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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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BACKGROUND: Focal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC). METHODS: The cytotoxic and radiochemosensitizing potential of targeting 10 RTK and ß1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts. RESULTS: Fibroblast growth factor receptor (FGFR 1-4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with ß1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions. CONCLUSIONS: This study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.
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Antineoplásicos , Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Integrina beta1/genética , Línea Celular Tumoral , Proteínas Tirosina Quinasas Receptoras/genética , Antineoplásicos/uso terapéutico , Receptores ErbB/metabolismo , Fenotipo , Transición Epitelial-Mesenquimal/genéticaRESUMEN
Phosphoinositide 3-kinase (PI3K)-α represents a key intracellular signal transducer involved in the regulation of key cell functions such as cell survival and proliferation. Excessive activation of PI3Kα is considered one of the major determinants of cancer therapy resistance. Despite preclinical and clinical evaluation of PI3Kα inhibitors in various tumor entities, including head and neck squamous cell carcinoma (HNSCC), it remains elusive how conventional radiochemotherapy can be enhanced by concurrent PI3K inhibitors and how PI3K deactivation mechanistically exerts its effects. Here, we investigated the radiochemosensitizing potential and adaptation mechanisms of four PI3K inhibitors, Alpelisib, Copanlisib, AZD8186, and Idelalisib in eight HNSCC models grown under physiological, three-dimensional matrix conditions. We demonstrate that Alpelisib, Copanlisib and AZD8186 but not Idelalisib enhance radio- and radiochemosensitivity in the majority of HNSCC cell models (= responders) in a manner independent of PIK3CA mutation status. However, Alpelisib promotes MAPK signaling in non-responders compared to responders without profound impact on Akt, NFκB, TGFß, JAK/STAT signaling and DNA repair. Bioinformatic analyses identified unique gene mutations associated with extracellular matrix to be more frequent in non-responder cell models than in responders. Finally, we demonstrate that targeting of the cell adhesion molecule ß1 integrin on top of Alpelisib sensitizes non-responders to radiochemotherapy. Taken together, our study demonstrates the sensitizing potential of Alpelisib and other PI3K inhibitors in HNSCC models and uncovers a novel ß1 integrin-dependent mechanism that may prove useful in overcoming resistance to PI3K inhibitors.
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Compuestos de Anilina , Cromonas , Neoplasias de Cabeza y Cuello , Fosfatidilinositol 3-Quinasas , Tiazoles , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello , Fosfatidilinositol 3-Quinasas/metabolismo , Integrina beta1/genética , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Fosfatidilinositol 3-Quinasa Clase I , Línea Celular TumoralRESUMEN
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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Constitutively active kinases play a crucial role in carcinogenesis, and their inhibition is a common target for molecular tumor therapy. We recently discovered the expression of two oncogenic isoforms of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC), Btk-p80 and BTK-p65. However, the precise role of BTK in HNSCC remains unclear. Analyses of a tissue microarray containing benign and malignant as well as inflammatory tissue samples of the head and neck region revealed the preferential expression of BTK-p80 in malignant tissue, whereas BTK-p65 expression was confirmed in over 80% of analyzed metastatic head and neck tumor cases. Therefore, processes associated with metastasis, like cancer stem cell (CSC) enrichment and the epithelial-mesenchymal transition (EMT), which in turn depend on an appropriate cytokine milieu, were analyzed. Treatment of HNSCC-derived cell lines cultured under 3D conditions with the BTK inhibitor AVL-292 caused reduced sphere formation, which was accompanied by reduced numbers of ALDH1A1+ CSCs as well as biological changes associated with the EMT. Moreover, we observed reduced NF-κB expression as well as altered NF-κB dependent pro-tumorigenic and EMT-associated cytokine release of IL-6, IFNγ, and TNFα when BTK activity was dampened. Therefore, an autocrine regulation of the oncogenic BTK-dependent process in HNSCC can be suggested, with BTK inhibition expected to be an effective treatment option for HNSCC.
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Agammaglobulinemia Tirosina Quinasa , Transición Epitelial-Mesenquimal , Neoplasias de Cabeza y Cuello , Humanos , Carcinogénesis , Citocinas , Neoplasias de Cabeza y Cuello/genética , Células Madre Neoplásicas , FN-kappa B , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Cancer presents as a highly heterogeneous disease with partly overlapping and partly distinct (epi)genetic characteristics. These characteristics determine inherent and acquired resistance, which need to be overcome for improving patient survival. In line with the global efforts in identifying druggable resistance factors, extensive preclinical research of the Cordes lab and others designated the cancer adhesome as a critical and general therapy resistance mechanism with multiple druggable cancer targets. In our study, we addressed pancancer cell adhesion mechanisms by connecting the preclinical datasets generated in the Cordes lab with publicly available transcriptomic and patient survival data. We identified similarly changed differentially expressed genes (scDEGs) in nine cancers and their corresponding cell models relative to normal tissues. Those scDEGs interconnected with 212 molecular targets from Cordes lab datasets generated during two decades of research on adhesome and radiobiology. Intriguingly, integrative analysis of adhesion associated scDEGs, TCGA patient survival and protein-protein network reconstruction revealed a set of overexpressed genes adversely affecting overall cancer patient survival and specifically the survival in radiotherapy-treated cohorts. This pancancer gene set includes key integrins (e.g. ITGA6, ITGB1, ITGB4) and their interconnectors (e.g. SPP1, TGFBI), affirming their critical role in the cancer adhesion resistome. In summary, this meta-analysis demonstrates the importance of the adhesome in general, and integrins together with their interconnectors in particular, as potentially conserved determinants and therapeutic targets in cancer.
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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.
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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
BRCA1 is a well-known breast cancer risk gene, involved in DNA damage repair via homologous recombination (HR) and replication fork protection. Therapy resistance was linked to loss and amplification of the BRCA1 gene causing inferior survival of breast cancer patients. Most studies have focused on the analysis of complete loss or mutations in functional domains of BRCA1. How mutations in non-functional domains contribute to resistance mechanisms remains elusive and was the focus of this study. Therefore, clones of the breast cancer cell line MCF7 with indels in BRCA1 exon 9 and 14 were generated using CRISPR/Cas9. Clones with successful introduced BRCA1 mutations were evaluated regarding their capacity to perform HR, how they handle DNA replication stress (RS), and the consequences on the sensitivity to MMC, PARP1 inhibition, and ionizing radiation. Unexpectedly, BRCA1 mutations resulted in both increased sensitivity and resistance to exogenous DNA damage, despite a reduction of HR capacity in all clones. Resistance was associated with improved DNA double-strand break repair and reduction in replication stress (RS). Lower RS was accompanied by increased activation and interaction of proteins essential for the S phase-specific DNA damage response consisting of HR proteins, FANCD2, and CHK1.
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Neoplasias de la Mama , Genes BRCA1 , Humanos , Femenino , Línea Celular Tumoral , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Recombinación Homóloga , Reparación del ADN/genética , Replicación del ADN , Daño del ADN , Neoplasias de la Mama/genética , Neoplasias de la Mama/tratamiento farmacológicoRESUMEN
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
Although radiation therapy has recently made great advances in cancer treatment, the majority of patients diagnosed with pancreatic cancer (PC) cannot achieve satisfactory outcomes due to intrinsic and acquired radioresistance. Identifying the molecular mechanisms that impair the efficacy of radiotherapy and targeting these pathways are essential to improve the radiation response of PC patients. Our goal is to identify sensitive targets for pancreatic cancer radiotherapy (RT) using the kinome-wide CRISPR-Cas9 loss-of-function screen and enhance the therapeutic effect through the development and application of targeted inhibitors combined with radiotherapy. We transduced pancreatic cancer cells with a protein kinase library; 2D and 3D library cells were irradiated daily with a single dose of up to 2 Gy for 4 weeks for a total of 40 Gy using an X-ray generator. Sufficient DNA was collected for next-generation deep sequencing to identify candidate genes. In this study, we identified several cell cycle checkpoint kinases and DNA damage related kinases in 2D- and 3D-cultivated cells, including DYRK1A, whose loss of function sensitizes cells to radiotherapy. Additionally, we demonstrated that the harmine-targeted suppression of DYRK1A used in conjunction with radiotherapy increases DNA double-strand breaks (DSBs) and impairs homologous repair (HR), resulting in more cancer cell death. Our results support the use of CRISPR-Cas9 screening to identify new therapeutic targets, develop radiosensitizers, and provide novel strategies for overcoming the tolerance of pancreatic cancer to radiotherapy.
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PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a cancer with unmet needs. The role of highly conformal radiation therapy is still under debate for PDAC. Owing to its desmoplastic nature, integrin-mediated interactions between PDAC cells and extracellular matrix (ECM) profoundly contribute to PDAC therapy resistance. In this study, we investigated the radiochemosensitizing potential of ß1 integrin targeting in therapy-naive and radioresistant PDAC cell cultures grown in 3-dimensional (3D) ECM. METHODS AND MATERIALS: In a panel of 3D, ECM-based PDAC cell cultures, ß1 integrin was inhibited by antibodies or siRNA-mediated knockdown. Together with x-ray irradiation and specific chemotherapies, we determined 3D colony formation capacity in therapy-naive and radioresistant PDAC cultures. We used kinome profiling, Western blotting, and immunofluorescence stainings to characterize these cell lines. Various siRNA screens were conducted to identify novel therapeutic targets. RESULTS: We found a significant radiosensitizing potential of ß1 integrin inhibition both in therapy-naive and radioresistant PDAC cell cultures. Kinome profiling upon ß1 integrin targeting identified a generally declined tyrosine and serine/threonine kinase activity, which presented less prominent in radioresistant than in therapy-naive PDAC cells. siRNA screens employing the top 34 deregulated kinases in combination with ß1 integrin inhibition revealed less efficacy and less radiosensitization in radioresistant relative to therapy-naive PDAC cell cultures. Triple inhibition of ß1 integrin, protein kinase D1, and rearranged during transfection turned out to be most effective in reducing 3D colony formation of radioresistant PDAC cells. CONCLUSIONS: Our study clearly shows that ß1 integrins are robust targets for overcoming radioresistance in PDAC. This seems to apply equally to therapy-sensitive and radioresistant cells. Concerning tumor heterogeneity, this dual therapy-sensitizing potential might be exploitable for a significant improvement of patient survival.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/radioterapia , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológicoRESUMEN
The authors wish to make the following corrections to this paper [...].
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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 authors wish to make the following corrections to this paper [...].
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For better tumor control, high-precision proton beam radiation therapy is currently being intensively discussed relative to conventional photon therapy. Here, we assumed that radiation type-specific molecular response profiles in more physiological 3D, matrix-based head and neck squamous cell carcinoma (HNSCC) cell cultures can be identified and therapeutically exploited. While proton irradiation revealed superimposable clonogenic survival and residual DNA double strand breaks (DSB) relative to photon irradiation, kinome profiles showed quantitative differences between both irradiation types. Pharmacological inhibition of a subset of radiation-induced kinases, predominantly belonging to the mitogen-activated protein kinase (MAPK) family, failed to sensitize HNSCC cells to either proton or photon irradiation. Likewise, inhibitors for ATM, DNA-PK and PARP did not discriminate between proton and photon irradiation but generally elicited a radiosensitization. Conclusively, our results suggest marginal cell line-specific differences in the radiosensitivity and DSB repair without a superiority of one radiation type over the other in 3D grown HNSCC cell cultures. Importantly, radiation-induced activity changes of cytoplasmic kinases induced during the first, acute phase of the cellular radiation response could neither be exploited for sensitization of HNSCC cells to photon nor proton irradiation.
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PURPOSE: The current status of German residency training in the field of radiation oncology is provided and compared to programmes in other countries. In particular, we present the DEGRO-Academy within the international context. METHODS: Certified courses from 2018 and 2019 were systematically assigned to the DEGRO-Curriculum, retrospectively for 2018 and prospectively for 2019. In addition, questionnaires of course evaluations were provided, answered by course participants and collected centrally. RESULTS: Our data reveal a clear increase in curriculum coverage by certified courses from 57.6% in 2018 to 77.5% in 2019. The analyses enable potential improvements in German curriculum-based education. Specific topics of the DEGRO-Curriculum are still underrepresented, while others decreased in representation between 2018 and 2019. It was found that several topics in the DEGRO-Curriculum require more attention because of a low DEGRO-curriculum coverage. Evaluation results of certified courses improved significantly with a median grade of 1.62 in 2018 to 1.47 in 2019 (p = 0.0319). CONCLUSION: The increase of curriculum coverage and the simultaneous improvement of course evaluations are promising with respect to educational standards in Germany. Additionally, the early integration of radiation oncology into medical education is a prerequisite for resident training because of rising demands on quality control and increasing patient numbers. This intensified focus is a requirement for continued high standards and quality of curriculum-based education in radiation oncology both in Germany and other countries.
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Curriculum , Internado y Residencia , Oncología por Radiación/educación , Curriculum/estadística & datos numéricos , Curriculum/tendencias , Alemania , Humanos , Evaluación de Programas y Proyectos de Salud , Garantía de la Calidad de Atención de Salud , Oncología por Radiación/tendencias , Encuestas y CuestionariosRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly therapy-resistant tumor entity of unmet needs. Over the last decades, radiotherapy has been considered as an additional treatment modality to surgery and chemotherapy. Owing to radiosensitive abdominal organs, high-precision proton beam radiotherapy has been regarded as superior to photon radiotherapy. To further elucidate the potential of combination therapies, we employed a more physiological 3D, matrix-based cell culture model to assess tumoroid formation capacity after photon and proton irradiation. Additionally, we investigated proton- and photon-irradiation-induced phosphoproteomic changes for identifying clinically exploitable targets. Here, we show that proton irradiation elicits a higher efficacy to reduce 3D PDAC tumoroid formation and a greater extent of phosphoproteome alterations compared with photon irradiation. The targeting of proteins identified in the phosphoproteome that were uniquely altered by protons or photons failed to cause radiation-type-specific radiosensitization. Targeting DNA repair proteins associated with non-homologous endjoining, however, revealed a strong radiosensitizing potential independent of the radiation type. In conclusion, our findings suggest proton irradiation to be potentially more effective in PDAC than photons without additional efficacy when combined with DNA repair inhibitors.
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The function of Keap1 (Kelch-like ECH-associated protein 1), a sensor of oxidative and electrophilic stress, in the radiosensitivity of cancer cells remains elusive. Here, we investigated the effects of pharmacological inhibition of Keap1 with ML344 on radiosensitivity, DNA double-strand break (DSB) repair and autophagy in head and neck squamous cell carcinoma (HNSCC) cell lines. Our data demonstrate that Keap1 inhibition enhances HNSCC cell radiosensitivity. Despite elevated, Nrf2-dependent activity of non-homologous end joining (NHEJ)-related DNA repair, Keap1 inhibition seems to impair DSB repair through delayed phosphorylation of DNA-PKcs. Moreover, Keap1 inhibition elicited autophagy and increased p62 levels when combined with X-ray irradiation. Our findings suggest HNSCC cell radiosensitivity, NHEJ-mediated DSB repair, and autophagy to be co-regulated by Keap1.
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Autofagia/fisiología , Proteína 1 Asociada A ECH Tipo Kelch/antagonistas & inhibidores , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Autofagia/genética , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteínas Nucleares/metabolismo , Tolerancia a Radiación/genéticaRESUMEN
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.