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In this study, proximal fleximer nucleos(t)ide analogues of Bemnifosbuvir were synthesized and evaluated for their potential to serve as antiviral therapeutics. The final parent flex-nucleoside and ProTide modified flex-nucleoside analogues were tested against several viral families including flaviviruses, filoviruses, and coronaviruses. Modest activity against Zaire Ebola virus was observed at 30 µM for compound ProTide modified analogue. Neither compound exhibited activity for any of the other viruses tested. The parent flex-nucleoside analogue was screened for toxicity in CD-1 mice and showed no adverse effects up to 300 mg/kg, the maximum concentration tested.
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BACKGROUND: Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered and exposure-based mouse models in different mouse strains and prostate lobes within each mouse strain, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. METHODS: In this study, we used single-cell RNA-sequencing (scRNA-seq) methods to assess the single-cell transcriptomes of 6-month-old mouse prostates from two commonly used mouse strains, friend virus B/NIH jackson (FVB/NJ) (N = 2) and C57BL/6J (N = 3). For each mouse, the lobes of the prostate were dissected (anterior, dorsal, lateral, and ventral), and individual scRNA-seq libraries were generated. In situ and pathological analyses were used to explore the spatial and anatomical distributions of novel cell types and molecular markers defining these cell types. RESULTS: Data dimensionality reduction and clustering analysis of scRNA-seq data revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Gene set enrichment analysis comparing luminal cells by strain showed enrichment of proto-Oncogene targets in FVB/NJ mice. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. CONCLUSIONS: Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.
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Células Endoteliais , Próstata , Masculino , Animais , Camundongos , Próstata/patologia , Camundongos Endogâmicos C57BL , Células Epiteliais , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismoRESUMO
One of the significant challenges to translation of intravenously administered nanomaterials has been complement-mediated infusion reactions which can be lethal. Slow infusions can reduce infusion reactions, but slow infusions are not always possible in applications like controlling bleeding following trauma. Thus, avoiding complement activation and infusion responses is essential to manage bleeding. We identified nanocapsules based on polyurethane as candidates that did not activate C5a and explored their PEGylation and functionalization with the GRGDS peptide to create a new class of hemostatic nanomaterials. Using the clinically relevant rotational thromboelastography (ROTEM), we determined that nanocapsules promote faster clotting than controls and maintain the maximum clot firmness, which is critical for reducing bleeding. Excitingly, these polyurethane-based nanocapsules did not activate complement or the major pro-inflammatory cytokines. This work provides critical evidence for the role of modulating the core material in developing safer nanomedicines for intravenous applications.
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Hemostáticos , Nanocápsulas , Hemorragia/tratamento farmacológico , Hemostasia , Hemostáticos/uso terapêutico , Humanos , TromboelastografiaRESUMO
The maintenance of cycling cell lineages relies on undifferentiated subpopulations consisting of stem and progenitor pools. Features that delineate these cell types are undefined for many lineages, including spermatogenesis, which is supported by an undifferentiated spermatogonial population. Here, we generated a transgenic mouse line in which spermatogonial stem cells are marked by expression of an inhibitor of differentiation 4 (Id4)-green fluorescent protein (Gfp) transgene. We found that Id4-Gfp(+) cells exist primarily as a subset of the type A(single) pool, and their frequency is greatest in neonatal development and then decreases in proportion during establishment of the spermatogenic lineage, eventually comprising â¼ 2% of the undifferentiated spermatogonial population in adulthood. RNA sequencing analysis revealed that expression of 11 and 25 genes is unique for the Id4-Gfp(+)/stem cell and Id4-Gfp(-)/progenitor fractions, respectively. Collectively, these findings provide the first definitive evidence that stem cells exist as a rare subset of the A(single) pool and reveal transcriptome features distinguishing stem cell and progenitor states within the mammalian male germline.
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Células Germinativas/citologia , Proteínas Inibidoras de Diferenciação/metabolismo , Células-Tronco/citologia , Testículo/citologia , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Inibidoras de Diferenciação/genética , Masculino , Camundongos , Camundongos Transgênicos , Espermatogênese/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismo , TranscriptomaRESUMO
Inflammation is involved in many prostate pathologies including infection, benign prostatic hyperplasia, and prostate cancer. Preclinical models are critical to our understanding of disease mechanisms, yet few models are genetically tractable. Here, we present a comparative quantitative proteomic analysis of urine from mice with and without prostate-specific inflammation induced by conditional prostate epithelial IL-1ß expression. Relative quantification and sample multiplexing was achieved using custom 4-plex N,N-dimethyl leucine (DiLeu) isobaric tags and nanoflow ultrahigh-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Each set of 4-plex DiLeu reagents allows four urine samples to be analyzed simultaneously, providing high-throughput and accurate quantification of urinary proteins. Proteins involved in the acute phase response, including haptoglobin, inter-α-trypsin inhibitor, and α1-antitrypsin 1-1, were differentially represented in the urine of mice with prostate inflammation. Mass spectrometry-based quantitative urinary proteomics represents a promising bioanalytical strategy for biomarker discovery and the elucidation of molecular mechanisms in urological research.
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Mediadores da Inflamação/urina , Marcação por Isótopo , Leucina/química , Próstata/metabolismo , Prostatite/urina , Proteoma , Proteômica/métodos , Animais , Biomarcadores/urina , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Leucina/análogos & derivados , Masculino , Camundongos Transgênicos , Próstata/patologia , Prostatite/genética , Prostatite/patologia , Espectrometria de Massas em Tandem , Fatores de Tempo , Urinálise , Fluxo de TrabalhoRESUMO
BACKGROUND: Elevated expression of the proinflammatory cytokine interleukin 1ß (IL-1ß) has been observed in expressed prostatic secretions of patients with chronic prostatitis/chronic pelvic pain syndrome, and genetic polymorphisms associated with the IL1B gene are linked to increased risk for aggressive prostate cancer. METHODS: To study the role of IL-1ß expression in prostate inflammation, we examined IL1B expression in human prostatic proliferative inflammatory atrophy (PIA) lesions and developed a tetracycline-regulated human IL1B transgene in the mouse prostate. RESULTS: Here, we demonstrate that IL1B expression is a common finding in human PIA lesions, which harbored focal IL1B expression in epithelial and stromal compartments. Human IL1B expression in the mouse prostate elicited acute and chronic inflammation. Penetrance and expressivity were variable and tunable by altering transgene dosage and the presence of an exogenous inducible marker antigen (green fluorescent protein). Inflammation was characterized by infiltration of CD4+ T cells, demonstrating an adaptive immune response. Chronic inflammation persisted after doxycycline (Dox) withdrawal. Reactive epithelia increased expression of downstream cytokines, and altered glandular architecture was observed upon sustained induction of IL1B. Immunohistochemical analyses revealed a higher proliferative index and decreased Nkx3.1 expression in inflamed mouse prostates. CONCLUSIONS: These data implicate IL-1ß in human prostate pathology and this model provides a versatile platform to interrogate molecular mechanisms of inflammation-associated prostate pathologies associated with episodic or sustained IL-1ß expression.
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Atrofia/imunologia , Linfócitos T CD4-Positivos/imunologia , Inflamação/imunologia , Interleucina-1beta/biossíntese , Próstata/imunologia , Doenças Prostáticas/imunologia , Animais , Doença Crônica , Modelos Animais de Doenças , Humanos , Interleucina-1beta/genética , Masculino , Camundongos , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Prostatite/imunologiaRESUMO
BACKGROUND: Advanced prostate cancers depend on protein synthesis for continued survival and accelerated rates of metabolism for growth. RNA polymerase I (Pol I) is the enzyme responsible for ribosomal RNA (rRNA) transcription and a rate-limiting step for ribosome biogenesis. We have shown using a specific and sensitive RNA probe for the 45S rRNA precursor that rRNA synthesis is increased in prostate adenocarcinoma compared to nonmalignant epithelium. We have introduced a first-in-class Pol I inhibitor, BMH-21, that targets cancer cells of multiple origins, and holds potential for clinical translation. METHODS: The effect of BMH-21 was tested in prostate cancer cell lines and in prostate cancer xenograft and mouse genetic models. RESULTS: We show that BMH-21 inhibits Pol I transcription in metastatic, castration-resistant, and enzalutamide treatment-resistant prostate cancer cell lines. The genetic abrogation of Pol I effectively blocks the growth of prostate cancer cells. Silencing of p53, a pathway activated downstream of Pol I, does not diminish this effect. We find that BMH-21 significantly inhibited tumor growth and reduced the Ki67 proliferation index in an enzalutamide-resistant xenograft tumor model. A decrease in 45S rRNA synthesis demonstrated on-target activity. Furthermore, the Pol I inhibitor significantly inhibited tumor growth and pathology in an aggressive genetically modified Hoxb13-MYC|Hoxb13-Cre|Ptenfl/fl (BMPC) mouse prostate cancer model. CONCLUSION: Taken together, BMH-21 is a novel promising molecule for the treatment of castration-resistant prostate cancer.
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Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , RNA Polimerase I/antagonistas & inibidores , Animais , Benzamidas , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Nitrilas , Células PC-3 , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Ribossômico/genética , Distribuição Aleatória , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Loss or mutation of PTEN alleles at 10q23 in combination with 8q24 amplification (encompassing MYC) are common findings in aggressive, human prostate cancer. Our group recently developed a transgenic murine model of prostate cancer involving prostate-specific Pten deletion and forced expression of MYC under the control of the Hoxb13 promoter. MYC overexpression cooperated with Pten loss to recapitulate lethal, human prostate cancer. METHOD: We now report on the generation of two mouse prostate cancer cell lines, BMPC1 and BMPC2, derived from a lymph node, and liver metastasis, respectively. RESULTS: Both cell lines demonstrate a phenotype consistent with adenocarcinoma and grew under standard tissue culture conditions. Androgen receptor (AR) protein expression is minimal (BMPC1) or absent (BMPC2) consistent with AR loss observed in the BMPC mouse model of invasive adenocarcinoma. Growth in media containing charcoal-stripped serum resulted in an increase in AR mRNA in BMPC1 cells with no effect on protein expression, unless androgens were added, in which case AR protein was stabilized, and showed nuclear localization. AR expression in BMPC2 cells was not effected by growth media or treatment with androgens. Treatment with an anti-androgen/castration or androgen supplemented media did not affect in vitro or in vivo growth of either cell line, irrespective of nuclear AR detection. DISCUSSION: These cell lines are a novel model of androgen-insensitive prostatic adenocarcinoma driven by MYC over-expression and Pten loss.
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Adenocarcinoma/patologia , Linhagem Celular Tumoral , PTEN Fosfo-Hidrolase/genética , Próstata/patologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Adenocarcinoma/genética , Alelos , Animais , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Masculino , Camundongos , Neoplasias da Próstata/genéticaRESUMO
Biodegradable polymeric nanoparticles (NPs) have demonstrated significant potential to improve the systemic delivery of RNA interference (RNAi) therapeutics, such as small interfering RNA (siRNA), for cancer therapy. However, the slow and inefficient siRNA release inside tumor cells generally observed for most biodegradable polymeric NPs may result in compromised gene silencing efficacy. Herein, a biodegradable and redox-responsive NP platform, composed of a solid poly(disulfide amide) (PDSA)/cationic lipid core and a lipid-poly(ethylene glycol) (lipid-PEG) shell for systemic siRNA delivery to tumor cells, is developed. This newly generated NP platform can efficiently encapsulate siRNA under extracellular environments and can respond to the highly concentrated glutathione (GSH) in the cytoplasm to induce fast intracellular siRNA release. By screening a library of PDSA polymers with different structures and chain lengths, the optimized NP platform shows the unique features of i) long blood circulation, ii) high tumor accumulation, iii) fast GSH-triggered intracellular siRNA release, and iv) exceptionally effective gene silencing. Together with the facile polymer synthesis technique and robust NP formulation enabling scale-up, this new redox-responsive NP platform may become an effective tool for RNAi-based cancer therapy.
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Nanopartículas/química , Citoplasma/metabolismo , Glutationa/química , Humanos , Oxirredução , Interferência de RNARESUMO
Dysfunction of the lower urinary tract commonly afflicts the middle-aged and aging male population. The etiology of lower urinary tract symptoms (LUTS) is multifactorial. Benign prostate hyperplasia, fibrosis, smooth muscle contractility, and inflammation likely contribute. Here we aim to characterize the urinary metabolomic profile associated with prostatic inflammation, which could inform future personalized diagnosis or treatment, as well as mechanistic research. Quantitative urinary metabolomics was conducted to examine molecular changes following induction of inflammation via conditional Interleukin-1ß expression in prostate epithelia using a novel transgenic mouse strain. To advance method development for urinary metabolomics, we also compared different urine normalization methods and found that normalizing urine samples based on osmolality prior to LC-MS most completely separated urinary metabolite profiles of mice with and without prostate inflammation via principal component analysis. Global metabolomics was combined with advanced machine learning feature selection and classification for data analysis. Key dysregulated metabolites and pathways were identified and were relevant to prostatic inflammation, some of which overlapped with our previous study of human LUTS patients. A binary classification model was established via the support vector machine algorithm to accurately differentiate control and inflammation groups, with an area-under-the-curve value of the receiver operating characteristic of 0.81, sensitivity of 0.974 and specificity of 0.995, respectively. This study generated molecular profiles of non-bacterial prostatic inflammation, which could assist future efforts to stratify LUTS patients and develop new therapies.
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BACKGROUND: The androgen-regulated homeodomain transcription factor NKX3.1 plays roles in early prostate development and functions as a prostate-specific tumor suppressor. Decreased expression of NKX3.1 protein is common in primary prostate cancer. Discordance between NKX3.1 mRNA and protein levels during prostate carcinogenesis suggested a key role for post-transcriptional modifications in regulating NKX3.1 protein levels in prostate epithelial cells. Subsequent studies revealed NKX3.1 to be modified post-translationally at multiple sites. METHODS: We reviewed published literature to identify and summarize post-translational modifications and structural elements critical in regulating NKX3.1 stability and levels in prostate epithelial cells. RESULTS: NKX3.1 is modified post-translationally at multiple sites by different protein kinases. These modifications together with several structural determinants were identified to play an important role in NKX3.1 stability and biology. CONCLUSIONS: In this review, we provide a comprehensive overview of the known post-translational modifications and structural features that impact NKX3.1. Defining factors that regulate NKX3.1 in prostate epithelial cells will extend our understanding of molecular changes that may contribute to prostate cancer initiation and progression.
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Proteínas de Homeodomínio/metabolismo , Neoplasias da Próstata/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Fatores de Transcrição/metabolismo , Genes Supressores de Tumor/fisiologia , Humanos , Masculino , Fosforilação , Neoplasias da Próstata/patologia , Estabilidade Proteica , UbiquitinaçãoRESUMO
Despite recent advancements in large-scale phosphoproteomics, methods to quantify kinase-specific phosphorylation stoichiometry of protein substrates are lacking. We developed a method to quantify kinase-specific phosphorylation stoichiometry by combining the reverse in-gel kinase assay (RIKA) with high-resolution liquid chromatography-mass spectrometry (LC-MS). Beginning with predetermined ratios of phosphorylated to nonphosphorylated protein kinase CK2 (CK2) substrate molecules, we employed 18O-labeled adenosine triphosphate (18O-ATP) as the phosphate donor in a RIKA, then quantified the ratio of 18O- versus 16O-labeled tryptic phosphopeptide using high mass accuracy mass spectrometry (MS). We demonstrate that the phosphorylation stoichiometry determined by this method across a broad percent phosphorylation range correlated extremely well with the predicted value (correlation coefficient = 0.99). This approach provides a quantitative alternative to antibody-based methods of determining the extent of phosphorylation of a substrate pool.
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Caseína Quinase II/química , Marcação por Isótopo , Fosfopeptídeos/análise , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Caseína Quinase II/metabolismo , Géis/química , Géis/metabolismo , Isótopos de Oxigênio , Fosfopeptídeos/metabolismo , FosforilaçãoRESUMO
OBJECTIVES: The objective of this study was to identify an injection strategy leading to repeatable nanoparticle deposition patterns in tumours and to quantify volumetric heat generation rate distribution based on micro-CT Hounsfield unit (HU) in magnetic nanoparticle hyperthermia. METHODS: In vivo animal experiments were performed on graft prostatic cancer (PC3) tumours in immunodeficient mice to investigate whether lowering ferrofluid infusion rate improves control of the distribution of magnetic nanoparticles in tumour tissue. Nanoparticle distribution volume obtained from micro-CT scan was used to evaluate spreading of the nanoparticles from the injection site in tumours. Heating experiments were performed to quantify relationships among micro-CT HU values, local nanoparticle concentrations in the tumours, and the ferrofluid-induced volumetric heat generation rate (q(MNH)) when nanoparticles were subject to an alternating magnetic field. RESULTS: An infusion rate of 3 µL/min was identified to result in the most repeatable nanoparticle distribution in PC3 tumours. Linear relationships have been obtained to first convert micro-CT greyscale values to HU values, then to local nanoparticle concentrations, and finally to nanoparticle-induced q(MNH) values. The total energy deposition rate in tumours was calculated and the observed similarity in total energy deposition rates in all three infusion rate groups suggests improvement in minimising nanoparticle leakage from the tumours. The results of this study demonstrate that micro-CT generated q(MNH) distribution and tumour physical models improve predicting capability of heat transfer simulation for designing reliable treatment protocols using magnetic nanoparticle hyperthermia.
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Hipertermia Induzida , Nanopartículas de Magnetita/administração & dosagem , Neoplasias/metabolismo , Neoplasias/terapia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Injeções , Nanopartículas de Magnetita/uso terapêutico , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/diagnóstico por imagem , Microtomografia por Raio-XRESUMO
MYC levels are tightly regulated in cells, and deregulation is associated with many cancers. In this report, we describe the existence of a MYC-protein kinase A (PKA)-polo-like kinase 1 (PLK1) signaling loop in cells. We report that sequential MYC phosphorylation by PKA and PLK1 protects MYC from proteasome-mediated degradation. Interestingly, short term pan-PKA inhibition diminishes MYC level, whereas prolonged PKA catalytic subunit α (PKACα) knockdown, but not PKA catalytic subunit ß (PKACß) knockdown, increases MYC. We show that the short term effect of pan-PKA inhibition on MYC is post-translational and the PKACα-specific long term effect on MYC is transcriptional. These data also reveal distinct functional roles among PKA catalytic isoforms in MYC regulation. We attribute this effect to differential phosphorylation selectivity among PKA catalytic subunits, which we demonstrate for multiple substrates. Further, we also show that MYC up-regulates PKACß, transcriptionally forming a proximate positive feedback loop. These results establish PKA as a regulator of MYC and highlight the distinct biological roles of the different PKA catalytic subunits.
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Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Domínio Catalítico , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Dados de Sequência Molecular , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Quinase 1 Polo-LikeRESUMO
Efavirenz (EFV) is a commonly used drug to treat human immunodeficiency virus infection and is known to exert adverse effects on the brain. Although it is known that EFV is associated with abnormal plasma lipid levels, the changes in the spatial localization of individual lipid molecules in brain tissue following EFV treatment are yet to be explored. In this study, we employed a matrix-assisted laser desorption/ionization mass spectrometry imaging approach to determine region-specific lipid alterations in mouse brains following EFV treatment. We detected unique spatial localization patterns of phosphatidylcholine (PC), sphingomyelin (SM), ceramide phosphoinositol (PI-Cer), and hexosylceramide (HexCer) molecules in the mouse brain. Interestingly, PC(32:0), PC(38:5), and SM(36:1;O2) showed high abundance in the hippocampus region, whereas PI-Cer(38:8) exhibited low abundance in the hippocampus region of the EFV-treated mouse brains. Additionally, we observed low abundance of PC(38:6), PC(40:6), and PI-Cer(40:3) in the thalamus region of the EFV-treated mouse brains. Furthermore, SM(40:1;O2), SM(42:2;O2), SM(42:1;O2), SM(43:2;O2), and SM(43:1;O2) exhibited their accumulation in the corpus callosum region of the EFV-treated mouse brains as compared to controls. However, HexCer(42:1;O3) exhibited depletion in the corpus callosum region in response to EFV treatment. To characterize the expression patterns of proteins, including lipid metabolizing enzymes, in response to EFV treatment, mass spectrometry-based proteomics was utilized. From these, the expression levels of 12 brain proteins were found to be significantly decreased following EFV treatment. Taken together, these multiomics data provide important insights into the effects of EFV on brain lipid metabolism.
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ABSTRACT: Provirus integration site for Moloney murine leukemia virus (PIM) family serine/threonine kinases perform protumorigenic functions in hematologic malignancies and solid tumors by phosphorylating substrates involved in tumor metabolism, cell survival, metastasis, inflammation, and immune cell invasion. However, a comprehensive understanding of PIM kinase functions is currently lacking. Multiple small-molecule PIM kinase inhibitors are currently being evaluated as cotherapeutics in patients with cancer. To further illuminate PIM kinase functions in cancer, we deeply profiled PIM1 substrates using the reverse in-gel kinase assay to identify downstream cellular processes targetable with small molecules. Pathway analyses of putative PIM substrates nominated RNA splicing and ribosomal RNA (rRNA) processing as PIM-regulated cellular processes. PIM inhibition elicited reproducible splicing changes in PIM-inhibitor-responsive acute myeloid leukemia (AML) cell lines. PIM inhibitors synergized with splicing modulators targeting splicing factor 3b subunit 1 (SF3B1) and serine-arginine protein kinase 1 (SRPK1) to kill AML cells. PIM inhibition also altered rRNA processing, and PIM inhibitors synergized with an RNA polymerase I inhibitor to kill AML cells and block AML tumor growth. These data demonstrate that deep kinase substrate knowledge can illuminate unappreciated kinase functions, nominating synergistic cotherapeutic strategies. This approach may expand the cotherapeutic armamentarium to overcome kinase inhibitor-resistant disease that limits durable responses in malignant disease.
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Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Humanos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Camundongos , Animais , Linhagem Celular Tumoral , Especificidade por Substrato , Splicing de RNA/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
How prostate cancer cells and their precursors mediate changes in the tumor microenvironment (TME) to drive prostate cancer progression is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we perform extensive single-cell RNA-sequencing (scRNA-seq) and molecular pathology of the comparative biology between human prostate cancer and key stages in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues reveal that cancer cell-intrinsic activation of MYC signaling is a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Cell communication network and pathway analyses in GEMMs show that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogram the TME during carcinogenesis, leading to a convergence of cell state alterations in neighboring epithelial, immune, and fibroblast cell types that parallel key findings in human prostate cancer.
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Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-myc , Microambiente Tumoral , Masculino , Microambiente Tumoral/genética , Humanos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Animais , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais , Análise de Célula Única , Modelos Animais de Doenças , Comunicação Celular , Carcinogênese/genética , Carcinogênese/patologia , Camundongos Transgênicos , RNA-SeqRESUMO
Reduced expression of the homeodomain transcription factor NKX3.1 is associated with prostate cancer initiation and progression. NKX3.1 turnover requires post-translational modifications including phosphorylation and ubiquitination. Here, we demonstrate the existence of a non-canonical mechanism for NKX3.1 turnover that does not require ubiquitination. Using a structure-function approach, we have determined that the conserved, C-terminal 21-amino acid domain of NKX3.1 (C21) is required for this novel ubiquitin-independent degradation mechanism. Addition of C21 decreased half-life of enhanced green fluorescence protein (EGFP) by 5-fold, demonstrating that C21 constitutes a portable degron. Point mutational analyses of C21 revealed that a conserved proline residue (Pro-221) is central to degron activity, and mutation to alanine (P221A) increased NKX3.1 half-life >2-fold. Proteasome inhibition and in vivo ubiquitination analyses indicated that degron activity is ubiquitin-independent. Evaluating degron activity in the context of a ubiquitination-resistant, lysine-null NKX3.1 mutant (NKX3.1(KO)) confirmed that P221A mutation conferred additional stability to NKX3.1. Treatment of prostate cancer cell lines with a C21-based peptide specifically increased the level of NKX3.1, suggesting that treatment with degron mimetics may be a viable approach for NKX3.1 restoration.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Homeodomínio/metabolismo , Peptídeos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitinação/efeitos dos fármacos , Substituição de Aminoácidos , Linhagem Celular Tumoral , Proteínas de Homeodomínio/genética , Humanos , Masculino , Mutação de Sentido Incorreto , Peptídeos/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Prolina/genética , Prolina/metabolismo , Prolina/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Complexo de Endopeptidases do Proteassoma/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação/genéticaRESUMO
Aurora-A is a serine/threonine kinase that has oncogenic properties in vivo. The expression and kinase activity of Aurora-A are up-regulated in multiple malignancies. Aurora-A is a key regulator of mitosis that localizes to the centrosome from the G2 phase through mitotic exit and regulates mitotic spindle formation as well as centrosome separation. Overexpression of Aurora-A in multiple malignancies has been linked to higher tumor grade and poor prognosis through mechanisms that remain to be defined. Using an unbiased proteomics approach, we identified the protein nuclear mitotic apparatus (NuMA) as a robust substrate of Aurora-A kinase. Using a small molecule Aurora-A inhibitor in conjunction with a reverse in-gel kinase assay (RIKA), we demonstrate that NuMA becomes hypo-phosphorylated in vivo upon Aurora-A inhibition. Using an alanine substitution strategy, we identified multiple Aurora-A phospho-acceptor sites in the C-terminal tail of NuMA. Functional analyses demonstrate that mutation of three of these phospho-acceptor sites significantly diminished cell proliferation. In addition, alanine mutation at these sites significantly increased the rate of apoptosis. Using confocal immunofluorescence microscopy, we show that the NuMA T1804A mutant mis-localizes to the cytoplasm in interphase nuclei in a punctate pattern. The identification of Aurora-A phosphorylation sites in NuMA that are important for cell cycle progression and apoptosis provides new insights into Aurora-A function.
Assuntos
Antígenos Nucleares/metabolismo , Proliferação de Células , Interfase , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Ativo do Núcleo Celular , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Antígenos Nucleares/genética , Apoptose , Aurora Quinases , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Humanos , Masculino , Mutagênese Sítio-Dirigida , Mutação , Proteínas Associadas à Matriz Nuclear/genética , Fosforilação , Piperazinas/farmacologia , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post-translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells. Pharmacologic Pim-1 inhibition using the small molecule inhibitor CX-6258 decreased steady state levels and half-life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S-proteasome, demonstrating that Pim-1 protects NKX3.1 from proteasome-mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim-1 phospho-acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N-terminal PEST domain is essential for Pim-1-mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim-1. Pim-1-mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells.