RESUMO
There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
Assuntos
Biomarcadores Tumorais/metabolismo , Vesículas Extracelulares/metabolismo , Neoplasias/diagnóstico , Animais , Biomarcadores Tumorais/sangue , Linhagem Celular , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Aprendizado de Máquina , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Neoplasias/metabolismo , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Sensibilidade e Especificidade , Tetraspanina 29/metabolismo , Proteínas rap de Ligação ao GTP/metabolismoRESUMO
Intercellular cross talk between cancer cells and stromal and immune cells is essential for tumor progression and metastasis. Extracellular vesicles and particles (EVPs) are a heterogeneous class of secreted messengers that carry bioactive molecules and that have been shown to be crucial for this cell-cell communication. Here, we highlight the multifaceted roles of EVPs in cancer. Functionally, transfer of EVP cargo between cells influences tumor cell growth and invasion, alters immune cell composition and function, and contributes to stromal cell activation. These EVP-mediated changes impact local tumor progression, foster cultivation of pre-metastatic niches at distant organ-specific sites, and mediate systemic effects of cancer. Furthermore, we discuss how exploiting the highly selective enrichment of molecules within EVPs has profound implications for advancing diagnostic and prognostic biomarker development and for improving therapy delivery in cancer patients. Altogether, these investigations into the role of EVPs in cancer have led to discoveries that hold great promise for improving cancer patient care and outcome.
Assuntos
Vesículas Extracelulares , Neoplasias , Comunicação Celular , Humanos , Imunoterapia , Neoplasias/patologia , Microambiente TumoralRESUMO
Primary tumors secrete a variety of factors to turn distant microenvironments into favorable and fertile 'soil' for subsequent metastases. Among these 'seeding' factors that initiate pre-metastatic niche (PMN) formation, tumor-derived extracellular vesicles (EVs) are of particular interest as tumor EVs can direct organotropism depending on their surface integrin profiles. In addition, EVs also contain versatile, bioactive cargo, which include proteins, metabolites, lipids, RNA, and DNA fragments. The cargo incorporated into EVs is collectively shed from cancer cells and cancer-associated stromal cells. Increased understanding of how tumor EVs promote PMN establishment and detection of EVs in bodily fluids highlight how tumor EVs could serve as potential diagnostic and prognostic biomarkers, as well as provide a therapeutic target for metastasis prevention. This review focuses on tumor-derived EVs and how they direct organotropism and subsequently modulate stromal and immune microenvironments at distal sites to facilitate PMN formation. We also outline the progress made thus far towards clinical applications of tumor EVs.
Assuntos
Vesículas Extracelulares , Neoplasias , Humanos , Neoplasias/metabolismo , Vesículas Extracelulares/metabolismo , Comunicação Celular , Microambiente TumoralRESUMO
Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.
Assuntos
Vesículas Extracelulares/ultraestrutura , Fixação de Tecidos/métodos , Animais , Carbodi-Imidas , Bovinos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Corpo Vítreo/ultraestruturaRESUMO
Autophagy, a pathway for lysosomal-mediated cellular degradation, has recently been described as a regulator of cell migration. Although the molecular mechanisms underlying autophagy-dependent motility are only beginning to emerge, new work demonstrates that selective autophagy mediated by the autophagy cargo receptor, NBR1, specifically promotes the dynamic turnover of integrin-based focal adhesion sites during motility. Here, we discuss the detailed mechanisms through which NBR1-dependent selective autophagy supports focal adhesion remodeling, and we describe the interconnections between this pathway and other established regulators of focal adhesion turnover, such as microtubules. We also highlight studies that examine the contribution of autophagy to selective degradation of proteins that mediate cellular tension and to integrin trafficking; these findings hint at further roles for autophagy in supporting adhesion and migration. Given the recently appreciated importance of selective autophagy in diverse cellular processes, we propose that further investigation into autophagy-mediated focal adhesion turnover will not only shed light onto how focal adhesions are regulated but will also unveil new mechanisms regulating selective autophagy.
Assuntos
Autofagia , Movimento Celular , Animais , Adesão Celular , Adesões Focais/metabolismo , Humanos , Integrinas/metabolismo , Transporte ProteicoRESUMO
Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aß42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aß42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aß42 levels.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Descoberta de Drogas , Macaca mulatta , Camundongos , Camundongos Transgênicos , Ratos , Ratos Sprague-DawleyRESUMO
The triazolyl amide γ-secretase modulators are potent alternatives to the cinnamyl amides that have entered the clinic for the treatment of Alzheimer's disease. Herein we build on the lead benzoazepinones described in our prior communication with imidazomethoxyarene moiety alternatives that offer opportunities to fine tune physical properties as well as address hERG binding and PK. Both half-life and bioavailability were significantly improved, especially in dog, with robust brain Aß42 lowering maintained in both transgenic mouse and rat.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/farmacocinética , Animais , Disponibilidade Biológica , Camundongos , Camundongos Transgênicos , RatosRESUMO
Scanning electron microscopy (SEM) offers an unparalleled view of the membrane topography of mammalian cells by using a conventional osmium (OsO4) and ethanol-based tissue preparation. However, conventional SEM methods limit optimal resolution due to ethanol and lipid interactions and interfere with visualization of fluorescent reporter proteins. Therefore, SEM correlative light and electron microscopy (CLEM) has been hindered by the adverse effects of ethanol and OsO4 on retention of fluorescence signals. To overcome this technological gap in achieving high-resolution SEM and retain fluorescent reporter signals, we developed a freeze-drying method with gaseous nitrogen (FDGN). We demonstrate that FDGN preserves cyto-architecture to allow visualization of detailed membrane topography while retaining fluorescent signals and that FDGN processing can be used in conjunction with a variety of high-resolution imaging systems to enable collection and validation of unique, high-quality data from these approaches. In particular, we show that FDGN coupled with high resolution microscopy provided detailed insight into viral or tumor-derived extracellular vesicle (TEV)-host cell interactions and may aid in designing new approaches to intervene during viral infection or to harness TEVs as therapeutic agents.
RESUMO
Synthesis, SAR, and evaluation of aryl triazoles as novel gamma secretase modulators (GSMs) are presented in this communication. Starting from the literature and in-house leads, we evaluated a range of five-membered heterocycles as replacements for olefins commonly found in non-acid GSMs. 1,2,3-C-aryl-triazoles were identified as suitable replacements which exhibited good modulation of γ-secretase activity, excellent pharmacokinetics and good central lowering of Aß42 in Sprague-Dawley rats.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Triazóis/síntese química , Triazóis/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Triazóis/metabolismoRESUMO
We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).
Assuntos
Líquidos Corporais/química , Centrifugação com Gradiente de Concentração , Vesículas Extracelulares/química , Fracionamento por Campo e Fluxo , Proteômica , Animais , Linhagem Celular , Humanos , CamundongosRESUMO
BACKGROUND & AIMS: The Notch signaling pathway is required for the expansion of undifferentiated pancreatic progenitor cells during embryonic development and has been implicated in the progression of pancreatic ductal adenocarcinoma (PDAC). The interaction of Notch ligands with their receptors promotes a gamma-secretase-dependent cleavage of the Notch receptor and release of the Notch intracellular domain, which translocates to the nucleus and activates transcription. We investigated the role of this pathway in PDAC progression. METHODS: We tested the effects of a gamma-secretase inhibitor (GSI) that blocks Notch signaling in PDAC cell lines and a genetically engineered mouse model of PDAC (Kras p53 L/+ mice). RESULTS: Notch signaling was activated in PDAC precursors and advanced tumors. The GSI inhibited the growth of premalignant pancreatic duct-derived cells in a Notch-dependent manner. Additionally, in a panel of over 400 human solid tumor-derived cell lines, PDAC cells, as a group, were more sensitive to the GSI than any other tumor type. Finally, the GSI completely inhibited tumor development in the genetically engineered model of invasive PDAC (P < .005, chi2 test; compared with mice exposed to vehicle). CONCLUSIONS: These results suggest that Notch signaling is required for PDAC progression. Pharmacologic targeting of this pathway offers therapeutic potential in this treatment-refractory malignancy.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores Notch/fisiologia , Transdução de Sinais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/fisiopatologia , Linhagem Celular , Linhagem Celular Tumoral , Óxidos S-Cíclicos/farmacologia , Progressão da Doença , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Tiadiazóis/farmacologiaRESUMO
We report herein a novel series of difluoropiperidine acetic acids as modulators of gamma-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective beta-difluorination with Selectfluor. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Abeta42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250mg/kg per day, AUC(0-24)=2100microMh) did not exhibit Notch-related effects.
Assuntos
Acetatos/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Flúor/química , Piperidinas/química , Acetatos/síntese química , Acetatos/farmacocinética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Compostos de Diazônio/química , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Piperidinas/síntese química , Piperidinas/farmacocinética , Ratos , Receptores Notch/metabolismoRESUMO
The development of a novel series of purines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based gamma-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Alphabeta42 in an APP-YAC transgenic mouse model.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Fragmentos de Peptídeos/antagonistas & inibidores , Purinas/química , Purinas/uso terapêutico , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Purinas/farmacologia , Receptores Notch/metabolismo , Relação Estrutura-AtividadeRESUMO
Although autophagy is being pursued as a therapeutic target in clinical oncology trials, its effects on metastasis, the principal cause of cancer mortality, remain unclear. Here, we utilize mammary cancer models to temporally delete essential autophagy regulators during carcinoma progression. Though genetic ablation of autophagy strongly attenuates primary mammary tumor growth, impaired autophagy promotes spontaneous metastasis and enables the outgrowth of disseminated tumor cells into overt macro-metastases. Transcriptomic analysis reveals that autophagy deficiency elicits a subpopulation of otherwise luminal tumor cells exhibiting basal differentiation traits, which is reversed upon preventing accumulation of the autophagy cargo receptor, Neighbor to BRCA1 (NBR1). Furthermore, pharmacological and genetic induction of autophagy suppresses pro-metastatic differentiation and metastatic outgrowth. Analysis of human breast cancer data reveal that autophagy gene expression inversely correlates with pro-metastatic differentiation signatures and predicts overall and distant metastasis-free survival. Overall, these findings highlight autophagy-dependent control of NBR1 as a key determinant of metastatic progression.
Assuntos
Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células MCF-7 , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , TranscriptomaRESUMO
The successful application of both solid and solution phase library synthesis, combined with tight integration into the medicinal chemistry effort, resulted in the efficient optimization of a novel structural series of selective HDAC1/HDAC2 inhibitors by the MRL-Boston Parallel Medicinal Chemistry group. An initial lead from a small parallel library was found to be potent and selective in biochemical assays. Advanced compounds were the culmination of iterative library design and possess excellent biochemical and cellular potency, as well as acceptable PK and efficacy in animal models.
Assuntos
Inibidores de Histona Desacetilases , Animais , Técnicas de Química Combinatória , Cães , Desenho de Fármacos , Histona Desacetilase 1 , Histona Desacetilase 2 , Humanos , Estrutura Molecular , Ratos , Proteínas Repressoras/antagonistas & inibidores , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor-derived extracellular vesicles promote metastasis by inducing functional changes in cells at pre-metastatic sites conducive for tumor cell colonization. In this issue of Cancer Cell, Ortiz and colleagues show that type I interferon regulates extracellular vesicle uptake and that modulating this pathway holds promise for treating metastasis.
Assuntos
Vesículas Extracelulares , Neoplasias , Oxisteróis , Humanos , InterferonsRESUMO
Metastasis, a critical phase of tumor progression, remains a primary challenge in treating cancer and a major cause of cancer mortality. Cell-cell communication via extracellular vesicles (exosomes and microvesicles) between primary tumor cells and the microenvironment of distant organs is crucial for pre-metastatic niche (PMN) formation and metastasis. Here, we review work on the contribution of exosome cargo to cancer progression, the role of exosomes in PMN establishment, and the function of exosomes in organotropic metastasis. We also describe the clinical utility of exosomes.
Assuntos
Exossomos/patologia , Metástase Neoplásica/patologia , Neoplasias/patologia , Animais , Comunicação Celular , Progressão da Doença , Exossomos/metabolismo , Fibroblastos/metabolismo , Humanos , Invasividade Neoplásica/patologia , Neoplasias/metabolismo , Neovascularização Patológica/patologia , Microambiente TumoralRESUMO
The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.
Assuntos
Neoplasias Encefálicas/genética , Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Hialuronoglucosaminidase/genética , Neovascularização Patológica/genética , Microambiente Tumoral/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Quimiocina CCL1/genética , Quimiocina CCL1/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Exossomos/patologia , Humanos , Hialuronoglucosaminidase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Metástase Neoplásica , Neovascularização Patológica/metabolismo , Neovascularização Patológica/mortalidade , Neovascularização Patológica/patologia , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A potent family of spirocyclic nicotinyl aminobenzamide selective HDAC1/HDAC2 inhibitors (SHI-1:2) is profiled. The incorporation of a biaryl zinc-binding motif into a nicotinyl scaffold resulted in enhanced potency and selectivity versus HDAC3, but also imparted hERG activity. It was discovered that increasing polar surface area about the spirocycle attenuates this liability. Compound 12 induced a 4-fold increase in acetylated histone H2B in an HCT-116 xenograft model study with acute exposure, and inhibited tumor growth in a 21-day efficacy study with qd dosing.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Inibidores de Histona Desacetilases , Niacinamida/síntese química , Niacinamida/farmacologia , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Animais , Antineoplásicos/química , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacologia , Técnicas de Química Combinatória , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Células HCT116 , Histona Desacetilases , Histonas/análise , Humanos , Camundongos , Camundongos Nus , Estrutura Molecular , Niacinamida/química , Isoformas de Proteínas , Compostos de Espiro/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.