RESUMEN
The paracrine actions of adipokine plasminogen activator inhibitor-1 (PAI-1) are implicated in obesity-associated tumorigenesis. Here, we show that PAI-1 mediates extracellular matrix (ECM) signaling via epigenetic repression of DKK1 in endometrial epithelial cells (EECs). While the loss of DKK1 is known to increase ß-catenin accumulation for WNT signaling activation, this epigenetic repression causes ß-catenin release from transmembrane integrins. Furthermore, PAI-1 elicits the disengagement of TIMP2 and SPARC from integrin-ß1 on the cell surface, lifting an integrin-ß1-ECM signaling constraint. The heightened interaction of integrin-ß1 with type 1 collagen (COL1) remodels extracellular fibrillar structures in the ECM. Consequently, the enhanced nanomechanical stiffness of this microenvironment is conducive to EEC motility and neoplastic transformation. The formation of extensively branched COL1 fibrils is also observed in endometrial tumors of patients with obesity. The findings highlight PAI-1 as a contributor to enhanced integrin-COL1 engagement and extensive ECM remodeling during obesity-associated neoplastic development.
Asunto(s)
Matriz Extracelular , Integrina beta1 , Obesidad , Inhibidor 1 de Activador Plasminogénico , beta Catenina , Humanos , Obesidad/metabolismo , Obesidad/patología , Femenino , Inhibidor 1 de Activador Plasminogénico/metabolismo , beta Catenina/metabolismo , Integrina beta1/metabolismo , Matriz Extracelular/metabolismo , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Animales , Osteonectina/metabolismo , Osteonectina/genética , Colágeno/metabolismo , Endometrio/metabolismo , Endometrio/patología , Colágeno Tipo I/metabolismo , Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Péptidos y Proteínas de Señalización IntercelularRESUMEN
In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity.
Asunto(s)
FN-kappa B , Neoplasias de la Próstata , Tolerancia a Radiación , Proteína Sequestosoma-1 , Transducción de Señal , Masculino , Animales , Humanos , Neoplasias de la Próstata/radioterapia , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/tratamiento farmacológico , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , FN-kappa B/metabolismo , FN-kappa B/genética , Transducción de Señal/efectos de los fármacos , Ratones , Tolerancia a Radiación/efectos de los fármacos , Línea Celular Tumoral , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/genéticaRESUMEN
Lung cancer is the leading cause of global cancer-related mortality resulting in â¼ 1.8 million deaths annually. Systemic, molecular targeted, and immune therapies have provided significant improvements of survival outcomes for patients. However, drug resistance usually arises and there is an urgent need for novel therapy screening and personalized medicine. 3D patient-derived organoid (PDO) models have emerged as a more effective and efficient alternative for ex vivo drug screening than 2D cell culture and patient-derived xenograft (PDX) models. In this review, we performed an extensive search of lung cancer PDO-based ex vivo drug screening studies. Lung cancer PDOs were successfully established from fresh or bio-banked sections and/or biopsies, pleural effusions and PDX mouse models. PDOs were subject to ex vivo drug screening with chemotherapy, targeted therapy and/or immunotherapy. PDOs consistently recapitulated the genomic alterations and drug sensitivity of primary tumors. Although sample sizes of the previous studies were limited and some technical challenges remain, PDOs showed great promise in the screening of novel therapy drugs. With the technical advances of high throughput, tumor-on-chip, and combined microenvironment, the drug screening process using PDOs will enhance precision care of lung cancer patients.
Asunto(s)
Antineoplásicos , Neoplasias Pulmonares , Humanos , Animales , Ratones , Medicina de Precisión/métodos , Antineoplásicos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Pulmón , Organoides/patología , Microambiente TumoralRESUMEN
While macrophage phagocytosis is an immune defense mechanism against invading cellular organisms, cancer cells expressing the CD47 ligand send forward signals to repel this engulfment. Here we report that the reverse signaling using CD47 as a receptor additionally enhances a pro-survival function of prostate cancer cells under phagocytic attack. Although low CD47-expressing cancer cells still allow phagocytosis, the reverse signaling delays the process, leading to incomplete digestion of the entrapped cells and subsequent tumor hybrid cell (THC) formation. Viable THCs acquire c-Myc from parental cancer cells to upregulate both M1- and M2-like macrophage polarization genes. Consequently, THCs imitating dual macrophage features can confound immunosurveillance, gaining survival advantage in the host. Furthermore, these cells intrinsically express low levels of androgen receptor and its targets, resembling an adenocarcinoma-immune subtype of metastatic castration-resistant prostate cancer. Therefore, phagocytosis-generated THCs may represent a potential target for treating the disease.
Asunto(s)
Antígeno CD47 , Macrófagos , Metástasis de la Neoplasia , Fagocitosis , Proteínas Proto-Oncogénicas c-myc , Escape del Tumor , Humanos , Masculino , Proteínas Portadoras , Antígeno CD47/metabolismo , Macrófagos/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Transducción de Señal , Escape del Tumor/genética , Escape del Tumor/inmunología , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/inmunología , Células Tumorales CultivadasRESUMEN
Tumor-associated macrophages (TAMs) are integral to the development of complex tumor microenvironments (TMEs) and can execute disparate cellular programs in response to extracellular cues. However, upstream signaling processes underpinning this phenotypic plasticity remain to be elucidated. Here, we report that concordant AXL-STAT3 signaling in TAMs is triggered by lung cancer cells or cancer-associated fibroblasts in the cytokine milieu. This paracrine action drives TAM differentiation toward a tumor-promoting "M2-like" phenotype with upregulation of CD163 and putative mesenchymal markers, contributing to TAM heterogeneity and diverse cellular functions. One of the upregulated markers, CD44, mediated by AXL-IL-11-pSTAT3 signaling cascade, enhances macrophage ability to interact with endothelial cells and facilitate formation of primitive vascular networks. We also found that AXL-STAT3 inhibition can impede the recruitment of TAMs in a xenograft mouse model, thereby suppressing tumor growth. These findings suggest the potential application of AXL-STAT3-related markers to quantitatively assess metastatic potential and inform therapeutic strategies in lung cancer.
Asunto(s)
Neoplasias Pulmonares , Macrófagos Asociados a Tumores , Humanos , Animales , Ratones , Células Endoteliales , Transducción de Señal , Diferenciación Celular , Microambiente Tumoral , Línea Celular TumoralRESUMEN
Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could be a significant contribution to rational drug design. Since the structural analysis suggests that gate opening is associated with a decrease in the content of α-helices and ß-sheets and an increase in random coil structures, we decided to explore the application of electronic circular dichroism (ECD) in the UV region to monitor the proteasome gating. A comparison of ECD spectra of wild type yeast 20S proteasome (predominantly closed) and an open-gate mutant (α3ΔN) revealed an increased intensity in the ECD band at 220 nm, which suggests increased contents of random coil and ß-turn structures. This observation was further supported by evaluating ECD spectra of human 20S treated with low concentration of SDS, known as a gate-opening reagent. Next, to evaluate the power of ECD to probe a ligand-induced gate status, we treated the proteasome with H2T4, a tetracationic porphyrin that we showed previously to induce large-scale protein conformational changes upon binding to h20S. H2T4 caused a significant increase in the ECD band at 220 nm, interpreted as an induced opening of the 20S gate. In parallel, we imaged the gate-harboring alpha ring of the 20S with AFM, a technique that we used previously to visualize the predominantly closed gate in latent human or yeast 20S and the open gate in α3ΔN mutant. The results were convergent with the ECD data and showed a marked decrease in the content of closed-gate conformation in the H2T4-treated h20S. Our findings provide compelling support for the use of ECD measurements to conveniently monitor proteasome conformational changes related to gating phenomena. We predict that the observed association of spectroscopic and structural results will help with efficient design and characterization of exogenous proteasome regulators.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Humanos , Dicroismo Circular , Complejo de la Endopetidasa Proteasomal/química , Conformación Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Microscopía de Fuerza AtómicaRESUMEN
Vascular mechanisms of Alzheimer's disease (AD) may constitute a therapeutically addressable biological pathway underlying dementia. We previously demonstrated that soluble pathogenic forms of tau (tau oligomers) accumulate in brain microvasculature of AD and other tauopathies, including prominently in microvascular endothelial cells. Here we show that soluble pathogenic tau accumulates in brain microvascular endothelial cells of P301S(PS19) mice modeling tauopathy and drives AD-like brain microvascular deficits. Microvascular impairments in P301S(PS19) mice were partially negated by selective removal of pathogenic soluble tau aggregates from brain. We found that similar to trans-neuronal transmission of pathogenic forms of tau, soluble tau aggregates are internalized by brain microvascular endothelial cells in a heparin-sensitive manner and induce microtubule destabilization, block endothelial nitric oxide synthase (eNOS) activation, and potently induce endothelial cell senescence that was recapitulated in vivo in microvasculature of P301S(PS19) mice. Our studies suggest that soluble pathogenic tau aggregates mediate AD-like brain microvascular deficits in a mouse model of tauopathy, which may arise from endothelial cell senescence and eNOS dysfunction triggered by internalization of soluble tau aggregates.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Ratones , Animales , Proteínas tau/genética , Proteínas tau/metabolismo , Células Endoteliales/metabolismo , Tauopatías/metabolismo , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Senescencia Celular , Ratones TransgénicosRESUMEN
The proteolytic active sites of the 26S proteasome are sequestered within the catalytic chamber of its 20S core particle (CP). Access to this chamber is through a narrow channel defined by the seven outer α subunits. In the resting state, the N-termini of neighboring α subunits form a gate blocking access to the channel. The attachment of the activators or regulatory particles rearranges the blocking α subunit N-termini facilitating the entry of substrates. By truncating or mutating each of the participating α N-termini, we report that whereas only a few N-termini are important for maintaining the closed gate, all seven N-termini participate in the open gate. Specifically, the open state is stabilized by a hydrogen bond between an invariant tyrosine (Y) in each subunit with a conserved aspartate (D) in its counterclockwise neighbor. The lone exception is the α1-α2 pair leaving a gap in the ring circumference. The third residue (X) of this YD(X) motif aligns with the open channel. Phenylalanine at this position in the α2 subunit comes in direct contact with the translocating substrate. Consequently, deletion of the α2 N-terminal tail attenuates proteolysis despite the appearance of an open gate state. In summary, the interlacing N-terminal YD(X) motifs regulate both the gating and translocation of the substrate.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Complejo de la Endopetidasa Proteasomal/metabolismo , Modelos Moleculares , ProteolisisRESUMEN
The proteasome has key roles in neuronal proteostasis, including the removal of misfolded and oxidized proteins, presynaptic protein turnover, and synaptic efficacy and plasticity. Proteasome dysfunction is a prominent feature of Alzheimer's disease (AD). We show that prevention of proteasome dysfunction by genetic manipulation delays mortality, cell death, and cognitive deficits in fly and cell culture AD models. We developed a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an AD mouse model, showed reduced mortality and cognitive deficits. To establish translational relevance, we developed a set of TAT-based proteasome-activating peptidomimetics that stably penetrated the blood-brain barrier and enhanced 20S/26S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. The protective effects of proteasome overexpression appear to be driven, at least in part, by the proteasome's increased turnover of the amyloid precursor protein along with the prevention of overall proteostatic dysfunction.
Asunto(s)
Enfermedad de Alzheimer , Disfunción Cognitiva , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster , Ratones , Ratones Transgénicos , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of "mechanical fitness" in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell-macrophage cocultures revealed that macrophages promoted epithelial-mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis. SIGNIFICANCE: The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.
Asunto(s)
Macrófagos/metabolismo , Células Neoplásicas Circulantes/metabolismo , Neoplasias de la Próstata/inmunología , Línea Celular Tumoral , Humanos , Masculino , FenotipoRESUMEN
The proteasome is a pivotal element of controlled proteolysis, responsible for the catabolic arm of proteostasis. By inducing apoptosis, small molecule inhibitors of proteasome peptidolytic activities are successfully utilized in treatment of blood cancers. However, the clinical potential of proteasome activation remains relatively unexplored. In this work, we introduce short TAT peptides derived from HIV-1 Tat protein and modified with synthetic turn-stabilizing residues as proteasome agonists. Molecular docking and biochemical studies point to the α1/α2 pocket of the core proteasome α ring as the binding site of TAT peptides. We postulate that the TATs' pharmacophore consists of an N-terminal basic pocket-docking "activation anchor" connected via a ß turn inducer to a C-terminal "specificity clamp" that binds on the proteasome α surface. By allosteric effects-including destabilization of the proteasomal gate-the compounds substantially augment activity of the core proteasome in vitro. Significantly, this activation is preserved in the lysates of cultured cells treated with the compounds. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.
Asunto(s)
Péptidos/química , Péptidos/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Regulación Alostérica , Sitios de Unión , Línea Celular Tumoral , Quimotripsina/química , Citoplasma/metabolismo , Humanos , Microscopía de Fuerza Atómica , Simulación del Acoplamiento Molecular , Péptido Hidrolasas/química , Péptidos/síntesis química , Complejo de la Endopetidasa Proteasomal/químicaRESUMEN
SULT2B1b (SULT2B) is a prostate-expressed hydroxysteroid sulfotransferase, which may regulate intracrine androgen homeostasis by mediating 3ß-sulfation of dehydroepiandrosterone (DHEA), the precursor for 5α-dihydrotestosterone (DHT) biosynthesis. The aldo-keto reductase (AKR)1C3 regulates androgen receptor (AR) activity in castration-resistant prostate cancer (CRPC) by promoting tumor tissue androgen biosynthesis from adrenal DHEA and also by functioning as an AR-selective coactivator. Herein we report that SULT2B-depleted CRPC cells, arising from stable RNA interference or gene knockout (KO), are markedly upregulated for AKR1C3, activated for ERK1/2 survival signal, and induced for epithelial-to-mesenchymal (EMT)-like changes. EMT was evident from increased mesenchymal proteins and elevated EMT-inducing transcription factors SNAI1 and TWIST1 in immunoblot and single-cell mass cytometry analyses. SULT2B KO cells showed greater motility and invasion in vitro; growth escalation in xenograft study; and enhanced metastatic potential predicted on the basis of decreased cell stiffness and adhesion revealed from atomic force microscopy analysis. While AR and androgen levels were unchanged, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several-fold higher in SULT2B-silenced cells. AKR1C3 silencing prevented ERK1/2 activation and SNAI1 induction in SULT2B-depleted cells. SULT2B was undetectable in nearly all CRPC metastases from 50 autopsy cases. Primary tumors showed variable and Gleason score (GS)-independent SULT2B levels. CRPC metastases lacking SULT2B expressed AKR1C3. Since AKR1C3 is frequently elevated in advanced prostate cancer, the inhibitory influence of SULT2B on AKR1C3 upregulation, ERK1/2 activation, EMT-like induction, and on cell motility and invasiveness may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for targeting SULT2B-deficient prostate cancer.
Asunto(s)
Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/metabolismo , Carcinoma/enzimología , Neoplasias de la Próstata/enzimología , Sulfotransferasas/metabolismo , Animales , Transición Epitelial-Mesenquimal , Humanos , Masculino , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Receptores Androgénicos/metabolismoRESUMEN
Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFß signaling and induced JAK1-STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1-STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFß, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFß, and JAK1-STAT3 signal activation in select tumors that may be targeted by combined AXL-JAK1 inhibition. SIGNIFICANCE: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Janus Quinasa 1/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Transición Epitelial-Mesenquimal/efectos de los fármacos , Estudios de Factibilidad , Femenino , Citometría de Flujo/métodos , Humanos , Janus Quinasa 1/metabolismo , Pulmón/patología , Neoplasias Pulmonares/patología , Masculino , Ratones , Persona de Mediana Edad , Nitrilos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteómica/métodos , Proteínas Proto-Oncogénicas/metabolismo , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , RNA-Seq , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de la Célula Individual/métodos , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor AxlRESUMEN
Allosteric regulators of clinically important enzymes are gaining popularity as alternatives to competitive inhibitors. This is also the case for the proteasome, a major intracellular protease and a target of anti-cancer drugs. All clinically used proteasome inhibitors bind to the active sites in catalytic chamber and display a competitive mechanism. Unfortunately, inevitable resistance associated with this type of inhibition drives the search for non-competitive agents. The multisubunit and multicatalytic "proteolytic machine" such as the proteasome is occasionally found to be affected by agents with other primary targets. For example the immunosuppressive agent rapamycin has been shown to allosterically inhibit the proteasome albeit at levels far higher than its mTOR related efficacy. As part of an ongoing program to search for novel proteasome-targeting pharmacophores, we identified the binding domain of rapamycin as required for proteasome inhibition even without the macrocyclic context of the parent compound. By subsequent structure-activity relationship studies, we generated a pipecolic ester derivative compound 3 representing a new class of proteasome inhibitors. Compound 3 affects the core proteasome activities and proliferation of cancer cells with low micromolar/high nanomolar efficacy. Molecular modeling, atomic force microscopy imaging and biochemical data suggest that compound 3 binds into one of intersubunit pockets in the proteasomal α ring and destabilizes the α face and the gate. The α face is used as a docking area for proteasome-regulating protein modules and the gate is critical for controlling access to the catalytic chamber. Thus, the pipecolic ester template elicits a new and attractive mechanism for proteasome inhibition distinct from classical competitive drugs.
Asunto(s)
Ésteres/química , Ácidos Pipecólicos/química , Ácidos Pipecólicos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacología , Dominio Catalítico , Diseño de Fármacos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Ácidos Pipecólicos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/metabolismoRESUMEN
Proline- and arginine-rich peptide PR11 is an allosteric inhibitor of 20S proteasome. We modified its sequence inter alia by introducing HbYX, RYX, or RHbX C-terminal extensions (Hb, hydrophobic moiety; R, arginine; Y, tyrosine; X, any residue). Consequently, we were able to improve inhibitory potency or to convert inhibitors into strong activators: the former with an aromatic penultimate Hb residue and the latter with the HbYX motif. The PR peptide activator stimulated 20S proteasome in vitro to efficiently degrade protein substrates, such as α-synuclein and enolase, but also activated proteasome in cultured fibroblasts. The positive and negative PR modulators differently influenced the proteasome conformational dynamics and affected opening of the substrate entry pore. The resolved crystal structure showed PR inhibitor bound far from the active sites, at the proteasome outer face, in the pocket used by natural activators. Our studies indicate the opportunity to tune proteasome activity by allosteric regulators based on PR peptide scaffold.
Asunto(s)
Péptidos/química , Complejo de la Endopetidasa Proteasomal/química , Regulación Alostérica , Secuencia de Aminoácidos , Arginina/química , Sitios de Unión , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Péptidos/síntesis química , Péptidos/metabolismo , Prolina/química , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an in vitro coculture system revealed transcriptional repression of GJA1 (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the GJA1 repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort (n = 141), DNA hypermethylation of GJA1 and related loci TJP2 and PRKCA was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap-junction intercellular communication and cell-cell interactions in vitro. Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of GJA1 and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients. SIGNIFICANCE: Studies reveal that adipose-derived stem cells in endometrial cancer pathogenesis influence epigenetic repression of gap junction loci, which suggests targeting of gap junction activity as a preventive strategy for obesity-associated endometrial cancer.
Asunto(s)
Adipoquinas/farmacología , Tejido Adiposo/patología , Comunicación Celular , Conexina 43/genética , Neoplasias Endometriales/patología , Represión Epigenética , Obesidad/complicaciones , Tejido Adiposo/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Conexina 43/metabolismo , Dieta Alta en Grasa/efectos adversos , Neoplasias Endometriales/etiología , Neoplasias Endometriales/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Uniones Comunicantes , Humanos , Masculino , Ratones , Ratones Noqueados , Obesidad/fisiopatología , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
The 20S proteasome is the main protease that directly targets intrinsically disordered proteins (IDPs) for proteolytic degradation. Mutations, oxidative stress, or aging can induce the buildup of IDPs resulting in incorrect signaling or aggregation, associated with the pathogenesis of many cancers and neurodegenerative diseases. Drugs that facilitate 20S-mediated proteolysis therefore have many potential therapeutic applications. We report herein the modulation of proteasome assembly by the small molecule TCH-165, resulting in an increase in 20S levels. The increase in the level of free 20S corresponds to enhanced proteolysis of IDPs, including α-synuclein, tau, ornithine decarboxylase, and c-Fos, but not structured proteins. Clearance of ubiquitinated protein was largely maintained by single capped proteasome complexes (19S-20S), but accumulation occurs when all 19S capped proteasome complexes are depleted. This study illustrates the first example of a small molecule capable of targeting disordered proteins for degradation by regulating the dynamic equilibrium between different proteasome complexes.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Ornitina Descarboxilasa/metabolismo , Ubiquitinación/efectos de los fármacos , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
The ubiquitin-proteasome pathway (UPP) is a major venue for controlled intracellular protein degradation in Eukaryota. The machinery of several hundred proteins is involved in recognizing, tagging, transporting, and cleaving proteins, all in a highly regulated manner. Short-lived transcription factors, misfolded translation products, stress-damaged polypeptides, or worn-out long-lived proteins, all can be found among the substrates of UPP. Carefully choreographed protein-protein interactions (PPI) are involved in each step of the pathway. For many of the steps small-molecule inhibitors have been identified and often they directly or indirectly target PPI. The inhibitors may destabilize intracellular proteostasis and trigger apoptosis. So far this is the most explored option used as an anticancer strategy. Alternatively, substrate-specific polyubiquitination may be regulated for a precise intervention aimed at a particular metabolic pathway. This very attractive opportunity is moving close to clinical application. The best known drug target in UPP is the proteasome: the end point of the journey of a protein destined for degradation. The proteasome alone is a perfect object to study the mechanisms and roles of PPI on many levels. This giant protease is built from multisubunit modules and additionally utilizes a service from transient protein ligands, for example, delivering substrates. An elaborate set of PPI within the highest-order proteasome assembly is involved in substrate recognition and processing. Below we will outline PPI involved in the UPP and discuss the growing prospects for their utilization in pharmacological interventions.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Humanos , Complejo de la Endopetidasa Proteasomal/química , Unión Proteica , Ubiquitinación , Ubiquitinas/química , Ubiquitinas/metabolismoRESUMEN
Older adults universally suffer from sarcopenia and approximately 60-70% are diabetic or prediabetic. Nonetheless, the mechanisms underlying these aging-related metabolic disorders are unknown. NFκB has been implicated in the pathogenesis of several aging-related pathologies including sarcopenia and type 2 diabetes and has been proposed as a target against them. NFκB also is thought to mediate muscle wasting seen with disuse, denervation, and some systemic diseases (e.g., cancer, sepsis). We tested the hypothesis that lifelong inhibition of the classical NFκB pathway would protect against aging-related sarcopenia and insulin resistance. Aged mice with muscle-specific overexpression of a super-repressor IκBα mutant (MISR) were protected from insulin resistance. However, MISR mice were not protected from sarcopenia; to the contrary, these mice had decreases in muscle mass and strength compared to wild-type mice. In MISR mice, NFκB suppression also led to an increase in proteasome activity and alterations in several genes and pathways involved in muscle growth and atrophy (e.g., myostatin). We conclude that the mechanism behind aging-induced sarcopenia is NFκB independent and differs from muscle wasting due to pathologic conditions. Our findings also indicate that, while suppressing NFκB improves insulin sensitivity in aged mice, this transcription factor is important for normal muscle mass maintenance and its sustained inhibition is detrimental to muscle function.
Asunto(s)
Envejecimiento/metabolismo , Resistencia a la Insulina , Músculo Esquelético/metabolismo , Miostatina/genética , FN-kappa B/genética , Sarcopenia/genética , Envejecimiento/patología , Animales , Glucemia/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Línea Celular , Ceramidas/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/patología , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Mioblastos/metabolismo , Mioblastos/patología , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Sarcopenia/metabolismo , Sarcopenia/patologíaRESUMEN
Cholesterol is an important risk factor of atherosclerosis, due to its active uptake by monocytes/macrophages. Monocyte recruitment from flowing blood to atherosclerotic foci is the key first step in the development of atherosclerosis. Cholesterol content alters cell membrane stiffness, and lateral lipid and protein diffusion. We hypothesized that cholesterol content will modulate the recruitment of monocytes to inflamed endothelial surface by altering the dynamics of adhesion receptors. We depleted or enriched the cellular cholesterol levels using methyl-ß-cyclodextran in freshly isolated human monocytes. We investigated the effect of these changes on the mechanics of monocyte rolling on E-selectin surfaces at 1 dyn/cm2 in microchannels. Using imaging flow cytometry and atomic force microscopy, we characterized the distribution of lipid rafts and the E-selectin counterreceptor CD44 on the monocyte surface. We observed that lower levels of cholesterol resulted in the uniform, CD44-mediated rolling of monocytes on the E-selectin-coated surfaces. We also observed that cells depleted of cholesterol had higher membrane fluidity, and more uniform distribution of CD44 counterreceptor, which resulted in smooth motion of the cells compared to cells enriched with cholesterol. This work demonstrates that cholesterol can modulate monocyte adhesion by regulating the receptor mobility, and our results provide insights into the biophysical regulation of inflammation for the better understanding of diseases like atherosclerosis and hypercholesterolemia.