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Numerous natural substances have anti-cancer properties. Especially indigenous people use aqueous plant extracts for tea or ointments including Dioscorea sansibarensis Pax to treat various diseases. The aim of this study was to evaluate the cytotoxic and radiosensitizing potential of aqueous extracts from Dioscorea sansibarensis Pax collected from Kenya in a panel of HPV-negative and -positive head and neck squamous cell carcinoma (HNSCC) cells grown in three-dimensional laminin-rich extracellular matrix (3D lrECM). The results show cytotoxicity, radiosensitization and increased levels of residual double strand breaks (DBS) by Dioscorea sansibarensis Pax extracts in HPV-negative and -positive HNSCC models in a concentration- and cell model-dependent manner. Application of ROS scavengers indicated an association between ROS-induced DSB and radiosensitization through Dioscorea sansibarensis Pax pretreatment. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based characterization of Dioscorea sansibarensis Pax identified the main components of the extract including camptothecin. Overall, Dioscorea sansibarensis Pax aqueous extracts alone and in combination with X-ray irradiation showed effective anticancer properties, which are worthy of further mechanistic investigation.
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Glioblastomas (GBM) are the most common primary brain tumors in adults and associated with poor clinical outcomes due to therapy resistances and destructive growth. Interactions of cancer cells with the extracellular matrix (ECM) play a pivotal role in therapy resistances and tumor progression. In this study, we investigate the functional dependencies between the discoidin domain receptor 1 (DDR1) and the integrin family of cell adhesion molecules for the radioresponse of human glioblastoma cells. By means of an RNA interference screen on DDR1 and all known integrin subunits, we identified co-targeting of DDR1/integrin ß3 to most efficiently reduce clonogenicity, enhance cellular radiosensitivity and diminish repair of DNA double strand breaks (DSB). Simultaneous pharmacological inhibition of DDR1 with DDR1-IN-1 and of integrins αVß3/αVß5 with cilengitide resulted in confirmatory data in a panel of 2D grown glioblastoma cultures and 3D gliospheres. Mechanistically, we found that key DNA repair proteins ATM and DNA-PK are altered upon DDR1/integrin αVß3/integrin αVß5 inhibition, suggesting a link to DNA repair mechanisms. In sum, the radioresistance of human glioblastoma cells can effectively be declined by co-deactivation of DDR1, integrin αVß3 and integrin αVß5.
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BACKGROUND: Glioblastoma (GBM) is a fast-growing primary brain tumor characterized by high invasiveness and resistance. This results in poor patient survival. Resistance is caused by many factors, including cell-extracellular matrix (ECM) interactions. Here, we addressed the role of adhesion protein integrin α2, which we identified in a high-throughput screen for novel potential targets in GBM cells treated with standard therapy consisting of temozolomide (TMZ) and radiation. METHODS: In our study, we used a range of primary/stem-like and established GBM cell models in vitro and in vivo. To identify regulatory mechanisms, we employed high-throughput kinome profiling, Western blotting, immunofluorescence staining, reporter, and activity assays. RESULTS: Our data showed that integrin α2 is overexpressed in GBM compared to normal brain and, that its deletion causes radiochemosensitization. Similarly, invasion and adhesion were significantly reduced in TMZ-irradiated GBM cell models. Furthermore, we found that integrin α2-knockdown impairs the proliferation of GBM cells without affecting DNA damage repair. At the mechanistic level, we found that integrin α2 affects the activity of activating transcription factor 1 (ATF1) and modulates the expression of extracellular signal-regulated kinase 1 (ERK1) regulated by extracellular signals. Finally, we demonstrated that integrin α2-deficiency inhibits tumor growth and thereby prolongs the survival of mice with orthotopically growing GBM xenografts. CONCLUSIONS: Taken together our data suggest that integrin α2 may be a promising target to overcome GBM resistance to radio- and chemotherapy. Thus, it would be worth evaluating how efficient and safe the adjuvant use of integrin α2 inhibitors is to standard radio(chemo)therapy in GBM.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Animales , Ratones , Glioblastoma/patología , Integrina alfa2/uso terapéutico , Resistencia a Antineoplásicos , Neoplasias Encefálicas/patología , Temozolomida/uso terapéutico , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Antineoplásicos Alquilantes/uso terapéuticoRESUMEN
Glioblastoma is a devastating malignant disease with poor patient overall survival. Strong invasiveness and resistance to radiochemotherapy have challenged the identification of molecular targets that can finally improve treatment outcomes. This study evaluates the influence of all six known p21-activated kinase (PAK) protein family members on the invasion capacity and radio-response of glioblastoma cells by employing a siRNA-based screen. In a panel of human glioblastoma cell models, we identified PAK4 as the main PAK isoform regulating invasion and clonogenic survival upon irradiation and demonstrated the radiosensitizing potential of PAK4 inhibition. Mechanistically, we show that PAK4 depletion and pharmacological inhibition enhanced the number of irradiation-induced DNA double-strand breaks and reduced the expression levels of various DNA repair proteins. In conclusion, our data suggest PAK4 as a putative target for radiosensitization and impairing DNA repair in glioblastoma, deserving further scrutiny in extended combinatorial treatment testing.
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Glioblastoma , Quinasas p21 Activadas , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de la radiación , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Humanos , ARN Interferente Pequeño , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Cell adhesion to extracellular matrix proteins mediates resistance to radio- and chemotherapy by activating integrin signaling. In addition, mutual and cooperative interactions between integrin and growth factor receptor signaling contribute to the cellular radiation response. Here, we investigate to which extend the crosstalk between ß1 integrins and growth factor receptor signaling determines the cellular radiation response of fibroblasts by assessing clonogenic survival and cell cycling. By utilizing growth factor signaling competent and either ß1 integrin wildtype GD25ß1A fibroblasts or ß1 integrin mutant, signaling incompetent GD25ß1B fibroblasts, we show basal clonogenic survival to depend on growth factor receptor but not integrin signaling. Our data further suggest the cooperation between ß1 integrins and growth factor receptors to be critical for enhancing the radiation-induced G2/M cell cycle block leading to improved clonogenic radiation survival. By pharmacological inhibition of EGFR and PI3K, we additionally show that the essential contribution of EGFR signaling to radiogenic G2/M cell cycle arrest depends on the co-activation of the ß1 integrin signaling axis, but occurs independent of PI3K. Taken together, elucidation of the signaling circuitry underlying the EGFR/ß1 integrin crosstalk may support the development of advanced molecular targeted therapies for radiation oncology.
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Integrina beta1 , Transducción de Señal , Animales , Ciclo Celular , Fibroblastos/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Receptores de Factores de Crecimiento/metabolismoRESUMEN
Glioblastoma is a tumor type of unmet need despite the development of multimodal treatment strategies. The main factors contributing to the poor prognosis of glioblastoma patients are diverse genetic and epigenetic changes driving glioblastoma persistence and recurrence. Complemented are these factors by extracellular cues mediated through cell surface receptors, which further aid in fostering pro-invasion and pro-survival signaling contributing to glioblastoma therapy resistance. The underlying mechanisms conferring this therapy resistance are poorly understood. Here, we show that the cytoskeleton regulator Lamellipodin (Lpd) mediates invasiveness, proliferation and radiosensitivity of glioblastoma cells. Phosphoproteome analysis identified the epidermal growth factor receptor (EGFR) signaling axis commonly hyperactive in glioblastoma to depend on Lpd. Mechanistically, EGFR signaling together with an interaction between Lpd and the Rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR) jointly regulate glioblastoma radiosensitivity. Collectively, our findings demonstrate an essential function of Lpd in the radiation response and invasiveness of glioblastoma cells. Thus, we uncover a novel Lpd-driven resistance mechanism, which adds an additional critical facet to the complex glioblastoma resistance network.
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The intermediate filament synemin has been previously identified as novel regulator of cancer cell therapy resistance and DNA double strand break (DSB) repair. c-Abl tyrosine kinase is involved in both of these processes. Using PamGene technology, we performed a broad-spectrum kinase activity profiling in three-dimensionally, extracellular matrix grown head and neck cancer cell cultures. Upon synemin silencing, we identified 86 deactivated tyrosine kinases, including c-Abl, in irradiated HNSCC cells. Upon irradiation and synemin inhibition, c-Abl hyperphosphorylation on tyrosine (Y) 412 and threonine (T) 735 was significantly reduced, prompting us to hypothesize that c-Abl tyrosine kinase is an important signaling component of the synemin-mediated radioresistance pathway. Simultaneous targeting of synemin and c-Abl resulted in similar radiosensitization and DSB repair compared with single synemin depletion, suggesting synemin as an upstream regulator of c-Abl. Immunoprecipitation assays revealed a protein complex formation between synemin and c-Abl pre- and post-irradiation. Upon pharmacological inhibition of ATM, synemin/c-Abl protein-protein interactions were disrupted implying synemin function to depend on ATM kinase activity. Moreover, deletion of the SH2 domain of c-Abl demonstrated a decrease in interaction, indicating the dependency of the protein-protein interaction on this domain. Mechanistically, radiosensitization upon synemin knockdown seems to be associated with an impairment of DNA repair via regulation of non-homologous end joining independent of c-Abl function. Our data generated in more physiological 3D cancer cell culture models suggest c-Abl as further key determinant of radioresistance downstream of synemin.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , Reparación del ADN , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Filamentos Intermediarios/genética , Proteínas Proto-Oncogénicas c-abl/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Roturas del ADN de Doble Cadena , ADN de Neoplasias/metabolismo , Embrión no Mamífero , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Proteínas de Filamentos Intermediarios/antagonistas & inhibidores , Proteínas de Filamentos Intermediarios/metabolismo , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-abl/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Tolerancia a Radiación/genética , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Rayos X , Pez CebraRESUMEN
The treatment resistance of cancer cells is a multifaceted process in which DNA repair emerged as a potential therapeutic target. DNA repair is predominantly conducted by nuclear events; yet, how extra-nuclear cues impact the DNA damage response is largely unknown. Here, using a high-throughput RNAi-based screen in three-dimensionally-grown cell cultures of head and neck squamous cell carcinoma (HNSCC), we identified novel focal adhesion proteins controlling DNA repair, including the intermediate filament protein, synemin. We demonstrate that synemin critically regulates the DNA damage response by non-homologous end joining repair. Mechanistically, synemin forms a protein complex with DNA-PKcs through its C-terminal tail domain for determining DNA repair processes upstream of this enzyme in an ATM-dependent manner. Our study discovers a critical function of the intermediate filament protein, synemin in the DNA damage response, fundamentally supporting the concept of cytoarchitectural elements as co-regulators of nuclear events.
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Therapy resistance of tumor cells is a major obstacle for efficient anticancer treatment approaches and has been attributed to tumor heterogeneity as well as genetic and epigenetic changes. Accumulating evidence demonstrates that tumor cell adhesion to the extracellular matrix acts as an additional essential factor conferring tumor cell resistance to both radio- and chemotherapeutic intervention. Our recent study demonstrates that DDR1 (discoidin domain receptor tyrosine kinase 1) elicits therapy resistance of glioblastoma multiforme (GBM) stem-like and bulk cells through its adhesion to extracellular matrix and the subsequent modulation of macroautophagy/autophagy. Mechanistically, DDR1 associates with a YWHA/14-3-3-BECN1-AKT1 multiprotein complex favoring pro-survival/anti-autophagic and resistance-mediating AKT-MTOR signaling. In turn, inhibition of DDR1 sensitizes glioblastoma cells to radio- and chemotherapy by inducing autophagy. Collectively, our study suggests that DDR1 may be a potential target for sensitizing glioblastoma cells to combination therapies through its efficient induction of autophagic cell death. Abbreviations: AKT1: AKT serine/threonine kinase 1; ATG14: autophagy related 14; BECN1: Beclin 1; DDR1: discoidin domain receptor tyrosine kinase 1; ECM: extracellular matrix; GBM: glioblastoma multiforme; MTOR: mechanistic target of rapamycin kinase; PDGFR: platelet derived growth factor receptor; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RPTOR: regulatory associated protein of MTOR complex 1; RICTOR: RPTOR independent companion of MTOR complex 2.
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Autofagia , Glioblastoma , Beclina-1 , Receptor con Dominio Discoidina 1 , Humanos , Transducción de SeñalRESUMEN
Glioblastoma (GBM) is highly refractory to therapy and associated with poor clinical outcome. Here, we reveal a critical function of the promitotic and adhesion-mediating discoidin domain receptor 1 (DDR1) in modulating GBM therapy resistance. In GBM cultures and clinical samples, we show a DDR1 and GBM stem cell marker co-expression that correlates with patient outcome. We demonstrate that inhibition of DDR1 in combination with radiochemotherapy with temozolomide in GBM models enhances sensitivity and prolongs survival superior to conventional therapy. We identify a 14-3-3-Beclin-1-Akt1 protein complex assembling with DDR1 to be required for prosurvival Akt and mTOR signaling and regulation of autophagy-associated therapy sensitivity. Our results uncover a mechanism driven by DDR1 that controls GBM therapy resistance and provide a rationale target for the development of therapy-sensitizing agents.
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Proteínas 14-3-3/metabolismo , Beclina-1/metabolismo , Neoplasias Encefálicas/metabolismo , Receptor con Dominio Discoidina 1/metabolismo , Glioblastoma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Autofagia , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Línea Celular , Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos , Femenino , Glioblastoma/tratamiento farmacológico , Glioblastoma/radioterapia , Humanos , Masculino , Ratones , Ratones Desnudos , Pronóstico , Tolerancia a Radiación , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.
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Esclerosis Amiotrófica Lateral/metabolismo , Daño del ADN , Neuronas Motoras/metabolismo , Mutación , Agregación Patológica de Proteínas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Transporte Activo de Núcleo Celular/genética , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Diferenciación Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Femenino , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Persona de Mediana Edad , Neuronas Motoras/patología , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Proteína FUS de Unión a ARN/genética , Transducción de SeñalRESUMEN
Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor ß1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with ß1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by ß1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a ß1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.
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Adaptación Biológica , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Integrina beta1/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Tolerancia a Radiación , Estrés Fisiológico , Animales , Neoplasias Encefálicas/radioterapia , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Movimiento Celular/efectos de la radiación , Quimioradioterapia , Ensamble y Desensamble de Cromatina , Reparación del ADN , Modelos Animales de Enfermedad , Glioma/mortalidad , Glioma/patología , Glioma/radioterapia , Histona Desacetilasas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Ratones , Modelos Biológicos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Estrés Fisiológico/efectos de la radiación , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Radiation and chemotherapy are the main pillars of the current multimodal treatment concept for cancer patients. However, tumor recurrences and resistances still hamper treatment success regardless of advances in radiation beam application, particle radiotherapy, and optimized chemotherapeutics. To specifically intervene at key recurrence- and resistance-promoting molecular processes, the development of potent and specific molecular-targeted agents is demanded for an efficient, safe, and simultaneous integration into current standard of care regimens. Potential targets for such an approach are integrins conferring structural and biochemical communication between cells and their microenvironment. Integrin binding to extracellular matrix activates intracellular signaling for regulating essential cellular functions such as survival, proliferation, differentiation, adhesion, and cell motility. Tumor-associated characteristics such as invasion, metastasis, and radiochemoresistance also highly depend on integrin function. Owing to their dual functionality and their overexpression in the majority of human malignancies, integrins present ideal and accessible targets for cancer therapy. In the following chapter, the current knowledge on aspects of the tumor microenvironment, the molecular regulation of integrin-dependent radiochemoresistance and current approaches to integrin targeting are summarized.
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Integrinas/antagonistas & inhibidores , Terapia Molecular Dirigida/métodos , Neoplasias/terapia , Oncología por Radiación/métodos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Anticuerpos Monoclonales/uso terapéutico , Quimioradioterapia , Humanos , Integrinas/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de la radiaciónRESUMEN
Integrin cell adhesion molecules play a crucial role in tumor cell resistance to radio- and chemotherapy and are therefore considered attractive targets for cancer therapy. Here, we assessed the role of ß1 integrin-interacting α integrin subunits in more physiological three-dimensional extracellular matrix grown head and neck squamous cell carcinoma (HNSCC) cell cultures for evaluating cytotoxic and radiosensitizing potential. α2, α3, α5 and α6 integrins, which are overexpressed in HNSCC according to Oncomine database analysis, were coprecipitated with ß1 integrin. More potently than α2, α5 or α6 integrin inhibition, siRNA-based α3 integrin targeting resulted in reduced clonogenic cell survival, induced apoptosis and enhanced radiosensitivity. These events were associated with diminished phosphorylation of Akt, Cortactin and Paxillin. Cell line-dependently, simultaneous α3 and ß1 integrin inhibition led to higher cytotoxicity and radiosensitization than α3 integrin blocking alone. Stable overexpression of wild-type and constitutively active forms of the integrin signaling mediator focal adhesion kinase (FAK) revealed FAK as a key determinant of α3 integrin depletion-mediated radiosensitization. Our findings show that α3 integrin is essentially involved in HNSCC cell radioresistance and critical for a modified cellular radiosensitivity along with ß1 integrins.
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Apoptosis/efectos de la radiación , Técnicas de Cultivo de Célula/métodos , Proliferación Celular/efectos de la radiación , Integrina alfa3/metabolismo , Apoptosis/genética , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Integrina alfa2/genética , Integrina alfa2/metabolismo , Integrina alfa3/genética , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Unión Proteica , Interferencia de ARN , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de la radiaciónRESUMEN
Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd's Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo.
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Movimiento Celular , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Xenopus/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sitios de Unión , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Células Epiteliales/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Melanocitos/citología , Melanocitos/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Noqueados , Células 3T3 NIH , Cresta Neural/citología , Cresta Neural/metabolismo , Pigmentación , Unión Proteica , Seudópodos/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Dominios Homologos srcRESUMEN
The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin-mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F-actin-dependent manner, suggesting that F-actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin-coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.
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Actinas/fisiología , Aciltransferasas/fisiología , Proteínas Portadoras/fisiología , Endocitosis/genética , Receptores ErbB/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Microfilamentos/fisiología , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiología , Actinas/genética , Actinas/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Células 3T3 NIH , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
The survival of cancer patients suffering from glioblastoma multiforme is limited to just a few months even after treatment with the most advanced techniques. The indefinable borders of glioblastoma cell infiltration into the surrounding healthy tissue prevent complete surgical removal. In addition, genetic mutations, epigenetic modifications and microenvironmental heterogeneity cause resistance to radio- and chemotherapy altogether resulting in a hardly to overcome therapeutic scenario. Therefore, the development of efficient therapeutic strategies to combat these tumors requires a better knowledge of genetic and proteomic alterations as well as the infiltrative behavior of glioblastoma cells and how this can be targeted. Among many cell surface receptors, members of the integrin family are known to regulate glioblastoma cell invasion in concert with extracellular matrix degrading proteases. While preclinical and early clinical trials suggested specific integrin targeting as a promising therapeutic approach, clinical trials failed to deliver improved cure rates up to now. Little is known about glioblastoma cell motility, but switches in invasion modes and adaption to specific microenvironmental cues as a consequence of treatment may maintain tumor cell resistance to therapy. Thus, understanding the molecular basis of integrin and protease function for glioblastoma cell invasion in the context of radiochemotherapy is a pressing issue and may be beneficial for the design of efficient therapeutic approaches. This review article summarizes the latest findings on integrins and extracellular matrix in glioblastoma and adds some perspective thoughts on how this knowledge might be exploited for optimized multimodal therapy approaches.
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Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Animales , Neoplasias Encefálicas/patología , Glioblastoma/patología , Humanos , Invasividad NeoplásicaRESUMEN
PURPOSE: Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. METHODS AND MATERIALS: Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks (γH2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg, ß1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. RESULTS: Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the ß1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. CONCLUSIONS: These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy.
Asunto(s)
Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Movimiento Celular/efectos de la radiación , Glioblastoma/patología , Glioblastoma/radioterapia , Invasividad Neoplásica , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Bromodesoxiuridina/análisis , Carbono , Ensayos de Migración Celular/métodos , Proliferación Celular/efectos de la radiación , Colágeno Tipo I , Roturas del ADN de Doble Cadena , Glioblastoma/genética , Glioblastoma/metabolismo , Histonas/análisis , Humanos , Integrina beta1/fisiología , Péptidos y Proteínas de Señalización Intracelular/análisis , MAP Quinasa Quinasa 4/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteína 1 de Unión al Supresor Tumoral P53 , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
BACKGROUND: Tight regulation of cell motility is essential for many physiological processes, such as formation of a functional nervous system and wound healing. Drosophila Abl negatively regulates the actin cytoskeleton effector protein Ena during neuronal development in flies, and it has been postulated that this may occur through an unknown intermediary. Lamellipodin (Lpd) regulates cell motility and recruits Ena/VASP proteins (Ena, Mena, VASP, EVL) to the leading edge of cells. However, the regulation of this recruitment has remained unsolved. RESULTS: Here we show that Lpd is a substrate of Abl kinases and binds to the Abl SH2 domain. Phosphorylation of Lpd positively regulates the interaction between Lpd and Ena/VASP proteins. Consistently, efficient recruitment of Mena and EVL to Lpd at the leading edge requires Abl kinases. Furthermore, transient Lpd phosphorylation by Abl kinases upon netrin-1 stimulation of primary cortical neurons positively correlates with an increase in Lpd-Mena coprecipitation. Lpd is also transiently phosphorylated by Abl kinases upon platelet-derived growth factor (PDGF) stimulation, regulates PDGF-induced dorsal ruffling of fibroblasts and axonal morphogenesis, and cooperates with c-Abl in an Ena/VASP-dependent manner. CONCLUSIONS: Our findings suggest that Abl kinases positively regulate Lpd-Ena/VASP interaction, Ena/VASP recruitment to Lpd at the leading edge, and Lpd-Ena/VASP function in axonal morphogenesis and in PDGF-induced dorsal ruffling. Our data do not support the suggested negative regulatory role of Abl for Ena. Instead, we propose that Lpd is the hitherto unknown intermediary between Abl and Ena/VASP proteins.
Asunto(s)
Axones/fisiología , Proteínas de Unión al ADN/fisiología , Fibroblastos/fisiología , Morfogénesis/fisiología , Proteínas Proto-Oncogénicas c-abl/fisiología , Animales , Western Blotting , Línea Celular , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiología , Inmunoprecipitación , Factores de Crecimiento Nervioso/fisiología , Netrina-1 , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/fisiología , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Rac1 and Rac2 GTPases transduce signals from multiple receptors leading to cell migration, adhesion, proliferation, and survival. In the absence of Rac1 and Rac2, B cell development is arrested at an IgD- transitional B cell stage that we term transitional type 0 (T0). We show that T0 cells cannot enter the white pulp of the spleen until they mature into the T1 and T2 stages, and that this entry into the white pulp requires integrin and chemokine receptor signaling and is required for cell survival. In the absence of Rac1 and Rac2, transitional B cells are unable to migrate in response to chemokines and cannot enter the splenic white pulp. We propose that loss of Rac1 and Rac2 causes arrest at the T0 stage at least in part because transitional B cells need to migrate into the white pulp to receive survival signals. Finally, we show that in the absence of Syk, a kinase that transduces B cell antigen receptor signals required for positive selection, development is arrested at the same T0 stage, with transitional B cells excluded from the white pulp. Thus, these studies identify a novel developmental checkpoint that coincides with B cell positive selection.