RESUMEN
Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.
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Células Endoteliales/metabolismo , Glucólisis , Neovascularización Fisiológica , Fosfofructoquinasa-2/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Células Endoteliales/citología , Femenino , Eliminación de Gen , Silenciador del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfofructoquinasa-2/genética , Seudópodos/metabolismo , Pez CebraRESUMEN
Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.
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Endocitosis/fisiología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Células Endoteliales/enzimología , Femenino , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.
Asunto(s)
Progresión de la Enfermedad , Neoplasias de la Próstata Resistentes a la Castración , Transducción de Señal , Masculino , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Humanos , Animales , Ratones , Transducción de Señal/efectos de los fármacos , Receptores Androgénicos/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proliferación Celular , Ensayos Antitumor por Modelo de Xenoinjerto , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
Aberrant androgen receptor (AR) signaling drives prostate cancer (PC), and it is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signaling and are refractory to the current repertoire of AR-targeting therapies, which together drive disease progression. There is an unmet clinical need, therefore, to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of coregulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signaling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesized that DNA-PKcs is critical for AR-V function. Using a proximity biotinylation approach, we demonstrated that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V-mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing and, via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicate that targeting DNA-PKcs attenuates AR-V signaling and provide evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V-positive patients with PC.
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Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Dominio Catalítico , Línea Celular Tumoral , Andrógenos/uso terapéutico , ADN , Regulación Neoplásica de la Expresión GénicaRESUMEN
Sunitinib is a potent and clinically approved tyrosine kinase inhibitor that can suppress tumour growth by inhibiting angiogenesis. However, conflicting data exist regarding the effects of this drug on the growth of metastases in preclinical models. Here we use 4T1 and RENCA tumour cells, which both form lung metastases in Balb/c mice, to re-address the effects of sunitinib on the progression of metastatic disease in mice. We show that treatment of mice with sunitinib prior to intravenous injection of tumour cells can promote the seeding and growth of 4T1 lung metastases, but not RENCA lung metastases, showing that this effect is cell line dependent. However, increased metastasis occurred only upon administration of a very high sunitinib dose, but not when lower, clinically relevant doses were used. Mechanistically, high dose sunitinib led to a pericyte depletion effect in the lung vasculature that correlated with increased seeding of metastasis. By administering sunitinib to mice after intravenous injection of tumour cells, we demonstrate that while sunitinib does not inhibit the growth of 4T1 lung tumour nodules, it does block the growth of RENCA lung tumour nodules. This contrasting response was correlated with increased myeloid cell recruitment and persistent vascularisation in 4T1 tumours, whereas RENCA tumours recruited less myeloid cells and were more profoundly devascularised upon sunitinib treatment. Finally, we show that progression of 4T1 tumours in sunitinib treated mice results in increased hypoxia and increased glucose metabolism in these tumours and that this is associated with a poor outcome. Taken together, these data suggest that the effects of sunitinib on tumour progression are dose-dependent and tumour model-dependent. These findings have relevance for understanding how anti-angiogenic agents may influence disease progression when used in the adjuvant or metastatic setting in cancer patients.
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Antineoplásicos/uso terapéutico , Indoles/uso terapéutico , Metástasis de la Neoplasia/tratamiento farmacológico , Pirroles/uso terapéutico , Animales , Ratones , Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones , Sunitinib , Tomografía Computarizada por Rayos XRESUMEN
Acquisition of epidermal barrier function occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. We investigated the molecular pathways active during epidermal barrier formation. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55alpha or B55alpha), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. Our data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. We therefore describe a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development.
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Epidermis/embriología , Epidermis/enzimología , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Diferenciación Celular , Células Cultivadas , Células Epidérmicas , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Queratinocitos/enzimología , Ratones , Fosforilación , Proteína Fosfatasa 2/genética , ARN Interferente Pequeño/genética , Ratas , Técnicas de Cultivo de TejidosRESUMEN
PURPOSE: Therapies targeting the androgen receptor (AR) have improved the outcome for patients with castration-sensitive prostate cancer (CSPC). Expression of the constitutively active AR splice variant-7 (AR-V7) has shown clinical utility as a predictive biomarker of AR-targeted therapy resistance in castration-resistant prostate cancer (CRPC), but its importance in CSPC remains understudied. EXPERIMENTAL DESIGN: We assessed different approaches to quantify AR-V7 mRNA and protein in prostate cancer cell lines, patient-derived xenograft (PDX) models, publicly available cohorts, and independent institutional clinical cohorts, to identify reliable approaches for detecting AR-V7 mRNA and protein and its association with clinical outcome. RESULTS: In CSPC and CRPC cohorts, AR-V7 mRNA was much less abundant when detected using reads across splice boundaries than when considering isoform-specific exonic reads. The RM7 AR-V7 antibody had increased sensitivity and specificity for AR-V7 protein detection by immunohistochemistry (IHC) in CRPC cohorts but rarely identified AR-V7 protein reactivity in CSPC cohorts, when compared with the EPR15656 AR-V7 antibody. Using multiple CRPC PDX models, we demonstrated that AR-V7 expression was exquisitely sensitive to hormonal manipulation. In CSPC institutional cohorts, AR-V7 protein quantification by either assay was associated neither with time to development of castration resistance nor with overall survival, and intense neoadjuvant androgen-deprivation therapy did not lead to significant AR-V7 mRNA or staining following treatment. Neither pre- nor posttreatment AR-V7 levels were associated with volumes of residual disease after therapy. CONCLUSIONS: This study demonstrates that further analytical validation and clinical qualification are required before AR-V7 can be considered for clinical use in CSPC as a predictive biomarker.
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Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Antagonistas de Andrógenos/uso terapéutico , Biomarcadores , Castración , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismoRESUMEN
Endocrine resistance (EnR) in advanced prostate cancer is fatal. EnR can be mediated by androgen receptor (AR) splice variants, with AR splice variant 7 (AR-V7) arguably the most clinically important variant. In this study, we determined proteins key to generating AR-V7, validated our findings using clinical samples, and studied splicing regulatory mechanisms in prostate cancer models. Triangulation studies identified JMJD6 as a key regulator of AR-V7, as evidenced by its upregulation with in vitro EnR, its downregulation alongside AR-V7 by bromodomain inhibition, and its identification as a top hit of a targeted siRNA screen of spliceosome-related genes. JMJD6 protein levels increased (P < 0.001) with castration resistance and were associated with higher AR-V7 levels and shorter survival (P = 0.048). JMJD6 knockdown reduced prostate cancer cell growth, AR-V7 levels, and recruitment of U2AF65 to AR pre-mRNA. Mutagenesis studies suggested that JMJD6 activity is key to the generation of AR-V7, with the catalytic machinery residing within a druggable pocket. Taken together, these data highlight the relationship between JMJD6 and AR-V7 in advanced prostate cancer and support further evaluation of JMJD6 as a therapeutic target in this disease. SIGNIFICANCE: This study identifies JMJD6 as being critical for the generation of AR-V7 in prostate cancer, where it may serve as a tractable target for therapeutic intervention.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/fisiología , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/genética , Empalme Alternativo , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Estudios de Cohortes , Inhibidores Enzimáticos/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/genética , Masculino , Terapia Molecular Dirigida , Oxigenasas/genética , Oxigenasas/fisiología , Pronóstico , Neoplasias de la Próstata Resistentes a la Castración/diagnóstico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/mortalidad , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Androgénicos/química , Receptores Androgénicos/metabolismo , Estudios RetrospectivosRESUMEN
Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC-regulated gene expression. In AR-SV-driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer. SIGNIFICANCE: Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies.See related commentary by Rasool et al., p. 1011.This article is highlighted in the In This Issue feature, p. 995.
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Antagonistas de Receptores Androgénicos/uso terapéutico , Imidazoles/uso terapéutico , Oxazoles/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Antagonistas de Receptores Androgénicos/farmacología , Animales , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Imidazoles/farmacología , Masculino , Ratones , Oxazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The microbiota comprises the microorganisms that live in close contact with the host, with mutual benefit for both counterparts. The contribution of the gut microbiota to the emergence of castration-resistant prostate cancer (CRPC) has not yet been addressed. We found that androgen deprivation in mice and humans promotes the expansion of defined commensal microbiota that contributes to the onset of castration resistance in mice. Specifically, the intestinal microbial community in mice and patients with CRPC was enriched for species capable of converting androgen precursors into active androgens. Ablation of the gut microbiota by antibiotic therapy delayed the emergence of castration resistance even in immunodeficient mice. Fecal microbiota transplantation (FMT) from CRPC mice and patients rendered mice harboring prostate cancer resistant to castration. In contrast, tumor growth was controlled by FMT from hormone-sensitive prostate cancer patients and Prevotella stercorea administration. These results reveal that the commensal gut microbiota contributes to endocrine resistance in CRPC by providing an alternative source of androgens.
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Andrógenos/biosíntesis , Bacterias/metabolismo , Microbioma Gastrointestinal/fisiología , Interacciones Microbiota-Huesped , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/microbiología , Anciano , Anciano de 80 o más Años , Antagonistas de Andrógenos/uso terapéutico , Animales , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Línea Celular Tumoral , Trasplante de Microbiota Fecal , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Neoplasias Experimentales , Prevotella/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Simbiosis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow-derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody-drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. SIGNIFICANCE: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials.
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Anticuerpos Monoclonales Humanizados/farmacología , Biomarcadores de Tumor/metabolismo , Camptotecina/análogos & derivados , Neurregulina-1/metabolismo , Organoides/patología , Neoplasias de la Próstata/patología , Receptor ErbB-3/antagonistas & inhibidores , Animales , Antineoplásicos Inmunológicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Camptotecina/farmacología , Proliferación Celular , Estudios de Seguimiento , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , Neurregulina-1/genética , Organoides/efectos de los fármacos , Organoides/metabolismo , Pronóstico , Estudios Prospectivos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Tasa de Supervivencia , Células Tumorales Cultivadas , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The mechanisms by which prostate cancer shifts from an indolent castration-sensitive phenotype to lethal castration-resistant prostate cancer (CRPC) are poorly understood. Identification of clinically relevant genetic alterations leading to CRPC may reveal potential vulnerabilities for cancer therapy. Here we find that CUB domain-containing protein 1 (CDCP1), a transmembrane protein that acts as a substrate for SRC family kinases (SFKs), is overexpressed in a subset of CRPC. Notably, CDCP1 cooperates with the loss of the tumor suppressor gene PTEN to promote the emergence of metastatic prostate cancer. Mechanistically, we find that androgens suppress CDCP1 expression and that androgen deprivation in combination with loss of PTEN promotes the upregulation of CDCP1 and the subsequent activation of the SRC/MAPK pathway. Moreover, we demonstrate that anti-CDCP1 immunoliposomes (anti-CDCP1 ILs) loaded with chemotherapy suppress prostate cancer growth when administered in combination with enzalutamide. Thus, our study identifies CDCP1 as a powerful driver of prostate cancer progression and uncovers different potential therapeutic strategies for the treatment of metastatic prostate tumors.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Neoplasias de la Próstata/metabolismo , Regulación hacia Arriba , Animales , Antígenos de Neoplasias/genética , Benzamidas , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Drosophila melanogaster , Humanos , Liposomas , Masculino , Nitrilos , Fosfohidrolasa PTEN/biosíntesis , Fosfohidrolasa PTEN/genética , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
BACKGROUND: Liquid biopsies have demonstrated that the constitutively active androgen receptor splice variant-7 (AR-V7) associates with reduced response and overall survival from endocrine therapies in castration-resistant prostate cancer (CRPC). However, these studies provide little information pertaining to AR-V7 expression in prostate cancer (PC) tissue. METHODS: Following generation and validation of a potentially novel AR-V7 antibody for IHC, AR-V7 protein expression was determined for 358 primary prostate samples and 293 metastatic biopsies. Associations with disease progression, full-length androgen receptor (AR-FL) expression, response to therapy, and gene expression were determined. RESULTS: We demonstrated that AR-V7 protein is rarely expressed (<1%) in primary PC but is frequently detected (75% of cases) following androgen deprivation therapy, with further significant (P = 0.020) increase in expression following abiraterone acetate or enzalutamide therapy. In CRPC, AR-V7 expression is predominantly (94% of cases) nuclear and correlates with AR-FL expression (P ≤ 0.001) and AR copy number (P = 0.026). However, dissociation of expression was observed, suggesting that mRNA splicing remains crucial for AR-V7 generation. AR-V7 expression was heterogeneous between different metastases from a patient, although AR-V7 expression was similar within a metastasis. Moreover, AR-V7 expression correlated with a unique 59-gene signature in CRPC, including HOXB13, a critical coregulator of AR-V7 function. Finally, AR-V7-negative disease associated with better prostate-specific antigen (PSA) responses (100% vs. 54%, P = 0.03) and overall survival (74.3 vs. 25.2 months, hazard ratio 0.23 [0.07-0.79], P = 0.02) from endocrine therapies (pre-chemotherapy). CONCLUSION: This study provides impetus to develop therapies that abrogate AR-V7 signaling to improve our understanding of AR-V7 biology and to confirm the clinical significance of AR-V7. FUNDING: Work at the University of Washington and in the Plymate and Nelson laboratories is supported by the Department of Defense Prostate Cancer Research Program (W81XWH-14-2-0183, W81XWH-12-PCRP-TIA, W81XWH-15-1-0430, and W81XWH-13-2-0070), the Pacific Northwest Prostate Cancer SPORE (P50CA97186), the Institute for Prostate Cancer Research, the Veterans Affairs Research Program, the NIH/National Cancer Institute (P01CA163227), and the Prostate Cancer Foundation. Work in the de Bono laboratory was supported by funding from the Movember Foundation/Prostate Cancer UK (CEO13-2-002), the US Department of Defense (W81XWH-13-2-0093), the Prostate Cancer Foundation (20131017 and 20131017-1), Stand Up To Cancer (SU2C-AACR-DT0712), Cancer Research UK (CRM108X-A25144), and the UK Department of Health through an Experimental Cancer Medicine Centre grant (ECMC-CRM064X).
Asunto(s)
Empalme Alternativo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/biosíntesis , Animales , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Ratones SCID , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Androgen receptor (AR) splice variants (AR-Vs) have been implicated in the development and progression of metastatic prostate cancer. AR-Vs are truncated isoforms of the AR, a subset of which lack a ligand-binding domain and remain constitutively active in the absence of circulating androgens, thus promoting cancer cell proliferation. Consequently, AR-Vs have been proposed to contribute not only to resistance to anti-androgen therapies but also to resistance to radiotherapy in patients receiving combination therapy by promoting DNA repair. AR-Vs, such as AR-V7, have been associated with unfavourable clinical outcomes in patients; however, attempts to specifically inhibit or prevent the formation of AR-Vs have, to date, been unsuccessful. Thus, novel therapeutic strategies are desperately needed to address the oncogenic effects of AR-Vs, which can drive lethal forms of prostate cancer. Disruption of alternative splicing through modulation of the spliceosome is one such potential therapeutic avenue; however, our understanding of the biology of the spliceosome and how it contributes to prostate cancer remains incomplete, as reflected in the dearth of spliceosome-targeted therapeutic agents. In this Review, the authors outline the current understanding of the role of the spliceosome in the progression of prostate cancer and explore the therapeutic utility of manipulating alternative splicing to improve patient care.
Asunto(s)
Carcinogénesis/genética , Neoplasias de la Próstata/genética , Isoformas de Proteínas/genética , Receptores Androgénicos/genética , Empalme Alternativo/genética , Proliferación Celular/genética , Reparación del ADN/genética , Progresión de la Enfermedad , Humanos , Masculino , Metástasis de la Neoplasia , Neoplasias de la Próstata/patología , Empalmosomas/genéticaRESUMEN
BACKGROUND: Androgen receptor splice variant 7 (AR-V7) has been implicated in resistance to abiraterone and enzalutamide treatment in men with metastatic castration-resistant prostate cancer (mCRPC). Tissue- or cell-based in situ detection of AR-V7, however, has been limited by lack of specificity. OBJECTIVE: To address current limitations in precision measurement of AR-V7 by developing a novel junction-specific AR-V7 RNA in situ hybridization (RISH) assay compatible with automated quantification. DESIGN, SETTING, AND PARTICIPANTS: We designed a RISH method to visualize single splice junctions in cells and tissue. Using the validated assay for junction-specific detection of the full-length AR (AR-FL) and AR-V7, we generated quantitative data, blinded to clinical data, for 63 prostate tumor biopsies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We evaluated clinical correlates of AR-FL/AR-V7 measurements, including association with prostate-specific antigen progression-free survival (PSA-PFS) and clinical and radiographic progression-free survival (PFS), in a subset of patients starting treatment with abiraterone or enzalutamide following biopsy. RESULTS AND LIMITATIONS: Quantitative AR-FL/AR-V7 data were generated from 56 of the 63 (88.9%) biopsy specimens examined, of which 44 were mCRPC biopsies. Positive AR-V7 signals were detected in 34.1% (15/44) mCRPC specimens, all of which also co-expressed AR-FL. The median AR-V7/AR-FL ratio was 11.9% (range 2.7-30.3%). Positive detection of AR-V7 was correlated with indicators of high disease burden at baseline. Among the 25 CRPC biopsies collected before treatment with abiraterone or enzalutamide, positive AR-V7 detection, but not higher AR-FL, was significantly associated with shorter PSA-PFS (hazard ratio 2.789, 95% confidence interval 1.12-6.95; p=0.0081). CONCLUSIONS: We report for the first time a RISH method for highly specific and quantifiable detection of splice junctions, allowing further characterization of AR-V7 and its clinical significance. PATIENT SUMMARY: Higher AR-V7 levels detected and quantified using a novel method were associated with poorer response to abiraterone or enzalutamide in prostate cancer.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Isoformas de Proteínas/genética , Receptores Androgénicos/genética , Anciano , Biopsia con Aguja , Supervivencia sin Enfermedad , Humanos , Inmunohistoquímica , Hibridación in Situ/métodos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Invasividad Neoplásica/patología , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Neoplasias de la Próstata Resistentes a la Castración/mortalidad , Neoplasias de la Próstata Resistentes a la Castración/patología , Análisis de SupervivenciaRESUMEN
Purpose: Persistent androgen receptor (AR) signaling drives castration-resistant prostate cancer (CRPC) and confers resistance to AR-targeting therapies. Novel therapeutic strategies to overcome this are urgently required. We evaluated how bromodomain and extra-terminal (BET) protein inhibitors (BETi) abrogate aberrant AR signaling in CRPC.Experimental Design: We determined associations between BET expression, AR-driven transcription, and patient outcome; and the effect and mechanism by which chemical BETi (JQ1 and GSK1210151A; I-BET151) and BET family protein knockdown regulates AR-V7 expression and AR signaling in prostate cancer models.Results: Nuclear BRD4 protein expression increases significantly (P ≤ 0.01) with castration resistance in same patient treatment-naïve (median H-score; interquartile range: 100; 100-170) and CRPC (150; 110-200) biopsies, with higher expression at diagnosis associating with worse outcome (HR, 3.25; 95% CI, 1.50-7.01; P ≤ 0.001). BRD2, BRD3, and BRD4 RNA expression in CRPC biopsies correlates with AR-driven transcription (all P ≤ 0.001). Chemical BETi, and combined BET family protein knockdown, reduce AR-V7 expression and AR signaling. This was not recapitulated by C-MYC knockdown. In addition, we show that BETi regulates RNA processing thereby reducing alternative splicing and AR-V7 expression. Furthermore, BETi reduce growth of prostate cancer cells and patient-derived organoids with known AR mutations, AR amplification and AR-V7 expression. Finally, BETi, unlike enzalutamide, decreases persistent AR signaling and growth (P ≤ 0.001) of a patient-derived xenograft model of CRPC with AR amplification and AR-V7 expression.Conclusions: BETi merit clinical evaluation as inhibitors of AR splicing and function, with trials demonstrating their blockade in proof-of-mechanism pharmacodynamic studies. Clin Cancer Res; 24(13); 3149-62. ©2018 AACR.
Asunto(s)
Antineoplásicos/uso terapéutico , Terapia Molecular Dirigida , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Empalme Alternativo , Antineoplásicos/farmacología , Biomarcadores de Tumor , Proteínas de Ciclo Celular , Línea Celular Tumoral , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Pronóstico , Neoplasias de la Próstata Resistentes a la Castración/mortalidad , Neoplasias de la Próstata Resistentes a la Castración/patología , Isoformas de Proteínas , ARN Interferente Pequeño/genética , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Resultado del TratamientoRESUMEN
Androgen receptor (AR) splice variants (SV) have been implicated in the development of metastatic castration-resistant prostate cancer and resistance to AR targeting therapies, including abiraterone and enzalutamide. Agents targeting AR-SV are urgently needed to test this hypothesis and further improve the outcome of patients suffering from this lethal disease. Clin Cancer Res; 22(17); 4280-2. ©2016 AACRSee related article by Yang et al., p. 4466.
Asunto(s)
Antagonistas de Receptores Androgénicos/uso terapéutico , Antineoplásicos/uso terapéutico , Terapia Molecular Dirigida , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/metabolismo , Empalme Alternativo , Antagonistas de Receptores Androgénicos/farmacología , Animales , Antineoplásicos/farmacología , Humanos , Masculino , Ratones , Neoplasias de la Próstata Resistentes a la Castración/genética , Dominios y Motivos de Interacción de Proteínas/genética , Receptores Androgénicos/genéticaRESUMEN
BACKGROUND: The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. OBJECTIVE: To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. DESIGN, SETTING, AND PARTICIPANTS: Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. RESULTS AND LIMITATIONS: Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5-90) compared to CRPC (HS 135, IQR 80-157.5; p<0.0001), and in biopsy tissue taken before (HS 80, IQR 30-136.3) compared to after (HS 140, IQR 105-167.5; p=0.007) abiraterone or enzalutamide treatment. Lower nuclear AR-V7 expression at CRPC biopsy was associated with longer OS (hazard ratio 1.012, 95% confidence interval 1.004-1.020; p=0.003). While this monoclonal antibody primarily binds to AR-V7 in PC biopsy tissue, it may also bind to other proteins. CONCLUSIONS: We provide the first evidence that nuclear AR-V7 expression increases with emerging CRPC and is prognostic for OS, unlike antibody staining for the AR N-terminal domain. These data indicate that AR-V7 is important in CRPC disease biology; agents targeting AR splice variants are needed to test this hypothesis and further improve patient outcome from CRPC. PATIENT SUMMARY: In this study we found that levels of the protein AR-V7 were higher in patients with advanced prostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time.
Asunto(s)
Anticuerpos Monoclonales , Inmunohistoquímica/métodos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/metabolismo , Anciano , Androstenos/uso terapéutico , Antineoplásicos/uso terapéutico , Benzamidas , Biopsia , Línea Celular Tumoral , Núcleo Celular/metabolismo , Resistencia a Antineoplásicos , Humanos , Masculino , Persona de Mediana Edad , Nitrilos , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/uso terapéutico , Pronóstico , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/inmunología , Estudios Retrospectivos , Tasa de Supervivencia , Factores de TiempoRESUMEN
Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR) and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear whether HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation. Cancer Res; 76(9); 2731-42. ©2016 AACR.
Asunto(s)
Benzamidas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoindoles/farmacología , Neoplasias de la Próstata/patología , Empalme del ARN/efectos de los fármacos , Receptores Androgénicos/genética , Animales , Western Blotting , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Inmunohistoquímica , Inmunoprecipitación , Masculino , Ratones , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , ARN Mensajero , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sunitinib and pazopanib are antiangiogenic tyrosine kinase inhibitors (TKI) used to treat metastatic renal cell carcinoma (RCC). However, the ability of these drugs to extend progression-free and overall survival in this patient population is limited by drug resistance. It is possible that treatment outcomes in RCC patients could be improved by rationally combining TKIs with other agents. Here, we address whether inhibition of the Ras-Raf-MEK-ERK1/2 pathway is a rational means to improve the response to TKIs in RCC. Using a xenograft model of RCC, we found that tumors that are resistant to sunitinib have a significantly increased angiogenic response compared with tumors that are sensitive to sunitinib in vivo. We also observed significantly increased levels of phosphorylated ERK1/2 in the vasculature of resistant tumors, when compared with sensitive tumors. These data suggested that the Ras-Raf-MEK-ERK1/2 pathway, an important driver of angiogenesis in endothelial cells, remains active in the vasculature of TKI-resistant tumors. Using an in vitro angiogenesis assay, we identified that the MEK inhibitor (MEKI) trametinib has potent antiangiogenic activity. We then show that, when trametinib is combined with a TKI in vivo, more effective suppression of tumor growth and tumor angiogenesis is achieved than when either drug is utilized alone. In conclusion, we provide preclinical evidence that combining a TKI, such as sunitinib or pazopanib, with a MEKI, such as trametinib, is a rational and efficacious treatment regimen for RCC.