RESUMEN
Quantitative proteomics/metabolomics investigation of laser-capture-microdissection (LCM) cell populations from clinical cohorts affords precise insights into disease/therapeutic mechanisms, nonetheless high-quality quantification remains a prominent challenge. Here, we devised an LC/MS-based approach allowing parallel, robust global-proteomics and targeted-metabolomics quantification from the same LCM samples, using biopsies from prostate cancer (PCa) patients as the model system. The strategy features: (i) an optimized molecular weight cutoff (MWCO) filter-based separation of proteins and small-molecule fractions with high and consistent recoveries; (ii) microscale derivatization and charge-based enrichment for ultrasensitive quantification of key androgens (LOQ = 5 fg/1k cells) with excellent accuracy/precision; (iii) reproducible/precise proteomics quantification with low-missing-data using a detergent-cocktail-based sample preparation and an IonStar pipeline for reproducible and precise protein quantification with excellent data quality. Key parameters enabling robust/reproducible quantification have been meticulously evaluated and optimized, and the results underscored the importance of surveying quantitative performances against key parameters to facilitate fit-for-purpose method development. As a proof-of-concept, high-quality quantification of the proteome and androgens in LCM samples of PCa patient-matched cancerous and benign epithelial/stromal cells was achieved (N = 16), which suggested distinct androgen distribution patterns across cell types and regions, as well as the dysregulated pathways involved in tumor-stroma crosstalk in PCa pathology. This strategy markedly leverages the scope of quantitative-omics investigations using LCM samples, and combining with IonStar, can be readily adapted to larger-cohort clinical analysis. Moreover, the capacity of parallel proteomics/metabolomics quantification permits precise corroboration of regulatory processes on both protein and small-molecule levels, with decreased batch effect and enhanced utilization of samples.
Asunto(s)
Metabolómica , Proteómica , Humanos , Captura por Microdisección con Láser , Rayos Láser , Masculino , Neoplasias de la Próstata , Proteoma , Espectrometría de Masas en TándemRESUMEN
UNLABELLED: BACKGROUND. A high throughput, high pressure liquid chromatographic (HPLC) method with triple quadrupole mass spectral detection (LC/MS/MS) was validated for the measurement of 5 endogenous androgens in human plasma and serum and applied to various in vivo and in vitro study samples to pursue a better understanding of the interrelationship of the androgen axis, intracrine metabolism, and castration-recurrent prostate cancer (CaP). METHODS: A Shimadzu HPLC system interfaced with a Sciex QTRAP 5500 mass spectrometer with electrospray ionization was used with in line column-switching. Samples were liquid/liquid extracted and chromatographed on a Luna C18(2) column at 60°C with a biphasic gradient using a 15-min run time. RESULTS: The method was validated for five androgens in human plasma and serum, and applied to four sets of samples. Plasma (n=188) and bone marrow aspirate (n=129) samples from patients with CaP, who received abiraterone acetate plus prednisone for up to 945 days(135 weeks), had undetectable androgens after 8 weeks of treatment. Plasma dehydroepiandrosterone(DHEA) concentrations were higher in African Americans than Caucasian Americans with newly diagnosed CaP. Analysis of prostate tumor tissue homogenates demonstrated reproducible testosterone (T) and dihydrotestosterone (DHT) concentrations with a minimal sample size of 1.02.0 mg of tissue. Finally, cell pellet and media samples from the LNCaP C4-2 cell line showed conversion of T to DHT. CONCLUSION: The proposed LC/MS/MS method was validated for quantitation of five endogenous androgens in human plasma and serum, and effectively profiles androgens in clinical specimens and cell culture samples.
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Andrógenos/metabolismo , Deshidroepiandrosterona/metabolismo , Dihidrotestosterona/metabolismo , Neoplasias de la Próstata/metabolismo , Espectrometría de Masas en Tándem/métodos , Testosterona/metabolismo , Andrógenos/sangre , Biomarcadores/sangre , Biomarcadores/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Deshidroepiandrosterona/sangre , Dihidrotestosterona/sangre , Humanos , Masculino , Neoplasias de la Próstata/sangre , Testosterona/sangreRESUMEN
BACKGROUND: Currently available 5α-reductase inhibitors are not completely effective for treatment of benign prostate enlargement, prevention of prostate cancer (CaP), or treatment of advanced castration-recurrent (CR) CaP. We tested the hypothesis that a novel 5α-reductase, 5α-reductase-3, contributes to residual androgen metabolism, especially in CR-CaP. METHODS: A new protein with potential 5α-reducing activity was expressed in CHO-K1 cellsandTOP10 E. coli for characterization. Protein lysates and total mRNA were isolated from preclinical and clinical tissues. Androgen metabolism was assessed using androgen precursors and thin layer chromatography or liquid chromatography tandem mass spectrometry. RESULTS: The relative mRNA expression for the three 5α-reductase enzymes in clinical samples of CR-CaP was 5α-reductase-3 â« 5α-reductase-1> 5α-reductase-2. Recombinant 5α-reductase-3 protein incubations converted testosterone, 4-androstene-3,17-dione (androstenedione) and 4-pregnene-3,20-dione (progesterone) to dihydrotestosterone, 5α-androstan-3,17-dione, and 5α-pregnan-3,20-dione, respectively. 5α-Reduced androgen metabolites were measurable in lysates from androgen-stimulated (AS) CWR22 and CR-CWR22 tumors and clinical specimens of AS-CaP and CR-CaP pre-incubated with dutasteride (a bi-specific inhibitor of 5α-reductase-1 and 2). CONCLUSION: Human prostate tissues contain a third 5α-reductase that was inhibited poorly by dutasteride at high androgen substrate concentration in vitro, and it may promote DHT formation in vivo, through alternative androgen metabolism pathways when testosterone levels are low.
Asunto(s)
Colestenona 5 alfa-Reductasa , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Andrógenos/metabolismo , Animales , Azaesteroides/farmacología , Células CHO , Colestenona 5 alfa-Reductasa/antagonistas & inhibidores , Colestenona 5 alfa-Reductasa/genética , Colestenona 5 alfa-Reductasa/metabolismo , Cricetulus , Dutasterida , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Expresión Génica , Xenoinjertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Trasplante de Neoplasias , Próstata , Hiperplasia Prostática/enzimología , Neoplasias de la Próstata Resistentes a la Castración , ARN Mensajero/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Prostate cancer (PCa) typically spreads to the bone, and this distribution is attributed to the central role of the microenvironment in progression. However, metastasis to the adrenal glands, while not as common, does occur. The biology that accounts for adrenal metastases may be attributed to the unique local steroid metabolome and co-clinical characterization may elucidate the role steroid biosynthesis plays in PCa progression. METHODS: Three patients with metastatic PCa who had archived tumor tissue from an adrenalectomy were retrospectively identified, and one adrenal metastasis was developed into a xenograft (MDA-PCa-250). The adrenal metastases were characterized by performing somatic DNA whole exome sequencing (WES), RNA-Seq, immunohistochemistry (IHC), and steroid metabolite quantitation. The influence of steroid metabolites on adrenal metastasis cells and tumor growth was tested in vitro and in vivo. RESULTS: Clinically, adrenalectomy was performed during castration-resistant oligometastatic disease, and two men experienced resensitization to leuprolide. Somatic DNA WES revealed heterogeneous alterations in tumor suppressor and DNA damage repair pathway genes. Adrenal metastases had active androgen receptor (AR) signaling by IHC, and RNA-Seq supported a potential role for adrenal androgen precursor metabolism in activating the AR. Steroid quantitation suggested the adrenal androgen precursors were converted into testosterone in these metastases, and stable isotope tracing of an organoid from MDA-PCa-250 confirmed the capability of adrenal metastases to biosynthesize testosterone from adrenal precursors. In vitro testing of a cell line derived from MDA-PCa-250 showed that testosterone and cortisol stimulated tumor cell growth. In vivo experiments demonstrated that MDA-PCa-250 grew in intact mice with circulating testosterone, but not in castrated mice. CONCLUSIONS: PCa adrenal metastases depend upon AR signaling driven by androgen precursors, androstenedione and dehydroepiandrosterone, available in the microenvironment, despite the presence of heterogeneous somatic DNA alterations. Moreover, MDA-PCa-250 provides a preclinical model that can recapitulate the unique androgen-dependence of adrenal metastases. CLINICAL TRIAL REGISTRATION: This study does not report the clinical results of a clinical trial, but it does use samples from a completed clinical trial that is registered with clinicaltrials.gov (NCT01254864).
Asunto(s)
Andrógenos , Neoplasias de la Próstata , Masculino , Humanos , Animales , Ratones , Andrógenos/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Estudios Retrospectivos , Esteroides/metabolismo , Testosterona/metabolismo , ADN , Línea Celular Tumoral , Microambiente TumoralRESUMEN
Functional implication of stromal heterogeneity in the prostate remains incompletely understood. Using lineage tracing and light-sheet imaging, we show that some fibroblast cells at the mouse proximal prostatic ducts and prostatic urethra highly express Lgr5. Genetic ablation of these anatomically restricted stromal cells, but not nonselective ablation of prostatic stromal cells, rapidly induces prostate epithelial turnover and dedifferentiation that are reversed following spontaneous restoration of the Lgr5+ stromal cells. RNA sequencing (RNA-seq) analysis indicates that ablating the Lgr5+ stromal cells activates a mechanosensory response. Ablating the Lgr5+ stromal cells impairs the control of prostatic ductal outlet, increases prostate tissue stiffness, and activates the mitogen-activated protein kinase (MAPK). Suppressing MAPK overrides the elevated epithelial proliferation. In summary, the Lgr5+ stromal cells regulate prostate tissue homeostasis and maintain its functional integrity in a long-distance manner. Our study implies that the cells at organ junctions most likely control organ homeostasis by sustaining a balanced mechanoforce.
Asunto(s)
Próstata , Células del Estroma , Animales , Homeostasis , Masculino , Ratones , Próstata/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Células del Estroma/metabolismoRESUMEN
BACKGROUND: A third isozyme of human 5α-steroid reductase, 5α-reductase-3, was identified in prostate tissue at the mRNA level. However, the levels of 5α-reductase-3 protein expression and its cellular localization in human tissues remain unknown. METHODS: A specific monoclonal antibody was developed, validated, and used to characterize for the first time the expression of 5α-reductase-3 protein in 18 benign and 26 malignant human tissue types using immunostaining analyses. RESULTS AND CONCLUSIONS: In benign tissues, 5α-reductase-3 immunostaining was high in conventional androgen-regulated human tissues, such as skeletal muscle and prostate. However, high levels of expression also were observed in non-conventional androgen-regulated tissues, which suggest either multiples target tissues for androgens or different functions of 5α-reductase-3 among human tissues. In malignant tissues, 5α-reductase-3 immunostaining was ubiquitous but particularly over-expressed in some cancers compared to their benign counterparts, which suggests a potential role for 5α-reductase-3 as a biomarker of malignancy. In benign prostate, 5α-reductase-3 immunostaining was localized to basal epithelial cells, with no immunostaining observed in secretory/luminal epithelial cells. In high-grade prostatic intraepithelial neoplasia (HGPIN), 5α-reductase-3 immunostaining was localized in both basal epithelial cells and neoplastic epithelial cells characteristic of HGPIN. In androgen-stimulated and castration-recurrent prostate cancer (CaP), 5α-reductase-3 immunostaining was present in most epithelial cells and at similar levels, and at levels higher than observed in benign prostate. Analyses of expression and functionality of 5α-reductase-3 in human tissues may prove useful for development of treatment for benign prostatic enlargement and prevention and treatment of CaP.
Asunto(s)
3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Isoenzimas/metabolismo , Próstata/enzimología , Hiperplasia Prostática/enzimología , Neoplasias de la Próstata/enzimología , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Western Blotting , Línea Celular Tumoral , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/enzimología , Células Epiteliales/patología , Humanos , Inmunohistoquímica , Isoenzimas/genética , Masculino , Músculo Esquelético/enzimología , Próstata/patología , Hiperplasia Prostática/genética , Hiperplasia Prostática/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Androgen deprivation therapy is the most common treatment option for advanced prostate cancer. Almost all prostate cancers recur during androgen deprivation therapy, and new evidence suggests that androgen receptor activation persists despite castrate levels of circulating androgens. Quantitation of tissue levels of androgens is critical to understanding the mechanism of recurrence of prostate cancer during androgen deprivation therapy. A liquid chromatography atmospheric pressure photoionization tandem mass spectrometric method was developed for quantitation of tissue levels of androgens. Quantitation of the saturated keto-steroids dihydrotestosterone and 5-alpha-androstanedione required detection of a novel parent ion, [M + 15](+). The nature of this parent ion was explored, and the method was applied to prostate tissue and cell culture with comparison to results achieved using electrospray ionization.
Asunto(s)
Andrógenos/análisis , Neoplasias de la Próstata/terapia , Espectrometría de Masas en Tándem/métodos , Presión Atmosférica , Cromatografía Líquida de Alta Presión/métodos , Dihidrotestosterona/análisis , Etiocolanolona/análogos & derivados , Etiocolanolona/análisis , Humanos , MasculinoRESUMEN
PURPOSE: Cabozantinib, an oral inhibitor of c-MET/VEGFR2 signaling, improved progression-free survival (mPFS) but not overall survival (OS) in metastatic castrate-resistant prostate cancer. We evaluated cabozantinib plus androgen deprivation therapy (ADT) in hormone-naïve metastatic prostate cancer (HNMPCa). PATIENTS AND METHODS: Patients received ADT plus cabozantinib starting at 60 mg daily. The primary endpoint was castrate-resistant PFS by radiographic criteria, clinical progression, or receipt of additional therapy. Secondary endpoints included OS, safety, radiographic responses, and biomarker modulation. RESULTS: Sixty-two patients received treatment. With a median follow-up of 31.2 months, the mPFS was 16.1 months (95% CI, 14.6-22.7 months), and mOS was not reached. Reductions in PSA ≥ 90%, bone-specific alkaline phosphatase ≥ 50%, and urine N-telopeptides ≥ 50% occurred in 83%, 87%, and 86% of evaluable patients, respectively. Responses in bone scan and measurable disease were observed in 81% of and 90% of evaluable patients, respectively. Most common grade 3 adverse events were hypertension (19%), diarrhea (6%), and thromboembolic events (6%), and dose reductions occurred in 85% of patients. Analysis of baseline cytokine and angiogenic factors (CAFs) revealed that higher plasma concentrations of Lumican, CXCL5, CD25, and CD30 were associated with shorter PFS as was high tumor expression of pFGFR1. CONCLUSIONS: Cabozantinib plus ADT has promising clinical activity in HNMPCa. CAF profiles and tissue markers suggest candidate prognostic and predictive markers of cabozantinib benefit and provide insights for rational therapy combinations.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Anilidas/uso terapéutico , Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Piridinas/uso terapéutico , Anciano , Anciano de 80 o más Años , Neoplasias Óseas/secundario , Quimioterapia Combinada , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Neoplasias de la Próstata Resistentes a la Castración/patología , Tasa de SupervivenciaRESUMEN
PURPOSE: Androgen receptor (AR) signaling affects prostate cancer (PCa) growth, metabolism, and progression. Often, PCa progresses from androgen-sensitive to castration-resistant prostate cancer (CRPC) following androgen-deprivation therapy. Clinicopathologic and genomic characterizations of CRPC tumors lead to subdividing CRPC into two subtypes: (1) AR-dependent CRPC containing dysregulation of AR signaling alterations in AR such as amplification, point mutations, and/or generation of splice variants in the AR gene; and (2) an aggressive variant PCa (AVPC) subtype that is phenotypically similar to small cell prostate cancer and is defined by chemotherapy sensitivity, gain of neuroendocrine or pro-neural marker expression, loss of AR expression, and combined alterations of PTEN, TP53, and RB1 tumor suppressors. Previously, we reported patient-derived xenograft (PDX) animal models that contain characteristics of these CRPC subtypes. In this study, we have employed the PDX models to test metabolic alterations in the CRPC subtypes. PROCEDURES: Mass spectrometry and nuclear magnetic resonance analysis along with in vivo hyperpolarized 1-[13C]pyruvate spectroscopy experiments were performed on prostate PDX animal models. RESULTS: Using hyperpolarized 1-[13C]pyruvate conversion to 1-[13C]lactate in vivo as well as lactate measurements ex vivo, we have found increased lactate production in AR-dependent CRPC PDX models even under low-hormone levels (castrated mouse) compared to AR-negative AVPC PDX models. CONCLUSIONS: Our analysis underscores the potential of hyperpolarized metabolic imaging in determining the underlying biology and in vivo phenotyping of CRPC.
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Ácido Láctico/metabolismo , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/métodos , Neoplasias de la Próstata Resistentes a la Castración/diagnóstico , Ácido Pirúvico/metabolismo , Receptores Androgénicos/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Xenoinjertos , Humanos , Aumento de la Imagen/métodos , Ácido Láctico/análisis , Masculino , Ratones , Ratones SCID , Invasividad Neoplásica , Próstata/química , Próstata/diagnóstico por imagen , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Ácido Pirúvico/análisis , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Androgen and androgen receptor (AR) are involved in growth of normal prostate and development of prostatic diseases including prostate cancer. Androgen deprivation therapy is used for treating advanced prostate cancer. This therapeutic approach focuses on suppressing the accumulation of potent androgens, testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), or inactivating the AR. Unfortunately, the majority of patients with prostate cancer eventually advance to androgen-independent states and no longer respond to the therapy. In addition to the potent androgens, 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), reduced from 5alpha-DHT through 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), activated signaling may represent a novel pathway responsible for the progression to androgen-independent prostate cancer. Androgen sensitive human prostate cancer LNCaP cells were used to compare 5alpha-DHT and 3alpha-diol activated androgenic effects. In contrast to 5alpha-DHT, 3alpha-diol regulated unique patterns of beta-catenin and Akt expression as well as Akt phosphorylation in parental and in AR-silenced LNCaP cells. More significantly, 3alpha-diol, but not 5alpha-DHT, supported AR-silenced LNCaP cells and AR negative prostate cancer PC-3 cell proliferation. 3alpha-diol-activated androgenic effects in prostate cells cannot be attributed to the accumulation of 5alpha-DHT, since 5alpha-DHT formation was not detected following 3alpha-diol administration. Potential accumulation of 3alpha-diol, as a result of elevated 3alpha-HSD expression in cancerous prostate, may continue to support prostate cancer growth in the presence of androgen deprivation. Future therapeutic strategies for treating advanced prostate cancer might need to target reductive 3alpha-HSD to block intraprostatic 3alpha-diol accumulation.
Asunto(s)
Androstano-3,17-diol/farmacología , Neoplasias de la Próstata/patología , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Dihidrotestosterona/farmacología , Progresión de la Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Relacionados con las Neoplasias , Humanos , Masculino , Modelos Biológicos , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Androgénicos/metabolismo , Factores de Tiempo , beta Catenina/metabolismoRESUMEN
Androgen receptor (AR) plays a crucial role in the development and progression of prostate cancer. AR expression has also been reported in other solid tumors, including renal cell carcinoma (RCC), but its biological role here remains unclear. Through integrative analysis of a reverse phase protein array, we discovered increased expression of AR in an RCC patient-derived xenograft model of acquired resistance to the receptor tyrosine kinase inhibitor (RTKi) sunitinib. AR expression was increased in RCC cell lines with either acquired or intrinsic sunitinib resistance in vitro An AR signaling gene array profiler indicated elevated levels of AR target genes in sunitinib-resistant cells. Sunitinib-induced AR transcriptional activity was associated with increased phosphorylation of serine 81 (pS81) on AR. Additionally, AR overexpression resulted in acquired sunitinib resistance and the AR antagonist enzalutamide-induced AR degradation and attenuated AR downstream activity in sunitinib-resistant cells, also indicated by decreased secretion of human kallikrein 2. Enzalutamide-induced AR degradation was rescued by either proteasome inhibition or by knockdown of the AR ubiquitin ligase speckle-type POZ protein (SPOP). In vivo treatment with enzalutamide and sunitinib demonstrated that this combination efficiently induced tumor regression in a RCC model following acquired sunitinib resistance. Overall, our results suggest the potential role of AR as a target for therapeutic interventions, in combination with RTKi, to overcome drug resistance in RCC.Significance: These findings highlight the therapeutic potential of targeting the androgen receptor to overcome RCC resistance to receptor tyrosine kinase inhibitors. Cancer Res; 78(11); 2886-96. ©2018 AACR.
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Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Fosforilación/efectos de los fármacos , Receptores Androgénicos/metabolismo , Sunitinib/farmacología , Animales , Benzamidas , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones SCID , Nitrilos , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Calicreínas de Tejido/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Although emerging evidence suggests a potential role of calcium/calmodulin-dependent kinase II (CaMKII) in prostate cancer, its role in prostate cancer tumorigenesis is largely unknown. Here, we examine whether the acetyl CoA-CaMKII pathway, first described in frog oocytes, promotes prostate cancer tumorigenesis. In human prostate cancer specimens, metastatic prostate cancer expressed higher levels of active CaMKII compared with localized prostate cancer. Correspondingly, basal CaMKII activity was significantly higher in the more tumorigenic PC3 and PC3-mm2 cells relative to the less tumorigenic LNCaP and C4-2B4 cells. Deletion of CaMKII by CRISPR/Cas9 in PC3-mm2 cells abrogated cell survival under low-serum conditions, anchorage-independent growth and cell migration; overexpression of constitutively active CaMKII in C4-2B4 cells promoted these phenotypes. In an animal model of prostate cancer metastasis, genetic ablation of CaMKII reduced PC3-mm2 cell metastasis from the prostate to the lymph nodes. Knockdown of the acetyl-CoA transporter carnitine acetyltransferase abolished CaMKII activation, providing evidence that acetyl-CoA generated from organelles is a major activator of CaMKII. Genetic deletion of the ß-oxidation rate-limiting enzyme ACOX family proteins decreased CaMKII activation, whereas overexpression of ACOXI increased CaMKII activation. Overall, our studies identify active CaMKII as a novel connection between organelle ß-oxidation and acetyl-CoA transport with cell survival, migration, and prostate cancer metastasis.Significance: This study identifies a cell metabolic pathway that promotes prostate cancer metastasis and suggests prostate cancer may be susceptible to ß-oxidation inhibitors. Cancer Res; 78(10); 2490-502. ©2018 AACR.
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Acetilcoenzima A/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Carcinogénesis/genética , Movimiento Celular/genética , Supervivencia Celular/genética , Neoplasias de la Próstata/patología , Acil-CoA Oxidasa/genética , Animales , Sistemas CRISPR-Cas/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Carnitina O-Acetiltransferasa/genética , Línea Celular Tumoral , Ácidos Grasos/metabolismo , Humanos , Metástasis Linfática/genética , Metástasis Linfática/patología , Masculino , Ratones , Ratones Noqueados , Ratones SCID , Oxidación-Reducción , Oxidorreductasas/genética , Células PC-3RESUMEN
Prostate stem cells (PSC) are characterized by their intrinsic resistance to androgen deprivation therapy (ADT), possibly due to the lack of androgen receptor (AR) expression. PSCs resistance to ADT and PSC expansion in castration resistant prostate cancer (CRPC) has sparked great interest in using differentiation therapy as an adjuvant to ADT. Understanding the mechanisms, by which PSCs maintain their undifferentiated phenotype, thus has important implications in differentiation therapy. In the prostate, the ATP binding cassette sub-family G member 2 (ABCG2) transporters, which enrich for AR-positive, ADT-resistant PSCs, play an important role in regulating the intracellular androgen levels by effluxing androgens. We hypothesized that the ABCG2-mediated androgen efflux is responsible for maintaining PSCs in an undifferentiated state. Using the HPr-1-AR (nontumorigenic) and CWR-R1 (tumorigenic) prostate cell lines, it was demonstrated that inhibiting the ABCG2-mediated androgen efflux, with Ko143 (ABCG2 inhibitor), increased the nuclear AR expression due to elevated intracellular androgen levels. Increased nuclear translocation of AR is followed by increased expression of AR regulated genes, a delayed cell growth response, and increased luminal differentiation. Furthermore, Ko143 reduced tumor growth rates in mice implanted with ABCG2-expressing CWR-R1 cells. In addition, Ko143-treated mice had more differentiated tumors as evidenced by an increased percentage of CK8+/AR+ luminal cells and decreased percentage of ABCG2-expressing cells. Thus, inhibiting ABCG2-mediated androgen efflux forces the PSCs to undergo an AR-modulated differentiation to an ADT-sensitive luminal phenotype. IMPLICATIONS: This study identifies the mechanism by which the prostate stem cell marker, ABCG2, plays a role in prostate stem cell maintenance and provides a rationale for targeting ABCG2 for differentiation therapy in prostate cancer. Mol Cancer Res; 15(2); 128-40. ©2016 AACR.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Andrógenos/metabolismo , Animales , Línea Celular Tumoral , Dicetopiperazinas/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/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/metabolismo , Testosterona/sangreRESUMEN
Therapies that target cancer cells may have unexpected effects on the tumor microenvironment that affects therapy outcomes or render therapy resistance. Prostate cancer (PCa) bone metastasis is uniquely associated with osteoblastic bone lesions and treatment with cabozantinib, a VEGFR-2 and MET inhibitor, leads to a reduction in number and/or intensity of lesions on bone scans. However, resistance to cabozantinib therapy inevitably occurs. We examined the effect of cabozantinib on osteoblast differentiation and secretion in the context of therapy resistance. We showed that primary mouse osteoblasts express VEGFR2 and MET and cabozantinib treatment decreased osteoblast proliferation but enhanced their differentiation. A genome-wide analysis of transcriptional responses of osteoblasts to cabozantinib identified a set of genes accounting for inhibition of proliferation and stimulation of differentiation, and a spectrum of secreted proteins induced by cabozantinib, including pappalysin, IGFBP2, WNT 16, and DKK1. We determined that these proteins were upregulated in the conditioned medium of cabozantinib-treated osteoblasts (CBZ-CM) compared to control CM. Treatment of C4-2B4 or PC3-mm2 PCa cells with CBZ-CM increased the anchorage-independent growth and migration of these PCa cells compared to cells treated with control CM. These results suggest that the effect of cabozantinib on the tumor microenvironment may increase tumor cell survival and cause therapy resistance.
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PURPOSE: Prostate cancer eventually recurs during androgen deprivation therapy despite castrate levels of serum androgens. Expression of androgen receptor and androgen receptor-regulated proteins suggests androgen receptor activation in recurrent prostate cancer. Many groups have pursued mechanisms of ligand-independent androgen receptor activation but we found high levels of testicular androgens in recurrent prostate cancer tissue using RIA. EXPERIMENTAL DESIGNS: Prostate specimens from 36 men were procured preserving blood flow to prevent ischemia and cyropreserved immediately. Recurrent prostate cancer specimens from 18 men whose cancer recurred locally during androgen deprivation therapy and androgen-stimulated benign prostate specimens from 18 men receiving no hormonal treatments were studied. Tissue levels of testosterone and dihydrotestosterone were measured in each specimen using liquid chromatography/electrospray tandem mass spectrometry. Testosterone and dihydrotestosterone levels were compared with clinical variables and treatment received. RESULTS: Testosterone levels were similar in recurrent prostate cancer (3.75 pmol/g tissue) and androgen-stimulated benign prostate (2.75 pmol/g tissue, Wilcoxon two-sided, P=0.30). Dihydrotestosterone levels decreased 91% in recurrent prostate cancer (1.25 pmol/g tissue) compared with androgen-stimulated benign prostate (13.7 pmol/g tissue; Wilcoxon two-sided, P < 0.0001) although dihydrotestosterone levels in most specimens of recurrent prostate cancer were sufficient for androgen receptor activation. Testosterone or dihydrotestosterone levels were not related to metastatic status, antiandrogen treatment, or survival (Wilcoxon rank sum, all P > 0.2). CONCLUSIONS: Recurrent prostate cancer may develop the capacity to biosynthesize testicular androgens from adrenal androgens or cholesterol. This surprising finding suggests intracrine production of dihydrotestosterone and should be exploited for novel treatment of recurrent prostate cancer.
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Dihidrotestosterona/análisis , Neoplasias de la Próstata/patología , Testosterona/análisis , Anciano , Anciano de 80 o más Años , Cromatografía Líquida de Alta Presión/métodos , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Neoplasias de la Próstata/metabolismo , Radioinmunoensayo/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Análisis de SupervivenciaRESUMEN
PURPOSE: Prostate cancer recurs during androgen deprivation therapy despite reduced circulating androgens. We showed that recurrent prostate cancer tissue has testosterone levels similar to androgen-stimulated benign prostate, whereas dihydrotestosterone levels were reduced 82% to 1.45 nmol/L, sufficient for androgen receptor activation. The altered testosterone/dihydrotestosterone ratio in recurrent prostate cancer suggests loss of 5alpha-reducing capability. The aim of this study was to characterize steroid 5alpha-reductase isozymes I (S5alphaRI) and II (S5alphaRII) in prostate tissues. EXPERIMENTAL DESIGN: A tissue microarray was constructed from 22 recurrent prostate cancer specimens and matched pairs of androgen-stimulated benign prostate and androgen-stimulated prostate cancer from 23 radical prostatectomy specimens. Immunoblots were constructed from eight recurrent prostate cancers, eight androgen-stimulated benign prostate, and eight androgen-stimulated prostate cancer specimens. Isozyme expression was examined in microarray sections and immunoblots using S5alphaRI and S5alphaRII polyclonal antibodies. Isozyme activities were measured in 12 recurrent prostate cancer, 12 androgen-stimulated benign prostate, and 12 androgen-stimulated prostate cancer specimens. RESULTS: Nuclear immunostaining exhibited higher S5alphaRI expression than S5alphaRII in recurrent prostate cancer, androgen-stimulated benign prostate, and androgen-stimulated prostate cancers (P < 0.0001); mean expression was 125, 150, and 115 for S5alphaRI versus 10, 29, and 37 for S5alphaRII, respectively. Cytoplasmic immunostaining was moderate and similar for both isozymes in the three tissue types (P > 0.05). Immunoblots confirmed immunohistochemistry; S5alphaRI was expressed in recurrent prostate cancer specimens and S5alphaRII was not detected. The activity of S5alphaRI (114.4 pmol/mg epithelial protein/minute) was 3.7-fold higher than S5alphaRII (30.7 pmol/mg epithelial protein/minute) in recurrent prostate cancer specimens. CONCLUSIONS: Expression levels and isozyme activity shifts from S5alphaRII toward S5alphaRI in recurrent prostate cancer. Dual inhibition of S5alphaRI and S5alphaRII should reduce dihydrotestosterone biosynthesis and may prevent or delay growth of recurrent prostate cancer.
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3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Isoenzimas/metabolismo , Neoplasias de la Próstata/patología , Anciano , Anciano de 80 o más Años , Núcleo Celular/enzimología , Citoplasma/enzimología , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Neoplasias de la Próstata/enzimología , Análisis de Matrices TisularesRESUMEN
Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) is a validated treatment target for the treatment of metastatic castration-resistant prostate cancer (CRPC). Abiraterone acetate (AA) inhibits both 17α-hydroxylase (hydroxylase) and 17,20-lyase (lyase) reactions catalyzed by CYP17A1 and thus depletes androgen biosynthesis. However, coadministration of prednisone is required to suppress the mineralocorticoid excess and cortisol depletion that result from hydroxylase inhibition. VT-464, a nonsteroidal small molecule, selectively inhibits CYP17A1 lyase and therefore does not require prednisone supplementation. Administration of VT-464 in a metastatic CRPC patient presenting with high tumoral expression of both androgen receptor (AR) and CYP17A1, showed significant reduction in the level of both dehydroepiandrosterone (DHEA) and serum PSA. Treatment of a CRPC patient-derived xenograft, MDA-PCa-133 expressing H874Y AR mutant with VT-464, reduced the increase in tumor volume in castrate male mice more than twice as much as the vehicle (P < 0.05). Mass spectrometry analysis of post-treatment xenograft tumor tissues showed that VT-464 significantly decreased intratumoral androgens but not cortisol. VT-464 also reduced AR signaling more effectively than abiraterone in cultured PCa cells expressing T877A AR mutant. Collectively, this study suggests that VT-464 therapy can effectively treat CRPC and be used in precision medicine based on androgen receptor mutation status.
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Naftalenos/administración & dosificación , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Triazoles/administración & dosificación , Acetato de Abiraterona/administración & dosificación , Andrógenos/biosíntesis , Animales , Biopsia , Línea Celular Tumoral , Deshidroepiandrosterona/química , Humanos , Hidrocortisona/sangre , Masculino , Espectrometría de Masas , Ratones , Ratones SCID , Trasplante de Neoplasias , Medicina de Precisión , Prednisona/administración & dosificación , Receptores Androgénicos/genética , Transducción de Señal , Esteroide 17-alfa-Hidroxilasa/metabolismoRESUMEN
A key feature in the molecular pathogenesis of liver fibrosis requires maintenance of the activated hepatic stellate cell (HSC) phenotype by both proliferation and inhibition of apoptosis. We provide evidence that leptin is a potent HSC mitogen and dramatically inhibits stellate cell apoptosis. Leptin proved to be as potent an HSC mitogen as platelet-derived growth factor (PDGF) as assessed by bromodeoxyuridine (BrdU) incorporation in isolated primary HSCs; data using fluorescent propidium iodide (PI) uptake revealed that leptin, like PDGF, increased HSC populations in the S- and G2/M-phases of the cell cycle. Leptin resulted in a robust increase in cyclin D1 expression. Using the chemical inhibitor of Janus kinase 2 (Jak2) activity, AG 490, and overexpression of the suppressor of cytokine signaling 3 (SOCS-3), we show that blockade of leptin receptor (Ob-Rb) phosphorylation blocks leptin-induced HSC proliferation. Leptin-associated phosphorylation of both extracellular regulated kinase (p44/p42, Erk) and Akt is also prohibited. Further, the PI-3 kinase inhibitor LY294002 and MAPK inhibitor PD98059 were found to significantly reduce leptin-induced HSC proliferation, thereby indicating that leptin induced HSC proliferation is Akt- and Erk-dependent. Akt was also protective against HSC apoptosis. Leptin abolished both cycloheximide-induced and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, demonstrated by reduced caspase-3 activity, HSC-TUNEL staining, and DNA fragmentation. We conclude that leptin acts as a direct hepatic stellate cell survival agonist. Importantly, we have demonstrated that leptin-induced HSC proliferation and survival by Ob-Rb phosphorylation are both Erk- and Akt-dependent.
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Apoptosis/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Leptina/metabolismo , Leptina/farmacología , Hígado/citología , Hígado/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Reguladoras de la Apoptosis , Caspasa 3 , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Ciclina D1/biosíntesis , Cicloheximida/farmacología , ADN/biosíntesis , ADN/genética , ADN/metabolismo , Replicación del ADN/efectos de los fármacos , Flavonoides/farmacología , Hígado/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Mitógenos/farmacología , Mitosis/efectos de los fármacos , Morfolinas/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Receptores de Leptina , Proteínas Represoras/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Ligando Inductor de Apoptosis Relacionado con TNF , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Cancer is a metabolic disease. Cancer cells, being highly proliferative, show significant alterations in metabolic pathways such as glycolysis, respiration, the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, lipid metabolism, and amino acid metabolism. Metabolites like peptides, nucleotides, products of glycolysis, the TCA cycle, fatty acids, and steroids can be an important read out of disease when characterized in biological samples such as tissues and body fluids like urine, serum, etc. The cancer metabolome has been studied since the 1960s by analytical techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Current research is focused on the identification and validation of biomarkers in the cancer metabolome that can stratify high-risk patients and distinguish between benign and advanced metastatic forms of the disease. In this review, we discuss the current state of prostate cancer metabolomics, the biomarkers that show promise in distinguishing indolent from aggressive forms of the disease, the strengths and limitations of the analytical techniques being employed, and future applications of metabolomics in diagnostic imaging and personalized medicine of prostate cancer.
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The peptidyl-prolyl isomerase Pin1 is over-expressed in several cancer tissues is a potential prognostic marker in prostate cancer, and Pin1 ablation can suppress tumorigenesis in breast and prostate cancers. Pin1 can co-operate with activated ErbB2 or Ras to enhance tumorigenesis. It does so by regulating the activity of proteins that are essential for gene expression and cell proliferation. Several targets of Pin1 such as c-Myc, the Androgen Receptor, Estrogen Receptor-alpha, Cyclin D1, Cyclin E, p53, RAF kinase and NCOA3 are deregulated in cancer. At the posttranscriptional level, emerging evidence indicates that Pin1 also regulates mRNA decay of histone mRNAs, GM-CSF, Pth, and TGFß mRNAs by interacting with the histone mRNA specific protein SLBP, and the ARE-binding proteins AUF1 and KSRP, respectively. To understand how Pin1 may affect mRNA abundance on a genome-wide scale in mammalian cells, we used RNAi along with DNA microarrays to identify genes whose abundance is significantly altered in response to a Pin1 knockdown. Functional scoring of differentially expressed genes showed that Pin1 gene targets control cell adhesion, leukocyte migration, the phosphatidylinositol signaling system and DNA replication. Several mRNAs whose abundance was significantly altered by Pin1 knockdown contained AU-rich element (ARE) sequences in their 3' untranslated regions. We identified HuR and AUF1 as Pin1 interacting ARE-binding proteins in vivo. Pin1 was also found to stabilize all core histone mRNAs in this study, thereby validating our results from a previously published study. Statistical analysis suggests that Pin1 may target the decay of essential mRNAs that are inherently unstable and have short to medium half-lives. Thus, this study shows that an important biological role of Pin1 is to regulate mRNA abundance and stability by interacting with specific RNA-binding proteins that may play a role in cancer progression.