Oncogenic EGFR Represses the TET1 DNA Demethylase to Induce Silencing of Tumor Suppressors in Cancer Cells

SummaryOncogene-induced DNA methylation-mediated transcriptional silencing of tumor suppressors frequently occurs in cancer, but the mechanism and functional role of this silencing in oncogenesis are not fully understood. Here, we show that oncogenic epidermal growth factor receptor (EGFR) induces silencing of multiple unrelated tumor suppressors in lung adenocarcinomas and glioblastomas by inhibiting the DNA demethylase TET oncogene family member 1 (TET1) via the C/EBPα transcription factor. After oncogenic EGFR inhibition, TET1 binds to tumor suppressor promoters and induces their re-expression through active DNA demethylation. Ectopic expression of TET1 potently inhibits lung and glioblastoma tumor growth, and TET1 knockdown confers resistance to EGFR inhibitors in lung cancer cells. Lung cancer samples exhibited reduced TET1 expression or TET1 cytoplasmic localization in the majority of cases. Collectively, these results identify a conserved pathway of oncogenic EGFR-induced DNA methylation-mediated transcriptional silencing of tumor suppressors that may have therapeutic benefits for oncogenic EGFR-mediated lung cancers and glioblastomas

Synergistic tumor suppression by combined inhibition of telomerase and CDKN1A

Tumor suppressor p53 plays an important role in mediating growth inhibition upon telomere dysfunction. Here, we show that loss of the p53 target gene cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21WAF1/CIP1) increases apoptosis induction following telomerase inhibition in a variety of cancer cell lines and mouse xenografts. This effect is highly specific to p21, as loss of other checkpoint proteins and CDK inhibitors did not affect apoptosis. In telomerase, inhibited cell loss of p21 leads to E2F1- and p53-mediated transcriptional activation of p53-upregulated modulator of apoptosis, resulting in increased apoptosis. Combined genetic or pharmacological inhibition of telomerase and p21 synergistically suppresses tumor growth. Furthermore, we demonstrate that simultaneous inhibition of telomerase and p21 also suppresses growth of tumors containing mutant p53 following pharmacological restoration of p53 activity. Collectively, our results establish that inactivation of p21 leads to increased apoptosis upon telomerase inhibition and thus identify a genetic vulnerability that can be exploited to treat many human cancers containing either wild-type or mutant p53

Nations within a nation: variations in epidemiological transition across the states of India, 1990–2016 in the Global Burden of Disease Study

18% of the world's population lives in India, and many states of India have populations similar to those of large countries. Action to effectively improve population health in India requires availability of reliable and comprehensive state-level estimates of disease burden and risk factors over time. Such comprehensive estimates have not been available so far for all major diseases and risk factors. Thus, we aimed to estimate the disease burden and risk factors in every state of India as part of the Global Burden of Disease (GBD) Study 2016

In-vivo-Hinweise für ein neues Initiationsmodell der Translation in Bakterien

The canonical initiation mode starts with the small ribosomal subunit, which with help of initiation factors and the initiator tRNA finds the start signal for protein synthesis on an mRNA. However, there are many observations in the literature that cannot easily be reconciled with this initiation mode. We found that a resolution of these difficulties can be provided with an alternative initiation mode, the so-called 70S-scanning mode. In this thesis we provide in vivo evidences supporting it. Towards this end we identify features that critically participate in this process. 1\. According to general wisdom the initiation factor IF1 binds to 30S subunits helping IF2 and IF3 to select the ribosomal binding site on the mRNA. The factor is believed to leave the 30S subunit upon association with the large ribosomal subunit. Therefore, it should never be present on 70S ribosomes. We determined the ribosome location of IF1 using cytosol-profiles on sucrose gradients and identified IF1 via Western blotting. It was strikingly found that IF1 is present specifically on 70S and polysomes rather than on 30S subunits. IF3 is thought to be an anti-association factor and thus should not be able to bind to 70S ribosomes. We found it on 30S subunits as expected, but 1/3 of the amount was present on 70S and significant amounts even on disomes. Obviously, the functional horizon of these initiation factors is wider than thought before. 2\. IF1 is an essential factor. To study its function in vivo we constructed a strain, where the chromosomal IF1 was knocked out and the IF1 gene present on a plasmid after an inducible AraB promoter (in the presence of arabinose IF1 is expressed, in the presence of glucose it is not). The effects of insufficient IF1 amounts can be summarized as follows: (i) A slowed growth rate with doubled generation time. (ii) Serious defects in 50S assembly leading to accumulation of 50S-precursors, accumulation of 30S subunit and strikingly poor polysome pattern. Obviously, the block in 50S formation reduces the formation of 70S and thus explains the indispensability of IF1 for bacterial cell survival. The specific defects of the 50S assembly are consistent with an assumption of involvement of IF1 in scanning ribosomes leading to stoichiometric synthesis of ribosomal proteins. 3\. Our assumption was that IF1 is essential for the 70S scanning initiation. To test this we constructed a bicistronic mRNA expressing Renilla and Firefly luciferase, respectively. The hypothesis predicts that the first cistron might be preferentially initiated in the canonical mode, whereas for the second expression the IF1-dependent scanning mechanism would be more important. Precisely this was observed: The expression of the second cistron was 4.5 times reduced, when the cells were starved for IF1. Remarkably, the expression of the first cistron was practically not affected at all by reducing IF1 amounts. Western blotting showed that this expression-reduction was accompanied by reducing the IF1 amounts for about 50%. This observation distinctly presents the evidence of involvement of IF1 in sliding 70S ribosomes, leading to translation initiation of second cistron. 4\. Eventually we demonstrated that the 70S-scanning type of initiation also exists for the expression of monocistronic mRNAs. With a strong secondary structure in the 5’-UTR abolishing the scanning mode we observed that the expression of the reference protein GFP was not affected by various levels of IF1, whereas without secondary structure allowing both initiation modes the expression was strongly IF1 dependent. In summary we provided strong evidence that IF1 is a 70S specific factor and participates crucially in the 70S-scanning type of initiation. Since the 70S-scanning mode is importantly involved in the expression of both mono- and polycistronic mRNAs it might be even the prevailing initiation mode in the bacterial cell.Das Standardmodell der Initiation beginnt mit der kleinen Untereinheit, die mit Hilfe der Initiationsfaktoren und der Initiator fMet-tRNA das Startsignal für die Proteinsynthese auf einer mRNA findet. Jedoch sind einige Beobachtungen nicht einfach mit dem Standardmodell in Einklang zu bringen. Wir prüfen in der vorliegenden Arbeit eine Hypothese, nach der neben dem Standardmodell eine zweite Initiationsart existiert, das sogenannte 70S- Scanning Modell, das die Ungereimtheiten befriedengend erklären kann. Die erzielten Ergebnisse der in vivo Untersuchungen unterstützen das Modell: 1\. Nach allgemeiner Ansicht bindet der Initiationsfaktor IF1 an die kleine Untereinheit und unterstützt die Faktoren IF2 und IF3, die ribosomale Bindungsstelle für den Start der Proteinsynthese auf einer mRNA zu finden. Die Annahme ist, dass IF1 die 30S Untereinheit nach Assoziation der großen verläßt. Daraus folgt, daß dieser Faktor nicht auf 70S Ribosomen anzutreffen sein soll. Wir bestimmten die IF1 Lokalisation auf ribosomalen Partikeln mittels Cytosol-Profilen auf Sucrose-Gradienten und anschließender IF1 Identifikation mittels Western-Blot. Zu unserer Überraschung fanden wir IF1 ausschließlich auf 70S und zu einem geringen Anteil auf Disomen, aber nicht auf der kleinen Untereinheit. IF3 als Antiassoziationsfaktor wird für einen spezifischen 30S Faktor gehalten, der nicht auf 70S Ribosomen anzutreffen sein soll. Unsere Western-Analyse zeigte, dass wie erwartet ein großer Teil des IF3 wie erwartet an die 30S Untereinheit bindet, aber etwa ein Drittel der Menge an 70S Ribosomen bindet. Es folgt daraus, dass der funktionelle Horizont beider Faktoren offenbar weiter reicht als allgemein angenommen. 2\. IF1 ist ein essentieller Faktor. Um dessen Funktionen in vivo zu testen, haben wir einen E. coli Stamm konstruiert, dem das chromosomale IF1-Gen fehlt und der Zelle auf einem Plasmid angeboten wird, dessen AraB Promoter die IF1 Synthese an- und ausschalten kann (in Gegenwart von Arabinose wird IF1 synthetisiert, während Glucose die Synthese abschaltet). Die Effekte einer IF1 Verarmung können folgendermaßen zusammengefaßt werden: (i) Eine 50% Reduktion der IF1 Menge halbiert die Wachstumsrate. (ii) Die Bildung der großen 50S Untereinheit ist schwer geschädigt, 50S Vorstufen werden angehäuft, 30S Untereinheiten akkumulieren mit dem Ergebnis, dass 70S Ribosomen sowie Polysomen deutlich vermindert sind. Die spezifischen Defekte des 50S Aufbaus können damit erklärt werden, daß IF1 an einem 70S-Scanning Initiations-modus beteiligt ist, was zu einer stöchiometrischen Synthese der ribosomalen Proteine führt. 3\. Unsere Annahme, dass IF1 wichtig für eine 70S-Scanning Initiation ist, wurde folgendermaßen getestet: wir konstruierten eine bi-cistronische mRNA, mit der die Renilla und die Feuerfliegen Luziferase exprimiert werden kann. Die produzierte Menge beider Luziferasen können in einem Ansatz ohne Überlappung getestet werden. Unsere Hypothese sagt voraus, dass das erste Cistron vornehmlich nach dem kanonischen 30S Modell initiiert wird, während bei der Expression des zweiten Cistrons der IF1 abhängige Scanning-Modus deutlicher beteiligt ist. Genau das wurde beobachtet: Eine IF1 Reduktion um 50% reduzierte die Translation des zweiten Cistrons um das 4,5 fache, während interessanterweise die Translationsleistung am ersten Cistron durch reduzierten IF1 Gehalt gar nicht beeinträchtigt wurde. Dieser Befund ist eine deutliche Unterstützung des Scanningmodells und belegt zum ersten Mal, dass IF1 wahrscheinlich ein spezifischer 70S Initiationsfaktor ist und eine geringe Rolle – wenn überhaupt – bei der kanonischen 30S Initiation spielt. 4\. Schließlich haben wir gezeigt, dass der 70S-Scanning Typ auch bei der Translation von mono-cistronischen mRNAs beteiligt ist. Bei einer mRNA mit einer starken Sekundärstruktur an der 5’-UTR, die 70S-Scanning verhindert aber eine 30S abhängige Initiation erlaubt, ist die Expression von GFP unabhängig von der IF1 Menge in der Zelle, während ohne Sekundärstruktur die GFP Synthese stark von der IF1 Menge abhängig war. Zusammengefasst, haben wir sehr starke Hinweise, dass IF1 ein spezifischer 70S-Scanning Initiationsfaktor ist, der eine bedeutende Rolle bei diesem neuen Typ der bakteriellen Initiation spielt. Da die 70S-Scanning Initiation sowohl bei der Initiation von poly- als auch mono-cistronischen mRNAs eine Rolle spielt, könnte dieser Initiationstyp sogar der in der bakteriellen Zelle vorherrschende sein

Interleukin 8 is a biomarker of telomerase inhibition in cancer cells

Abstract Background Telomerase activity is required for both initiation and maintenance of tumorigenesis and over 90% cancers overexpress telomerase. Therefore, telomerase targeting has emerged as a potential strategy for cancer treatment. In agreement with this, several telomerase inhibitors are being tested for cancer treatment and have shown some promise. However, because of the variability in response between the cancer patients, it is important to identify biomarkers that allow for distinguishing cancers that are responsive to telomerase inhibition from the cancers that are not. Therefore, in this study we performed experiments to identify a biomarker that can be used to predict telomerase inhibition induced tumor growth inhibition. Methods In our study, we have performed transcriptome-wide gene expression analysis on multiple ovarian and colon cancer cell lines that were treated with telomerase inhibitor imetelstat and were responsive to telomerase inhibition-induced tumor growth attenuation. Results We demonstrate that telomerase inhibition by telomerase inhibitor imetelstat results in decreased expression of interleukin 8 (IL8) in all telomerase responsive cancer cell lines. This phenomenon is of general occurrence because we find that multiple ovarian and colon cell lines show decrease in IL8 mRNA and protein levels after telomerase inhibition. Additionally, we find loss of IL8 phenocopy Telomerase inhibition mediated growth inhibitory effect in cancer cells. Conclusion Taken together, our results show that IL8 is a biomarker that predict telomerase inhibition mediated growth attenuation of cancer cells and its loss phenocopy telomerase inhibition. Therefore, IL8 expression can be utilized as a biomarker for telomerase targeted cancer therapies to potentially predict therapeutic response

The BRD9/7 Inhibitor TP-472 Blocks Melanoma Tumor Growth by Suppressing ECM-Mediated Oncogenic Signaling and Inducing Apoptosis

Melanoma accounts for the majority of all skin cancer-related deaths and only 1/3rd of melanoma patients with distal metastasis survive beyond five years. However, current therapies including BRAF/MEK targeted therapies or immunotherapies only benefit a subset of melanoma patients due to the emergence of intrinsic or extrinsic resistance mechanisms. Effective treatment of melanoma will thus require new and more effective therapeutic agents. Towards the goal of identifying new therapeutic agents, we conducted an unbiased, druggable epigenetic drug screen using a library of 32 epigenetic inhibitors obtained from the Structural Genome Consortium that targets proteins encoding for epigenetic regulators. This chemical genetic screening identified TP-472, which targets bromodomain-7/9, as the strongest inhibitor of melanoma growth in both short- and long-term survival assays and in mouse models of melanoma tumor growth. Mechanistically, using a transcriptome-wide mRNA sequencing profile we identified TP-472 treatment downregulates genes encoding various extracellular matrix (ECM) proteins, including integrins, collagens, and fibronectins. Reactome-based functional pathway analyses revealed that many of the ECM proteins are involved in extracellular matrix interactions required for cancer cell growth and proliferation. TP-472 treatment also upregulated several pro-apoptotic genes that can inhibit melanoma growth. Collectively, our results identify BRD7/9 inhibitor TP-472 as a potentially useful therapeutic agent for melanoma therapy

DESIGN AND STATISTICAL EVALUATION OF A MULTIUNIT DELIVERY SYSTEM CONTAINING NISOLDIPINE-SOLUPLUS® SOLID DISPERSION FOR HYPERTENSION CHRONOTHERAPY

Objective: To study the mechanism and factors affecting the design of an industrially scalable formulation in a combined drug delivery module containing solid dispersion (SD) multiunit pellets with novel polymer Soluplus® in a modified release system to address chronotherapeutic needs of hypertension therapy.Methods: Nisoldipine-Soluplus® SD pellet formulations were prepared using the central composite design of experiments (CCD) to study the effect of inert core level and drug to polymer ratio. The solid dispersions were formed on inert pellets surface by fluidized bed coating and characterized by dissolution efficiency and time for 90% drug release. The data was statistically analyzed to develop a response surface for optimum SD formulation in pellets. The SD pellets were characterized by FTIR, DSC and SEM. The optimum formulation of SD coated pellets was further coated with Eudragit S100-L100 polymer mix and characterized for dissolution in multimedia and two-step dissolution for lag time.Results: A response surface was developed for highest dissolution efficiency (%DE) and least time to release 90% drug (T90). The model was significant, and the role of core pellets was found to be more significant than the drug-polymer ratio. The study of the desirability function indicated that a polymer content of 75% and inert core level to yield 23% net weight gain, provided optimum dissolution enhanced SD pellets. The drug was found to exist in amorphous form. The final capsules containing Eudragit S100-L100 coated delayed release SD pellets showed a lag time of 2 h and a definite pH-gradient towards drug release.Conclusion: The findings from this study helped to understand the mechanism, design and factors affecting drug release from a delayed release SD system for a poorly soluble drug for potential hypertension chronotherapy

Negative pressure wound therapy in the treatment of ulcerated infantile haemangioma

Background: Infantile haemangioma is a common benign tumour of infancy. Ulceration is the most common complication and is often painful and difficult to treat. Propranolol therapy is widely used to induce involution in rapidly growing or ulcerated lesions, or those in anatomically awkward locations. The ideal dressing regimen for these lesions would provide effective analgesia, act as a wound dressing, and aid involution of the primary lesion. To date, no ideal regimen has been established. Negative pressure wound therapy (NPWT) has been used in adult and paediatric populations to help improve wound healing in a variety of settings. It may provide a useful alternative to traditional dressing regimens in ulcerated infantile haemangioma. Methods: Six consecutive patients with ulcerating infantile haemangioma presenting to the Royal Children’s Hospital vascular anomalies clinic were included in the study. Each patient was treated with a combination of NPWT and propranolol. Outcomes including time to wound healing, perceived ease of dressing management, and analgesia were recorded. Results: Complete wound healing was obtained in all cases. Patient outcomes in terms of analgesia, comfort, and ease of wound dressing were improved following application of NPWT. Discussion/Conclusions: We propose that this regimen represents a novel therapy for management of ulcerated infantile haemangioma. Possible mechanisms for healing effect, and improved analgesia are discussed. Further investigation is required to determine if negative pressure wound therapy results in faster healing times compared to traditional dressing regimens