RESUMO
Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.
Assuntos
Neoplasias Encefálicas/imunologia , Epigênese Genética , Glioblastoma/imunologia , Evasão da Resposta Imune/imunologia , Células Mieloides/imunologia , Células-Tronco Neoplásicas/imunologia , Microambiente Tumoral/imunologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Metilação de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Mieloides/metabolismo , Células Mieloides/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cell adhesion to macromolecules in the microenvironment is essential for the development and maintenance of tissues, and its dysregulation can lead to a range of disease states, including inflammation, fibrosis, and cancer. The biomechanical and biochemical mechanisms that mediate cell adhesion rely on signaling by a range of effector proteins, including kinases and associated scaffolding proteins. The intracellular trafficking of these must be tightly controlled in space and time to enable effective cell adhesion and microenvironmental sensing and to integrate cell adhesion with, and compartmentalize it from, other cellular processes, such as gene transcription, protein degradation, and cell division. Delivery of adhesion receptors and signaling proteins from the plasma membrane to unanticipated subcellular locales is revealing novel biological functions. Here, we review the expected and unexpected trafficking, and sites of activity, of adhesion and growth factor receptors and intracellular kinase partners as we begin to appreciate the complexity and diversity of their spatial regulation.
Assuntos
Adesão Celular/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Transporte Proteico/genética , Receptores de Fatores de Crescimento/genética , Membrana Celular/genética , Núcleo Celular/genética , Endossomos/genética , Humanos , Fosfotransferases/genéticaRESUMO
Focal adhesion kinase (FAK) promotes anti-tumor immune evasion. Specifically, the kinase activity of nuclear-targeted FAK in squamous cell carcinoma (SCC) cells drives exhaustion of CD8(+) T cells and recruitment of regulatory T cells (Tregs) in the tumor microenvironment by regulating chemokine/cytokine and ligand-receptor networks, including via transcription of Ccl5, which is crucial. These changes inhibit antigen-primed cytotoxic CD8(+) T cell activity, permitting growth of FAK-expressing tumors. Mechanistically, nuclear FAK is associated with chromatin and exists in complex with transcription factors and their upstream regulators that control Ccl5 expression. Furthermore, FAK's immuno-modulatory nuclear activities may be specific to cancerous squamous epithelial cells, as normal keratinocytes do not have nuclear FAK. Finally, we show that a small-molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, also drives depletion of Tregs and promotes a CD8(+) T cell-mediated anti-tumor response. Therefore, FAK inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic opportunities.
Assuntos
Carcinoma de Células Escamosas/imunologia , Quimiocina CCL5/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias Cutâneas/imunologia , Linfócitos T Reguladores/imunologia , Evasão Tumoral , Aminopiridinas/administração & dosagem , Animais , Carcinoma de Células Escamosas/metabolismo , Quimiocina CCL5/imunologia , Modelos Animais de Doenças , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Nus , Neoplasias Cutâneas/metabolismo , Transcrição GênicaRESUMO
Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.
Assuntos
Proteínas Aviárias/química , Proteína-Tirosina Quinases de Adesão Focal/química , Membranas/química , Multimerização Proteica , Animais , Proteínas Aviárias/metabolismo , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Humanos , Membranas/enzimologia , Relação Estrutura-AtividadeRESUMO
OBJECTIVE: Immunotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC) has shown limited efficacy. Poor CD8 T-cell infiltration, low neoantigen load and a highly immunosuppressive tumour microenvironment contribute to this lack of response. Here, we aimed to further investigate the immunoregulatory function of focal adhesion kinase (FAK) in PDAC, with specific emphasis on regulation of the type-II interferon response that is critical in promoting T-cell tumour recognition and effective immunosurveillance. DESIGN: We combined CRISPR, proteogenomics and transcriptomics with mechanistic experiments using a KrasG12Dp53R172H mouse model of pancreatic cancer and validated findings using proteomic analysis of human patient-derived PDAC cell lines and analysis of publicly available human PDAC transcriptomics datasets. RESULTS: Loss of PDAC cell-intrinsic FAK signalling promotes expression of the immunoproteasome and Major Histocompatibility Complex class-I (MHC-I), resulting in increased antigen diversity and antigen presentation by FAK-/- PDAC cells. Regulation of the immunoproteasome by FAK is a critical determinant of this response, optimising the physicochemical properties of the peptide repertoire for high affinity binding to MHC-I. Expression of these pathways can be further amplified in a STAT1-dependent manner via co-depletion of FAK and STAT3, resulting in extensive infiltration of tumour-reactive CD8 T-cells and further restraint of tumour growth. FAK-dependent regulation of antigen processing and presentation is conserved between mouse and human PDAC, but is lost in cells/tumours with an extreme squamous phenotype. CONCLUSION: Therapies aimed at FAK degradation may unlock additional therapeutic benefit for the treatment of PDAC through increasing antigen diversity and promoting antigen presentation.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Humanos , Animais , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Apresentação de Antígeno , Evasão da Resposta Imune , Proteômica , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Microambiente Tumoral , Linhagem Celular TumoralRESUMO
Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell-matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease.
Assuntos
Neoplasias Ósseas , Endoglina/metabolismo , Sarcoma de Ewing , Neoplasias Ósseas/genética , Endoglina/genética , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Proteômica , Receptores de Fatores de Crescimento , Sarcoma de Ewing/patologia , Transdução de SinaisRESUMO
Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Angiopoietina-1/biossíntese , Angiopoietina-1/genética , Linhagem Celular , Cromatina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Humanos , Cadeias alfa de Integrinas/biossíntese , Cadeias alfa de Integrinas/genética , Cadeias beta de Integrinas/biossíntese , Cadeias beta de Integrinas/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/biossíntese , Fator de Crescimento Transformador beta3/genéticaRESUMO
Reverse-phase protein array (RPPA) technology is a high-throughput antibody- and microarray-based approach for the rapid profiling of levels of proteins and protein posttranslational modifications in biological specimens. The technology consumes small amounts of samples, can sensitively detect low-abundance proteins and posttranslational modifications, enables measurements of multiple signaling pathways in parallel, has the capacity to analyze large sample numbers, and offers robust interexperimental reproducibility. These features of RPPA experiments have motivated and enabled the use of RPPA technology in various biomedical, translational, and clinical applications, including the delineation of molecular mechanisms of disease, profiling of druggable signaling pathway activation, and search for new prognostic markers. Owing to the complexity of many of these applications, such as developing multiplex protein assays for diagnostic laboratories or integrating posttranslational modification-level data using large-scale proteogenomic approaches, robust and well-validated data are essential. There are many distinct components of an RPPA workflow, and numerous possible technical setups and analysis parameter options exist. The differences between RPPA platform setups around the world offer opportunities to assess and minimize interplatform variation. Crossplatform validation may also aid in the evaluation of robust, platform-independent protein markers of disease and response to therapy.
Assuntos
Análise Serial de Proteínas , Proteômica , Biomarcadores/análise , Humanos , Análise Serial de Proteínas/normas , Proteínas/química , Reprodutibilidade dos TestesRESUMO
We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5ß1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-ß1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-ß1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5ß1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking.
Assuntos
Anticorpos Monoclonais Murinos/química , Epitopos/química , Fibronectinas/química , Integrina alfa5beta1/química , Modelos Moleculares , Oligopeptídeos/química , Regulação Alostérica/imunologia , Anticorpos Monoclonais Murinos/imunologia , Epitopos/genética , Epitopos/imunologia , Fibronectinas/genética , Fibronectinas/imunologia , Humanos , Integrina alfa5beta1/genética , Integrina alfa5beta1/imunologia , Células Jurkat , Oligopeptídeos/genética , Oligopeptídeos/imunologiaRESUMO
Glomerular disease often features altered histologic patterns of extracellular matrix (ECM). Despite this, the potential complexities of the glomerular ECM in both health and disease are poorly understood. To explore whether genetic background and sex determine glomerular ECM composition, we investigated two mouse strains, FVB and B6, using RNA microarrays of isolated glomeruli combined with proteomic glomerular ECM analyses. These studies, undertaken in healthy young adult animals, revealed unique strain- and sex-dependent glomerular ECM signatures, which correlated with variations in levels of albuminuria and known predisposition to progressive nephropathy. Among the variation, we observed changes in netrin 4, fibroblast growth factor 2, tenascin C, collagen 1, meprin 1-α, and meprin 1-ß. Differences in protein abundance were validated by quantitative immunohistochemistry and Western blot analysis, and the collective differences were not explained by mutations in known ECM or glomerular disease genes. Within the distinct signatures, we discovered a core set of structural ECM proteins that form multiple protein-protein interactions and are conserved from mouse to man. Furthermore, we found striking ultrastructural changes in glomerular basement membranes in FVB mice. Pathway analysis of merged transcriptomic and proteomic datasets identified potential ECM regulatory pathways involving inhibition of matrix metalloproteases, liver X receptor/retinoid X receptor, nuclear factor erythroid 2-related factor 2, notch, and cyclin-dependent kinase 5. These pathways may therefore alter ECM and confer susceptibility to disease.
Assuntos
Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Nefropatias/genética , Glomérulos Renais/metabolismo , Albuminúria/genética , Albuminúria/metabolismo , Animais , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Matriz Extracelular/ultraestrutura , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Predisposição Genética para Doença , Membrana Basal Glomerular/ultraestrutura , Nefropatias/metabolismo , Receptores X do Fígado , Masculino , Metaloproteinases da Matriz/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos , Fator 2 Relacionado a NF-E2/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Netrinas , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Nucleares Órfãos/metabolismo , RNA/análise , Fatores Sexuais , Transdução de Sinais , Tenascina/genética , Tenascina/metabolismoRESUMO
Cell migration makes a fundamental contribution to both normal physiology and disease pathogenesis. Integrin engagement with extracellular ligands spatially controls, via the cyclical activation and deactivation of the small GTPase Rac1, the dynamic membrane protrusion and cytoskeletal reorganization events that are required for directional migration. Although the pathways that control integrin-mediated Rac1 activation are reasonably well defined, the mechanisms that are responsible for switching off activity are poorly understood. Here, proteomic analysis of activated integrin-associated complexes suggests filamin-A and IQ-motif-containing GTPase-activating protein 1 (IQGAP1) as candidates that link ß1 integrin to Rac1. siRNA-mediated knockdown of either filamin-A or IQGAP1 induced high, dysregulated Rac1 activity during cell spreading on fibronectin. Using immunoprecipitation and immunocytochemistry, filamin-A and IQGAP1 were shown to be part of a complex that is recruited to active ß1 integrin. Mass spectrometric analysis of individual filamin-A, IQGAP1 and Rac1 pull-downs and biochemical analysis, identified RacGAP1 as a novel IQGAP1 binding partner. Further immunoprecipitation and immunocytochemistry analyses demonstrated that RacGAP1 is recruited to IQGAP1 and active ß1 integrin, and that suppression of RacGAP1 expression triggered elevated Rac1 activity during spreading on fibronectin. Consistent with these findings, reduced expression of filamin-A, IQGAP1 or RacGAP1 triggered unconstrained membrane protrusion and disrupted directional cell migration on fibrillar extracellular matrices. These findings suggest a model whereby integrin engagement, followed by filamin-A, IQGAP1 and RacGAP1 recruitment, deactivates Rac1 to constrain its activity spatially and thereby coordinate directional cell migration.
Assuntos
Integrina beta1/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Movimento Celular , Filaminas , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteômica , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Glomérulos Renais/metabolismo , Proteoma/química , Adulto , Colágeno Tipo VI/química , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/isolamento & purificação , Ontologia Genética , Humanos , Glomérulos Renais/química , Glomérulos Renais/citologia , Lipocalinas/química , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Proteoma/genéticaRESUMO
The glomerular basement membrane (GBM) is a specialized extracellular matrix (ECM) compartment within the glomerulus that contains tissue-restricted isoforms of collagen IV and laminin. It is integral to the capillary wall and therefore, functionally linked to glomerular filtration. Although the composition of the GBM has been investigated with global and candidate-based approaches, the relative contributions of glomerular cell types to the production of ECM are not well understood. To characterize specific cellular contributions to the GBM, we used mass spectrometry-based proteomics to analyze ECM isolated from podocytes and glomerular endothelial cells in vitro. These analyses identified cell type-specific differences in ECM composition, indicating distinct contributions to glomerular ECM assembly. Coculture of podocytes and endothelial cells resulted in an altered composition and organization of ECM compared with monoculture ECMs, and electron microscopy revealed basement membrane-like ECM deposition between cocultured cells, suggesting the involvement of cell-cell cross-talk in the production of glomerular ECM. Notably, compared with monoculture ECM proteomes, the coculture ECM proteome better resembled a tissue-derived glomerular ECM dataset, indicating its relevance to GBM in vivo. Protein network analyses revealed a common core of 35 highly connected structural ECM proteins that may be important for glomerular ECM assembly. Overall, these findings show the complexity of the glomerular ECM and suggest that both ECM composition and organization are context-dependent.
Assuntos
Proteínas da Matriz Extracelular/fisiologia , Matriz Extracelular/fisiologia , Glomérulos Renais/fisiologia , Receptor Cross-Talk/fisiologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Proteínas da Matriz Extracelular/biossíntese , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Fenótipo , Podócitos/fisiologia , Mapas de Interação de ProteínasRESUMO
Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.
Assuntos
Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Matriz Extracelular/metabolismo , Proteômica/métodos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Análise por Conglomerados , Meios de Cultivo Condicionados/química , Células-Tronco Embrionárias/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células Alimentadoras , Fibrilina-1 , Fibrilinas , Fibroblastos/citologia , Fibronectinas/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Integrinas/metabolismo , Cariotipagem , Camundongos , Proteínas dos Microfilamentos/metabolismoRESUMO
HER2 (human epidermal growth factor receptor 2)-targeted therapy in breast cancer is one of the earliest and arguably most successful examples of the modern class of targeted drugs. Initially identified in the 1980s, the observation that HER2 acts as an independent predictor of poor prognosis in the 20% of breast cancer cases carrying a gene amplification or protein overexpression cemented its place at the forefront of research in this field. The outlook for patients with HER2-positive breast cancer has been revolutionized by the introduction of HER2-targeted agents, such as trastuzumab and lapatinib, yet resistance is frequently encountered and multiple different resistance mechanisms have been identified. We have explored resistance to a novel pan-HER inhibitor, AZD8931, and we examine mechanisms of resistance common to trastuzumab, lapatinib and AZD8931, and discuss the current problems associated with translating the wealth of pre-clinical data into clinical benefit.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Lapatinib , Quinazolinas/uso terapêutico , TrastuzumabRESUMO
Kindler syndrome (KS) is a rare genodermatosis resulting from loss-of-function mutations in FERMT1, the gene that encodes Kindlin-1. KS patients have a high propensity to develop aggressive and metastatic cutaneous squamous cell carcinoma (cSCC). Here we show in non-KS-associated patients that elevation of FERMT1 expression is increased in actinic keratoses compared to normal skin, with a further increase in cSCC supporting a pro-tumorigenic role in this population. In contrast, we show that loss of Kindlin-1 leads to increased SCC tumor growth in vivo and in 3D spheroids, which was associated with the development of a hypoxic tumor environment and increased glycolysis. The metalloproteinase Mmp13 was upregulated in Kindlin-1-depleted tumors, and increased expression of MMP13 was responsible for driving increased invasion of the Kindlin-1-depleted SCC cells. These results provide evidence that Kindlin-1 loss in SCC can promote invasion through the upregulation of MMP13, and offer novel insights into how Kindlin-1 loss leads to the development of a hypoxic environment that is permissive for tumor growth.
RESUMO
Glioblastoma (GBM) is driven by malignant neural stem-like cells that display extensive heterogeneity and phenotypic plasticity, which drive tumor progression and therapeutic resistance. Here, we show that the extracellular matrix-cell adhesion protein integrin-linked kinase (ILK) stimulates phenotypic plasticity and mesenchymal-like, invasive behavior in a murine GBM stem cell model. ILK is required for the interconversion of GBM stem cells between malignancy-associated glial-like states, and its loss produces cells that are unresponsive to multiple cell state transition cues. We further show that an ILK/STAT3 signaling pathway controls the plasticity that enables transition of GBM stem cells to an astrocyte-like state in vitro and in vivo. Finally, we find that ILK expression correlates with expression of STAT3-regulated proteins and protein signatures describing astrocyte-like and mesenchymal states in patient tumors. This work identifies ILK as a pivotal regulator of multiple malignancy-associated GBM phenotypes, including phenotypic plasticity and mesenchymal state.
RESUMO
The cell microenvironment has a profound influence on the behaviour, growth and survival of cells. The extracellular matrix (ECM) provides not only mechanical and structural support to cells and tissues but also binds soluble ligands and transmembrane receptors to provide spatial coordination of signalling processes. The ability of cells to sense the chemical, mechanical and topographical features of the ECM enables them to integrate complex, multiparametric information into a coherent response to the surrounding microenvironment. Consequently, dysregulation or mutation of ECM components results in a broad range of pathological conditions. Characterization of the composition of ECM derived from various cells has begun to reveal insights into ECM structure and function, and mechanisms of disease. Proteomic methodologies permit the global analysis of subcellular systems, but extracellular and transmembrane proteins present analytical difficulties to proteomic strategies owing to the particular biochemical properties of these molecules. Here, we review advances in proteomic approaches that have been applied to furthering our understanding of the ECM microenvironment. We survey recent studies that have addressed challenges in the analysis of ECM and discuss major outcomes in the context of health and disease. In addition, we summarize efforts to progress towards a systems-level understanding of ECM biology.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Proteômica , Animais , Adesão Celular , Humanos , Espectrometria de Massas , Modelos BiológicosRESUMO
Introduction: The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development. Methods: Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry. Results: We identified 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue, and we defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively. Discussion: These data chart extensive remodelling of the lung extracellular niche and reveal tumour matrisome signatures in human NSCLC.
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Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes, play key roles in fundamental cellular processes, including the sensing and transduction of mechanical cues. Here, we investigate systems-level changes in the integrin adhesome in patient-derived cutaneous squamous cell carcinoma cells and identify the actin regulatory protein Mena as a key node in the adhesion complex network. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We uncover an unexpected role for Mena at the nuclear membrane, where it controls nuclear architecture, chromatin repositioning and gene expression. Our findings identify an adhesion protein that regulates gene transcription via direct signalling across the nuclear envelope.