RESUMO
Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM-integrin-cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, "stiff-like" spreading on soft is determined by a balance of a cell's contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.
Assuntos
Matriz Extracelular , Integrinas , Humanos , Adesão Celular , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Citoesqueleto/metabolismo , Transdução de SinaisRESUMO
TrackMate is an automated tracking software used to analyze bioimages and is distributed as a Fiji plugin. Here, we introduce a new version of TrackMate. TrackMate 7 is built to address the broad spectrum of modern challenges researchers face by integrating state-of-the-art segmentation algorithms into tracking pipelines. We illustrate qualitatively and quantitatively that these new capabilities function effectively across a wide range of bio-imaging experiments.
Assuntos
Algoritmos , Software , Processamento de Imagem Assistida por Computador/métodosRESUMO
During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias Pulmonares/secundário , Pulmão/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/fisiopatologia , Neoplasias da Mama/virologia , Permeabilidade Capilar , Proliferação de Células , Proteínas de Ligação a DNA , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemorragia/etiologia , Hemorragia/prevenção & controle , Humanos , Leucócitos/imunologia , Leucócitos/patologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/prevenção & controle , Depleção Linfocítica , Camundongos Transgênicos , Células Mieloides/imunologia , Células Mieloides/patologia , Proteínas de Neoplasias/genética , Neovascularização Patológica/etiologia , Neovascularização Patológica/prevenção & controle , Infiltração de Neutrófilos , Polyomavirus/patogenicidade , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sobrevida , Fatores de Transcrição , Carga TumoralRESUMO
Intravital microscopy represents a more physiologically relevant method for assessing therapeutic response. However, the movement into an in vivo setting brings with it several additional considerations, the primary being the context in which drug activity is assessed. Microenvironmental factors, such as hypoxia, pH, fibrosis, immune infiltration and stromal interactions have all been shown to have pronounced effects on drug activity in a more complex setting, which is often lost in simpler two- or three-dimensional assays. Here we present a practical guide for the application of intravital microscopy, looking at the available fluorescent reporters and their respective expression systems and analysis considerations. Moving in vivo, we also discuss the microscopy set up and methods available for overlaying microenvironmental context to the experimental readouts. This enables a smooth transition into applying higher fidelity intravital imaging to improve the drug discovery process.
Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia Intravital/métodos , Animais , Humanos , Retalhos Cirúrgicos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVß3 and α5ß1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration.
Assuntos
Anexina A6/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Qa-SNARE/metabolismo , Animais , Anexina A6/antagonistas & inibidores , Anexina A6/genética , Células CHO , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Movimento Celular , Células Cultivadas , Cricetulus , Endossomos/ultraestrutura , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Camundongos , Microscopia Confocal , Microscopia de Vídeo , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/genética , Interferência de RNA , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Imagem com Lapso de TempoRESUMO
Basic in vitro systems can be used to model and assess complex diseases, such as cancer. Recent advances in this field include the incorporation of multiple cell types and extracellular matrix proteins into three-dimensional (3D) models to recapitulate the structure, organization and functionality of live tissue in situ. Cells within such a 3D environment behave very differently from cells on two-dimensional (2D) substrates, as cell-matrix interactions trigger signalling pathways and cellular responses in 3D, which may not be observed in 2D. Thus, the use of 3D systems can be advantageous for the assessment of disease progression over 2D set-ups alone. Here, we highlight the current advantages and challenges of employing 3D systems in the study of cancer and provide an overview to guide the appropriate use of distinct models in cancer research.
Assuntos
Comunicação Celular , Modelos Biológicos , Neoplasias/patologia , Microambiente Tumoral , Animais , Técnicas de Cocultura , Humanos , Transdução de SinaisRESUMO
The progression of noninvasive ductal carcinoma in situ to invasive ductal carcinoma for patients with breast cancer results in a significantly poorer prognosis and is the precursor to metastatic disease. In this work, we have identified insulin-like growth factor-binding protein 2 (IGFBP2) as a potent adipocrine factor secreted by healthy breast adipocytes that acts as a barrier against invasive progression. In line with this role, adipocytes differentiated from patient-derived stromal cells were found to secrete IGFBP2, which significantly inhibited breast cancer invasion. This occurred through binding and sequestration of cancer-derived IGF-II. Moreover, depletion of IGF-II in invading cancer cells using small interfering RNAs or an IGF-II-neutralizing antibody ablated breast cancer invasion, highlighting the importance of IGF-II autocrine signaling for breast cancer invasive progression. Given the abundance of adipocytes in the healthy breast, this work exposes the important role they play in suppressing cancer progression and may help expound upon the link between increased mammary density and poorer prognosis.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Adipócitos , Anticorpos Neutralizantes , Mama , Fator de Crescimento Insulin-Like IIRESUMO
Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.
Assuntos
Técnicas Biossensoriais , Proteínas Proto-Oncogênicas c-akt , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Técnicas Biossensoriais/métodosRESUMO
Tissue architecture and function are orchestrated by an intricate repertoire of cellular adhesion and signalling receptors, and by the surrounding extracellular matrix (ECM). The essential role of cell-tissue interactions in guiding organogenesis was identified in experimental embryology studies over a century ago, and in 1954 Grobstein laid down the fundamental concept of ECM being the ultimate integrator of cellular systems. Long before the main cell adhesion receptors were identified, Abercrombie and colleagues proposed in 1971 that cell attachment to the ECM substratum was mediated through electron-dense plaques containing longitudinal cytoplasmic filaments that localise to areas of the ventral cell membrane that lie close to the substratum. In 1982, Bissell and co-workers proposed "the minimum required unit for expression of tissue specific functions", a model depicting a structure in which the nucleus links to the ECM via cytoskeletal filament bundles that connect to a hypothetical transmembrane ECM adhesion receptor.
Assuntos
Citoesqueleto , Matriz Extracelular , Proteínas de Transporte , Adesão Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Humanos , Integrinas/metabolismoRESUMO
Current evidence indicates that resistance to the tyrosine kinase-type cell surface receptor (HER2)-targeted therapies is frequently associated with HER3 and active signaling via HER2-HER3 dimers, particularly in the context of breast cancer. Thus, understanding the response to HER2-HER3 signaling and the regulation of the dimer is essential to decipher therapy relapse mechanisms. Here, we investigate a bidirectional relationship between HER2-HER3 signaling and a type-1 transmembrane sorting receptor, sortilin-related receptor (SorLA; SORL1). We demonstrate that heregulin-mediated signaling supports SorLA transcription downstream of the mitogen-activated protein kinase pathway. In addition, we demonstrate that SorLA interacts directly with HER3, forming a trimeric complex with HER2 and HER3 to attenuate lysosomal degradation of the dimer in a Ras-related protein Rab4-dependent manner. In line with a role for SorLA in supporting the stability of the HER2 and HER3 receptors, loss of SorLA compromised heregulin-induced cell proliferation and sensitized metastatic anti-HER2 therapy-resistant breast cancer cells to neratinib in cancer spheroids in vitro and in vivo in a zebrafish brain xenograft model.
Assuntos
Neoplasias da Mama/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Xenoenxertos , Humanos , Camundongos , Neuregulina-1/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Peixe-Zebra , Proteínas rab4 de Ligação ao GTP/genéticaRESUMO
Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.
Assuntos
Neoplasias da Mama/patologia , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose , Integrina beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Transporte Biológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Clatrina/genética , Dinaminas/genética , Feminino , Humanos , Integrina beta1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flowinduced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.
RESUMO
Microtubule-associated serine/threonine-protein kinase-like (MASTL) is a mitosis-accelerating kinase with emerging roles in cancer progression. However, possible cell cycle-independent mechanisms behind its oncogenicity remain ambiguous. Here, we identify MASTL as an activator of cell contractility and MRTF-A/SRF (myocardin-related transcription factor A/serum response factor) signaling. Depletion of MASTL increased cell spreading while reducing contractile actin stress fibers in normal and breast cancer cells and strongly impairing breast cancer cell motility and invasion. Transcriptome and proteome profiling revealed MASTL-regulated genes implicated in cell movement and actomyosin contraction, including Rho guanine nucleotide exchange factor 2 (GEF-H1, ARHGEF2) and MRTF-A target genes tropomyosin 4.2 (TPM4), vinculin (VCL), and nonmuscle myosin IIB (NM-2B, MYH10). Mechanistically, MASTL associated with MRTF-A and increased its nuclear retention and transcriptional activity. Importantly, MASTL kinase activity was not required for regulation of cell spreading or MRTF-A/SRF transcriptional activity. Taken together, we present a previously unknown kinase-independent role for MASTL as a regulator of cell adhesion, contractility, and MRTF-A/SRF activity.
Assuntos
Citoesqueleto de Actina/enzimologia , Adesão Celular/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica , Humanos , Integrinas/genética , Integrinas/metabolismo , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteoma/metabolismo , RNA Interferente Pequeno , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fibras de Estresse/genética , Fibras de Estresse/metabolismo , Transativadores/genética , Transcriptoma/genética , Tropomiosina/genética , Tropomiosina/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMO
Annexin A6 (AnxA6), a member of the calcium (Ca2+ ) and membrane binding annexins, is known to stabilize and establish the formation of multifactorial signaling complexes. At the plasma membrane, AnxA6 is a scaffold for protein kinase Cα (PKCα) and GTPase-activating protein p120GAP to promote downregulation of epidermal growth factor receptor (EGFR) and Ras/mitogen-activated protein kinase (MAPK) signaling. In human squamous A431 epithelial carcinoma cells, which overexpress EGFR, but lack endogenous AnxA6, restoration of AnxA6 expression (A431-A6) promotes PKCα-mediated threonine 654 (T654)-EGFR phosphorylation, which inhibits EGFR tyrosine kinase activity. This is associated with reduced A431-A6 cell growth, but also decreased migration and invasion in wound healing, matrigel, and organotypic matrices. Here, we show that A431-A6 cells display reduced EGFR activity in vivo, with xenograft analysis identifying increased pT654-EGFR levels, but reduced tyrosine EGFR phosphorylation compared to controls. In contrast, PKCα depletion in A431-A6 tumors is associated with strongly reduced pT654 EGFR levels, yet increased EGFR tyrosine phosphorylation and MAPK activity. Moreover, tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib) more effectively inhibit cell viability, clonogenic growth, and wound healing of A431-A6 cells compared to controls. Likewise, the ability of AnxA6 to inhibit A431 motility and invasiveness strongly improves TKI efficacy in matrigel invasion assays. This correlates with a greatly reduced invasion of the surrounding matrix of TKI-treated A431-A6 when cultured in 3D spheroids. Altogether, these findings implicate that elevated AnxA6 scaffold levels contribute to improve TKI-mediated inhibition of growth and migration, but also invasive properties in EGFR overexpressing human squamous epithelial carcinoma.
Assuntos
Anexina A6/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Anexina A6/genética , Apoptose , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Camundongos , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Fosforilação , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance and wound healing. In order for cells to migrate, they must interact with their environment using adhesion receptors, such as integrins, and form specialized adhesion complexes that mediate responses to different extracellular cues. In this review, we discuss the role of integrin adhesion complexes (IACs) in cell migration, highlighting the layers of regulation that are involved, including intracellular signalling cascades, mechanosensing and reciprocal feedback to the extracellular environment. We also discuss the role of IACs in extracellular matrix remodeling and how they impact upon cell migration.
Assuntos
Movimento Celular/fisiologia , Junções Célula-Matriz/metabolismo , Actinas/metabolismo , Animais , Matriz Extracelular/metabolismo , Humanos , Integrinas/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Chronic inflammation is a hallmark of obesity and is linked to the development of numerous diseases. The activation of toll-like receptor 4 (TLR4) by long-chain saturated fatty acids (lcSFAs) is an important process in understanding how obesity initiates inflammation. While experimental evidence supports an important role for TLR4 in obesity-induced inflammation in vivo, via a mechanism thought to involve direct binding to and activation of TLR4 by lcSFAs, several lines of evidence argue against lcSFAs being direct TLR4 agonists. Using multiple orthogonal approaches, we herein provide evidence that while loss-of-function models confirm that TLR4 does, indeed, regulate lcSFA-induced inflammation, TLR4 is not a receptor for lcSFAs. Rather, we show that TLR4-dependent priming alters cellular metabolism, gene expression, lipid metabolic pathways, and membrane lipid composition, changes that are necessary for lcSFA-induced inflammation. These results reconcile previous discordant observations and challenge the prevailing view of TLR4's role in initiating obesity-induced inflammation.
Assuntos
Inflamação/metabolismo , Macrófagos/metabolismo , Obesidade/metabolismo , Palmitatos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Humanos , Inflamação/etiologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Obesidade/complicações , Transdução de SinaisRESUMO
Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Animais , Benzamidas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Hipóxia , Microscopia Intravital/métodos , Camundongos , Camundongos Endogâmicos BALB C , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Nanopartículas/química , Nitroimidazóis/farmacologia , Nitroimidazóis/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Mostardas de Fosforamida/farmacologia , Mostardas de Fosforamida/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas , Transdução de Sinais/efeitos dos fármacos , Transplante Heterólogo , Microambiente TumoralRESUMO
MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer. Global phosphoproteomic analysis of immortalised breast MCF10A cells engineered to overexpressed MASTL revealed disruption to desmosomes, actin cytoskeleton, PI3K/AKT/mTOR and p38 stress kinase signalling pathways. Notably, these pathways were also disrupted in patient samples that overexpress MASTL. In MCF10A cells, these alterations corresponded with a loss of contact inhibition and partial epithelial-mesenchymal transition, which disrupted migration and allowed cells to proliferate uncontrollably in 3D culture. Furthermore, MASTL overexpression increased aberrant mitotic divisions resulting in increased micronuclei formation. Mathematical modelling indicated that this delay was due to continued inhibition of PP2A-B55, which delayed timely mitotic exit. This corresponded with an increase in DNA damage and delayed transit through interphase. There were no significant alterations to replication kinetics upon MASTL overexpression, however, inhibition of p38 kinase rescued the interphase delay, suggesting the delay was a G2 DNA damage checkpoint response. Importantly, knockdown of MASTL, reduced cell proliferation, prevented invasion and metastasis of MDA-MB-231 breast cancer cells both in vitro and in vivo, indicating the potential of future therapies that target MASTL. Taken together, these results suggest that MASTL overexpression contributes to chromosome instability and metastasis, thereby decreasing breast cancer patient survival.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Instabilidade Cromossômica/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Serina-Treonina Quinases/genética , Citoesqueleto de Actina/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Dano ao DNA/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
Annexin A6 (AnxA6) belongs to a highly conserved protein family characterized by their calcium (Ca2+)-dependent binding to phospholipids. Over the years, immunohistochemistry, subcellular fractionations, and live cell microscopy established that AnxA6 is predominantly found at the plasma membrane and endosomal compartments. In these locations, AnxA6 acts as a multifunctional scaffold protein, recruiting signaling proteins, modulating cholesterol and membrane transport and influencing actin dynamics. These activities enable AnxA6 to contribute to the formation of multifactorial protein complexes and membrane domains relevant in signal transduction, cholesterol homeostasis and endo-/exocytic membrane transport. Hence, AnxA6 has been implicated in many biological processes, including cell proliferation, survival, differentiation, inflammation, but also membrane repair and viral infection. More recently, we and others identified roles for AnxA6 in cancer cell migration and invasion. This review will discuss how the multiple scaffold functions may enable AnxA6 to modulate migratory cell behavior in health and disease.
Assuntos
Anexina A6/genética , Adesão Celular/genética , Invasividade Neoplásica/genética , Neoplasias/genética , Anexina A6/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Invasividade Neoplásica/patologia , Metástase Neoplásica , Neoplasias/patologia , Fosfolipídeos/genética , Ligação Proteica , Transdução de SinaisRESUMO
Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.