, thus inducing the ubiquitination of POI and further degradation through the proteasome pathway. Here, the development of oligonucleotide-based PROTAC (O'PROTACs), a class of noncanonical PROTACs in which a TF-recognizing double-stranded oligonucleotide is incorporated as a binding moiety of POI is reported. It is demonstrated that O'PROTACs of lymphoid enhancer-binding factor 1 (LEF1) and ETS-related gene (ERG), two highly cancer-related transcription factors, successfully promote degradation of these proteins, impede their transcriptional activity, and inhibit cancer cell growth in vitro and in vivo. The programmable nature of O'PROTACs indicates that this approach is also applicable to destruct other TFs. O'PROTACs not only can serve as a research tool but also can be harnessed as a therapeutic arsenal to target DNA binding proteins for effective treatment of diseases such as cancer.
Asunto(s)
Proteínas de Unión al ADN/genética , Factor de Unión 1 al Potenciador Linfoide/genética , Neoplasias/genética , Oligonucleótidos/genética , Humanos , Neoplasias/terapia , Oligonucleótidos/farmacología , Complejo de la Endopetidasa Proteasomal/genética , Proteolisis/efectos de los fármacos , Regulador Transcripcional ERG/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Acquisition of resistance to phosphatidylinositol 3-kinase (PI3K)/AKT-targeted monotherapy implies the existence of common resistance mechanisms independent of cancer type. Here, we demonstrate that PI3K/AKT inhibitors cause glycolytic crisis, acetyl-coenzyme A (CoA) shortage, and a global decrease in histone acetylation. In addition, PI3K/AKT inhibitors induce drug resistance by selectively augmenting histone H3 lysine 27 acetylation (H3K27ac) and binding of CBP/p300 and BRD4 proteins at a subset of growth factor and receptor (GF/R) gene loci. BRD4 occupation at these loci and drug-resistant cell growth are vulnerable to both bromodomain and histone deacetylase (HDAC) inhibitors. Little or no occupation of HDAC proteins at the GF/R gene loci underscores the paradox that cells respond equivalently to the two classes of inhibitors with opposite modes of action. Targeting this unique acetyl-histone-related vulnerability offers two clinically viable strategies to overcome PI3K/AKT inhibitor resistance in different cancers.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Neoplasias/tratamiento farmacológico , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Células HCT116 , Humanos , Masculino , Ratones , Ratones SCID , Neoplasias/enzimología , Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Superficie Celular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The tumor-suppressor protein RB acts as a transcription repressor via interaction of its pocket domain with an LXCXE motif in histone deacetylase (HDAC) proteins such as HDAC1. Here, we demonstrate that HDAC5 deficient for the LXCXE motif interacts with both RB-N (via an FXXXV motif) and RB-C segments, and such interactions are diminished by phosphorylation of RB serine-249/threonine-252 and threonine-821. HDAC5 was frequently downregulated or deleted in human cancers such as prostate cancer. Loss of HDAC5 increased histone H3 lysine 27 acetylation (H3K27-ac) and circumvented RB-mediated repression of cell-cycle-related pro-oncogenic genes. HDAC5 loss also conferred resistance to CDK4/6 inhibitors such as palbociclib in prostate and breast cancer cells in vitro and prostate tumors in vivo, but this effect was overcome by the BET-CBP/p300 dual inhibitor NEO2734. Our findings reveal an unknown role of HDAC5 in RB-mediated histone deacetylation and gene repression and define a new mechanism modulating CDK4/6 inhibitor therapeutic sensitivity in cancer cells. SIGNIFICANCE: This study defines a previously uncharacterized role of HDAC5 in tumor suppression and provides a viable strategy to overcome CDK4/6 inhibitor resistance in HDAC5-deficent cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Histona Desacetilasas/genética , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas de Unión a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Acetilación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Masculino , Fosforilación , Piperazinas/farmacología , Piperazinas/uso terapéutico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Piridonas/farmacología , Piridonas/uso terapéutico , RNA-SeqRESUMEN
Mutations in SPOP E3 ligase gene are reportedly associated with genome-wide DNA hypermethylation in prostate cancer (PCa) although the underlying mechanisms remain elusive. Here, we demonstrate that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured PCa cells and primary PCa specimens. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated PCa cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Our findings reveal the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated PCa. They suggest that SPOP mutation could be a biomarker for effective treatment of PCa with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics.
Asunto(s)
Metilación de ADN/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas Nucleares/genética , Neoplasias de la Próstata/genética , Proteínas Represoras/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Azacitidina/farmacología , Azacitidina/uso terapéutico , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/efectos de los fármacos , Docetaxel/farmacología , Docetaxel/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Supresores de Tumor , Humanos , Masculino , Ratones , Mutación , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Estabilidad Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Represoras/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Androgen receptor-positive prostate cancer (PCa) and estrogen receptor-positive luminal breast cancer (BCa) are generally less responsive to immunotherapy compared with certain tumor types such as melanoma. However, the underlying mechanisms are not fully elucidated. In this study, we found that FOXA1 overexpression inversely correlated with interferon (IFN) signature and antigen presentation gene expression in PCa and BCa patients. FOXA1 bound the STAT2 DNA-binding domain and suppressed STAT2 DNA-binding activity, IFN signaling gene expression, and cancer immune response independently of the transactivation activity of FOXA1 and its mutations detected in PCa and BCa. Increased FOXA1 expression promoted cancer immuno- and chemotherapy resistance in mice and PCa and BCa patients. These findings were also validated in bladder cancer expressing high levels of FOXA1. FOXA1 overexpression could be a prognostic factor to predict therapy resistance and a viable target to sensitize luminal PCa, BCa, and bladder cancer to immuno- and chemotherapy.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/inmunología , Factor Nuclear 3-alfa del Hepatocito/inmunología , Interferones/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Transducción de Señal/inmunología , Animales , Femenino , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , Interferones/genética , Masculino , Ratones , Proteínas de Neoplasias/genética , Neoplasias/genética , Transducción de Señal/genéticaRESUMEN
Protein B23/nucleophosmin/numatrin (B23) is a key nucleolar/nuclear matrix-associated protein required for cell growth-related functions, such as rRNA synthesis. Protein kinase CK2 (CK2) (formerly casein kinase 2, a protein Ser/Thr kinase signal that is involved in cell growth and cell death) mediates phosphorylation of B23, thereby influencing its functional activity. Here we have delineated the dynamics of B23 and its link to CK2 status in response to altered growth stimuli and induction of apoptosis in cultured prostate cells and in rat prostate cells in vivo. Our studies employing PC-3 and ALVA-41 prostate cancer cells demonstrated colocalization of CK2 and B23 in the nucleus. Further, CK2 and B23 underwent coordinate modulation in the nucleus related to their nucleocytoplasmic shuttling in response to induction of apoptotic activity in cells caused by downregulation of CK2 or by treatment with other apoptosis-inducing agents. These alterations in nuclear association of B23 occurred in the absence of a significant change in the level of cytoplasmic B23. Similar studies in the in vivo model of rat prostate epithelial cells subjected to androgen deprivation (that resulted in loss of nuclear CK2 and induction of apoptosis) demonstrated dynamic modulation of nuclear matrix-associated B23 without a significant change in its cytoplasmic level. These changes were reversed by androgen-mediated growth response in the prostate. Our results suggest that CK2-mediated phosphorylation of B23 is essential for its retention in the nucleus and that coordinated nuclear localization of B23 and CK2 is dynamically regulated in response to altered growth status in the cell.
Asunto(s)
Apoptosis , Quinasa de la Caseína II/metabolismo , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Próstata/citología , Animales , Línea Celular Tumoral , Núcleo Celular/enzimología , Proliferación Celular , Masculino , Nucleofosmina , Próstata/enzimología , Próstata/metabolismo , Transporte de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
Rationale: The Polycomb group (PcG) protein EZH2 is implicated in cancer progression due to its frequent overexpression in many cancer types and therefore is a promising therapeutic target. Forkhead box transcription factor-1 (FOXO1) is a tumor suppressor that is often transcriptionally downregulated in human cancers such as prostate cancer although the underlying regulatory mechanisms remain elusive. Methods: Analysis of EZH2 ChIP-seq and ChIP-on-chip data in various cell types was performed. ChIP-qPCR, RT-qPCR, and western blot analyses were conducted to determine the mechanism by which EZH2 represses FOXO1 expression. Immunohistochemistry was employed to assess the correlation between EZH2 and FOXO1 protein expression in prostate cancer patient specimens. In vitro MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and animal experiments were performed to determine the anti-cancer efficacy of EZH2 inhibitor alone or in combination of docetaxel, a chemotherapy agent of the taxane family, and dependency of the efficacy on FOXO1 expression. Results: We demonstrated that EZH2 binds to the FOXO1 gene promoter. EZH2 represses FOXO1 gene expression at the transcriptional level. EZH2 protein level inversely correlated with FOXO1 protein expression in prostate cancer patient specimens. This repression requires the methyltransferase activity and the functional PRC2 complex. While effectively inducing loss of viability of PTEN-positive 22Rv1 prostate cancer cells, EZH2 inhibitor failed to inhibit growth of PTEN-negative C4-2 prostate cancer cells. Co-treatment with docetaxel overcame EZH2 inhibitor resistance in PTEN-negative cancer cells in vitro and in mice. This effect was largely mediated by docetaxel-induced nuclear localization and activation of FOXO1. Conclusions: This study identifies FOXO1 as a bona fide repression target of EZH2 and an essential mediator of EZH2 inhibition-induced cell death. Our findings suggest that EZH2 repression of FOXO1 can be targeted by EZH2 inhibitor as a monotherapy for PTEN-proficient cancers or in combination with taxane for treatment of cancers with PTEN mutation or deletion.
Asunto(s)
Antineoplásicos/uso terapéutico , Hidrocarburos Aromáticos con Puentes/uso terapéutico , Resistencia a Antineoplásicos , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Taxoides/uso terapéutico , Sales de Tetrazolio/uso terapéutico , Tiazoles/uso terapéutico , Animales , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica , Hidrocarburos Aromáticos con Puentes/administración & dosificación , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Ratones SCID , Mutación , Fosfohidrolasa PTEN/genética , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Unión Proteica , Taxoides/administración & dosificación , Sales de Tetrazolio/administración & dosificación , Tiazoles/administración & dosificaciónRESUMEN
Rationale: The overall success rate of prostate cancer (PCa) diagnosis and therapy has been improved over the years. However, genomic and phenotypic heterogeneity remains a major challenge for effective detection and treatment of PCa. Efforts to better classify PCa into functional subtypes and elucidate the molecular mechanisms underlying prostate tumorigenesis and therapy resistance are warranted for further improvement of PCa outcomes. Methods: We generated Cre+;Runx2-cTg;Ptenp/+ (Runx2-Pten double mutant) mice by crossbreeding Cre+;Runx2-cTg males with Pten conditional (Ptenp/p) females. By using Hematoxylin and Eosin (H&E) staining, SMA and Masson's Trichrome staining, we investigated the effect of PTEN haploinsufficiency in combination with Runx2 overexpression on prostate tumorigenesis. Moreover, we employed immunohistochemistry (IHC) to stain Ki67 for cell proliferation, cleaved caspase 3 for apoptosis and AKT phosphorylation for signaling pathway in prostate tissues. Chromatin immunoprecipitation coupled quantitative PCR (ChIP-qPCR), reverse transcription coupled quantitative PCR (RT-qPCR), western blot (WB) analyses and immunofluorescence (IF) were conducted to determine the underlying mechanism by which RUNX2 regulates CXCR7 and AKT phosphorylation in PCa cells. Results: We demonstrated that mice with prostate-specific Pten heterozygous deletion and Runx2 overexpression developed high-grade prostatic intraepithelial neoplasia (HGPIN) and cancerous lesions at age younger than one year, with concomitant high level expression of Akt phosphorylation and the chemokine receptor Cxcr7 in malignant glands. RUNX2 overexpression induced CXCR7 transcription and membrane location and AKT phosphorylation in PTEN-deficient human PCa cell lines. Increased expression of RUNX2 also promoted growth of PCa cells and this effect was largely mediated by CXCR7. CXCR7 expression also positively correlated with AKT phosphorylation in PCa patient specimens. Conclusions: Our results reveal a previously unidentified cooperative role of RUNX2 overexpression and PTEN haploinsufficiency in prostate tumorigenesis, suggesting that the defined RUNX2-CXCR7-AKT axis can be a viable target for effective treatment of PCa.