RESUMO
Single-cell RNA sequencing (scRNA-seq) distinguishes cell types, states and lineages within the context of heterogeneous tissues. However, current single-cell data cannot directly link cell clusters with specific phenotypes. Here we present Scissor, a method that identifies cell subpopulations from single-cell data that are associated with a given phenotype. Scissor integrates phenotype-associated bulk expression data and single-cell data by first quantifying the similarity between each single cell and each bulk sample. It then optimizes a regression model on the correlation matrix with the sample phenotype to identify relevant subpopulations. Applied to a lung cancer scRNA-seq dataset, Scissor identified subsets of cells associated with worse survival and with TP53 mutations. In melanoma, Scissor discerned a T cell subpopulation with low PDCD1/CTLA4 and high TCF7 expression associated with an immunotherapy response. Beyond cancer, Scissor was effective in interpreting facioscapulohumeral muscular dystrophy and Alzheimer's disease datasets. Scissor identifies biologically and clinically relevant cell subpopulations from single-cell assays by leveraging phenotype and bulk-omics datasets.
Assuntos
Melanoma , Análise de Célula Única , Perfilação da Expressão Gênica , Humanos , Melanoma/genética , Fenótipo , Análise de Sequência de RNARESUMO
Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia-inducible factor 1 alpha (HIF1α). Therefore, HIF1α is considered as a promising therapeutic target. However, HIF inhibitors have not proven to be effective in clinical testing. The underlying mechanism is unclear. We report that oncogenic protein ID1 is upregulated in hypoxia by HIF1α shRNA or pharmacological inhibitors. In turn, ID1 supports tumor growth in hypoxia in vitro and in xenografts in vivo, conferring adaptive survival response and resistance. Mechanistically, ID1 proteins interfere HIF1-mediated gene transcription activation, thus ID1 protein degradation is accelerated by HIF1α-dependent mechanisms in hypoxia. Inhibitions of HIF1α rescues ID1, which compensates the loss of HIF1α by the upregulation of GLS2 and glutamine metabolism, thereby switching the metabolic dependency of HIF1α -inhibited cells from glucose to glutamine.
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The discovery that androgens play an important role in the progression of prostate cancer led to the development of androgen deprivation therapy (ADT) as a first line of treatment. However, paradoxical growth inhibition has been observed in a subset of prostate cancer upon administration of supraphysiologic levels of testosterone (SupraT), both experimentally and clinically. Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive prostate cancer cells. This was initiated by the induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activated nucleic acid sensors converge on NFκB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT resulted in increased migration of cytotoxic immune cells to tumor beds in xenograft models and patient tumors. Collectively, these findings indicate that SupraT may inhibit a subset of prostate cancer by activating nucleic acid sensors and downstream immune signaling. SIGNIFICANCE: This study demonstrates that supraphysiologic testosterone induces two parallel autophagy-mediated processes, ferritinophagy and nucleophagy, which then activate nucleic acid sensors to drive immune signaling pathways in prostate cancer.
Assuntos
Androgênios/farmacologia , Autofagia , Ferroptose , Neoplasias da Próstata/imunologia , Testosterona/farmacologia , Animais , Apoptose , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Nus , Prognóstico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The transcription factor c-MYC (MYC thereafter) is a global regulator of gene expression. It is overexpressed or deregulated in human cancers of diverse origins and plays a key role in the development of cancers. Hypoxia-inducible factors (HIFs), a central regulator for cells to adapt to low cellular oxygen levels, is also often overexpressed and activated in many human cancers. HIF mediates the primary transcriptional response of a wide range of genes in response to hypoxia. Earlier studies focused on the inhibition of MYC by HIF during hypoxia, when MYC is expressed at physiological level, to help cells survive under low oxygen conditions. Emerging evidence suggests that MYC and HIF also cooperate to promote cancer cell growth and progression. This review will summarize the current understanding of the complex molecular interplay between MYC and HIF.
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Despite recent advances, the efficacy of androgen/androgen receptor (AR)-targeted therapy remains limited for many patients with metastatic prostate cancer. This is in part because prostate cancers adaptively switch to the androgen/AR-independent pathway for survival and growth, thereby conferring therapy resistance. Tumor hypoxia is considered as a major cause of treatment resistance. However, the exact mechanism is largely unclear. Here we report that chronic-androgen deprivation therapy (ADT) in the condition of hypoxia induces adaptive androgen/AR-independence, and therefore confers resistance to androgen/AR-targeted therapy, e.g., enzalutamide. Mechanistically, this is mediated by glucose-6-phosphate isomerase (GPI), which is transcriptionally repressed by AR in hypoxia, but restored and increased by AR inhibition. In turn, GPI maintains glucose metabolism and energy homeostasis in hypoxia by redirecting the glucose flux from androgen/AR-dependent pentose phosphate pathway (PPP) to hypoxia-induced glycolysis pathway, thereby reducing the growth inhibitory effect of enzalutamide. Inhibiting GPI overcomes the therapy resistance in hypoxia in vitro and increases enzalutamide efficacy in vivo.
Assuntos
Androgênios/farmacologia , Resistencia a Medicamentos Antineoplásicos , Terapia de Alvo Molecular , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Hipóxia Tumoral/efeitos dos fármacos , Benzamidas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias da Próstata/genética , Transcrição Gênica/efeitos dos fármacos , Hipóxia Tumoral/genética , Regulação para Cima/efeitos dos fármacosRESUMO
FOSL1 is frequently overexpressed in multiple types of human cancers including invasive breast cancers and implicated in cancer invasion and metastasis. However, how FOSL1 is overexpressed in cancers remains to be elucidated. Several microRNAs (miRNAs) have been shown to target FOSL1 and are downregulated in human cancers. Here, we report that miR-130a is a novel FOSL1 targeting miRNA. Using gene expression microarray analysis, we found that FOSL1 is among the most up-regulated genes in cells transfected with miR-130a inhibitors. Transient transfection-immunoblot, RNA-immunoprecipitation, and luciferase reporter assays revealed that miR-130a directly targets FOSL1 mRNA at its 3'-UTR. Overexpression of miR-130a significantly reduced the levels of FOSL1 in invasive breast cancer MDA-MB-231 and Hs578T cell lines and suppresses their migration and invasion. This inhibition can be rescued by ectopic expression of miR-130a-resistant FOSL1. Interestingly, we show that overexpression of miR-130a increased the levels of tight-junction protein ZO-1 while inhibition of miR-130a reduced the levels of ZO-1. We further show that miR-130a expression is significantly reduced in cancer tissues from triple-negative breast cancer (TNBC) patients, correlating significantly with the upregulation of FOSL1 expression, compared to non-TNBC tissues. Together, our results reveal that miR-130a directly targets FOSL1 and suppresses the inhibition of ZO-1, thus inhibiting cancer cell migration and invasion, in TNBCs.
Assuntos
Movimento Celular , Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA Neoplásico/biossíntese , Neoplasias de Mama Triplo Negativas/metabolismo , Regulação para Cima , Proteína da Zônula de Oclusão-1/biossíntese , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-fos/genética , RNA Neoplásico/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína da Zônula de Oclusão-1/genéticaRESUMO
cAMP-response element binding protein (CREB) is a nuclear transcription factor activated by multiple extracellular signals including growth factors and hormones. These extracellular cues activate CREB through phosphorylation at Ser133 by various protein serine/threonine kinases. Once phosphorylated, it promotes its association with transcription coactivators CREB-binding protein (CBP) and its paralog p300 to activate CREB-dependent gene transcription. Tumor tissues of different origins have been shown to present overexpression and/or overactivation of CREB, indicating CREB as a potential cancer drug target. We previously identified 666-15 as a potent inhibitor of CREB with efficacious anti-cancer activity both in vitro and in vivo. Herein, we investigated the specificity of 666-15 and evaluated its potential in vivo toxicity. We found that 666-15 was fairly selective in inhibiting CREB. 666-15 was also found to be readily bioavailable to achieve pharmacologically relevant concentrations for CREB inhibition. Furthermore, the mice treated with 666-15 showed no evidence of changes in body weight, complete blood count, blood chemistry profile, cardiac contractility and tissue histologies from liver, kidney and heart. For the first time, these results demonstrate that pharmacological inhibition of CREB is well-tolerated in vivo and indicate that such inhibitors should be promising cancer therapeutics.
Assuntos
Anilidas , Antineoplásicos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Naftalenos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Anilidas/efeitos adversos , Anilidas/farmacocinética , Anilidas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HEK293 , Humanos , Camundongos , Naftalenos/efeitos adversos , Naftalenos/farmacocinética , Naftalenos/farmacologia , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
Recent studies have shown that nuclear transcription factor cyclic adenosine monophosphate response element binding protein (CREB) is overexpressed in many different types of cancers. Therefore, CREB has been pursued as a novel cancer therapeutic target. Naphthol AS-E and its closely related derivatives have been shown to inhibit CREB-mediated gene transcription and cancer cell growth. Previously, we identified naphthamide 3a as a different chemotype to inhibit CREB's transcription activity. In a continuing effort to discover more potent CREB inhibitors, a series of structural congeners of 3a was designed and synthesized. Biological evaluations of these compounds uncovered compound 3i (666-15) as a potent and selective inhibitor of CREB-mediated gene transcription (IC50 = 0.081 ± 0.04 µM). 666-15 also potently inhibited cancer cell growth without harming normal cells. In an in vivo MDA-MB-468 xenograft model, 666-15 completely suppressed the tumor growth without overt toxicity. These results further support the potential of CREB as a valuable cancer drug target.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Naftalenos/química , Naftalenos/uso terapêutico , Ativação Transcricional/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Naftalenos/farmacologiaRESUMO
HIF-1α is degraded by oxygen-dependent mechanisms but stabilized in hypoxia to form transcriptional complex HIF-1, which transactivates genes promoting cancer hallmarks. However, how HIF-1α is specifically regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase SET9 promotes HIF-1α protein stability in hypoxia and enhances HIF-1 mediated glycolytic gene transcription, thereby playing an important role in mediating cancer cell adaptation and survival to hypoxic stress. Specifically, SET9 interacts with HIF-1α and promotes HIF-1α protein stability in hypoxia. Silencing SET9 by siRNA reduces HIF-1α protein stability in hypoxia, and attenuates the hypoxic induction of HIF-1 target genes mediating hypoxic glycolysis. Mechanistically, we find that SET9 is enriched at the hypoxia response elements (HRE) within promoters of the HIF-1-responsive glycolytic genes. Silencing SET9 reduces HIF-1α levels at these HREs in hypoxia, thereby attenuating HIF-1-mediated gene transcription. Further, silencing SET9 by siRNA reduces hypoxia-induced glycolysis and inhibits cell viability of hypoxic cancer cells. Our findings suggest that SET9 enriches at HRE sites of HIF-1 responsive glycolytic genes and stabilizes HIF-1α at these sites in hypoxia, thus establishes an epigenetic mechanism of the metabolic adaptation in hypoxic cancer cells.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisina/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina/metabolismo , Citoproteção , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicólise/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Regiões Promotoras Genéticas/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Elementos de Resposta/genética , Ativação Transcricional/genéticaRESUMO
Carbohydrate-conjugated fluorescent silica nanoprobes were prepared and used as a platform for galectin-1 and prostate cancer cell detection. The nanoparticles were efficiently conjugated using native, unprotected carbohydrate structures following a photochemical approach, resulting in lactose- and cellobiose-conjugated probes, respectively. The probes were used to challenge binding to galectin-1, an overexpressed galactose-selective lectin at prostate cancer cell surfaces, and the results show that lactose-conjugated nanoprobes exhibit differential binding behavior with prostate cancer cells and normal prostate cells. In particular, lactose-conjugated fluorescent silica nanoparticles showed specific binding to PC3 cells pre-treated with a reducing agent. The results indicate that galectin-1 expression and galectin-1-selective nanoparticles are potentially useful for sensitive and selective detection of prostate cancer.
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BACKGROUND: Resistance to chemotherapy represents a significant obstacle in prostate cancer therapeutics. Novel mechanistic understandings in cancer cell chemotherapeutic sensitivity and resistance can optimize treatment and improve patient outcome. Molecular alterations in the metabolic pathways are associated with cancer development; however, the role of these alterations in chemotherapy efficacy is largely unknown. METHODS: In a bed-side to bench-side reverse translational approach, we used cDNA microarray and qRT-PCR to identify genes that are associated with biochemical relapse after chemotherapy. Further, we tested the function of these genes in cell proliferation, metabolism, and chemosensitivity in prostate cancer cell lines. RESULTS: We report that the gene encoding mitochondrial malate dehydrogenase 2 (MDH2) is overexpressed in clinical prostate cancer specimens. Patients with MDH2 overexpression had a significantly shorter period of relapse-free survival (RFS) after undergoing neoadjuvant chemotherapy. To understand the molecular mechanism underlying this clinical observation, we observed that MDH2 expression was elevated in prostate cancer cell lines compared to benign prostate epithelial cells. Stable knockdown of MDH2 via shRNA in prostate cancer cell lines decreased cell proliferation and increased docetaxel sensitivity. Further, MDH2 shRNA enhanced docetaxel-induced activations of JNK signaling and induced metabolic inefficiency. CONCLUSION: Taken together, these data suggest a novel function for MDH2 in prostate cancer development and chemotherapy resistance, in which MDH2 regulates chemotherapy-induced signal transduction and oxidative metabolism.
Assuntos
Antineoplásicos/uso terapêutico , Resistência a Medicamentos/genética , Metabolismo Energético/genética , Sistema de Sinalização das MAP Quinases/genética , Malato Desidrogenase/metabolismo , Neoplasias da Próstata/metabolismo , Taxoides/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Intervalo Livre de Doença , Docetaxel , Humanos , Malato Desidrogenase/genética , Masculino , Consumo de Oxigênio/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Lysine acetylation regulates protein stability and function. p300 is a component of the HIF-1 transcriptional complex and positively regulates the transactivation of HIF-1. Here, we show a novel molecular mechanism by which p300 facilitates HIF-1 activity. p300 increases HIF-1α (HIF1α) protein acetylation and stability. The regulation can be opposed by HDAC1, but not by HDAC3, and is abrogated by disrupting HIF1α-p300 interaction. Mechanistically, p300 specifically acetylates HIF1α at Lys-709, which increases the protein stability and decreases polyubiquitination in both normoxia and hypoxia. Compared with the wild-type protein, a HIF1α K709A mutant protein is more stable, less polyubiquitinated, and less dependent on p300. Overexpression of the HIF1α wild-type or K709A mutant in cancer cells lacking the endogenous HIF1α shows that the K709A mutant is transcriptionally more active toward the HIF-1 reporter and some endogenous target genes. Cancer cells containing the K709A mutant are less sensitive to hypoxia-induced growth arrest than the cells containing the HIF1α wild-type. Taken together, these data demonstrate a novel biological consequence upon HIF1α-p300 interaction, in which HIF1α can be stabilized by p300 via Lys-709 acetylation.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Substituição de Aminoácidos , Pontos de Checagem do Ciclo Celular/fisiologia , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Lisina/genética , Lisina/metabolismo , Mutação de Sentido Incorreto , Estabilidade Proteica , Fatores de Transcrição de p300-CBP/genéticaRESUMO
Hypoxia-inducible factor 1 α (HIF1α) is an essential part of the HIF-1 transcriptional complex that regulates angiogenesis, cellular metabolism, and cancer development. In von Hippel-Lindau (VHL)-null kidney cancer cell lines, we reported previously that HIF1α proteins can be acetylated and inhibited by histone deacetylase (HDAC) inhibitors or specific siRNA against HDAC4. To investigate the mechanism and biological consequence of the inhibition, we have generated stable HDAC4 knockdown via shRNA in VHL-positive normal and cancer cell lines. We report that HDAC4 regulates HIF1α protein acetylation and stability. Specifically, the HIF1α protein acetylation can be increased by HDAC4 shRNA and decreased by HDAC4 overexpression. HDAC4 shRNA inhibits HIF1α protein stability. In contrast, HDAC1 or HDAC3 shRNA has no such inhibitory effect. Mutations of the first five lysine residues (lysine 10, 11, 12, 19, and 21) to arginine within the HIF1α N terminus reduce protein acetylation but render the mutant HIF1α protein resistant to HDAC4 and HDACi-mediated inhibition. Functionally, in VHL-positive cancer cell lines, stable inhibition of HDAC4 decreases both the HIF-1 transcriptional activity and a subset of HIF-1 hypoxia target gene expression. On the cellular level, HDAC4 inhibition reduces the hypoxia-related increase of glycolysis and resistance to docetaxel chemotherapy. Taken together, the novel biological relationship between HDAC4 and HIF1α presented here suggests a potential role for the deacetylase enzyme in regulating HIF-1 cancer cell response to hypoxia and presents a more specific molecular target of inhibition.
Assuntos
Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , Lisina/química , Mutação , Proteínas Repressoras/metabolismo , Acetilação , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Genes Reporter , Glicólise , Células HEK293 , Humanos , Inibidores da Síntese de Proteínas/farmacologia , RNA Interferente Pequeno/metabolismoRESUMO
Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials.
Assuntos
Carcinoma de Células Renais/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dasatinibe , Resistencia a Medicamentos Antineoplásicos , Humanos , Imuno-Histoquímica , Imunoprecipitação , Técnicas In Vitro , Inibidores de Proteínas Quinases , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologiaRESUMO
Recent insights into the regulation of the androgen receptor (AR) activity led to novel therapeutic targeting of AR function in prostate cancer patients. Docetaxel is an approved chemotherapy for treatment of castration-resistant prostate cancer; however, the mechanism underlying the action of this tubulin-targeting drug is not fully understood. This study investigates the contribution of microtubules and the cytoskeleton to androgen-mediated signaling and the consequences of their inhibition on AR activity in human prostate cancer. Tissue microarrays from docetaxel-treated and untreated prostate cancer patients were comparatively analyzed for prostate-specific antigen (PSA) and AR immunoreactivity. The AR transcriptional activity was determined in prostate cancer cells in vitro, based on PSA mRNA expression and the androgen response element reporter activity. The interaction of AR with tubulin was examined by immunoprecipitation and immunofluorescence. Treatment of prostate cancer patients with docetaxel led to a significant translocation of AR. In untreated specimens, 50% prostate tumor cells exhibited nuclear accumulation of AR, compared with docetaxel-treated tumors that had significantly depleted nuclear AR (38%), paralleled by an increase in cytosolic AR. AR nuclear localization correlated with PSA expression. In vitro, exposure of prostate cancer cells to paclitaxel (1 µmol/L) or nocodazole (5 µg/mL) inhibited androgen-dependent AR nuclear translocation by targeting AR association with tubulin. Introduction of a truncated AR indicated the requirement of the NH(2)-terminal domain for AR-tubulin interaction. Our findings show that in addition to blocking cell division, docetaxel impairs AR signaling, evidence that enables new insights into the therapeutic efficacy of microtubule-targeting drugs in prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Tubulina (Proteína)/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Linhagem Celular Tumoral , Docetaxel , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Análise em Microsséries , Mitoxantrona/uso terapêutico , Mitoxantrona/toxicidade , Antígeno Prostático Específico/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxoides/uso terapêutico , Taxoides/toxicidadeRESUMO
To identify potential mechanisms underlying prostate cancer chemotherapy response and resistance, we compared the gene expression profiles in high-risk human prostate cancer specimens before and after neoadjuvant chemotherapy and radical prostatectomy. Among the molecular signatures associated with chemotherapy, transcripts encoding inhibitor of DNA binding 1 (ID1) were significantly upregulated. The patient biochemical relapse status was monitored in a long-term follow-up. Patients with ID1 upregulation were found to be associated with longer relapse-free survival than patients without ID1 increase. This in vivo clinical association was mechanistically investigated. The chemotherapy-induced ID1 upregulation was recapitulated in the prostate cancer cell line LNCaP. Docetaxel dose-dependently induced ID1 transcription, which was mediated by ID1 promoter E-box chromatin modification and c-Myc binding. Stable ID1 overexpression in LNCaP increased cell proliferation, promoted G(1) cell cycle progression, and enhanced docetaxel-induced cytotoxicity. These changes were accompanied by a decrease in cellular mitochondria content, an increase in BCL2 phosphorylation at serine 70, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. In contrast, ID1 siRNA in the LNCaP and C42B cell lines reduced cell proliferation and decreased docetaxel-induced cytotoxicity by inhibiting cell death. ID1-mediated chemosensitivity enhancement was in part due to ID1 suppression of p21. Overexpression of p21 in LNCaP-ID1-overexpressing cells restored the p21 level and reversed ID1-enhanced chemosensitivity. These molecular data provide a mechanistic rationale for the observed in vivo clinical association between ID1 upregulation and relapse-free survival. Taken together, it shows that ID1 expression has a novel therapeutic role in prostate cancer chemotherapy and prognosis.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína 1 Inibidora de Diferenciação/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Taxoides/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/química , Docetaxel , Relação Dose-Resposta a Droga , Fase G1 , Humanos , Masculino , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
BACKGROUND: Metastatic prostate cancer is either inherently resistant to chemotherapy or rapidly acquires this phenotype after chemotherapy exposure. In this study, we identified a docetaxel-induced resistance mechanism centered on CCL2. METHODS: We compared the gene expression profiles in individual human prostate cancer specimens before and after exposure to chemotherapy collected from previously untreated patients who participated in a clinical trial of preoperative chemotherapy. Subsequently, we used the gain- and loss-of-function approach in vitro to identify a potential mechanism underlying chemotherapy resistance. RESULTS: Among the molecular signatures associated with treatment, several genes that regulate the inflammatory response and chemokine activity were upregulated including a significant increase in transcripts encoding the CC chemokine CCL2. Docetaxel increased CCL2 expression in prostate cancer cell lines in vitro. CCL2-specific siRNA inhibited LNCaP and LAPC4 cell proliferation and enhanced the growth inhibitory effect of low-dose docetaxel. In contrast, overexpression of CCL2 or recombinant CCL2 protein stimulated prostate cancer cell proliferation and rescued cells from docetaxel-induced cytotoxicity. This protective effect of CCL2 was associated with activation of the ERK/MAP kinase and PI3K/AKT, inhibition of docetaxel-induced Bcl2 phosphorylation at serine 70, phosphorylation of Bad, and activation of caspase-3. The addition of a PI3K/AKT inhibitor Ly294002 reversed the CCL2 protection and was additive to docetaxel-induced toxicity. CONCLUSION: These results support a mechanism of chemotherapy resistance mediated by cellular stress responses involving the induction of CCL2 expression and suggest that inhibiting CCL2 activity could enhance therapeutic responses to taxane-based therapy.
Assuntos
Antineoplásicos/farmacologia , Quimiocina CCL2/genética , Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Taxoides/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/farmacologia , Docetaxel , Resistencia a Medicamentos Antineoplásicos/genética , Quimioterapia Combinada , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Mitoxantrona/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/farmacologia , Regulação para CimaRESUMO
HIF-1 is a heterodimeric transcription factor that mediates adaptive responses to hypoxia and plays critical roles in cancer progression. Using a cell-based screening assay we have identified acriflavine as a drug that binds directly to HIF-1alpha and HIF-2alpha and inhibits HIF-1 dimerization and transcriptional activity. Pretreatment of mice bearing prostate cancer xenografts with acriflavine prevented tumor growth and treatment of mice bearing established tumors resulted in growth arrest. Acriflavine treatment inhibited intratumoral expression of angiogenic cytokines, mobilization of angiogenic cells into peripheral blood, and tumor vascularization. These results provide proof of principle that small molecules can inhibit dimerization of HIF-1 and have potent inhibitory effects on tumor growth and vascularization.
Assuntos
Acriflavina/farmacologia , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Acriflavina/farmacocinética , Substituição de Aminoácidos , Inibidores da Angiogênese/farmacocinética , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Dimerização , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Técnicas In Vitro , Masculino , Camundongos , Camundongos SCID , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Neovascularização Patológica/prevenção & controle , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/patologia , Estrutura Quaternária de Proteína/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transplante HeterólogoRESUMO
PURPOSE: To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia. EXPERIMENTAL DESIGN: Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays composed of 6,200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative reverse transcription-PCR. RESULTS: Comparative analyses identified 954 transcript alterations associated with cancer (q < 0.01%), including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy use, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of androgen receptor expression changes was noted. In exploratory analyses, androgen receptor down-regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy. CONCLUSIONS: Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation.