10,893 research outputs found

    Converging organoids and extracellular matrix::New insights into liver cancer biology

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    Overcoming drug resistance: targeting the BCL-2 family and the long non-coding RNA HCP5 in medulloblastoma and colorectal cancer

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    Colorectal cancer (CRC) is one of the most common cancers in the UK and medulloblastoma is a common cancer found in children. While there has been a progressive improvement in treatment outcomes, success has been marred by drug resistance and severe side effects. Therefore, this project focused on two aspects of chemotherapeutic drug resistance, the first using the antimitotic agent vincristine in combination with inhibitors of the anti-apoptotic Bcl-2 family proteins, while the second investigated the role of the long non-coding RNA (lncRNA), HCP5 in the resistance of cells to genotoxic agents. In the first part, three medulloblastoma cell lines (DAOY, MB03, ONS76) were analysed for the expression of Bcl-xL and ONS76 cells found to have the highest level of this anti-apoptotic protein. Subsequent results indicated that Bcl-xL encourages mitotic slippage and stemness and that knockdown of Bcl-xL in the high expressing ONS76 cells, reduces these and sensitizes the cells to the anti-mitotic agent vincristine. Thus, pharmacological inhibition of Bcl-xL should sensitize medulloblastoma cells to low doses of vincristine. Regarding the lncRNA HCP5, results showed that HCP5 was generally more highly expressed in a panel of CRC cell lines than the three medulloblastoma cell lines, corroborating data from an in-silico analysis for the corresponding tumours. One function of HCP5 is to translocate the multifunctional YB-1 protein from the cytoplasm to the nucleus where it carries out many of its functions. Knockdown of HCP5 followed by immunofluorescence indicated a reduction in the amount of YB-1 in the nucleus, confirming this function. Subsequently, HCP5 silencing sensitized all cell lines tested to the DNA damaging agents, cisplatin, oxaliplatin and tert-butyl hydroperoxide and also resulted in an increase in double-strand breaks as determined by H2AX formation. Finally, fluorescence activated cell sorting using Annexin V and propidium iodide confirmed a decrease in cell viability in HCP5 knockdown cells following treatment with genotoxic agents and that this was mirrored by an increased apoptotic fraction. Together, these studies indicate the possibilities of using novel therapeutics to increase the functionality of existing treatments to combat acquired drug resistance in cancer patients

    Targeting pre-mRNA splicing in cancers: roles, inhibitors, and therapeutic opportunities

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    Accumulating evidence has indicated that pre-mRNA splicing plays critical roles in a variety of physiological processes, including development of multiple diseases. In particular, alternative splicing is profoundly involved in cancer progression through abnormal expression or mutation of splicing factors. Small-molecule splicing modulators have recently attracted considerable attention as a novel class of cancer therapeutics, and several splicing modulators are currently being developed for the treatment of patients with various cancers and are in the clinical trial stage. Novel molecular mechanisms modulating alternative splicing have proven to be effective for treating cancer cells resistant to conventional anticancer drugs. Furthermore, molecular mechanism-based combination strategies and patient stratification strategies for cancer treatment targeting pre-mRNA splicing must be considered for cancer therapy in the future. This review summarizes recent progress in the relationship between druggable splicing-related molecules and cancer, highlights small-molecule splicing modulators, and discusses future perspectives of splicing modulation for personalized and combination therapies in cancer treatment

    Dysregulation of Transcription Factor Networks Unveils Different Pathways in Human Papillomavirus 16-Positive Squamous Cell Carcinoma and Adenocarcinoma of the Uterine Cervix

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    Squamous cell carcinoma (SCC) and adenocarcinoma (ADC) are the most common histological types of cervical cancer (CC). The worse prognosis of ADC cases highlights the need for better molecular characterization regarding differences between these CC types. RNA-Seq analysis of seven SCC and three ADC human papillomavirus 16-positive samples and the comparison with public data from non-tumoral human papillomavirus-negative cervical tissue samples revealed pathways exclusive to each histological type, such as the epithelial maintenance in SCC and the maturity-onset diabetes of the young (MODY) pathway in ADC. The transcriptional regulatory network analysis of cervical SCC samples unveiled a set of six transcription factor (TF) genes with the potential to positively regulate long non-coding RNA genes DSG1-AS1, CALML3-AS1, IGFL2-AS1, and TINCR. Additional analysis revealed a set of MODY TFs regulated in the sequence predicted to be repressed bymiR-96-5p ormiR-28-3p in ADC. These microRNAs were previously described to target LINC02381, which was predicted to be positively regulated by two MODY TFs upregulated in cervical ADC. Therefore, we hypothesize LINC02381might act by decreasing the levels ofmiR-96-5p andmiR-28-3p, promoting the MODY activation in cervical ADC. The novel TF networks here described should be explored for the development of more efficient diagnostic tools

    Microtubule-Associated Proteins MAP7 and MAP7D1 Promote 53BP1-Mediated DNA Double-Stranded Break Repair in the G1 Cell Cycle Phase

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    The DNA damage response is a complex signalling network that guards cellular genomic integrity and protects organisms from a number of diseases including cancer. Quantitative proteomics revealed that the microtubule-associated proteins, MAP7 and MAP7D1 which regulate kinesin-1 recruitment to microtubules, interact with several DNA repair proteins including the double-stranded break repair proteins BRCA1 and RAD50. The interactions of MAP7 with BRCA1 and RAD50 are partly dependent on MAP7’s ability to bind phosphorylated Ser1336 in BRCA1 and phosphorylated Thr690 in RAD50. Using a phospho-specific antibody against BRCA1 pSer1336, it was established that the kinase which phosphorylates BRCA1 at Ser1336 is casein kinase 2. Furthermore, co-immunoprecipitation and domain mapping experiments revealed that MAP7 interacts with the DNA repair proteins through amino acids 144 to 300 located in its N-terminus. Additionally, these experiments showed that other proteins bridge the interactions between MAP7 and the DNA damage response proteins. Experiments with siRNA-mediated down-regulation of MAP7 and MAP7D1 elucidated that decreased levels of MAP7 and MAP7D1 lead to increased phosphorylation of p53 at Ser15 after γ-irradiation treatment. Moreover, the down-regulation of MAP7D1 leads to a strong G1 phase cell cycle arrest. The knock-down of both MAP7 and MAP7D1 in cells arrested in the G1 phase negatively affects DNA double-stranded break repair and the localisation of 53BP1 to the site of damage. In addition, cells with depleted MAP7 or MAP7D1 exhibit decreased localisation of RAD50 to the chromatin before and after γ-irradiation treatment. Taken together, these findings describe for the first time a novel function of MAP7 and MAP7D1 in the cellular response to DNA damage caused by γ-irradiation

    Molecular analysis of the primary neoplasms of the posterior pituitary lobe

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    Die primĂ€ren Tumoren des Hypophysenhinterlappens, Pituizytom (PITUI), Granularzelltumor der Neurohypophyse (GCT) und Spindelzellonkozytom (SCO), gehören zu den seltenen Erkrankungen. Eine Meta-Analyse aus dem Jahr 2019 gibt 270 in der Literatur beschriebene Exemplare an. Seit ihrer mutmaßlich erstmaligen Beschreibung im spĂ€ten 19. Jahrhundert hat es wenig Fortschritte in der Diagnostik und Therapie dieser niedriggradigen Gliome gegeben. Die Entwicklung immunhistochemischer und molekularer Techniken in den letzten beiden Jahrzehnten hat jedoch zu neuen Entdeckungen gefĂŒhrt. Dies waren zum einen die Expression des thyreoidalen Transkriptionsfaktors 1 (TTF1) in allen drei EntitĂ€ten sowie in Pituizyten, zum anderen wurden in EinzelfĂ€llen Alterationen des MAPK Signalweges beschrieben. Wir haben in unserer Arbeit 47 dieser Tumoren zusammengetragen (14 PITUI; 12 GCT; 21 SCO) und ihre DNA-Methylierungsprofile, ihren Mutationsstatus in 479 krebsrelevanten Genen und ihre chromosomalen Kopiezahlprofile analysiert, um sie molekular zu charakterisieren und herauszufinden, ob sich klinisch relevante Subgruppen ableiten lassen. Wir haben zwei Hauptgruppen in unsupervidierten Clusterings der DNA-Methylierungsdaten nachweisen können. Allerdings sind die Unterschiede zwischen den einzelnen Gruppen gering. Die grĂ¶ĂŸere der beiden Methylierungs-Gruppen enthĂ€lt 23 der 47 Tumoren und setzt sich aus PITUIs und einem Teil der SCOs zusammen und ist angereichert fĂŒr Tumoren mit pathogenen Mutationen innerhalb des MAPK/PI3K Signalweges. Zwölf der 17 sequenzierten Tumoren (71%), in denen sich pathogene Mutationen nachweisen ließen, fielen in diese Gruppe. Es wurden FGFR1 (3), HRAS (3), BRAF (2), NF1 (2), CBL (1), MAP2K2 (1), PTEN (1) Mutationen nachgewiesen. Die zweite Gruppe bestand aus 16 Tumoren, hauptsĂ€chlich vom Typ des GCT sowie auch einzelne SCO, die allesamt zumeist keine krebsrelevanten Mutationen aufwiesen. Unsere Überlebenszeitanalyse ergab, dass chromosomale Imbalancen in der Gruppe der PITUI und SCO signifikant mit einem reduzierten progressionsfreien Überleben assoziiert waren (p=0,031). Zusammenfassend sprechen die nur geringen Unterschiede in den DNA- Methylierungsprofilen dieser TumorentitĂ€ten dafĂŒr, dass sie am besten als Subtypen einer einzigen EntitĂ€t betrachtet werden sollten. Allerdings zeigen unsere Ergebnisse deutliche Unterschiede in der Verteilung der Mutationen und im klinischen Outcome. Daher schlagen wir eine kombinierte histomolekulare Subklassifikation in drei Subtypen vor. Subtyp 1 ist charakterisiert durch GCT-Histologie und zeigt ĂŒberwiegend keine molekularen Alterationen. Subtyp 2 zeigt PITUI oder SCO Histologie, keine chromosomalen Imbalancen und hat ein ĂŒberwiegend gutes klinisches Outcome. Subtyp 3 zeigt eine PITUI oder SCO Histologie, weist chromosomale Imbalancen auf und es finden sich hĂ€ufiger Rezidive. Beide der letztgenannten Subtypen haben hĂ€ufig MAPK/PI3K Mutationen, die mögliche therapeutische Targets darstellen.The primary tumours of the posterior lobe of the pituitary gland, pituicytoma (PITUI), granular cell tumour of the neurohypophysis (GCT) and spindle cell oncocytoma (SCO), are rare diseases. A meta-analysis (published in 2019) reported 270 cases described in the literature. Since their presumed first description in the late 19th century, there has been little progress in the diagnosis and treatment of these low-grade gliomas. However, the development of immunohistochemical and molecular techniques in the last two decades has led to new discoveries: Firstly the expression of thyroid transcription factor 1 (TTF1) in all three entities as well as in pituicytes. Secondly, alterations of the MAPK signalling pathway were described in single cases. In our work, we collected 47 of these tumours (14 PITUI; 12 GCT; 21 SCO) and evaluated their DNA methylation profiles, their mutation status in 479 cancer-relevant genes and their copy number profiles in order to characterise them molecularly and to find out whether clinically relevant subgroups can be derived. We were able to indetify two main groups in unsupervised clusterings of the DNA methylation data. However, the differences between the groups are small. The larger of the two methylation groups contains 23 of the 47 tumours and is composed of PITUIs and a subset of SCOs and was enriched for tumours with pathogenic mutations within the MAPK/PI3K pathway. Twelve of the 17 sequenced tumours (71%) in which such pathogenic mutations could be detected fell into this group. FGFR1 (3), HRAS (3), BRAF (2), NF1 (2), CBL (1), MAP2K2 (1), PTEN (1) mutations were detected. The second group consisted of 16 tumours, mainly of the GCT type as well as single SCO, which, like the other tumours in this group, mostly had no driver mutations. Our survival time analysis showed that chromosomal imbalances in the PITUI and SCO group were significantly associated with reduced progression-free survival (p=0.031). In summary, the only minor differences in the DNA methylation profiles of these tumours suggest that they are best considered as subtypes of a single entity. However, our results show marked differences in mutation distribution and clinical outcome. Therefore, we propose a combined histomolecular subclassification into three subtypes. Subtype 1 is characterised by GCT histology and shows predominantly no molecular alterations. Subtype 2 shows PITUI or SCO histology, no chromosomal imbalances and has a good clinical outcome. Subtype 3 shows PITUI or SCO histology, has chromosomal imbalances and recurrences are more common. Both of the latter subtypes often have MAPK/PI3K mutations, which are potential therapeutic targets

    Comparison of pharmacological inhibitors of lysine-specific demethylase 1 in glioblastoma stem cells reveals inhibitor-specific efficacy profiles

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    IntroductionImproved therapies for glioblastoma (GBM) are desperately needed and require preclinical evaluation in models that capture tumor heterogeneity and intrinsic resistance seen in patients. Epigenetic alterations have been well documented in GBM and lysine-specific demethylase 1 (LSD1/KDM1A) is amongst the chromatin modifiers implicated in stem cell maintenance, growth and differentiation. Pharmacological inhibition of LSD1 is clinically relevant, with numerous compounds in various phases of preclinical and clinical development, but an evaluation and comparison of LSD1 inhibitors in patient-derived GBM models is lacking.MethodsTo assess concordance between knockdown of LSD1 and inhibition of LSD1 using a prototype inhibitor in GBM, we performed RNA-seq to identify genes and biological processes associated with inhibition. Efficacy of various LSD1 inhibitors was assessed in nine patient-derived glioblastoma stem cell (GSC) lines and an orthotopic xenograft mouse model.ResultsLSD1 inhibitors had cytotoxic and selective effects regardless of GSC radiosensitivity or molecular subtype. In vivo, LSD1 inhibition via GSK-LSD1 led to a delayed reduction in tumor burden; however, tumor regrowth occurred. Comparison of GBM lines by RNA-seq was used to identify genes that may predict resistance to LSD1 inhibitors. We identified five genes that correlate with resistance to LSD1 inhibition in treatment resistant GSCs, in GSK-LSD1 treated mice, and in GBM patients with low LSD1 expression.ConclusionCollectively, the growth inhibitory effects of LSD1 inhibition across a panel of GSC models and identification of genes that may predict resistance has potential to guide future combination therapies

    Exposure of wild Caspian seals (Pusa caspica) to parasites, bacterial and viral pathogens, evaluated via molecular and serological assays

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    Disease surveillance of marine mammal populations is essential to understand the causes of strandings, identify potential threats to animal health, and to support development of conservation strategies. Here we report the first large multi-pathogen screening of prevalence for viruses, bacteria and parasites in a sample of 177 live, healthy, wild Caspian seals (Pusa caspica), captured and released during satellite telemetry studies 2007-2017. Employing molecular and serological assays we assess prevalence of pathogens known to be of significance for marine mammal health worldwide, and evaluate the results in relation to Caspian seal health and conservation. RT-PCR, and PCR assays find evidence for infection by Canine Distemper Virus (CDV), Phocine herpes virus, phocine adenovirus and Influenza A at prevalences of 5%, 6.4%, 21.7%, and 4% respectively. The genomes of CDV isolates collected in 2008 showed 99.59% identity with the 2000 Caspian seal CDV epizootic strain. A partial coding sequence for the Us2 gene from the Caspian seal herpes virus was identical to PhHV-1 isolate PB84, previously reported from a harbor seal (Phoca vitulina), while amplicon sequences for the adenovirus polymerase gene indicated a novel strain. ELISA assays detected exposure to Influenza A (55% of tested samples), adenovirus (25%), coronavirus (6%), CDV (8%), herpes virus (94%), Toxoplasma gondii (2.6%) and heartworm (1%). Hemagglutination inhibition (HI) tests detected exposure to Influenza B at a prevalence of 20%, and Leptospira microscopic agglutination tests detected suspected exposure to Leptospira serovars in 9% of tested samples. Overall, the risks, profile and prevalence of pathogens in Caspian seals appear comparable to other wild phocid seal populations. Our results suggest Caspian seals have exposure pathways to pathogens with epizootic potential or ability to cause significant morbidity, and that disease impacts could reduce the resilience of the population to other conservation threats. Caspian seals are listed as Endangered by the International Union for Conservation of Nature (IUCN), and we recommend that resources are invested to support further surveillance programs and to understand how anthropogenic pressures may influence future disease risks. A translated version of this abstract is available in Russian and Kazakh in the Supplementary Material (Presentation 1 and Presentation 2

    Prototype Foamy Virus Capsid – Nucleic Acid Interactions: Mechanistic Insights & Application for Efficient RNA Transfer

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    Foamy viruses (FV) represent a distinct genus in the retrovirus family and separate themselves from the large group of orthoretroviruses by various distinct features in their replication cycle (reviewed in Lindemann & Rethwilm, 2011). In gene therapy retroviruses are commonly used as vectors to deliver genetic information into target cells and also FV has been successfully used for example in a canine genetic disease model (Trobridge et al., 2009). Here we investigated the interactions between the FV capsid-forming protein ‘Gag’ and nucleic acids. We found that prototype FV (PFV) Gag binds various cellular mRNAs, incorporates them into the nascent particle and thereby enables their transfer into the cytosol of target cells. There these mRNAs can serve as template for protein translation. This feature seems uniquely efficient for PFV and we developed it further into a “RNA transfer vector system” allowing efficient transgene mRNA transfer into target cells, as showed in proof-of-principle experiments in vitro and in vivo (Hamann et al., 2014a). In parallel we started investigating the specificity in viral RNA genome packaging (Hamann et al., 2014b). To date little is known how PFV selects its RNA genome over the vast excess of cellular RNAs present in the cytosol. Elevated fundamental knowledge of this mechanism could help to make the “RNA transfer vector system” even more efficient since it would allow enrichment of certain specific “designer-RNAs” in virus particles

    Emerging radiopharmaceuticals for PET-imaging gliomas. A multi-: radiopharmaceutical, camera, modality, model, and modelling assessment

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    Gliomas, which are a type of brain tumour derived from the non-neuronal and nutrient-supplying glial cells of the brain, are particularly devastating disease due to the importance and delicate nature of cerebral matter. Surgical removal, chemotherapy, and radiation therapy often have unwanted consequences depending on a variety of physiological and probability factors. With the human life expectancy averaging 12-15 months after clinical diagnosis (with treatment) for aggressive brain tumours, accurately detecting and characterizing these tumours non-invasively is important for treatment planning. Currently, the highest anatomical resolution imaging modality available for brain imaging is magnetic resonance imaging (MRI), but this lacks biochemical information. Positron emission tomography paired with computed tomography for anatomical reference (PET-CT) divulges quantifiable biochemical information. By selecting imaging radiopharmaceuticals for PET imaging that have relevance to tumour surface proteins or other cellular metabolic processes it is possible to not only aid in detecting or delineating gliomas, but also gain specific biochemical-property insight into these lesions. The aim of these studies was to evaluate the two emerging radiopharmaceuticals (2S, 4R)-4-[18F]fluoroglutamine ([18F]FGln) and Al[18F]F-NOTA-Folate ([18F]FOL) and to directly compare them with routinely clinically-used radiopharmaceuticals 2-deoxy-2-[18F]fluoro-ᮅ-glucose ([18F]FDG) and ʟ-[11C]methionine ([11C]Met) for the PET imaging of gliomas in animal models. Other parameters, such as the in vivo stability, ex vivo biodistribution, in vitro binding and blocking, and the presence of relevant receptors on human tissue samples were investigated in to divulge additional information. The results demonstrated that both [18F]FGln and [18F]FOL provided an enhanced level of contrast between tumour and adjacent non-tumour brain tissue versus that of the clinically used radiopharmaceuticals [18F]FDG and [11C]Met in animal models.Uudet radiolÀÀkeaineet glioomien PET-kuvantamiseen. Tutkimuksia radiolÀÀkeaineista, modaliteeteista, kameroista, kokeellisista malleista ja mallintamisesta Glioomat ovat aivokasvaimia, jotka syntyvĂ€t ravinteiden kuljetusta hoitavista glia- eli hermotukisoluista. Ne ovat erityisen tuhoisia sairauksia aivokudoksen tĂ€rkeyden ja herkkyyden vuoksi. Kirurgisella leikkauksella, kemoterapialla, ja sĂ€dehoidolla on usein ei-toivottuja seurauksia riippuen fysiologisista ja todennĂ€köisyystekijöistĂ€. Koska elinajanodote aggressiivisen aivokasvaimen diagnoosin jĂ€lkeen on keskimÀÀrin 12–15 kuukautta (hoidon kanssa), ei-invasiivinen tarkka havaitseminen ja karakterisointi on tĂ€rkeÀÀ hoidon suunnittelussa. TĂ€llĂ€ hetkellĂ€ parhaat työkalut aivojen kuvantamiseen ovat magneettikuvaus (MRI), joka mahdollistaa parhaimman anatomisen tarkkuuden, ja positroniemissiotomografia (PET), joka paljastaa biokemiallisen informaation. Valitsemalle PET-kuvantamiseen radiolÀÀkeaine, joka kiinnittyy syöpĂ€solun pintaproteiineihin tai liittyy solun aineenvaihduntaprosessiin on mahdollista paitsi havaita tai rajata glioomia, myös saada erityistĂ€ biokemiallista tietoa nĂ€istĂ€ leesioista. TĂ€mĂ€n tutkimuksen tavoitteena oli arvioida kahta uutta radiolÀÀkeainetta; (2S, 4R)-4-[18F]fluoriglutamiinia ([18F]FGln) ja Al[18F]F-NOTA-folaattia ([18F]FOL) ja verrata niitĂ€ kliinisessĂ€ kĂ€ytössĂ€ oleviin 2-deoxy-2-[18F]fluori-ᮅ-glukoosiin ([18F]FDG) ja ʟ-[11C]metioniiniin ([11C]Met) glioomien PET-kuvantamisessa. Stabiilisuutta, biologista jakautumista, sitoutumista ja sitoutumisen salpautumista, sekĂ€ farmakokineettista mallintamista tutkittiin in vivo, ex vivo ja in vitro olosuhteissa elĂ€inmalleissa ja kudosnĂ€ytteillĂ€. Tulokset osoittivat, ettĂ€ elĂ€inmalleissa sekĂ€ [18F]FGln ettĂ€ [18F]FOL mahdollistavat paremman kontrastin tuumorin ja viereisen tuumorittoman aivokudoksen vĂ€lillĂ€ verrattuna kliinisessĂ€ kĂ€ytössĂ€ oleviin [18F]FDG ja [11C]Met radiolÀÀkeaineisiin
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