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Polo-like kinase 1 (PLK1) inhibitors have had limited antitumor efficacy as single agents, and a 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 anti-apoptotic protein SURVIVIN, and that this was mitigated by IPA. Studies in three PTEN deficient prostate cancer xenograft models showed that co-treatment with IPA and ONV led to significant tumor growth inhibition compared to 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.
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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 , TriazóisRESUMO
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.
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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/farmacologiaRESUMO
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.
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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/metabolismoRESUMO
Abiraterone is a standard treatment for metastatic castrate-resistant prostate cancer (mCRPC) that slows disease progression by abrogating androgen synthesis and antagonizing the androgen receptor (AR). Here we report that inhibitors of the mitotic regulator polo-like kinase-1 (Plk1), including the clinically active third-generation Plk1 inhibitor onvansertib, synergizes with abiraterone in vitro and in vivo to kill a subset of cancer cells from a wide variety of tumor types in an androgen-independent manner. Gene-expression analysis identified an AR-independent synergy-specific gene set signature upregulated upon abiraterone treatment that is dominated by pathways related to mitosis and the mitotic spindle. Abiraterone treatment alone caused defects in mitotic spindle orientation, failure of complete chromosome condensation, and improper cell division independently of its effects on AR signaling. These effects, although mild following abiraterone monotherapy, resulted in profound sensitization to the antimitotic effects of Plk1 inhibition, leading to spindle assembly checkpoint-dependent mitotic cancer cell death and entosis. In a murine patient-derived xenograft model of abiraterone-resistant metastatic castration-resistant prostate cancer (mCRPC), combined onvansertib and abiraterone resulted in enhanced mitotic arrest and dramatic inhibition of tumor cell growth compared with either agent alone. Overall, this work establishes a mechanistic basis for the phase II clinical trial (NCT03414034) testing combined onvansertib and abiraterone in mCRPC patients and indicates this combination may have broad utility for cancer treatment. SIGNIFICANCE: Abiraterone treatment induces mitotic defects that sensitize cancer cells to Plk1 inhibition, revealing an AR-independent mechanism for this synergistic combination that is applicable to a variety of cancer types.
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Neoplasias de Próstata Resistentes à Castração , Receptores Androgênicos , Masculino , Humanos , Animais , Camundongos , Receptores Androgênicos/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Androgênios , MitoseRESUMO
Testosterone is the canonical growth factor of prostate cancer but can paradoxically suppress its growth when present at supraphysiological levels. We have previously demonstrated that the cyclical administration of supraphysiological androgen (SPA), termed bipolar androgen therapy (BAT), can result in tumor regression and clinical benefit for patients with castration-resistant prostate cancer. However, predictors and mechanisms of response and resistance have been ill defined. Here, we show that growth inhibition of prostate cancer models by SPA required high androgen receptor (AR) activity and were driven in part by downregulation of MYC. Using matched sequential patient biopsies, we show that high pretreatment AR activity predicted downregulation of MYC, improved clinical response, and prolonged progression-free and overall survival for patients on BAT. BAT induced strong downregulation of AR in all patients, which is shown to be a primary mechanism of acquired resistance to SPA. Acquired resistance was overcome by alternating SPA with the AR inhibitor enzalutamide, which induced adaptive upregulation of AR and resensitized prostate cancer to SPA. This work identifies high AR activity as a predictive biomarker of response to BAT and supports a treatment paradigm for prostate cancer involving alternating between AR inhibition and activation.
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Neoplasias de Próstata Resistentes à Castração , Receptores Androgênicos , Masculino , Humanos , Receptores Androgênicos/metabolismo , Androgênios/farmacologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Antagonistas de Receptores de Andrógenos/uso terapêutico , Nitrilas , Testosterona/farmacologia , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular TumoralRESUMO
Resistance to AR signaling inhibitors (ARSis) in a subset of metastatic castration-resistant prostate cancers (mCRPCs) occurs with the emergence of AR- neuroendocrine prostate cancer (NEPC) coupled with mutations/deletions in PTEN, TP53, and RB1 and the overexpression of DNMTs, EZH2, and/or SOX2. To resolve whether the lack of AR is the driving factor for the emergence of the NE phenotype, molecular, cell, and tumor biology analyses were performed on 23 xenografts derived from patients with PC, recapitulating the full spectrum of genetic alterations proposed to drive NE differentiation. Additionally, phenotypic response to CRISPR/Cas9-mediated AR KO in AR+ CRPC cells was evaluated. These analyses document that (a) ARSi-resistant NEPC developed without androgen deprivation treatment; (b) ARS in ARSi-resistant AR+/NE+ double-positive "amphicrine" mCRPCs did not suppress NE differentiation; (c) the lack of AR expression did not necessitate acquiring a NE phenotype, despite concomitant mutations/deletions in PTEN and TP53, and the loss of RB1 but occurred via emergence of an AR-/NE- double-negative PC (DNPC); (d) despite DNPC cells having homogeneous genetic driver mutations, they were phenotypically heterogeneous, expressing basal lineage markers alone or in combination with luminal lineage markers; and (e) AR loss was associated with AR promoter hypermethylation in NEPCs but not in DNPCs.
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Carcinoma Neuroendócrino/genética , Transformação Celular Neoplásica/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias de Próstata Resistentes à Castração/genética , Acetato de Abiraterona/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Carcinoma Neuroendócrino/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Masculino , Camundongos , Transplante de Neoplasias , Nitrilas/uso terapêutico , PTEN Fosfo-Hidrolase/genética , Feniltioidantoína/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética , Proteínas de Ligação a Retinoblastoma/genética , Fatores de Transcrição SOXB1/genética , Tioidantoínas/uso terapêutico , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The role of truncated androgen receptor splice variant-7 (AR-V7) in prostate cancer biology is an unresolved question. Is it simply a marker of resistance to 2nd-generation androgen receptor signaling inhibitors (ARSi) like abiraterone acetate (Abi) and enzalutamide (Enza) or a functional driver of lethal resistance via its ligand-independent transcriptional activity? To resolve this question, the correlation between resistance to ARSi and genetic chances and expression of full length AR (AR-FL) vs. AR-V7 were evaluated in a series of independent patient-derived xenografts (PDXs). While all PDXs lack PTEN expression, there is no consistent requirement for mutation in TP53, RB1, BRCA2, PIK3CA, or MSH2, or expression of SOX2 or ERG and ARSi resistance. Elevated expression of AR-FL alone is sufficient for Abi but not Enza resistance, even if AR-FL is gain-of-function (GOF) mutated. Enza resistance is consistently correlated with enhanced AR-V7 expression. In vitro and in vivo growth responses of Abi-/Enza-resistant LNCaP-95 cells in which CRISPR-Cas9 was used to knockout AR-FL or AR-V7 alone or in combination were evaluated. Combining these growth responses with RNAseq analysis demonstrates that both AR-FL- and AR-V7-dependent transcriptional complementation are needed for Abi/Enza resistance.
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Antagonistas de Androgênios/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptores Androgênicos/genética , Acetato de Abiraterona/farmacologia , Acetato de Abiraterona/uso terapêutico , Idoso , Antagonistas de Androgênios/uso terapêutico , Animais , Benzamidas , Biópsia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Feniltioidantoína/uso terapêutico , Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Splicing de RNA , RNA-Seq , Receptores Androgênicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PRX302 is a highly potent, mutant bacterial pore-forming biologic protoxin engineered for selective activation by PSA, a serine protease expressed by benign and malignant prostate epithelial cells. Although being developed as a local therapy for benign prostatic hyperplasia and localized prostate cancer, PRX302 cannot be administered systemically as a treatment for metastatic disease due to binding to ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored proteins, which leads to poor accumulation within the tumor microenvironment. To overcome this limitation, poly-lactic-co-glycolic acid (PLGA) microparticles encapsulating the protoxin were developed, which are known to accumulate in the liver, a major site of metastasis for prostate cancer and other solid tumors. A highly sensitive and reproducible sandwich ELISA to quantify PRX302 released from microparticles was developed. Utilizing this assay, PRX302 release from different microparticle formulations was assessed over multiple days. Hemolysis assays documented PSA-dependent pore formation and lytic potential (i.e., function) of the released protoxin. MTT assays demonstrated that conditioned supernatant from PRX302-loaded, but not blank (i.e., unloaded), PLGA microparticles was highly cytotoxic to PC3 and DU145 human prostate cancer cells in the presence of exogenous PSA. Microparticle encapsulation prevented PRX302 from immediately interacting with GPI-anchored proteins as demonstrated in a competition assay, which resulted in an increased therapeutic index and significant antitumor efficacy following a single dose of PRX302-loaded microparticles in a preclinical model of prostate cancer liver metastasis with no obvious toxicity. These results document that PRX302 released from PLGA microparticles demonstrate in vivo antitumor efficacy in a clinically relevant preclinical model of metastatic prostate cancer.
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Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Composição de Medicamentos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Neoplasias de Próstata Resistentes à Castração/patologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Toxinas Bacterianas/metabolismo , Produtos Biológicos/administração & dosagem , Produtos Biológicos/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Composição de Medicamentos/métodos , Hemólise/efeitos dos fármacos , Humanos , Masculino , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ligação Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Liver metastasis is not uncommon in men with metastatic castration-resistant prostate cancer (mCRPC), estimated at ~20% to 60% of advanced late-stage patients. Liver and other visceral metastases are associated with worse overall survival. Recent evidence suggests the frequency of visceral metastases may be increasing for reasons that are unclear but may be related to selective pressures induced by modern therapies, including second-generation antiandrogen receptor signaling inhibitors such as enzalutamide and abiraterone. Consequently, robust models to study the pathobiology of prostate cancer liver metastases and their response to therapy are urgently needed. METHODS: Hemi-spleen injection of human (LN95, PC3, VCaP, and MDA-PCa-2b) or syngeneic (Myc-CaP) prostate cancer cells (1 × 106 ) was performed to seed liver metastases via the splenic vessels. Plasma levels of prostate-specific antigen (PSA) were monitored longitudinally in human androgen receptor-positive (AR+) models. Immunohistochemical staining of AR and HoxB13 was performed to document the prostatic origin of hepatic lesions. RESULTS: LN95, PC3, and Myc-CaP produced distinct liver micrometastases that progressed to macrometastases by ~2 to 4 weeks postinoculation, while inoculation of MDA-PCa-2b and VCaP only produced occasional micrometastases and seeding of individual cells adjacent to blood vessels, respectively, at the time points analyzed. All lesions are characterized by positive staining for nuclear AR and/or the prostate-specific differentiation marker HoxB13 depending on the model. Circulating PSA levels are strongly correlated with overall tumor burden in mice seeded with LN95. Histologic micrometastases and low levels of circulating PSA are detected in mice seeded with MDA-PCa-2b at ~60 days postinoculation, but no circulating PSA was detected in animals inoculated with VCaP up to ~75 days despite the presence of rare AR+ cells in the liver. CONCLUSION: The studies reported herein establish intrasplenic injection as a robust model of mCRPC liver metastasis. In addition, circulating PSA was validated as a noninvasive biomarker to longitudinally monitor overall tumor burden when using PSA+ models. Therefore, this model can be used to interrogate the pathophysiology of prostate cancer liver metastases, the microenvironmental factors permissive to such growth, immunologic variables, and the response of hepatic lesions to therapy.
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Neoplasias Hepáticas/secundário , Neoplasias de Próstata Resistentes à Castração/patologia , Baço/patologia , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Calicreínas/sangue , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias/métodos , Antígeno Prostático Específico/sangue , Neoplasias de Próstata Resistentes à Castração/sangue , Transplante HeterólogoRESUMO
Accurate modeling of angiogenesis in vitro is essential for guiding the preclinical development of novel anti-angiogenic agents and treatment strategies. The formation of new blood vessels is a multifactorial and multi-stage process dependent upon paracrine factors produced by stromal cells in the local microenvironment. Mesenchymal stem cells (MSCs) are multipotent cells in adults that can be recruited to sites of inflammation and tissue damage where they aid in wound healing through regenerative, trophic, and immunomodulatory properties. Primary stromal cultures derived from human bone marrow, normal prostate, or prostate cancer tissue are highly enriched in MSCs and stromal progenitors. Using conditioned media from these primary cultures, a robust pro-angiogenic response was observed in a physiologically-relevant three-dimensional fibrin matrix assay. To evaluate the utility of this assay, the allosteric HDAC4 inhibitor tasquinimod and the anti-VEGF monoclonal antibody bevacizumab were used as model compounds with distinct mechanisms of action. While both agents had a profound inhibitory effect on endothelial sprouting, only bevacizumab induced significant regression of established vessels. Additionally, the pro-angiogenic properties of MSCs derived from prostate cancer patients provides further evidence that selective targeting of this population may be of therapeutic benefit.
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Células-Tronco Mesenquimais/fisiologia , Neovascularização Fisiológica/fisiologia , Próstata/citologia , Inibidores da Angiogênese/farmacologia , Bevacizumab/farmacologia , Células Cultivadas , Fibrina , Humanos , Masculino , Quinolonas/farmacologia , Nicho de Células-TroncoRESUMO
PURPOSE: The current standard of care for patients with locally advanced prostate cancer is a combination of androgen deprivation and radiation therapy. Radiation is typically given with androgen suppression when testosterone levels are at their nadir. Recent reports have shown that androgen stimulation of androgen-deprived prostate cancer cells leads to formation of double-strand breaks (DSB). Here, we exploit this finding and investigate the extent and timing of androgen-induced DSBs and their effect on tumor growth following androgen stimulation in combination with ionizing radiation (IR). EXPERIMENTAL DESIGN: Androgen-induced DNA damage was assessed by comet assays and γH2A.X foci formation. Effects of androgen stimulation and radiation were determined in vitro and in vivo with xenograft models. RESULTS: We document that androgen treatment of androgen-deprived prostate cancer cell lines resulted in a dose- and time-dependent induction of widespread DSBs. Generation of these breaks was dependent on androgen receptor and topoisomerase II beta but not on cell-cycle progression. In vitro models demonstrated a synergistic interaction between IR and androgen stimulation when IR is given at a time point corresponding with high levels of androgen-induced DSB formation. Furthermore, in vivo studies showed a significant improvement in tumor growth delay when radiation was given shortly after androgen repletion in castrated mice. CONCLUSIONS: These results suggest a potential cooperative effect and improved tumor growth delay with androgen-induced DSBs and radiation with implications for improving the therapeutic index of prostate cancer radiation therapy. Clin Cancer Res; 22(13); 3310-9. ©2016 AACRSee related commentary by Chua and Bristow, p. 3124.
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Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Neoplasias da Próstata/radioterapia , Receptores Androgênicos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Masculino , Camundongos , Orquiectomia/métodos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Radiação Ionizante , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tasquinimod, an orally active quinoline-3-carboxamide, binds with high affinity to HDAC4 and S100A9 in cancer and infiltrating host cells within compromised tumor microenvironment inhibiting adaptive survival pathways needed for an angiogenic response. Clinical trials document that as low as 0.5-1mg tasquinimod/day is therapeutic against castrate resistant metastatic prostate cancer. Tasquinimod is metabolized via cytochrome P4503A4, but ketoconazole at a dose which completely inhibits CYP3A metabolism does not affect tasquinimod's ability to inhibit endothelial "sprouting" in vitro or anti-cancer efficacy against human prostate cancer xenografts in vivo. Tasquinimod's potency is facilitated by its reversible binding (Kd < 35 µM) to the IIA subdomain of albumin (Sudlow's site I). As blood vessels within the compromised cancer microenvironment are characterized by a higher degree of leakiness than those in normal tissues, this results in an enhanced uptake of tasquinimod bound to albumin in cancer tissue via a tumor specific process known as the "enhanced permeability and retention" (i.e., EPR) effect. Thus, despite plasma levels of < 1 µM, the EPR effect results in intracellular drug concentrations of 2-3 µM, levels several-fold higher than needed for inhibition of endothelial sprouting (IC50 ~ 0.5 µM) or for inhibition of HDAC4 and S100A9 mediated tumor growth.
Assuntos
Inibidores da Angiogênese/farmacocinética , Neoplasias de Próstata Resistentes à Castração/irrigação sanguínea , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Quinolinas/farmacocinética , Albumina Sérica/metabolismo , Microambiente Tumoral , Administração Oral , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/sangue , Animais , Disponibilidade Biológica , Biotransformação , Permeabilidade Capilar , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Orquiectomia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Ligação Proteica , Quinolinas/administração & dosagem , Quinolinas/sangue , Quinolonas , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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/genéticaRESUMO
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-DawleyRESUMO
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.".
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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-IdadeRESUMO
Tasquinimod is an orally active antiangiogenic drug that is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer. However, the target of this drug has remained unclear. In this study, we applied diverse strategies to identify the histone deacetylase HDAC4 as a target for the antiangiogenic activity of tasquinimod. Our comprehensive analysis revealed allosteric binding (Kd 10-30 nmol/L) to the regulatory Zn(2+) binding domain of HDAC4 that locks the protein in a conformation preventing HDAC4/N-CoR/HDAC3 complex formation. This binding inhibited colocalization of N-CoR/HDAC3, thereby inhibiting deacetylation of histones and HDAC4 client transcription factors, such as HIF-1α, which are bound at promoter/enhancers where epigenetic reprogramming is required for cancer cell survival and angiogenic response. Through this mechanism, tasquinimod is effective as a monotherapeutic agent against human prostate, breast, bladder, and colon tumor xenografts, where its efficacy could be further enhanced in combination with a targeted thapsigargin prodrug (G202) that selectively kills tumor endothelial cells. Together, our findings define a mechanism of action of tasquinimod and offer a perspective on how its clinical activity might be leveraged in combination with other drugs that target the tumor microenvironment. Cancer Res; 73(4); 1386-99. ©2012 AACR.
Assuntos
Histona Desacetilases/metabolismo , Neoplasias/tratamento farmacológico , Quinolinas/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Acetilação/efeitos dos fármacos , Regulação Alostérica , Animais , Western Blotting , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Histona Desacetilases/química , Histona Desacetilases/genética , Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Nus , Modelos Moleculares , Neoplasias/genética , Neoplasias/patologia , Pró-Fármacos/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Quinolonas , Interferência de RNA , Proteínas Repressoras/química , Proteínas Repressoras/genética , Tapsigargina/farmacologia , Microambiente Tumoral/genéticaRESUMO
Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.