RESUMO
Cellular stress and toxicity are often associated with the formation of protein multimers, or aggregates. Numerous degenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, prion-propagated disease, amyotrophic lateral sclerosis, cardiac amyloidosis, and diabetes, are characterized by aggregated protein deposits. Current methods are limited in the ability to assess multimer size along with multimer quantitation and to incorporate one or more ancillary traits, including target specificity, operative simplicity, and process speed. Here, we report development of a microparticle immunocapture assay that combines the advantages inherent to a monoclonal antibody:protein interaction with highly quantitative flow cytometry analysis. Using established reagents to build our platform, and aggregation-prone amyloid beta 1-42 peptide (Aß42) and alpha-synuclein to demonstrate proof of principle, our results indicate that this assay is a highly adaptable method to measure multimer size and quantity at the same time in a technically streamlined workflow applicable to laboratory and clinical samples.
Assuntos
Amiloidose , Doença de Huntington , Doenças Priônicas , Humanos , Peptídeos beta-Amiloides/metabolismo , Amiloidose/metabolismo , Doenças Priônicas/metabolismoRESUMO
Advances in genomics and proteomics drive precision medicine by providing actionable genetic alterations and molecularly targeted therapies, respectively. While genomic analysis and medicinal chemistry have advanced patient stratification with treatments tailored to the genetic profile of a patient's tumor, proteomic targeting has the potential to enhance the therapeutic index of drugs like poly(ADP-ribose) polymerase (PARP) inhibitors. PARP inhibitors in breast and ovarian cancer patients with BRCA1/2 mutations have shown promise. About 10% of the patients who received Olaparib (PARP inhibitor) showed adverse side effects including neutropenia, thrombocytopenia and in some cases resulted in myelodysplastic syndrome, indicating that off-target effects were substantial in these patients. Through proteomic analysis, our lab previously identified plectin, a cytolinker protein that mislocalized onto the cell surface during malignant transformation of healthy ovarian tissue. This cancer specific phenotype allowed us to image pancreatic cancer successfully using plectin targeted peptide (PTP) conjugated to nanoparticles or displayed on capsid protein of adeno-associated virus (AAV) particles. Objective: The goal of this study was to integrate the available pharmacogenomics and proteomic data to develop effective anti-tumor therapies using a targeted drug delivery approach. Methods: Plectin expression and localization in human ovarian tumor specimens were analyzed followed by in vitro confirmation of cell surface plectin localization in healthy and ovarian cancer cell lines. PTP-conjugated liposomes were prepared and their specificity for plectin+ cells was determined in vitro and in vivo. A remote loading method was employed to encapsulate a PARP inhibitor (AZ7379) into liposomes. An ideal buffer exchange method and remote loading conditions were determined based on the amount of lipid and drug recovered at the end of a remote loading process. Finally, in vivo tumor growth studies were performed to determine the efficacy of PTP liposomes in preventing PARP activity in mice bearing OVCAR8 (high grade epithelial ovarian cancer (EOC)) tumors. Results: PTP liposomal AZ7379 delivery not only enhanced PARP inhibition but also resulted in decelerated tumor growth in mice bearing subcutaneous and intraperitoneal OVCAR8 tumors. In mice bearing subcutaneous or intraperitoneal tumors, treatment with PTP liposomes resulted in a 3- and 1.7-fold decrease in tumor volume, respectively, compared to systemic drug treatment. Conclusion: Targeted drug delivery assisted by genomic and proteomic data provides an adaptable model system that can be extended to effectively treat other cancers and diseases.
Assuntos
Antineoplásicos/administração & dosagem , Lipossomos/química , Nanopartículas/química , Neoplasias Ovarianas/tratamento farmacológico , Plectina/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Lipossomos/efeitos adversos , Camundongos , Camundongos Nus , Nanopartículas/efeitos adversos , Peptídeos/química , Peptídeos/farmacocinética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacocinética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Ligação ProteicaRESUMO
While it has long been recognized that mononuclear phagocytes play a significant role in determining breast tumor progression, the molecular factors that contribute to these events are not fully understood. In this report, we sought to determine whether focal adhesion kinase (FAK) expression in this cell population influences primary breast tumor initiation and growth. Using the MMTV-polyoma middle T (PyVmT) murine model of spontaneous breast cancer, we found that FAK expression in mononuclear phagocytes accelerates tumor initiation/progression during the early stages of PyVmT tumor growth but subsequently restricts tumor growth once the tumors have transitioned to malignancy. Mononuclear phagocytes accumulated at the site of developing tumors in a FAK-independent manner. However, once in the tumor, our data suggest that FAK expression is upregulated in the tumor-associated myeloid cells, and its activity in this population of cells may influence the immune landscape of the tumor by supporting the recruitment and/or survival of NK cells. Together, these data support a model in which FAK expression in the mononuclear phagocyte compartment positively regulates the early steps of tumor progression but subsequently functions to restrict tumor growth as the tumors transition to invasive carcinoma.
RESUMO
Sustained angiogenesis is essential for the development of solid tumors and metastatic disease. Disruption of signaling pathways that govern tumor vascularity provide a potential avenue to thwart cancer progression. Through phage display-based functional proteomics, immunohistochemical analysis of human pancreatic ductal carcinoma (PDAC) specimens, and in vitro validation, we reveal that hornerin, an S100 fused-type protein, is highly expressed on pancreatic tumor endothelium in a vascular endothelial growth factor (VEGF)-independent manner. Murine-specific hornerin knockdown in PDAC xenografts results in tumor vessels with decreased radii and tortuosity. Hornerin knockdown tumors have significantly reduced leakiness, increased oxygenation, and greater apoptosis. Additionally, these tumors show a significant reduction in growth, a response that is further heightened when therapeutic inhibition of VEGF receptor 2 (VEGFR2) is utilized in combination with hornerin knockdown. These results indicate that hornerin is highly expressed in pancreatic tumor endothelium and alters tumor vessel parameters through a VEGF-independent mechanism.Angiogenesis is essential for solid tumor progression. Here, the authors interrogate the proteome of pancreatic cancer endothelium via phage display and identify hornerin as a critical protein whose expression is essential to maintain the pancreatic cancer vasculature through a VEGF-independent mechanism.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Carcinoma Ductal Pancreático/genética , Proteínas de Filamentos Intermediários/genética , Neovascularização Patológica/genética , Neoplasias Pancreáticas/genética , Animais , Apoptose/genética , Permeabilidade Capilar/genética , Carcinoma Ductal Pancreático/irrigação sanguínea , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Pancreáticas/irrigação sanguínea , Compostos de Fenilureia/farmacologia , Quinolinas/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Monocytes are short-lived myeloid cells that perform functions essential for tissue homeostasis and disease resolution. However, the cellular mechanisms controlling the maintenance and turnover of monocyte populations are largely undefined. Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase that regulates numerous immune cell functions, but its role in monocytes is currently unknown. In this study, we sought to characterize the expression and function of Pyk2 in lineage-committed monocyte populations. Here, we report that Pyk2 protein expression is increased in the Ly6C- monocyte population. Using a Pyk2 knockout mouse model (Pyk2-/-), we show that Pyk2 regulates the relative proportion of monocyte subsets normally represented in the bone marrow (BM) at steady state. In support of this conclusion, a similar phenotype was observed in the peripheral blood and spleen. Data from reciprocal BM chimera experiments indicate that the alterations in monocyte populations exhibited by Pyk2-/- mice are due to factors intrinsic to the monocytes. Lineage-tracing of monocyte populations suggests that Pyk2 promotes apoptosis in BM monocytes, thereby acting as an important homeostatic regulator of turnover in these short-lived, innate immune cells.
Assuntos
Apoptose/imunologia , Quinase 2 de Adesão Focal/imunologia , Monócitos/imunologia , Animais , Apoptose/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Transplante de Medula Óssea , Quinase 2 de Adesão Focal/genética , Camundongos , Camundongos Knockout , Monócitos/citologia , Quimeras de TransplanteRESUMO
Tissue homeostasis requires a complete repertoire of functional macrophages in peripheral tissues. Recent evidence indicates that many resident tissue macrophages are seeded during embryonic development and persist through adulthood as a consequence of localized proliferation. Mononuclear phagocytes are also produced during adult hematopoiesis; these cells are then recruited to sites throughout the body, where they function in tissue repair and remodeling, resolution of inflammation, maintenance of homeostasis, and disease progression. The focus of this review is on mononuclear phagocytes that comprise the nonresident monocyte/macrophage populations in the body. Key features of monocyte differentiation are presented, focusing primarily on the developmental hierarchy that is established through this process, the markers used to identify discrete cell populations, and novel, functional attributes of these cells. These features are then explored in the context of the tumor microenvironment, where mononuclear phagocytes exhibit extensive plasticity in phenotype and function.
Assuntos
Hematopoese , Macrófagos/imunologia , Monócitos/imunologia , Animais , Antígenos Ly/análise , Receptor 1 de Quimiocina CX3C , Linhagem da Célula , Movimento Celular , Citocinas/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Homeostase , Humanos , Sistema Imunitário/embriologia , Sistema Imunitário/imunologia , Imunidade Inata , Inflamação , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/citologia , Camundongos , Modelos Biológicos , Monócitos/classificação , Monócitos/citologia , Metástase Neoplásica , Neoplasias/imunologia , Neoplasias/patologia , Especificidade de Órgãos , Receptores de Quimiocinas/fisiologia , Fatores de Transcrição/fisiologia , Microambiente TumoralRESUMO
Osteoclasts are highly specialized cells that resorb bone and contribute to bone remodeling. Diseases such as osteoporosis and osteolytic bone metastasis occur when osteoclast-mediated bone resorption takes place in the absence of concurrent bone synthesis. Considerable effort has been placed on identifying molecules that regulate the bone resorption activity of osteoclasts. To this end, we investigated unique and overlapping functions of members of the FAK family (FAK and Pyk2) in osteoclast functions. With the use of a conditional knockout mouse model, in which FAK is selectively targeted for deletion in osteoclast precursors (FAK(Δmyeloid)), we found that loss of FAK resulted in reduced bone resorption by osteoclasts in vitro, coincident with impaired signaling through the CSF-1R. However, bone architecture appeared normal in FAK(Δmyeloid) mice, suggesting that Pyk2 might functionally compensate for reduced FAK levels in vivo. This was supported by data showing that podosome adhesion structures, which are essential for bone degradation, were significantly more impaired in osteoclasts when FAK and Pyk2 were reduced than when either molecule was depleted individually. We conclude that FAK contributes to cytokine signaling and bone resorption in osteoclasts and partially compensates for the absence of Pyk2 to maintain proper adhesion structures in these cells.
Assuntos
Reabsorção Óssea/enzimologia , Quinase 1 de Adesão Focal/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Osteoclastos/enzimologia , Osteoclastos/patologia , Animais , Reabsorção Óssea/patologia , Osso e Ossos/enzimologia , Osso e Ossos/patologia , Imunofluorescência , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
Prostasin, a trypsinlike serine peptidase, is highly expressed in prostate, kidney, and lung epithelia, where it is bound to the cell surface, secreted, or both. Prostasin activates the epithelial sodium channel (ENaC) and suppresses invasion of prostate and breast cancer cells. The studies reported here establish mechanisms of membrane anchoring and secretion in kidney and lung epithelial cells and demonstrate a critical role for prostasin in regulating epithelial monolayer function. We report that endogenous mouse prostasin is glycosylphosphatidylinositol (GPI) anchored to the cell surface and is constitutively secreted from the apical surface of kidney cortical collecting duct cells. Using site-directed mutagenesis, detergent phase separation, and RNA interference approaches, we show that prostasin secretion depends on GPI anchor cleavage by endogenous GPI-specific phospholipase D1 (Gpld1). Secretion of prostasin by kidney and lung epithelial cells, in contrast to prostate epithelium, does not depend on COOH-terminal processing at conserved Arg(322). Using stably transfected M-1 cells expressing wild-type, catalytically inactive, or chimeric transmembrane (not GPI)-anchored prostasins we establish that prostasin regulates transepithelial resistance, current, and paracellular permeability by GPI anchor- and protease activity-dependent mechanisms. These studies demonstrate a novel role for prostasin in regulating epithelial monolayer resistance and permeability in kidney epithelial cells and, furthermore, show specifically that prostasin is a critical regulator of transepithelial ion transport in M-1 cells. These functions depend on the GPI anchor as well as the peptidase activity of prostasin. These studies suggest that cell-specific Gpld1- or peptidase-dependent pathways for prostasin secretion may control prostasin functions in a tissue-specific manner.