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1.
Prostate ; 84(10): 909-921, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38619005

RESUMO

INTRODUCTION: Lysine-specific demethylase 1 (LSD1) is emerging as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Neuroendocrine prostate cancer (NEPC) is increasingly recognized as an adaptive mechanism of resistance in mCRPC patients failing androgen receptor axis-targeted therapies. Safe and effective LSD1 inhibitors are necessary to determine antitumor response in prostate cancer models. For this reason, we characterize the LSD1 inhibitor bomedemstat to assess its clinical potential in NEPC as well as other mCRPC pathological subtypes. METHODS: Bomedemstat was characterized via crystallization, flavine adenine dinucleotide spectrophotometry, and enzyme kinetics. On-target effects were assessed in relevant prostate cancer cell models by measuring proliferation and H3K4 methylation using western blot analysis. In vivo, pharmacokinetic (PK) and pharmacodynamic (PD) profiles of bomedemstat are also described. RESULTS: Structural, biochemical, and PK/PD properties of bomedemstat, an irreversible, orally-bioavailable inhibitor of LSD1 are reported. Our data demonstrate bomedemstat has >2500-fold greater specificity for LSD1 over monoamine oxidase (MAO)-A and -B. Bomedemstat also demonstrates activity against several models of advanced CRPC, including NEPC patient-derived xenografts. Significant intra-tumoral accumulation of orally-administered bomedemstat is measured with micromolar levels achieved in vivo (1.2 ± 0.45 µM at the 7.5 mg/kg dose and 3.76 ± 0.43 µM at the 15 mg/kg dose). Daily oral dosing of bomedemstat at 40 mg/kg/day is well-tolerated, with on-target thrombocytopenia observed that is rapidly reversible following treatment cessation. CONCLUSIONS: Bomedemstat provides enhanced specificity against LSD1, as revealed by structural and biochemical data. PK/PD data display an overall safety profile with manageable side effects resulting from LSD1 inhibition using bomedemstat in preclinical models. Altogether, our results support clinical testing of bomedemstat in the setting of mCRPC.


Assuntos
Histona Desmetilases , Neoplasias de Próstata Resistentes à Castração , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/metabolismo , Masculino , Humanos , Animais , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Camundongos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/farmacocinética , Benzamidas , Piperazinas , Triazóis
2.
Prostate ; 84(1): 100-110, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37796107

RESUMO

BACKGROUND: Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. However, castration-resistant prostate cancer (CRPC) tumors can resist AR signaling inhibitors through AR amplification and AR splice variants in AR-positive CRPC (ARPC), and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We have shown previously that DNPC can bypass AR-dependence through fibroblast growth factor receptor (FGFR) signaling. However, the role of the FGFR pathway in other CRPC phenotypes has not been elucidated. METHODS: RNA-Seq analysis was conducted on patient metastases, LuCaP patient-derived xenograft (PDX) models, and CRPC cell lines. Cell lines (C4-2B, VCaP, and 22Rv1) and ex vivo LuCaP PDX tumor cells were treated with enzalutamide (ENZA) and FGFR inhibitors (FGFRi) alone or in combination and sensitivity was determined using cell viability assays. In vivo efficacy of FGFRi in ARPC, DNPC, and SCNPC were evaluated using PDX models. RESULTS: RNA-Seq analysis of FGFR signaling in metastatic specimens, LuCaP PDX models, and CRPC cell lines revealed significant FGF pathway activation in AR-low PC (ARLPC), DNPC, and SCNPC tumors. In vitro/ex vivo analysis of erdafitinib and CH5183284 demonstrated robust and moderate growth suppression of ARPC, respectively. In vivo studies using four ARPC PDX models showed that combination ENZA and CH5183284 significantly suppressed tumor growth. Additional in vivo studies using four ARPC PDX models revealed that erdafitinib monotherapy was as effective as ENZA in suppressing tumor growth, and there was limited combination benefit. Furthermore, two of three DNPC models and two of four SCNPC models responded to CH5183284 monotherapy, suggesting FGFRi responses were model dependent. RNA-Seq and gene set enrichment analysis of end-of-study ARPC tumors treated with FGFRi displayed decreased expression of E2F and MYC target genes and suppressed G2M checkpoint genes, whereas end-of-study SCNPC tumors had heterogeneous transcriptional responses. CONCLUSIONS: Although FGFRi treatments suppressed tumor growth across CRPC phenotypes, our analyses did not identify a single pathway or biomarker that would identify tumor response to FGFRi. This is very likely due to the array of FGFR1-4 expression and tumor phenotypes present in CRPC. Nevertheless, our data nominate the FGFR pathway as a clinically actionable target that promotes tumor growth in diverse phenotypes of treatment-refractory metastatic CRPC.


Assuntos
Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Androgênios/farmacologia , Transdução de Sinais , Linhagem Celular Tumoral , Nitrilas/farmacologia
3.
Prostate ; 83(15): 1470-1493, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37559436

RESUMO

BACKGROUND: The quinoline-3-carboxamide, Tasquinimod (TasQ), is orally active as a maintenance therapy with an on-target mechanism-of-action via allosteric binding to HDAC4. This prevents formation of the HDAC4/NCoR1/HDAC3 complex, disrupting HIF-1α transcriptional activation and repressing MEF-2 target genes needed for adaptive survival signaling in the compromised tumor micro environment. In phase 3 clinical testing against metastatic castration-resistant prostate cancer(mCRPC), TasQ (1 mg/day) increased time-to-progression, but not overall survival. METHODS: TasQ analogs were chemically synthesized and tested for activity compared to the parental compound. These included HDAC4 enzymatic assays, qRT-PCR and western blot analyses of gene and protein expression following treatment, in vitro and in vivo efficacy against multiple prostate cancer models including PDXs, pharmacokinetic analyses,AHR binding and agonist assays, SPR analyses of binding to HDAC4 and NCoR1, RNAseq analysis of in vivo tumors, 3D endothelial sprouting assays, and a targeted kinase screen. Genetic knockout or knockdown controls were used when appropriate. RESULTS: Here, we document that, on this regimen (1 mg/day), TasQ blood levels are 10-fold lower than the optimal concentration (≥2 µM) needed for anticancer activity, suggesting higher daily doses are needed. Unfortunately, we also demonstrate that TasQ is an arylhydrocarbon receptor (AHR) agonist, which binds with an EC50 of 1 µM to produce unwanted off-target side effects. Therefore, we screened a library of TasQ analogsto maximize on-target versus off-target activity. Using this approach, we identified ESATA-20, which has ~10-fold lower AHR agonism and 5-fold greater potency against prostate cancer patient-derived xenografts. CONCLUSION: This increased therapeuticindex nominates ESATA-20 as a lead candidate forclinical development as an orally active third generation quinoline-3-carboxamide analog thatretains its on-target ability to disrupt HDAC4/HIF-1α/MEF-2-dependent adaptive survival signaling in the compromisedtumor microenvironment found in mCRPC.


Assuntos
Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Western Blotting , Linhagem Celular Tumoral , Microambiente Tumoral , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo
4.
Molecules ; 26(24)2021 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-34946547

RESUMO

Søren Brøgger Christensen isolated and characterized the cell-penetrant sesquiterpene lactone Thapsigargin (TG) from the fruit Thapsia garganica. In the late 1980s/early 1990s, TG was supplied to multiple independent and collaborative groups. Using this TG, studies documented with a large variety of mammalian cell types that TG rapidly (i.e., within seconds to a minute) penetrates cells, resulting in an essentially irreversible binding and inhibiting (IC50~10 nM) of SERCA 2b calcium uptake pumps. If exposure to 50-100 nM TG is sustained for >24-48 h, prostate cancer cells undergo apoptotic death. TG-induced death requires changes in the cytoplasmic Ca2+, initiating a calmodulin/calcineurin/calpain-dependent signaling cascade that involves BAD-dependent opening of the mitochondrial permeability transition pore (MPTP); this releases cytochrome C into the cytoplasm, activating caspases and nucleases. Chemically unmodified TG has no therapeutic index and is poorly water soluble. A TG analog, in which the 8-acyl groups is replaced with the 12-aminododecanoyl group, afforded 12-ADT, retaining an EC50 for killing of <100 nM. Conjugation of 12-ADT to a series of 5-8 amino acid peptides was engineered so that they are efficiently hydrolyzed by only one of a series of proteases [e.g., KLK3 (also known as Prostate Specific Antigen); KLK2 (also known as hK2); Fibroblast Activation Protein Protease (FAP); or Folh1 (also known as Prostate Specific Membrane Antigen)]. The obtained conjugates have increased water solubility for systemic delivery in the blood and prevent cell penetrance and, thus, killing until the TG-prodrug is hydrolyzed by the targeting protease in the vicinity of the cancer cells. We summarize the preclinical validation of each of these TG-prodrugs with special attention to the PSMA TG-prodrug, Mipsagargin, which is in phase II clinical testing.


Assuntos
Antineoplásicos , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Pró-Fármacos , Tapsigargina , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Humanos , Pró-Fármacos/farmacocinética , Pró-Fármacos/uso terapêutico , Tapsigargina/farmacocinética , Tapsigargina/uso terapêutico
5.
Prostate ; 79(3): 320-330, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30488530

RESUMO

BACKGROUND: Prostate cancer is characterized by T-cell exclusion, which is consistent with their poor responses to immunotherapy. In addition, T-cells restricted to the adjacent stroma and benign areas are characterized by anergic and immunosuppressive phenotypes. In order for immunotherapies to produce robust anti-tumor responses in prostate cancer, this exclusion barrier and immunosuppressive microenvironment must first be overcome. We have previously identified mesenchymal stem cells (MSCs) in primary and metastatic human prostate cancer tissue. METHODS: An Opal Multiplex immunofluorescence assay based on CD73, CD90, and CD105 staining was used to identify triple-labeled MSCs in human prostate cancer tissue. T-cell suppression assays and flow cytometry were used to demonstrate the immunosuppressive potential of primary MSCs expanded from human bone marrow and prostate cancer tissue from independent donors. RESULTS: Endogenous MSCs were confirmed to be present at sites of human prostate cancer. These prostate cancer-infiltrating MSCs suppress T-cell proliferation in a dose-dependent manner similar to their bone marrow-derived counterparts. Also similar to bone marrow-derived MSCs, prostate cancer-infiltrating MSCs upregulate expression of PD-L1 and PD-L2 on their cell surface in the presence of IFNγ and TNFα. CONCLUSION: Prostate cancer-infiltrating MSCs suppress T-cell proliferation similar to canonical bone marrow-derived MSCs, which have well-documented immunosuppressive properties with numerous effects on both innate and adaptive immune system function. Thus, we hypothesize that selective depletion of MSCs infiltrating sites of prostate cancer should restore immunologic recognition and elimination of malignant cells via broad re-activation of cytotoxic pro-inflammatory pathways.


Assuntos
Células-Tronco Mesenquimais/imunologia , Neoplasias da Próstata/imunologia , Microambiente Tumoral/imunologia , Comunicação Celular/imunologia , Humanos , Tolerância Imunológica , Ativação Linfocitária , Masculino , Células-Tronco Mesenquimais/patologia , Células PC-3 , Neoplasias da Próstata/patologia , Linfócitos T/imunologia
6.
Prostate ; 79(11): 1326-1337, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31212368

RESUMO

BACKGROUND: Serially transplantable patient-derived xenografts (PDXs) are invaluable preclinical models for studying tumor biology and evaluating therapeutic agents. As these models are challenging to establish from prostate cancer specimens, the ability to preserve them through cryopreservation has several advantages for ongoing research. Despite this, there is still uncertainty about the ability to cryopreserve PDXs of prostate cancer. This study compared three different cryopreservation protocols to identify a method that can be used to reproducibly cryopreserve a diverse cohort of prostate cancer PDX models. METHODS: One serially transplantable prostate cancer PDX from the Melbourne Urological Research Alliance cohort was used to compare three cryopreservation protocols: slow freezing in fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO), FCS with 10% DMSO supplemented with the Rho-associated kinase (ROCK) inhibitor Y-27632 and vitrification. The efficiency of the slow freezing protocols was then assessed in 17 additional prostate cancer PDXs. Following cryopreservation, PDXs were re-established in host mice that were either intact and supplemented with testosterone or castrated. Graft take rate, tumor growth, histological features, and transcriptome profiles before and after cryopreservation were compared. RESULTS: Slow freezing maintained the viability and histological features of prostate cancer PDXs, and the addition of a ROCK inhibitor increased their growth following cryopreservation. Using the slow freezing method, we re-established 100% of PDXs grown in either testosterone-supplemented or castrated host mice. Importantly, the long-term tumor growth rate and transcriptome profile were maintained following cryopreservation. CONCLUSION: This study has identified a protocol to reliably cryopreserve and re-establish a diverse cohort of serially transplantable PDXs of prostate cancer. This study has the potential to significantly improve the practicality of maintaining PDX models. Cryopreservation may also increase the accessibility of these important resources and provide new opportunities for preclinical studies on a broader spectrum of prostate tumors.


Assuntos
Criopreservação/métodos , Xenoenxertos , Transplante de Neoplasias/métodos , Neoplasias da Próstata/patologia , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Transplante de Neoplasias/patologia
7.
Prostate ; 78(9): 646-654, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29575112

RESUMO

BACKGROUND: Presently, ∼85 serially transplantable human prostate cancer xenografts spanning the phenotypic, epigenetic, and genetic heterogeneity seen clinically are available in a variety of laboratories throughout the world. If distributed to the prostate cancer research community, these can provide an experimental platform for resolving the specificity versus generalizability of basic cancer biology principles (eg, credentialing of therapeutic molecular targets) and for validating translational approaches for prevention, diagnosis, and therapy. Thus, there is an urgent need to distribute the already established serially transplantable human prostate cancer xenografts and to develop robust methods for establishing new ones. METHODS: To accelerate the development of such additional xenografts, particularly from patients treated with the newer standard of care agents (ie, abiraterone, enzalutamide, cabazitaxel, alpharadin, etc), a historic review of the field will be presented. RESULTS: Over the last 50 years, multiple groups throughout the world have developed methods for the successful establishment of serially transplantable human prostate cancer xenografts using a variety of immune deficient mice. These are summarized chronologically. CONCLUSIONS AND FUTURE: With the ever growing appreciation of the value of personalized medicine (aka precision medicine), methods need to be developed that allow efficient and timely growth of primary patient derived prostate cancer xenografts (PDXs), which can be used as "avatars" for defining optimal therapy for that specific patient. Such development should be based upon the leads obtained from the successful establishment of serially transplantable prostate cancer xenografts described in this review.


Assuntos
Modelos Animais de Doenças , Neoplasias da Próstata , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Materiais Biocompatíveis , Colágeno , Combinação de Medicamentos , Humanos , Laminina , Masculino , Camundongos Endogâmicos , Proteoglicanas
8.
Prostate ; 78(9): 655-663, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29572902

RESUMO

BACKGROUND: The SH-group at Cys-34 of human serum albumin (HSA) is a unique and accessible functional group that can be exploited for efficient linkage of a maleimide containing cytotoxic drug derivative to albumin. The specific maleimide chemistry used for production of the maleimide-linked albumin drug (MAD) is critical, however, to minimize the plasma concentration of "free" cytotoxic drug spontaneously released from albumin carrier thus decreasing dose-limiting host toxicity while enhancing the plasma half-life from minutes to days (ie, pharmacokinetic effect) and tissue concentration of the MAD in the extracellular cellular fluid at sites of cancer (ie, EPR effect). METHODS: To accomplish this goal, a chemical synthesis was developed using 2-fluoro-5-maleimidobenzoic acid to stably link the potent cytotoxic chemically modified analogue of the naturally occurring sesquiterpene γ-lactone, thapsigargin, 8-O-(12-aminododecanoyl)-8-O-debutanoyl thapsigargin (12ADT), to Cys-34 of albumin to produce 12ADT-MAD. RESULTS: Using FITC-labeling, LC/MS analysis, and in vitro growth and clonogenic survival assays on a series of 6 human prostate cancer lines (LNCaP, LAPC-4, VCap, CWR22Rv 1, PC3, and Du145), we documented that 12ADT-MAD is endocytosed by prostate cancer cells where it is degraded into its amino acids liberating cysteinyl-maleimide-12ADT which is both chemically stable at the acidic pH of 5.5 present in the endosome while retaining its high killing ability (IC50 50 nM) via SERCA inhibition. CONCLUSIONS: Based upon these positive in vitro validation results, the in vivo efficacy versus host toxicity of this 12-ADT-MAD approach is presently being evaluated against a series of patient derived androgen responsive and castration resistant human xenografts in immune-deficient mice.


Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Lactonas/farmacocinética , Maleimidas/farmacologia , Antígeno Prostático Específico/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Sesquiterpenos/farmacocinética , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Citotoxinas/síntese química , Citotoxinas/química , Citotoxinas/farmacologia , Citotoxinas/uso terapêutico , Líquido Extracelular/química , Líquido Extracelular/efeitos dos fármacos , Humanos , Lactonas/síntese química , Lactonas/química , Lactonas/uso terapêutico , Masculino , Maleimidas/síntese química , Maleimidas/química , Maleimidas/uso terapêutico , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/secundário , Albumina Sérica Humana/farmacologia , Albumina Sérica Humana/uso terapêutico , Sesquiterpenos/síntese química , Sesquiterpenos/química , Sesquiterpenos/uso terapêutico
9.
Prostate ; 78(16): 1262-1282, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30073676

RESUMO

BACKGROUND: While it has been challenging to establish prostate cancer patient-derived xenografts (PDXs), with a take rate of 10-40% and long latency time, multiple groups throughout the world have developed methods for the successful establishment of serially transplantable human prostate cancer PDXs using a variety of immune deficient mice. In 2014, the Movember Foundation launched a Global Action Plan 1 (GAP1) project to support an international collaborative prostate cancer PDX program involving eleven groups. Between these Movember consortium members, a total of 98 authenticated human prostate cancer PDXs were available for characterization. Eighty three of these were derived directly from patient material, and 15 were derived as variants of patient-derived material via serial passage in androgen deprived hosts. A major goal of the Movember GAP1 PDX project was to provide the prostate cancer research community with a summary of both the basic characteristics of the 98 available authenticated serially transplantable human prostate cancer PDX models and the appropriate contact information for collaborations. Herein, we report a summary of these PDX models. METHODS: PDX models were established in immunocompromised mice via subcutaneous or subrenal-capsule implantation. Dual-label species (ie, human vs mouse) specific centromere and telomere Fluorescence In Situ Hybridization (FISH) and immuno-histochemical (IHC) staining of tissue microarrays (TMAs) containing replicates of the PDX models were used for characterization of expression of a number of phenotypic markers important for prostate cancer including AR (assessed by IHC and FISH), Ki67, vimentin, RB1, P-Akt, chromogranin A (CgA), p53, ERG, PTEN, PSMA, and epithelial cytokeratins. RESULTS: Within this series of PDX models, the full spectrum of clinical disease stages is represented, including androgen-sensitive and castration-resistant primary and metastatic prostate adenocarcinomas as well as prostate carcinomas with neuroendocrine differentiation. The annotated clinical characteristics of these PDXs were correlated with their marker expression profile. CONCLUSION: Our results demonstrate the clinical relevance of this series of PDXs as a platform for both basic science studies and therapeutic discovery/drug development. The present report provides the prostate cancer community with a summary of the basic characteristics and a contact information for collaborations using these models.


Assuntos
Xenoenxertos , Transplante de Neoplasias/métodos , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Biomarcadores Tumorais/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Neoplasias da Próstata/metabolismo
10.
Prostate ; 77(4): 374-384, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27859428

RESUMO

BACKGROUND: There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. METHODS: A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. RESULTS: Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. CONCLUSIONS: The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic "drivers" for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374-384, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Células Epiteliais/metabolismo , Próstata/citologia , Próstata/metabolismo , Telomerase/biossíntese , Animais , Linhagem Celular Transformada , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Expressão Gênica , Humanos , Masculino , Camundongos , Telomerase/genética
11.
N Engl J Med ; 371(11): 1028-38, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25184630

RESUMO

BACKGROUND: The androgen-receptor isoform encoded by splice variant 7 lacks the ligand-binding domain, which is the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone. METHODS: We used a quantitative reverse-transcriptase-polymerase-chain-reaction assay to evaluate AR-V7 in circulating tumor cells from prospectively enrolled patients with metastatic castration-resistant prostate cancer who were initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status (positive vs. negative) and prostate-specific antigen (PSA) response rates (the primary end point), freedom from PSA progression (PSA progression-free survival), clinical or radiographic progression-free survival, and overall survival. RESULTS: A total of 31 enzalutamide-treated patients and 31 abiraterone-treated patients were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in circulating tumor cells. Among men receiving enzalutamide, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 53%, P=0.004) and shorter PSA progression-free survival (median, 1.4 months vs. 6.0 months; P<0.001), clinical or radiographic progression-free survival (median, 2.1 months vs. 6.1 months; P<0.001), and overall survival (median, 5.5 months vs. not reached; P=0.002). Similarly, among men receiving abiraterone, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 68%, P=0.004) and shorter PSA progression-free survival (median, 1.3 months vs. not reached; P<0.001), clinical or radiographic progression-free survival (median, 2.3 months vs. not reached; P<0.001), and overall survival (median, 10.6 months vs. not reached, P=0.006). The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of full-length androgen receptor messenger RNA. CONCLUSIONS: Detection of AR-V7 in circulating tumor cells from patients with castration-resistant prostate cancer may be associated with resistance to enzalutamide and abiraterone. These findings require large-scale prospective validation. (Funded by the Prostate Cancer Foundation and others.).


Assuntos
Androstenóis/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/genética , RNA Neoplásico/análise , Receptores Androgênicos/genética , Androstenos , Benzamidas , Humanos , Masculino , Morfinanos/análise , Nitrilas , Feniltioidantoína/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida
12.
Prostate ; 76(6): 552-64, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26732992

RESUMO

BACKGROUND: Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. METHODS: Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (<25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-ß measured by ELISA. RESULTS: MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-ß concentrations. All conditions generated populations with an average cell diameter >15 µm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. CONCLUSIONS: Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping properties. The lack of adipogenesis in stromal cultures derived from normal prostates suggests they have a lineage-restricted progenitor phenotype. The retention of adipogenic differentiation in cultures from a subset of prostate cancer patients suggests the active recruitment of less committed progenitors or MSCs from the bone marrow as a function of disease progression. This recruitment can potentially be exploited for prognostic purposes or a cell-based platform for the systemic delivery of cytotoxic agents to sites of prostate cancer.


Assuntos
Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Próstata/patologia , Neoplasias da Próstata/patologia , Microambiente Tumoral/fisiologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Fibroblastos/patologia , Citometria de Fluxo/métodos , Humanos , Masculino , Próstata/metabolismo , Neoplasias da Próstata/metabolismo
13.
Am J Pathol ; 185(3): 614-6, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25700984

RESUMO

This commentary highlights the article by Ge et al, which proposes the use of methylation and expression of SRD5A2 as a gene signature to tailor therapies for prostatic diseases.


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Envelhecimento/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Animais , Humanos , Masculino
15.
Prostate ; 74(11): 1118-31, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24913829

RESUMO

INTRODUCTION: Physiologic testosterone continuously stimulates prostate stromal cell secretion of paracrine growth factors (PGFs), which if unopposed would induce hyperplastic overgrowth of normal prostate epithelial cells (PrECs). METHODS: Lentiviral shRNA stable knock down of c-MYC, ß-catenin, or TCF-4 completely inhibits normal (i.e., non-transformed) human PrECs growth. c-MYC enhancer driven reporter expression and growth is inhibited by two chemically distinct molecules, which prevent ß-catenin signaling either by blocking TCF-4 binding (i.e., toxoflavin) or by stimulating degradation (i.e., AVX939). Recombinant DKK1 protein at a dose, which inhibits activation of canonical Wnt signaling does not inhibit PrEC growth. Nuclear ß-catenin translocation and PrEC growth is prevented by both lack of PGFs or Akt inhibitor-I. Growth inhibition induced by lack of PGFs, toxoflavin, or Akt inhibitor-I is overcome by constitutive c-MYC transcription. RESULTS: In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen binding to AR suppressing c-MYC transcription, resulting in G0 arrest/terminal differentiation independent of Rb, p21, p27, FoxP3, or down regulation of growth factors receptors and instead involves androgen-induced formation of AR/ß-catenin/TCF-4 complexes, which suppress c-MYC transcription. Such suppression does not occur when AR is mutated in its zinc-finger binding domain. DISCUSSION: Proliferation of non-transformed human PrECs is dependent upon c-MYC transcription via formation/binding of ß-catenin/TCF-4 complexes at both 5' and 3' c-MYC enhancers stimulated by Wnt-independent, PGF induced Akt signaling. In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen-induced formation of AR/ß-catenin/TCF-4 complexes, which retains binding to 3' c-MYC enhancer, but now suppresses c-MYC transcription.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Proliferação de Células , Células Epiteliais/patologia , Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Receptores Androgênicos/fisiologia , Fatores de Transcrição/fisiologia , beta Catenina/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células Cultivadas , Fatores de Transcrição Forkhead/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , Hiperplasia Prostática/prevenção & controle , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/fisiologia , Fator de Transcrição 4 , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Transcrição Gênica/fisiologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/efeitos dos fármacos , beta Catenina/genética
16.
Prostate ; 74(13): 1308-19, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25053236

RESUMO

BACKGROUND: As carcinoma progresses, the stroma undergoes a variety of phenotypic changes, including the presence of carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). FAP is a post-prolyl endopeptidase whose expression in a healthy adult is largely restricted to the cancer-associated stroma. FAP-targeted prodrugs with a 100-fold greater therapeutic window over the parent compound were previously generated. METHODS: Prodrugs and non-cleavable controls were incubated in the presence of FAP. Plasma and tumor half-lives (t1/2) of the full-length and active forms of the prodrugs were determined using LCMS. Biodistribution studies of prodrug activation were performed. Histopathological analysis of tissues from treated animals were compared to vehicle-treated controls. Toxicity and efficacy studies were performed in human breast (MDA-MB-231 and MCF-7) and prostate (LNCaP) cancer xenografts models. RESULTS: These FAP-activated prodrugs have a significantly slower clearance from tumor tissue than the circulation (∼12 vs. ∼4.5 hr). Micromolar concentrations of active drug persist in the tumor. Active drug is detected in non-target tissues; however, histopathologic evaluation reveals no evidence of drug-induced toxicity. A FAP-activated prodrug (ERGETGP-S12ADT) inhibits tumor growth in multiple human breast and prostate cancer xenograft models. The anti-tumor effect is comparable to that observed with docetaxel, but results in significantly less toxicity. CONCLUSION: FAP-activated prodrugs are a viable strategy for the management of prostate and other cancers. These prodrugs exhibit less toxicity than a commonly used chemotherapeutic agent. Further refinement of the FAP cleavage site for greater specificity may reduce prodrug activation in non-target tissues and enhance clinical benefit.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacocinética , Gelatinases/farmacocinética , Proteínas de Membrana/farmacocinética , Pró-Fármacos/farmacocinética , Neoplasias da Próstata/tratamento farmacológico , Serina Endopeptidases/farmacocinética , Adenocarcinoma/patologia , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Endopeptidases , Gelatinases/efeitos adversos , Gelatinases/uso terapêutico , Humanos , Masculino , Proteínas de Membrana/efeitos adversos , Proteínas de Membrana/uso terapêutico , Camundongos , Pró-Fármacos/efeitos adversos , Pró-Fármacos/uso terapêutico , Neoplasias da Próstata/patologia , Serina Endopeptidases/efeitos adversos , Serina Endopeptidases/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Prostate ; 74(8): 805-10, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24677180

RESUMO

BACKGROUND: It has been reported that significant hypoxia may occur in the rat prostate following androgen deprivation (AD). It is well known that hypoxia substantially reduces radiation sensitivity of cells both in vitro and in vivo. Given that contemporary management of men with intermediate and high-risk prostate cancer includes the use of neoadjuvant androgen suppression and radiation, AD-induced hypoxia in the prostate could result in suboptimal therapeutic results. Given this concern, we fully investigate possible AD-induced hypoxia in the ventral prostate (VP) of adult rats by two independent methods. METHODS: Tissue pO2 levels in the VP of adult Spraque-Dawley rats were evaluated prior to and at various time points following castration by two independent techniques. First, an Oxylab tissue oxygen monitor with a 240 µm probe was used for quantitative monitoring of global VP oxygenation. Second, fluorescence immunohistochemistry using the hypoxia marker EF5, known to be metabolically activated by hypoxic cells, was used to evaluate cell-to-cell variation in hypoxia at various days post-castration. RESULTS: Neither the oxygen probe nor EF5 method demonstrate any substantive change in pO2 levels in the rat VP at any time point post-castration. CONCLUSIONS: We find no evidence that the rat VP becomes hypoxic at any point following castration using an animal model that closely mimics the human prostate. These data are in contrast to previous reports suggesting prostatic hypoxia occurs following AD and provide assurance that our present therapeutic strategy of neoadjuvant AD followed by radiation is not compromised by AD-induced tissue hypoxia.


Assuntos
Androgênios/metabolismo , Hipóxia Celular , Orquiectomia , Consumo de Oxigênio/fisiologia , Próstata/metabolismo , Androgênios/deficiência , Animais , Hipóxia Celular/fisiologia , Masculino , Próstata/patologia , Ratos , Ratos Sprague-Dawley
18.
Acta Crystallogr Sect E Struct Rep Online ; 70(Pt 3): o297-8, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24765003

RESUMO

The title compound, C20H17F3N2O4, named tasquinimod, is a second-generation oral quinoline-3-carboxamide analogue, which is currently in phase III clinical trials for the treatment of metastatic prostate cancer. The quinoline unit is almost planar (r.m.s. deviation of fitted atoms = 0.0075 Å). The carboxamide side chain, substituted at position 3, is tilted by 88.07 (7)° to the quinoline plane. Both the methyl and carbonyl groups of this carboxamide side chain are in a syn conformation. The 4-(tri-fluoro-meth-yl)phenyl plane is inclined at 50.62 (17)° to the plane of the carboxamide side chain, and at 87.14 (4)° to the plane of the quinoline ring system. The 4-hy-droxy H atom acts as a double proton donor in an intra-molecular hydrogen bond to the 5-position meth-oxy O atom and in an inter-molecular contact to the 2-oxo group, generating a chain along [010] in the crystal structure.

19.
Mol Cancer Ther ; 23(10): 1404-1417, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-38894678

RESUMO

Polo-like kinase 1 (PLK1) inhibitors have had limited antitumor efficacy as single agents, and focus of current efforts is on combination therapies. We initially confirmed that the PLK1-specific inhibitor onvansertib (ONV) could enhance responses to a PARP inhibitor (olaparib) in prostate cancer xenografts. To identify more effective combinations, we screened a library of bioactive compounds for efficacy in combination with ONV in LNCaP prostate cancer cells, which identified a series of compounds including multiple AKT inhibitors. We confirmed in vitro synergy between ONV and the AKT inhibitor ipatasertib (IPA) and found that the combination increased apoptosis. Mechanistic studies showed that ONV increased expression of the antiapoptotic protein SURVIVIN and that this was mitigated by IPA. Studies in three PTEN-deficient prostate cancer xenograft models showed that cotreatment with IPA and ONV led to significant tumor growth inhibition compared with monotherapies. Together, these in vitro and in vivo studies demonstrate that the efficacy of PLK1 antagonists can be enhanced by PARP or AKT inhibition and support further development of these combination therapies.


Assuntos
Proteínas de Ciclo Celular , Quinase 1 Polo-Like , Neoplasias da Próstata , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Masculino , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Camundongos , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Sinergismo Farmacológico , Pteridinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Piperazinas , Pirimidinas
20.
Prostate ; 73(12): 1316-25, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23775398

RESUMO

BACKGROUND: Immune deficient male mice bearing human prostate cancer xenografts are used to evaluate therapeutic response to novel androgen ablation approaches and the results compared to surgical castration based upon assumption that testosterone microenvironment in intact and castrated adult male mice mimics eugonadal and castrated aging adult human males. METHODS: To test these assumptions, serum total testosterone (TT) and free testosterone (FT) were determined longitudinally in groups (n > 20) of intact versus castrated adult male nude, NOG, and immune competent C57BL/6 mice. RESULTS: In adult male mice, TT and FT varies by 30- to 100-fold within the same animal providing a microenvironment that is only equivalent to hypogonadal, not eugonadal, adult human males (TT is 1.7 ± 1.2 ng/ml [5.8 ± 4.1 nM] in nude and 2.5 ± 1.3 ng/ml [8.7 ± 4.4 nM] in NOG mice versus >4.2 ng/ml [14.7 nM] in eugonadal humans). This was confirmed based upon enhanced growth of androgen dependent human prostate cancer xenografts inoculated into mice supplemented with exogenous testosterone to elevate and chronically maintain serum TT at a level (5 ng/ml [18 nM]) equivalent to a 50-year-old eugonadal human male. In castrated mice, TT and FT range from 2 to 20 pg/ml (7-70 pM) and <0.8 pg/ml (<2.6 pM), respectively, which is equivalent to castrate resistant prostate cancer (CRPC) patients treated with abiraterone. This was confirmed based upon the inability of another CYP17A1 inhibitor, ketoconazole, to inhibit the growth of CRPC xenografts in castrated mice. CONCLUSIONS: Adult male mice supplemented with testosterone mimic eugonadal human males, while unsupplemented animals mimic standard androgen ablation and castrated animals mimic abiraterone treated patients. These studies confirm what is claimed in Robert Burns' poem "To a Mouse" that "The best laid schemes of mice and men/often go awry.".


Assuntos
Envelhecimento/sangue , Androstenóis/farmacologia , Hipogonadismo/sangue , Orquiectomia , Testosterona/sangue , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/efeitos dos fármacos , Androstenos , Androstenóis/toxicidade , Animais , Linhagem Celular Tumoral , Humanos , Hipogonadismo/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Pessoa de Meia-Idade
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