Photoswitchable Azo- and Diazocine-Functionalized Derivatives of the VEGFR-2 Inhibitor Axitinib

In this study, we aimed at the application of the concept of photopharmacology to the approved vascular endothelial growth factor receptor (VEGFR)-2 kinase inhibitor axitinib. In a previous study, we found out that the photoisomerization of axitinib's stilbene-like double bond is unidirectional in aqueous solution due to a competing irreversible [2+2]-cycloaddition. Therefore, we next set out to azologize axitinib by means of incorporating azobenzenes as well as diazocine moieties as photoresponsive elements. Conceptually, diazocines (bridged azobenzenes) show favorable photoswitching properties compared to standard azobenzenes because the thermodynamically stable Z-isomer usually is bioinactive, and back isomerization from the bioactive E-isomer occurs thermally. Here, we report on the development of different sulfur-diazocines and carbon-diazocines attached to the axitinib pharmacophore that allow switching the VEGFR-2 activity reversibly. For the best sulfur-diazocine, we could verify in a VEGFR-2 kinase assay that the Z-isomer is biologically inactive (IC50 >> 10,000 nM), while significant VEGFR-2 inhibition can be observed after irradiation with blue light (405 nm), resulting in an IC50 value of 214 nM. In summary, we could successfully develop reversibly photoswitchable kinase inhibitors that exhibit more than 40-fold differences in biological activities upon irradiation. Moreover, we demonstrate the potential advantage of diazocine photoswitches over standard azobenzenes

Analyzing the influence of kinase inhibitors on DNA repair by differential proteomics of chromatin-interacting proteins and nuclear phospho-proteins

The combination of radiotherapy and pharmacological inhibition of cellular signal transduction pathways offers promising strategies for enhanced cancer cell inactivation. However, the molecular effects of kinase inhibitors especially on DNA damage detection and repair after X-irradiation have to be understood to facilitate the development of efficient and personalized treatment regimens. Therefore, we applied differential proteomics for analyzing inhibitor-induced changes in either chromatin-bound or phosphorylated nuclear proteins. The effect of the multi kinase inhibitor sorafenib on DNA repair, chromatin binding and phosphorylation of nuclear proteins was analyzed in UT-SCC 42B head and neck cancer cells using metabolic labeling based differential proteomics (SILAC). Sorafenib significantly inhibited DNA repair but failed to significantly affect chromatin interactions of 90 quantified proteins. In contrast, analyzing nuclear phospho-proteins following sorafenib treatment, we detected quantitative changes in 9 out of 59 proteins, including DNA-repair proteins. In conclusion, the analysis of nuclear phospho-proteins by differential proteomics is an effective tool for determining the molecular effects of kinase inhibitors on X-irradiated cells. Analyzing chromatin binding might be less promising

Light-controlled inhibition of BRAFV600E kinase

Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAFV600E mutant kinase. However, systemic inhibition of BRAF by small molecule drugs in cancer patients results - paradoxically - in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAFV600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors containing an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approximately 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAFV600E inhibitor with high spatial and temporal resolution, thus limiting the damage to other tissues

Investigation of the influence of the nonstructural protein 5A of hepatitis C virus on cell proliferation, apoptosis and carcinogenesis in vitro and in vivo

Titelblatt und Inhaltsverzeichnis Einleitung Zielsetzung Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Literatur VerzeichnisseDas Hepatitis C Virus ist ein membranumhülltes Virus mit einem +SS-RNA Genom aus der Familie der Flaviviridae. Sein Genom codiert für die Strukturproteine Core, E1 und E2, das p7-Protein und für die Nicht-Strukturproteine NS2, NS3, NS4A, NS4B, NS5A und NS5B. Da das Nicht-Strukturpotein 5A unter anderem in der Lage ist, mit zentralen Signalwegen für Zellwachstum und Differenzierung zu interferieren, wird dem Protein eine Rolle bei der HCV-assoziierten Entstehung von Leberpathologien bis hin zur HCC-Bildung zugeschrieben. Im ersten Teil dieser Arbeit wurde der Einfluss der HCV-Replicon-Zelllinien huh9-13 und huh ET auf die rezeptorvermittelte Apoptose untersucht. Dabei konnte mit Hilfe von Western Blot-Analysen und durchflusszytometrischen Quantifizierungen apoptotischer Zellen, eine gesteigerte Sensitivität von huh9-13 aber nicht von huh ET -Zellen im Bezug auf TNFα\- und TRAIL-vermittelte Apoptose, verglichen mit der Ausganszelllinie huh7, beobachtet werden. Diese Unterschiede spiegelten sich auch im Expressionsmuster von apoptoserelevanten Genen wieder, wie durch Membranen-Arrays beobachtet werden konnte. Nach Entfernung der HCV- Replicon-RNA aus den Zellen mittels IFNα-Behandlung zeigten so behandelte huh9-13 Zellen keine gesteigerten Sensitivitäten mehr, was für einen Einfluss des entsprechenden Replicons auf die rezeptorvermittelter Apoptose offenbart. Im Falle der huh ET Zellen hatte die IFN-Behandlung keinen Effekt. Zudem konnte durch die Verwendung von Etoposide und Actinomycin D gezeigt werden, dass der intrinsische Apoptoseweg durch die HCV-Replicons nicht beeinflusst wurde, jedoch wurde ein starker Einfluss des jeweiligen Zellhintergrundes beobachtet. Im zweiten Teil dieser Arbeit wurde der Effekt von NS5A auf den Raf-1/ERK-abhängigen MAP-Kinasesignalweg näher untersucht. Bei der Verwendung von phosphospezifischen Antikörpern konnte nach NS5A-Expression eine gesteigerte Phophorylierung von Raf-1 an dem Serinrest 338 beobachtet werden. Jedoch wurde keine Steigerung der MEK1/2-Phosphorylierung an den Serinresten 217/221 festgestellt. Durch die Verwendung von NS5A-Deletionsmutanten konnte die Region AS 302-449 als ausreichend für die gesteigerte Raf-1 Phosphorylierung identifiziert werden. Dabei korrelierte die Lokalisation der Deletionsmutanten mit der Art des Effektes auf die Raf-1-Phosphorylierung. Durch die Verwendung verschiedener Inhibitoren konnte zudem gezeigt werden, dass die Raf-1-Phosphorylierung unabhängig von der PKC-Aktivität war. Ein dritter Schwerpunkt dieser Arbeit lag auf der Charakterisierung einer neuen NS5A-transgenen Maus zur Untersuchung einer möglichen Rolle von NS5A bei der HCV-assoziierten Leberschädigung. Mit Hilfe von Western Blot-Analysen konnte eine robuste, leberspezifische und über die Zeit stabile NS5A-Expression in männlichen sowie in weiblichen Tieren festgestellt werden. Zudem war die Expressionsstärke bei zwei verschiedenen founder-Linien vergleichbar. Es konnte keine Tumorentstehung oder Zellschädigung in den Lebern transgener Tiere festgestellt werden, auch nicht nach der Bestrahlung der Tiere mit einer subletalen Dosis von 3 Gy. Um Transgen-bedingte Änderungen der Genexpression zu untersuchen, wurden Microarray- und Proteomanalysen von transgenen und Kontrolltieren durchgeführt. Dabei konnte sowohl auf mRNA-Ebene als auch auf Protein-Ebene gezeigt werden, dass die Expression unterschiedlicher Gene durch NS5A beeinflusst wurde. Um den postulierten inhibitorischen Effekt von NS5A auf die IFN-Antwort zu untersuchen, wurden transgene und WT-Mäuse mit LCMV infiziert. Dabei konnte im Falle der transgenen Tieren eine inhibierte Viruseliminierung und eine gesteigerte GPT-Aktivität in den Seren nachgewiesen werden. Zudem wurde in transgenen Lebern eine verminderte Induktion von 2´,5´-OAS und PKR festgestellt. Diese Daten sprechen für einen inhibitorischen Einfluss von NS5A auf die IFN-abhängige antivirale Antwort der Zellen, was zu einer Chronifizierung der HCV-Infektion beitragen könnte.The hepatitis C virus (HCV) is an enveloped positive-strand RNA virus which belongs to the family of the Flaviviridea. The genome codes for the structural proteins core, E1 and E2 and the nonstructural proteins p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. Because the nonstructural protein 5A is able to deregulate important signalling cascades for cell proliferation and differantiation, the protein is thought to play a major role in the development of HCV-associated HCC progression. In the first part of this work the influence of two subgenomic HCV replicons, huh9-13 and huh ET cells, on death receptor-mediated and intrinsic apoptosis was investigated. Based on Western blot experiments and FACS analysis an increased sensitivity of huh9-13 but not of huh ET replicon cells towards TNFα\- and TRAIL-mediated apoptosis compared to huh7 control cells was observed. This difference in the sensitivity to death receptor-mediated apoptosis is reflected in a variety of differentially regulated genes involved in apoptosis induction as determined by a membrane array. After elimination of the replicon RNA by IFNα treatment, the increased sensitivity of huh9-13 cells was abolished elucidating an influence of the HCV replicon on receptor mediated apoptosis for huh9-13 cells. In the case of huh ET cells the interferon treatment did not restore the sensitivity to TNFα-dependent apoptosis. Using etoposide and actinomycin D for the induction of apoptosis, no effect of the replicon RNA on the intrinsic apoptotic pathway could be detected, however strong effects of the cellular background have been detected. In the second part of this work the effect of NS5A on MAPK signalling was examined. Using phophospecific antibodies an increased Raf-1 phosphorylation at serin 338 was observed, which was caused by NS5A expression. Unexpectedly, the increased Raf-1 phosphorylation had no effect on MEK1/2 phosphorylation at serin 217/221. Investigating different NS5A deletion mutants, a region between amino acid 302-449 was identified to be sufficient for increased Raf-1 phosphorylation. Thereby a correlation of NS5A mutant localisation and measured effect on Raf-1 phosphorylation has been observed. Inhibition of different kinases involved in MAPK signalling revealed no participation of PKC. In the third part of this work a new NS5A transgenic mouse was characterized to elucidate the potential role of NS5A for HCV- associated liver damage. The mice were genotyped using PCR. Robust, liver specific and long term transgene expression was monitored by Western blot analysis showing an equivalent NS5A expression in two different founder strains. In transgenic mice no tumour formation or liver damage was detected not even after irradiation of mice with sub-lethal doses of 3 Gy gamma radiations. To investigate alterations in gene expression triggered by NS5A, transgenic mice and wild type littermates were subjected to microarray and proteome analysis detecting several genes altered on mRNA or protein level. To control the postulated effect of NS5A on the interferon response the mice were infected with LCMV. In the transgenic mice a reduced virus elimination and elevated GPT-activity in the sera was observed. On the molecular level an impaired induction of 2´,5`-OAS and of PKR was observed in transgenic liver samples. These results argue for an inhibitory effect of NS5A on interferon- mediated antiviral response which could mediate a chronic progression in case of HCV infection

Knockdown of AKT3 Activates HER2 and DDR Kinases in Bone-Seeking Breast Cancer Cells, Promotes Metastasis In Vivo and Attenuates the TGFβ/CTGF Axis

Bone metastases frequently occur in breast cancer patients and lack appropriate treatment options. Hence, understanding the molecular mechanisms involved in the multistep process of breast cancer bone metastasis and tumor-induced osteolysis is of paramount interest. The serine/threonine kinase AKT plays a crucial role in breast cancer bone metastasis but the effect of individual AKT isoforms remains unclear. Therefore, AKT isoform-specific knockdowns were generated on the bone-seeking MDA-MB-231 BO subline and the effect on proliferation, migration, invasion, and chemotaxis was analyzed by live-cell imaging. Kinome profiling and Western blot analysis of the TGFβ/CTGF axis were conducted and metastasis was evaluated by intracardiac inoculation of tumor cells into NOD scid gamma (NSG) mice. MDA-MB-231 BO cells exhibited an elevated AKT3 kinase activity in vitro and responded to combined treatment with AKT- and mTOR-inhibitors. Knockdown of AKT3 significantly increased migration, invasion, and chemotaxis in vitro and metastasis to bone but did not significantly enhance osteolysis. Furthermore, knockdown of AKT3 increased the activity and phosphorylation of pro-metastatic HER2 and DDR1/2 but lowered protein levels of CTGF after TGFβ-stimulation, an axis involved in tumor-induced osteolysis. We demonstrated that AKT3 plays a crucial role in bone-seeking breast cancer cells by promoting metastatic potential without facilitating tumor-induced osteolysis

A Subset of Histone H2B Genes Produces Polyadenylated mRNAs under a Variety of Cellular Conditions

<div><p>Unlike other metazoan mRNAs, replication-dependent histone gene transcripts are not polyadenylated but instead have a conserved stem-loop structure at their 3′ end. Our previous work has shown that under certain conditions replication-dependent histone genes can produce alternative transcripts that are polyadenylated at the 3′ end and, in some cases, spliced. A number of microarray studies examining the expression of polyadenylated mRNAs identified changes in the levels of histone transcripts e.g. during differentiation and tumorigenesis. However, it remains unknown which histone genes produce polyadenylated transcripts and which conditions regulate this process. In the present study we examined the expression and polyadenylation of the human histone H2B gene complement in various cell lines. We demonstrate that H2B genes display a distinct expression pattern that is varies between different cell lines. Further we show that the fraction of polyadenylated <i>HIST1H2BD</i> and <i>HIST1H2AC</i> transcripts is increased during differentiation of human mesenchymal stem cells (hMSCs) and human fetal osteoblast (hFOB 1.19). Furthermore, we observed an increased fraction of polyadenylated transcripts produced from the histone genes in cells following ionizing radiation. Finally, we show that polyadenylated transcripts are transported to the cytoplasm and found on polyribosomes. Thus, we propose that the production of polyadenylated histone mRNAs from replication-dependent histone genes is a regulated process induced under specific cellular circumstances.</p></div

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure

Polyadenylation of histone H2B genes assigned using polyadenylation and alternative polyadenylation (APA) map.

<p>Number of reads at polyA sites on different replication- dependent histone H2B genes which are mapped using xPAD server (<a href="http://johnlab.org/xpad/" target="_blank">http://johnlab.org/xpad/</a>) in (A) MCF7 (human breast cancer cell line), (B) MCF10A (immortalized human mammary epithelial cell line) cells and (C) normal and breast tumor tissues.</p

Radiation induced elevated expression of spliced histone transcripts.

<p>A549 cells were exposed to gamma-irradiation (6 Gy) and incubated for 24 hours. RNA was extracted and analyzed by qRT-PCR for (A) spliced and (B) total <i>HIST1H2BD</i> and <i>HIS1H2AC</i>. Values were normalized to <i>RPLP0</i>. Mean±SD, n = 3. (C) Expression of spliced <i>HIST1H2BD</i> and <i>HIST1H2AC</i> transcripts was normalized to the total <i>HIST1H2BD</i> and <i>HIST1H2AC</i> levels. P-values were calculated and statistical significance is represented as follows (*P≤0.05; **P≤0.01).</p

Expression of normal and PolyA⁺<i>HIST1H2BD</i> and <i>HIST1H2AC</i> transcripts in HCT116 cells.

<p>(A) Cells were treated with Nutlin-3a as in Fig. 2. RNA was reverse transcribed into cDNA using both random and poly-T primers to check the mRNA levels of <i>HIST1H2BD</i> and <i>HIST1H2AC</i>, total and polyadenylated transcripts respectively. Values were normalized to <i>RPLP0</i> expression. Mean±SD, n = 3. (B) Enrichment for polyadenylated histone transcripts using PolyATtract® mRNA Isolation System III. Total RNA was used to isolate polyadenylated RNA and reverse transcribed using poly-T primers. Expression of total and polyA⁺<i>HIST1H2BD</i> and <i>HIST1H2AC</i> transcripts was analyzed by qRT-PCR. Values were normalized to <i>RPLP0</i> expression. Mean±SD, n = 3. P-values were calculated and statistical significance was represented as follows (**P≤0.01; ***P≤0.001).</p