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Item Open Access Die Rolle des p53-Status für die Sensitivität von Tumoren gegenüber unterschiedlichen p53 Aktivatoren(2014) Weilbacher, Andrea; Peter Scheurich (Prof. Dr.)Die tumorsuppressiven Eigenschaften von p53 gelten als zentral bei der Aufrechterhaltung der genomischen Stabilität. Auf Grund dessen spielt p53 eine Schlüsselrolle bei der Reaktion auf genomischen Stress welcher unter anderem durch klassische Chemotherapeutika, wie beispielsweise Cisplatin, induziert wird. In der vorliegenden Studie wurde die Rolle des p53-Status für die Sensitivität gegenüber p53-aktivierenden Substanzen untersucht. Hierfür wurde eine Auswahl von Zelllinien aus verschiedenen Entitäten und mit unterschiedlichen p53-Genotypen sowie Tumor-assoziierte Fibroblasten aus der humanen Lunge und PBMNCs von gesunden Donoren verwendet. Als p53-Aktivatoren wurden neben dem klassischen DNA-modifizierenden Molekül Cisplatin die direkten Aktivatoren von p53, Nutlin-3 und RITA eingesetzt. Die Behandlung mit Nutlin-3 führte selektiv in wtp53-exprimierenden Zellen zu einem G1-Arrest. Dieser trat auch in primären, nichtmalignen Zellen auf. Nutlin-3 agiert somit selektiv in wtp53-exprimierenden Zellen, nicht aber tumorselektiv. Die wtp53-selektive Wirkungsweise konnte weder nach Cisplatin- noch nach RITA-Behandlung nachgewiesen werden. Beide Substanzen induzierten Zelltod auch in mtp53-Systemen oder im Falle von RITA auch in der p53-null-Zelllinie OVCAR5. Der durch Cisplatin und RITA induzierte Zelltod in der wtp53-exprimierenden Zelllinie NTERA-2D1 konnte auf die Aktivierung von wtp53 zurückgeführt werden. Hingegen war der in den mtp53-exprimierenden Zelllinien OVCAR3 und OVCAR4 induzierte Zelltod im Falle einer Behandlung mit Cisplatin oder RITA unabhängig von mtp53. Zudem führte die siRNA-vermittelte Depletion von p63 und p73 zu keiner Verminderung des Zelltods. Cisplatin und RITA können somit unabhängig von der p53-Superfamilie Zelltod in p53-defekten Systemen induzieren. Dieser war für beide Substanzen auf die Aktivierung der mitochondrialen Effektoren BAX und BAK zurückzuführen. Die Induktion von Zelltod nach Cisplatin-Behandlung konnte weiterhin auf die Aktivierung der pro-apoptotischen Bcl-2-Proteine NOXA und PUMA zurückgeführt werden. Die Analyse der konstitutiven Expression der Bcl-2-Proteine in der gesamten Zelllinienauswahl zeigte eine signifikante Korrelation des Verhältnisses aus pro- und anti-apoptotischen Bcl-2-Proteinen gegenüber der Cisplatin-Sensitivität. NOXA und Bcl-w wurden in diesem Ansatz als prädiktive Marker der Cisplatin-Sensitivität innerhalb der Zellauswahl identifiziert. Die Kombinationsbehandlung von Cisplatin mit dem BH3-mimetic ABT-737 führte zu einer Sensitivierung von Cisplatin-insensitiven Zellen. Im Falle der Behandlung mit RITA konnte keine Korrelation zwischen dem Verhältnis aus pro- und anti-apoptotischen Bcl-2-Proteinen und dem durch RITA induzierten Zelltod festgestellt werden. Jedoch erwies sich die Herunterregulation von anti-apoptotischen Bcl-2-Proteinen nach RITA-Behandlung als wichtig für die Induktion von Apoptose. Infolgedessen führte die Kombinationsbehandlung von RITA mit ABT-737 zu einer Verstärkung des RITA-induzierten Zelltods in RITA-sensitiven Zellen. RITA-insensitive Zellen blieben dabei unbeeinflusst. Weiterhin konnte der durch RITA vermittelte Zelltod in p53-defekten Systemen auf die Aktivierung des JNK- und p38-Signaltransduktionsweges zurückgeführt werden. Insbesondere JNK1 erwies sich als entscheidend für die Induktion von Apoptose nach RITA-Behandlung. Im Vergleich der drei Substanzen zeigte sich überraschenderweise eine größere Ähnlichkeit von RITA zu Cisplatin als zu Nutlin-3. Cisplatin, als klassischer über DNA-Schädigung wirkender p53-Aktivator, führte zur Induktion von Zelltod in Zelllinien welche nahezu alle auch sensitiv gegenüber der Behandlung mit RITA waren. Potentiell könnte somit auch RITA über DNA-Schädigungen Zelltod induzieren. Intrazellulär führen jedoch beide Substanzen zu unterschiedlichen Effekten. Während Cisplatin zu einer Hochregulation von pro-apoptotischen BH3-only-Proteinen führt, induziert die Behandlung mit RITA eine Reduktion anti-apoptotischer Bcl-2-Proteine. Im Falle von Cisplatin konnte die Proteinkonzentration von NOXA und Bcl-w als Marker für die Sensitivität innerhalb der Zellauswahl identifiziert werden. RITA hingegen induzierte Zelltod nur in einer bestimmten Gruppe von Zellen, weshalb der Transport von RITA ein potentieller Marker für die RITA-Sensitivität darstellen könnte. Zusammenfassend konnte in der verwendeten Zellauswahl sowohl nach Nutlin-3-, als auch nach Cisplatin- oder RITA-Behandlung ein Einfluss von wtp53 für die Sensitivität nachgewiesen werden. Allerdings konnten durch Cisplatin und RITA auch Effekte unabhängig von p53 vermittelt werden. Interessanterweise führte die Behandlung mit RITA zu einer von der p53-Superfamilie und von der Aktivierung des JNK-Signaltransduktionsweges unabhängigen Regulation von p53-Zielgenen. Dementsprechend können im p53-defekten System p53-Zielgene sowie typische p53-Funktionen durch die Aktivierung p53-unabhängiger Signalwege vermittelt werden.Item Open Access Comparative analysis of the nuclear receptors CAR, PXR and PPARα in the regulation of hepatic energy homeostasis and xenobiotic metabolism(2014) Kandel, Benjamin; Zanger, Ulrich (Prof. Dr.)Nuclear receptors (NRs), most notably the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR), regulate the transcription of several drug metabolizing enzymes and transporters (DMET) and thus represent important regulators of drug metabolism in the liver. Accordingly, the ligand dependent activation of these NRs by drugs and other xenobiotics contributes to the intra- and inter-individual variability of the drug detoxifying system. CAR and PXR were further shown to regulate the transcription of key enzymes involved in lipid and glucose metabolism. The NR peroxisome proliferator-activated receptor alpha (PPARa), a key regulator of fatty acid catabolism and target of lipid lowering fibrates, was recently identified as a direct regulator of cytochrome P450 3A4 (CYP3A4) and also potentially of other DMET genes. In this respect, CAR, PXR and PPARa are determinants of an overlapping number of liver functions including drug metabolism and energy homeostasis and are therefore associated with adverse drug reactions as well as liver disease like steatosis. Until now there have been no comparative studies investigating the transcriptomes of CAR, PXR and PPARa in humans. Therefore, a major focus of this study was to assess the genome-wide transcriptional changes provoked by these NRs in primary human hepatocytes (PHHs). To investigate human liver-specific gene expression and its regulation PHHs represent the most suitable available in vitro cell system. To identify the CAR-, PXR- and PPARa-specific genome-wide expression changes, hepatocyte cultures from six individual donors were treated with the prototypical ligands for CAR (CITCO), PXR (rifampicin) and PPARa (WY-14643) as well as DMSO (vehicle control). Afterwards, the mRNA expression in these samples was determined utilizing Affymetrix® microarrays. The obtained expression data were statistically evaluated to identify the genes that showed a differential expression in response to the agonist treatments and to investigate to which metabolic functions these genes contribute. The results of these experiments confirmed that CAR, PXR and PPARa regulated a highly overlapping but distinct set of genes coding for DMET. For example, according to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses expression of 10 DMET genes were shown to be regulated by all three NRs, whereas other DMET genes responded exclusively to the activation of one of the NRs. In addition several DMET related genes previously not shown to be regulated by CAR [like CYP2E1, sulfotransferase 1B1 (SULT1B1), UDP-glucuronosyltransferase 2B4 (UGT2B4) and cytochrome P450 reductase (POR)], PXR [like CYP2E1, alcohol dehydrogenases (ADHs), flavin containing monooxygenase 5 (FMO5) and glutathione peroxidase 2 (GPX2)] or PPARa like UBT2B4, ADH1s and FMO5) were identified to respond to the respective agonists. For PXR and CAR, this extends the list of genes by which these NRs influence drug metabolism and potentially contribute to drug-drug interactions (DDIs). The results obtained further specify the role of PPARa as a regulator of drug metabolism in vitro by increasing expression of, e.g., CYP3A4, 2B6, 2C8 and UGT1A1, thus pointing to a potential role of PPARa in adverse drug reactions in vivo. Furthermore, several genes coding for proteins involved in energy homeostasis, were identified as differentially expressed in response to PXR activation [e.g., pyruvate dehydrogenase kinase 4 (PDK4), glycogen synthase 2 (GYS2), carnitine palmitoyltransferase 2 (CPT2)], where such a relation was not reported so far. These results further expanded the knowledge of how PXR potentially impact fatty acid catabolism, gluconeogenesis and lipid de novo synthesis and provide interesting starting points to investigate how PXR activation contributes to altered glucose and lipid levels or disease like hepatic steatosis. Besides ligand-dependent regulation of nuclear receptors, post-translational modification has also been shown to influence the activity of liver-enriched NRs and expression of their target genes. In this context, protein kinase A (PKA) had been shown to repress CYP3A4 expression via PXR in a species-dependent manner, whereas the influence of PKA on the expression of other DMET genes had not been investigated in detail so far. The second part of this work therefore investigated the impact of PKA activation on the expression and activity of important drug metabolizing enzymes in a PXR- as well as a CAR-dependent manner. In this work PKA activation in primary human hepatocytes was identified as a determinant of drug metabolism in vitro by repressing PXR- and CAR-mediated or reducing basal expression and activity of CYP1A1, CYP2B6, CYP2C8 and CYP3A4, but also expression of ATP-binding cassette B1 (ABCB1) and UGT1A1. Using reporter gene assays, these observed effects could be linked to PKA-mediated repression of PXR and CAR activity that may involve phosphorylation of these NRs. It could be further shown that expression of DMET genes was also repressed by the fasting hormone glucagon, a physiologically relevant activator of PKA signaling, which was not investigated in humans so far. Due to the promiscuous ligand-specificity of PXR, which includes numerous compounds, drug treatment often leads to PXR activation, even with so-called “natural” compounds like St. John’s wort (SJW). It would thus be highly desirable to develop strategies in drug development to assess or circumvent the activation of NRs without compromising the pharmacological effects. Therefore, the last part of this work consists of an in vitro study to investigate synthetic acylated phloroglucinols, designed as substitutes for hyperforin, regarding their potential to activate PXR. Hyperforin the major active constituent of the plant SJW used to treat depressions was shown to exert its antidepressant properties via indirect inhibition of serotonin reuptake by selectively activating the canonical transient receptor potential channel 6 (TRPC6). In addition, hyperforin is associated with clinically relevant drug-drug interactions in patients that had taken SJW concomitantly with other drugs due to potent activation of the nuclear receptor PXR by hyperforin. The phloroglucinol derivatives investigated in this thesis had previously been evaluated for their bioactivity. It had been reported that five of the nine synthetic acylated phloroglucinols activate TRPC6 with similar potency as hyperforin. In this work, all these nine synthetic phloroglucinol derivatives were investigated in comparison to hyperforin and rifampicin for their potential to activate PXR. Hyperforin and rifampicin treatment of HepG2 cells co-transfected with a human PXR expression vector and a CYP3A4 promoter reporter construct resulted in potent PXR-dependent induction, while all TRPC6-activating compounds failed to show any PXR activation or to antagonize rifampicin-mediated CYP3A4 promoter induction. Hyperforin and rifampicin treatment of primary human hepatocytes resulted in highly correlated induction of PXR target genes, whereas treatment with the phloroglucinol derivatives elicited moderate gene expression changes that only weakly correlated to those of rifampicin treatment. The herein observed lack of PXR activation by the TRPC6 activating phloroglucinols was further supported by in silico pharmacophore modeling that did not indicate potent agonist or antagonist interactions for the TRPC6 activating derivatives and docking studies that suggested interaction of only one of these compounds. These in silico studies performed by Prof. Sean Ekins are published together with the results presented in this work (Kandel et al., 2014). This approach shows that strategies avoiding PXR activation are conceivable in drug development in order to prevent DDIs and improve drug safety. Taken together, these results further increase the number of genes by which CAR, PXR, and PPARa contribute to the regulation of drug metabolism and energy homeostasis. Moreover it was demonstrated that the PKA, which is involved in the transduction of the effects of, e.g., the hormone glucagon, represents a determinant of the drug detoxifying system in humans. Furthermore, a strategy could be presented, taking the example of the hyperforin derivates, which can be used to investigate and avoid DDIs in drug development. Such information will become imperative in future personalized medicine and the ever-present polypharmacy in order to handle intra- and inter-individual variability and to minimize drug failure or drug-drug interactions.Item Open Access Role of inflammatory cytokine signaling in the regulation of detoxifying functions in human hepatocytes and liver(2014) Klein, Marcus; Zanger, Ulrich (Prof. Dr.)During inflammation, circulating pro-inflammatory cytokines such as TNFα, IL-1ß, and IL-6, which are produced by, e.g., Kupffer cells, macrophages, or tumor cells, play important roles in hepatocellular signalling pathways and in the regulation of cellular homeostasis. In particular, these cytokines are responsible for the acute phase response (APR) but also for a dramatic reduction of drug detoxification capacity due to impaired expression of numerous genes coding for drug metabolic enzymes and transporters (DMETs). Several pathways are known to be activated by IL-6 such as the JAK/STAT, MAPK/ERK, and PI3K/AKT pathways. Earlier work by others has shown that downregulation of CYP3A4 is independent of the JAK/STAT and MAPK/ERK pathways. However, there is evidence that MAPKs are able to phosphorylate nuclear receptors (NRs) such as RXR-α, which alters their function. Moreover, AKT, downstream of PI3K, may induce nuclear translocation of NF-κB which antagonizes RXR-α and other NRs. RXR-α, which heterodimerizes with subfamily 1 NRs (e.g., CAR and PXR), is an important regulator of detoxifying functions in liver. Inhibition of RXR-α or other NRs could therefore explain the simultaneous downregulation of large gene batteries including many DMET genes. The contributing signaling events and mechanisms remained, however, largely unexplained. Therefore, the major focus of this thesis was the investigation of the impact of the major inflammatory mediator IL-6 on the regulation of detoxifying functions in human liver. For this purpose, a large-scale investigation of DMET gene expression changes in IL-6-stimulated primary human hepatocytes (PHH) was carried out. Many important DMET genes were found to be downregulated in response to IL-6 stimulation of PHH. Most significantly suppressed were genes coding for cytochrome P450s (e.g., CYP1A2, 2C9, 2D6, and 3A4) and ATP-binding cassette (e.g., ABCB1 and ABCC2) and solute carrier (e.g., SLC10A1 and SLCO1B1) drug transporters. The average phase II metabolism gene expression appeared to be only moderately affected by IL-6, showing much stronger variability in gene expression, including genes with a trend towards upregulation (SULTs). Most notably, CYPs appeared to be highly downregulated in a coordinated fashion, demonstrating the broad suppressive potency of IL-6 towards this particular family of drug metabolizing enzymes (DMEs). Moreover, determination of metabolite formation rates in IL-6-treated PHH revealed impaired metabolic functionality of the major CYP isoenzymes CYP1A2, 2B6, 2C8, 2C9, 2C19, and 3A4. Therefore, it was shown that IL-6 signaling extensively interferes with drug detoxification capacity in human hepatocytes. Phosphoprotein analyses revealed activation of the JAK/STAT, MAPK, and PI3K cascades by IL-6. Whereas individual chemical inhibition of the two latter pathways attenuated many IL-6-mediated effects on DMET gene expression, co-inhibition almost completely abolished these effects. Inhibition of JAK/STAT signaling barely affected IL-6-mediated effects. Notably, activation of PI3K and knock-down (KD) of RXR-α demonstrated strikingly similar DMET gene expression patterns compared to IL-6 stimulation. Therefore, these data indicated a MAPK/ERK- and PI3K/AKT-dependent but JAK/STAT-independent downregulation of DMET genes in response to IL-6, possibly via interference with RXR-α. In conclusion, these data suggest that MAPKs and AKT-activated NF-κB antagonize NR signaling, causing a coordinated downregulation of DMET genes. The investigation of sensitive regulatory mechanisms is complicated by the interindividual variability of PHH. The human hepatocellular carcinoma derived HepaRG cell line has been shown to retain many functional characteristics of PHH, including the expression of functional DMETs, but the influence of inflammation has not been investigated so far. Thus, HepaRG cells were tested for their robustness and suitability in studying the inflammation-mediated impact on the drug detoxification capacity in human liver. Indeed, IL-6 stimulation of HepaRG cells led to highly induced expression of acute phase (AP) genes (e.g., CRP) and significantly repressed DMET gene expression in a coordinated fashion. The selectivity and magnitude of these effects were strikingly similar to those observed in IL-6-exposed PHH, with only few exceptions (e.g., CYP2E1 and SULTs). This was further supported by a strong positive correlation of IL-6-mediated expression changes of DMET and critical modifier genes in both cell models. Moreover, decreased protein expression and activity of major P450s could be determined in HepaRG cells, comparable to PHH. Exposure of HepaRG cells to different cytokines resulted in moderately different gene expression patterns, indicating specific responsiveness to particular pro-inflammatory cytokines. These data indicate that HepaRG cells retain the regulatory mechanisms that are responsible for the downregulation of the liver’s drug detoxification capacity during inflammation. This cell line may therefore provide a good alternative model for detailed mechanistic analyses during such conditions. The inflammation-mediated transcriptional changes that have major effects on drug detoxification in the liver have not been analyzed on a transcriptome-wide scale so far. Therefore, the last part of this work focused on the unbiased assessment of genome-wide transcriptional changes in response to inflammatory signaling in the human liver. For this purpose, microarray analysis was carried out in IL-6-stimulated PHH and compared to transcriptome data, previously acquired in samples from a liver cohort, including patients having undergone an APR (elevated CRP). Remarkably, major human-relevant CYPs, 2C8, 3A4, and 2A6 were the most strongly downregulated genes in IL-6-challenged PHH. Their transcription was at least 4-fold repressed. A total of 40 DMET genes were identified as significantly altered, of which 30 were downregulated, including almost all transcripts of major CYPs of importance in humans (e.g., 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 3A4 and 3A5), phase II drug metabolizing enzymes (e.g., GSTAs, SULTs and UGTs), and drug transporters (ABCs and SLCs). In liver samples from patients with elevated CRP, 29 DMET genes were downregulated including important genes coding for phase I/II drug metabolizing enzymes (e.g., ADHs, ALDHs, CYPs, GSTs and UGTs) and drug transporters (e.g., ABCG2 and SLCs). In both studies, gene term enrichment analyses indicated a very strong influence on xenobiotic metabolic and related processes, containing mostly downregulated DMET genes. Moreover, pathway enrichment (KEGG) analyses revealed that drug and xenobiotic metabolic signaling pathways were the most strongly impacted reaction networks, clearly demonstrating that the drug detoxification system in the liver is largely affected during inflammation. Gene annotation analysis also identified enriched processes related to diverse lipid metabolic processes such as fatty-acid and steroid metabolism. Moreover, enriched biological processes and regulatory pathways related to amino acid metabolism were found, particularly in the retrospective study. The data indicated a conservation and allocation of specific amino acids, possibly in favor of acute phase protein (APP) synthesis. Taken together, these findings highlight the scale on which the human liver transcriptome is affected during inflammation. Extensive reorganization related to xenobiotic, lipid, and amino acid metabolism takes place. It appears that the liver devotes its transcriptional machinery to the immune response while other major liver functions are shut down. This may help to pave the way towards a better understanding of how the liver organizes its many responsibilities in different conditions.Item Open Access Entwicklung eines autologen Fettgewebeersatzes mit angiogenen Eigenschaften auf Basis einer adipogenen Alginatmatrix in Kombination mit humanen mesenchymalen Stammzellen(2013) Handel, Marina; Pfizenmaier, Klaus (Prof. Dr.)Die Rekonstruktion von subkutanem Fettgewebe stellt die Plastische Chirurgie noch immer vor eine große Herausforderung, denn für großflächige Gewebeschäden steht meist nicht ausreichend patienteneigenes Gewebe zur Verfügung. In der Regenerativen Medizin arbeitet man daher an künstlichem Fettgewebeersatz. Aufgrund der schlechten Vaskularisierung weisen diese Ansätze jedoch eine unzureichende Nährstoffversorgung auf, woraus eine Transplantatvolumenreduktion von bis zu 60 % resultiert. In dieser Arbeit wurde daher der Ansatz des "Adipose Tissue Engineering" (ATE) verfolgt, einer neuen Variante zur Rekonstruktion von subkutanem Fettgewebe. Geeignete Trägerstrukturen (z.B. textile Implantate) wurden mit patienteneigenen Vorläuferzellen besiedelt und zu Fettzellen differenziert, um autologes Fettgewebe in situ - de novo zu generieren. Dieser Fettgewebeersatz auf Basis humaner Stammzellen aus Fettgewebe (hASC) zeigte dabei ausgeprägte angiogene Eigenschaften. Das adipogene Differenzierungspotenzial sowie die Sekretion pro-angiogener Zytokine der hASC wurde ermittelt. Ein spezifischer VEGF-Knockdown bestätigte VEGF als potentes pro-angiogenes Zytokin, weshalb die VEGF-Sekretion stellvertretend für weitere pro-angiogene Zytokine humaner ASC ermittelt wurde. Standardisierte in vitro Angiogenese-Modelle belegten ein erhöhtes angiogenes Potenzial hASC besiedelter Implantate. Diese in vitro Ergebnisse wurden mit dem in vivo CAM Angiogenese Assay bestätigt. Die Kombination von Implantatmaterialien mit hASC zeigte auch hierbei stets ein verbessertes angiogenes Potenzial. Mit fortschreitender adipogener Differenzierung nahm die VEGF-Sekretion humaner ASC hingegen rapide ab. Um das pro-angiogene Potenzial der Vorläuferzellen post transplantationem nutzen zu können, ist daher zu empfehlen, native hASC zu verwenden. Hierzu wurden erste adipogene Alginatstrukturen entwickelt, welche über integrierte Differenzierungsfaktoren eine adipogene Differenzierung immobilisierter hASC induzierten. Die Ergebnisse dieses Ansatz des ATE zeigten auf, dass eine Differenzierung der Vorläuferzellen durch das Scaffoldmaterial post transplantationem induziert werden kann, was aus therapeutischer Sicht sowohl die erforderliche VEGF-Sekretion humaner ASC und somit das angiogene Potenzial zum Zeitpunkt der Implantation sicherstellen, als auch die Zeitspanne vor einer Geweberekonstruktion deutlich verkürzen würde.Item Open Access Chemosensitivity of testicular germ cell tumors is based on high constitutive Noxa protein levels and a functional p53 response(2013) Gutekunst, Matthias; Scheurich, Peter (Prof. Dr.)In contrast to the majority of tumors, testicular germ cell tumors (TGCTs) can be cured by chemotherapy even in advanced metastatic stages. Thus, these neoplasms are considered a paradigm of chemosensitive tumors. It is well accepted that their intrinsic susceptibility to apoptosis determines the unique responsiveness to Cisplatin-based chemotherapy. Although p53 has been implicated in the chemotherapeutic response of TGCTs, the role of this tumor suppressor remains controversial to date. In this study, RNAi-mediated silencing of p53 is shown to completely abrogate Cisplatin hypersensitivity of TGCTs. The central role of p53 is further demonstrated by the finding that the amount of p53 protein induced by Cisplatin treatment is tightly correlated with apoptosis induction. In this context, a reduced capacity to repair damaged DNA is rather unlikely to account for hypersensitivity, a conjecture that is strengthened by the finding that TGCT cells are capable of removing Cisplatin adducts from DNA if apoptosis induction is blocked by caspase inhibition. On the contrary, the hypersensitive phenotype is a result of the unique responsiveness of TGCTs to p53 activation in general, since treatment with the nongenotoxic p53 activators Nutlin-3 or Bortezomib leads to p53-dependent apoptosis to a similar extent as does Cisplatin treatment. Although functional p53 is a mandatory requirement for Cisplatin hypersensitivity in TGCTs, the proapoptotic character of the p53 response is dependent on the pluripotent cellular context of TGCTs mediated by Oct-4. Differentiation or silencing of Oct-4 expression abrogates Cisplatin sensitivity and reduces apoptosis induction by Nutlin-3 treatment. This is not due to differential activation of p53 since no substantial alterations in Cisplatin-induced target gene activation are observed. Furthermore, p53 accumulation is unaffected by differentiation or Oct-4 depletion. Rather, examination of the Bcl-2 family profile in pluripotent vs. differentiated or Oct-4-depleted cells revealed that high constitutive Noxa protein levels dictate an extremely low apoptotic threshold in TGCT cells. Oct-4 status and Noxa protein levels are correlated to Cisplatin sensitivity in a panel of TGCT cell lines as well as in TGCT patient samples. Importantly, this correlation is also seen with other genotoxic agents such as Etoposide or Doxorubicin and the non-genotoxic p53 activator Nutlin-3. Since Oct-4 depletion leads to a concomitant loss of Noxa protein and NOXA/PMAIP1 transcript, an Oct-4-dependent transcriptional mechanism might account for the high Noxa protein levels. RNAi experiments show that Puma is another important determinant of TGCT hypersensitivity. In contrast to Noxa, Puma is not present at high levels in TGCT cells but exerts its proapoptotic function upon activation by p53. In conclusion, this study demonstrates that pluripotent TGCTs are primed for apoptosis by Oct-4-mediated high constitutive Noxa protein levels. Thus, in response to genotoxic or other stresses p53 activation results in an efficient induction of apoptosis mediated by transcriptional target genes such as PUMA/BBC3.Item Open Access Regulation of the BH3-only protein NOXA in mantle cell lymphoma : relevance for novel treatment concepts(2014) Dengler, Michael; Scheurich, Peter (Prof. Dr.)Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma and characterized by only transient responses to chemotherapy and relatively short survival. Extensive research is ongoing to improve current therapy and identify novel targets for treatment of this B cell malignancy. The central role of BH3-only proteins in mediating the response of tumor cells to anticancer drugs has been shown by many studies. The present work aimed at the investigation of the role of the BH3-only protein NOXA in the decision between life and death in MCL. Astonishingly, basal NOXA mRNA and NOXA protein expression levels were found in this study to be extremely discrepant in MCL cell lines as well as primary cells from MCL patients. The malignant B cells express very high constitutive levels of NOXA mRNA. In strong contrast, NOXA protein levels are very low in MCL cell lines and even hardly detectable in primary MCL cells. It could be demonstrated in this study that chronic active B-cell receptor (BCR)-signaling and Cyclin D1 overexpression, the hallmark of MCL, contribute to the maintenance of the high constitutive transcript levels of the pro-apoptotic Bcl-2 family member. Furthermore, the phoshatidyl-inositol-3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway was determined as the major mediator responsible for the high NOXA mRNA expression downstream of the BCR and surprisingly also Cyclin D1. Overexpression of the cell cycle regulatory protein Cyclin D1 contributes to enhanced NOXA transcription by exerting a positive feedback loop on the PI3K/AKT/mTOR signaling. Increased expression of the pro-apoptotic Bcl-2 family member NOXA is generally implicated in induction of cell death. Intriguingly, the high basal transcript levels of the BH3-only protein do not impair viability of MCL cells. The malignant B cells adapt to this permanent pro-apoptotic signal by extensive polyubiquitination and rapid proteasome-mediated degradation of NOXA (T½ < 30 min). Importantly, the resulting phenotype of high NOXA mRNA and low NOXA protein expression appears to constitute a druggable Achilles heel of MCL. Exposure of the cells to the proteasome inhibitor Bortezomib accumulates NOXA protein and efficiently induces apoptosis. In addition to Bortezomib, the neddylation inhibitor MLN4924 and the fatty acid synthase inhibitor Orlistat were identified in the present study as effective inducers of NOXA protein and NOXA-dependent apoptosis in MCL. Such as Bortezomib, both inhibitors target rapid NOXA protein turnover thereby stabilizing the pre-existing pool of the pro-apoptotic protein. Interestingly, in contrast to the proteasome inhibitor, MLN4924 as well as Orlistat interfere with ubiquitination of NOXA and stabilize the BH3-only protein in a proteasome independent manner. Therefore, these substances not only represent alternatives to Bortezomib treatment but may also offer new approaches to treat Bortezomib resistant MCL cells. Indeed, results from the present work demonstrate that Bortezomib resistant MCL cells were still sensitive to Orlistat and MLN4924. Remarkably, active PI3K/AKT/mTOR signaling is needed for effective accumulation of NOXA and induction of cell death upon treatment with Bortezomib, Orlistat or MLN4924 indicating that the high constitutive NOXA mRNA levels are essential for the sensitivity of MCL cells to inhibitors targeting NOXA turnover. Especially fatty acid metabolism appears to represent a very promising target for treatment of MCL. Constitutive Cyclin D1 overexpression in MCL was found to have an inhibitory effect on cellular metabolism thereby rendering MCL cells susceptible to drugs targeting the fatty acid synthase such as Orlistat. Furthermore, inhibition of fatty acid metabolism may not only constitute a new and selective strategy to kill MCL cells but also sensitize the malignant B cells to other anticancer drugs. In summary, the present study highlights a critical role of the BH3-only protein NOXA in MCL and provides evidence that the regulation of the pro-apoptotic protein might represent an Achilles heel of this aggressive tumor. Targeting the phenotype of high NOXA mRNA/low NOXA protein expression by inhibitors inferring with different steps of the ubiquitin-proteasome system may provide a novel therapeutic approach to kill MCL cells and improve clinical outcome of this lymphoid neoplasm.