Nicole Forster

Publications

• 4.09

  Impact points

  Notch signaling mediates p63-induced quiescence: a new facet of p63/Notch crosstalk.

  Nicole Forster, Leif W Ellisen

  Cell cycle (Georgetown, Tex.). 11/2011; 10(21):3632-3.
• 7.54

  Impact points

  Physical association of HDAC1 and HDAC2 with p63 mediates transcriptional repression and tumor maintenance in squamous cell carcinoma.

  Matthew R Ramsey, Lei He, Nicole Forster, Benjamin Ory, Leif W Ellisen

  Cancer research. 04/2011; 71(13):4373-9.

  Squamous cell carcinoma (SCC) is a treatment-refractory subtype of human cancer arising from stratified epithelium of the skin, lung, esophagus, oropharynx, and other tissues. A unifying feature of SCC is high-level expression of the p53-related protein p63 (TP63) in 80% of cases. The major protein ... [more] Squamous cell carcinoma (SCC) is a treatment-refractory subtype of human cancer arising from stratified epithelium of the skin, lung, esophagus, oropharynx, and other tissues. A unifying feature of SCC is high-level expression of the p53-related protein p63 (TP63) in 80% of cases. The major protein isoform of p63 expressed in SCC is ΔNp63α, an N-terminally truncated form which functions as a key SCC cell survival factor by mechanisms that are unclear. In this study, we show that ΔNp63α associates with histone deacetylase 1 (HDAC1) and HDAC2 to form an active transcriptional repressor complex that can be targeted to therapeutic advantage. Repression of proapoptotic Bcl-2 family member genes including p53 upregulated modulator of apoptosis (PUMA) by p63/HDAC is required for survival of SCC cells. Cisplatin chemotherapy, a mainstay of SCC treatment, promotes dissociation of p63 and HDAC from the PUMA promoter, leading to increased histone acetylation, PUMA activation, and apoptosis. These effects are recapitulated upon targeting the p63/HDAC complex selectively with class I/II HDAC inhibitors using both in vitro and in vivo models. Sensitivity to HDAC inhibition is directly correlated with p63 expression and is abrogated in tumor cells that overexpress endogenous Bcl-2. Together, our results elucidate a mechanism of p63-mediated transcriptional repression and they identify the ΔNp63α/HDAC complex as an essential tumor maintenance factor in SCC. In addition, our findings offer a rationale to apply HDAC inhibitors for SCC treatment.
• 7.54

  Impact points

  The integrin alpha(v)beta(3-5) ligand MFG-E8 is a p63/p73 target gene in triple-negative breast cancers but exhibits suppressive functions in ER(+) and erbB2(+) breast cancers.

  Chuanwei Yang, Tetsu Hayashida, Nicole Forster, Cuiqi Li, Dejun Shen, Shyamala Maheswaran, Li Chen, Karen S Anderson, Leif W Ellisen, Dennis Sgroi, Emmett V Schmidt

  Cancer research. 02/2011; 71(3):937-45.

  The progression from preinvasive lesion to invasive carcinoma is a critical step contributing to breast cancer lethality. We identified downregulation of milk fat globule-EGF factor 8 (MFG-E8) as a contributor to breast cancer progression using microarray analysis of laser capture microdissected (LC... [more] The progression from preinvasive lesion to invasive carcinoma is a critical step contributing to breast cancer lethality. We identified downregulation of milk fat globule-EGF factor 8 (MFG-E8) as a contributor to breast cancer progression using microarray analysis of laser capture microdissected (LCM) tissues. We first identified MFG-E8 downregulation in invasive lesions in transgenic mammary tumor models, which were confirmed in LCM-isolated human invasive ductal carcinomas compared with patient-matched normal tissues. In situ analyses of MFG-E8 expression in estrogen receptor (ER) positive cases confirmed its downregulation during breast cancer progression and small inhibitory MFG-E8 RNAs accelerated ER(+) breast cancer cell proliferation. MFG-E8 also decreased in erbB2(+) human cancers and erbB2 transgenic mice lacking MFG-E8 showed accelerated tumor formation. In contrast, MFG-E8 expression was present at high levels in triple-negative (ER(-), PgR(-), erbB2(-)) breast cancers, cell lines, and patient sera. Knockdown, chromatin immunoprecipitation, and reporter assays all showed that p63 regulates MFG-E8 expression, and MFG-E8 knockdowns sensitized triple-negative breast cancers to cisplatin treatment. Taken together, our results show that MFG-E8 is expressed in triple-negative breast cancers as a target gene of the p63 pathway, but may serve a suppressive function in ER(+) and erbB2(+) breast cancers. Its potential use as a serum biomarker that contributes to the pathogenesis of triple-negative breast cancers urges continued evaluation of its differential functions.
• 15.39

  Impact points

  A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma.

  Benjamin Ory, Matthew R Ramsey, Catherine Wilson, Douangsone D Vadysirisack, Nicole Forster, James W Rocco, S Michael Rothenberg, Leif W Ellisen

  The Journal of clinical investigation. 01/2011; 121(2):809-20.

  The p53 tumor suppressor, a central mediator of chemosensitivity in normal cells, is functionally inactivated in many human cancers. Therefore, a central challenge in human cancer therapy is the identification of pathways that control tumor cell survival and chemosensitivity in the absence of functi... [more] The p53 tumor suppressor, a central mediator of chemosensitivity in normal cells, is functionally inactivated in many human cancers. Therefore, a central challenge in human cancer therapy is the identification of pathways that control tumor cell survival and chemosensitivity in the absence of functional p53. The p53-related transcription factors p63 and p73 exhibit distinct functions—p73 mediates chemosensitivity while p63 promotes proliferation and cell survival—and are both overexpressed in squamous cell carcinomas (SCCs). However, how p63 and p73 interact functionally and govern the balance between prosurvival and proapoptotic programs in SCC remains elusive. Here, we identify a microRNA-dependent mechanism of p63/p73 crosstalk that regulates p53-independent survival of both human and murine SCC. We first discovered that a subset of p63-regulated microRNAs target p73 for inhibition. One of these, miR-193a-5p, expression of which was repressed by p63, was activated by proapoptotic p73 isoforms in both normal cells and tumor cells in vivo. Chemotherapy caused p63/p73-dependent induction of this microRNA, thereby limiting chemosensitivity due to microRNA-mediated feedback inhibition of p73. Importantly, inhibiting miR-193a interrupted this feedback and thereby suppressed tumor cell viability and induced dramatic chemosensitivity both in vitro and in vivo. Thus, we have identified a direct, microRNA-dependent regulatory circuit mediating inducible chemoresistance, whose inhibition may provide a new therapeutic opportunity in p53-deficient tumors.
• 8.99

  Impact points

  Zbtb4 represses transcription of P21CIP1 and controls the cellular response to p53 activation.

  Axel Weber, Judith Marquardt, David Elzi, Nicole Forster, Sven Starke, Andre Glaum, Daisuke Yamada, Pierre-Antoine Defossez, Jeffrey Delrow, Robert N Eisenman, Holger Christiansen, Martin Eilers

  The EMBO journal. 07/2008; 27(11):1563-74.

  In response to stimuli that activate p53, cells can undergo either apoptosis or cell cycle arrest, depending on the precise pattern of p53 target genes that is activated. We show here that Zbtb4, a transcriptional repressor protein, associates with the Sin3/histone deacetylase co-repressor and repre... [more] In response to stimuli that activate p53, cells can undergo either apoptosis or cell cycle arrest, depending on the precise pattern of p53 target genes that is activated. We show here that Zbtb4, a transcriptional repressor protein, associates with the Sin3/histone deacetylase co-repressor and represses expression of P21CIP1 as part of a heterodimeric complex with Miz1. In vivo, expression of ZBTB4 is downregulated in advanced stages of multiple human tumours. In cell culture, depletion of ZBTB4 promotes cell cycle arrest in response to activation of p53 and suppresses apoptosis through regulation of P21CIP1, thereby promoting long-term cell survival. Our data suggest that Zbtb4 is a critical determinant of the cellular response to p53 activation and reinforce the notion that p21Cip1 can provide an essential survival signal in cells with activated p53.
• 2.46

  Impact points

  Post-SELEX chemical optimization of a trypanosome-specific RNA aptamer.

  Annette Adler, Nicole Forster, Matthias Homann, H Ulrich Göringer

  Combinatorial chemistry & high throughput screening. 02/2008; 11(1):16-23.

  African trypanosomes are the causative agent of sleeping sickness. The therapeutics used to control and treat the disease are very ineffective and thus, the development of improved drugs is urgently needed. Recently, new strategies for the design of novel trypanocidals have been put forward. Among t... [more] African trypanosomes are the causative agent of sleeping sickness. The therapeutics used to control and treat the disease are very ineffective and thus, the development of improved drugs is urgently needed. Recently, new strategies for the design of novel trypanocidals have been put forward. Among them are techniques that rely on parasite-specific RNA aptamers. One approach involves the aptamer-directed transport of lytic compounds to the lysosome of the parasite. The aptamer has been termed 2-16 RNA and here we report the optimization of the RNA for its applications in vivo. To convert aptamer 2-16 into a serum-stable reagent 2'-deoxy-2'-F- and/or 2'-deoxy-2'-NH(2)-uridine- and cytidine-substituted RNAs were generated. While 2'-NH(2)-dC/dU-modified RNAs were RNase-resistant, they were functionally inactive. By contrast, 2'-F-dC/dU-substituted 2-16 RNA retained its ability to bind to live trypanosomes (K(d)=45 nM) and was routed to the lysosome identically to unmodified RNA. 2'-F-dC/dU-substituted 2-16 RNA is thermostable (T(m)=75 degrees C) and has a serum half-life of 3.4 days. Furthermore, aptamer 2-16 was site-specifically PEGylated to increase its serum retention time. Conjugation with PEG polymers < or = 10 kDa only marginally impacted the binding characteristics of the RNA, while the addition of higher molecular mass PEG molecules resulted in non-functional aptamers. Together, the data provide optimized conjugation chemistries for the large-scale production of substituted aptamer 2-16 preparations with improved in vivo functionality.
• 5.65

  Impact points

  p300 protein acetyltransferase activity suppresses systemic lupus erythematosus-like autoimmune disease in mice.

  Nicole Forster, Sven Gallinat, Jadwiga Jablonska, Siegfried Weiss, Hans-Peter Elsässer, Werner Lutz

  Journal of immunology (Baltimore, Md. : 1950). 07/2007; 178(11):6941-8.

  Conditional knock-in mice expressing a histone acetyltransferase-deficient version of the transcriptional coregulator p300 exclusively in B lymphocytes die prematurely with full penetrance. The mice develop an autoimmune disease similar to systemic lupus erythematosus in its pathological manifestati... [more] Conditional knock-in mice expressing a histone acetyltransferase-deficient version of the transcriptional coregulator p300 exclusively in B lymphocytes die prematurely with full penetrance. The mice develop an autoimmune disease similar to systemic lupus erythematosus in its pathological manifestations, such as splenomegaly, glomerulonephritis, vasculitis, deposition of immune complexes, and production of autoantibodies against dsDNA. Aged mice show a severe reduction of transitional and marginal zone B cells and generate aberrant mature B cells. These B cells show diminished proliferation in response to stimulation of the BCR, but respond normally to other stimuli. Yet, the mice mount a normal primary immune response against a T-dependent Ag. In contrast, the memory response is impaired. In addition, serum Ig levels, in particular IgG2b, are increased. We conclude that p300 acetyltransferase activity is essential for maintaining self-tolerance of B lymphocytes. These findings support the concept of treating lupus with inhibitors of protein deacetylases and point to B cells as a critical target of these drugs.

• Die p300 Protein Acetyltransferaseaktivität supprimiert eine dem humanen Systemischen Lupus Erythematosus ähnliche Autoimmunerkrankung in Mäusen

  Nicole Forster

  p300, als eine von 15 beschriebenen Acetyltransferasen der Mammalia, besitzt zahlreiche Interaktionspartnern, die gleichzeitig als Substrate der Acetylierung fungieren. Unter diesen sind Proteine, die in der Hämatopoese eine essentielle Rolle spielen. Eine konditionelle Knock-In-Maus mit heterozygot... [more] p300, als eine von 15 beschriebenen Acetyltransferasen der Mammalia, besitzt zahlreiche Interaktionspartnern, die gleichzeitig als Substrate der Acetylierung fungieren. Unter diesen sind Proteine, die in der Hämatopoese eine essentielle Rolle spielen. Eine konditionelle Knock-In-Maus mit heterozygoter Expression eines Acetyltransferase-defizienten p300 (p300AS) in der Keimbahn ist embryonal letal, wohingegen heterozygote Knock-Out-Mäuse für p300 und embryonale Stammzellen, die homozygot für die Mutation waren, lebensfähig sind. Des Weiteren sind mehrere Mutationen der p300-Acetyltransferaseaktivität in Leukämien bekannt. Zusammenfassend lassen diese Daten eine Rolle der p300-Acetyltransferaseaktivität in der Hämatopoese der Mäuse vermuten. Daher wurde mit dieser Arbeit die Funktion der p300-Acetyltransferaseaktvität während der B-Zell-Entwicklung und -Differenzierung untersucht. In der vorliegenden Arbeit konnte ich zeigen, dass Mäuse mit Expression eines Acetyltransferase-defizienten p300 ausschließlich in B-Zellen aufgrund der Entwicklung einer Autoimmunerkrankung frühzeitig sterben. Diese Erkrankung ist in ihrer Pathologie ähnlich dem humanen Systemischen Lupus Erythematosus (SLE). Die pathologischen Erscheinungen der Mäuse umfassen eine Splenomegalie, Glomerulonephritis, Vaskulitis, Einlagerungen von Immunglobulinkomplexen in verschiedenen Organen und die Produktion von Autoantikörpern gegen dsDNA und andere nukleäre Antigene. Das frühere Sterben der Weibchen gegenüber den Männchen ist ein weiteres Merkmal des humanen SLE, bei dem über 90% der Patienten Frauen sind. Die fehlende p300-Acetyltransferaseaktivität in den B-Zellen führt neben einer reduzierten Anzahl an transitionellen und Marginalzonen-B-Zellen in der Milz zu einem partiellen Verlust der B-Zell-Differenzierung in den Mäusen. Mäuse mit einer induzierten ubiquitären Expression des mutierten p300 entwickeln ebenfalls eine SLE-ähnliche Autoimmunerkrankung. Daraus lässt sich schließen, dass die p300-Acetyltransferaseaktivität notwendig für die Regulation der Selbsttoleranz der B-Zellen sowie deren Differenzierung ist. Ebenso zeigt dies, dass die Acetyltransferaseaktivität von p300 die Ausbildung einer SLE-ähnlichen Autoimmunerkrankung supprimiert. Neben diesem Phänotyp der B-Zellen konnte ich zeigen, dass die B-Zellen mit Expression des Acetyltransferase-defizienten p300 dennoch in der Lage sind, in vivo Immunglobuline zu bilden, welche insbesondere für die Isotypen IgG2b und IgM erhöht sind. FACS-Analysen zeigen, dass die erhöhten Mengen an Immunglobulinen mit einer höheren Anzahl an aktivierten B-Zellen in der Milz dieser Mäuse korrelieren. Eine Analyse der humoralen Immunantwort gegenüber dem T-Zell-abhängigen Antigen Ovalbumin verdeutlicht eine bereits erhöhte Anzahl an Ovalbumin-spezifischen Antikörpern vor der Immunisierung. Wohingegen 14 Tage nach der Immunisierung die Primärantwort der Mäuse mit Acetyltransferase-defizientem p300 ähnlich der von den Kontrollmäusen ist. Im Gegensatz dazu wird bei der Erinnerungsantwort eine schwächere Bildung von Ovalbumin-spezifischen Antikörpern detektiert. Da die Mäuse mit B-Zell-spezifischer Expression von Acetyltransferase-defizientem p300 nach Gabe des Antigens Ovalbumin eine B-Zell-Aktivierung zeigen, wird zusätzlich die Proliferation der B-Zellen ex vivo nach Aktivierung verschiedener Signalwege untersucht. Alleinig nach Induktion des BCR-Signalweges durch Zugabe von α-IgM zur Quervernetzung des BCR proliferieren die B-Zellen schwächer. Die reduzierte Anzahl proliferativer Zellen wird dabei nicht durch eine erhöhte Apoptose verursacht. Daher deutet dies auf eine Hemmung der Proliferation durch die fehlende Acetyltransferaseaktivität von p300 hin, welches unter anderem durch eine im cDNA-Microarray detektierte Deregulation des BCR-Signalweges verdeutlicht wird. Biochemische Analysen des Effektes von Acetyltransferase-defizientem p300 auf den Acetylierungsstatus der Proteine zeigen keine globalen oder promotorspezifischen Veränderungen der Histon H3K18-Acetylierung in B-Zellen mit reduzierter p300-Acetyltransferaseaktivität auf. Allerdings ist eine reduzierte Acetylierung bei größeren Proteinen als Histonen nachweisbar. Dies deutet darauf hin, dass vermutlich andere Proteine als Histone die kritischen Substrate der p300-Acetyltransferaseaktivität in vivo sind. Für weitere Analysen stellte ich eine B-Zelllinie her, in die durch homologe Rekombination das mutierte p300AS eingeführt wird. Diese B-Zelllinie synthetisiert nachweislich das mutierte p300AS, welches in vitro eine reduzierte Acetyltransferase-aktivität aufweist. Mit Hilfe der Zelllinie sind nun weitere Versuche, wie Proteomanalysen, zur Identifikation der Substrate, die auf die p300-Acetyltransferaseaktivität angewiesen sind, möglich. p300, one of 15 mammalian acetyltransferases known, interacts with a huge number of proteins which directly function as substrates for its acetyltransferase activity. Among them are several proteins involved in hematopoiesis. A conditional knock-in mouse with heterozygous expression of an acetyltransferase-deficient p300 (p300AS) in the germline is embryonic lethal, whereas heterozygous p300 knock-out mice and embryonic stem cells homozygous for the p300 mutation are viable. Moreover, several mutations of p300 acetyltransferase activity were found in different leukemia patients. All together, these data suggest a function of p300 acetyltransferase activity in hematopoiesis. Therefore, the task of my thesis work was to define the role of p300 acetyltransferase activity during B cell development and differentiation. During my studies, I showed that mice expressing an acetyltransferase-deficient p300 only in B cells develop an autoimmune disease similar to the Systemic Lupus Erythematosus (SLE) in human and undergo premature death. The pathological manifestations of these mice include splenomegaly, glomerulonephritis, vasculitis, immune complex depositions in different organs, and production of autoantibodies against dsDNA and other nuclear antigens. A premature death of females in comparison to males was observed, reminiscent to another hallmark of human SLE which affects over 90% of women. In addition, the deficient acetyltransferase activity of p300 in B cells leads to a reduced number of transitional and marginal zone B cells in the spleen besides a partial loss of B cell maturation in these mice. Similar results are obtained by using mice having an inducible and ubiquitously expressed form of mutant p300. These results suggest that the p300 acetyltransferase activity is necessary for the regulation of self tolerance in B cells as well as for their differentiation. Moreover, the acetyltransferase activity of p300 suppresses the development of an SLE-like autoimmune disease. In addition to the observed B cell phenotype, I prove that B cells expressing acetyltransferase-deficient p300 are still able to produce in vivo immunoglobulins with a specific increase for the isotypes IgG2b and IgM. FACS analysis show that the increased levels of total immunoglobulins are correlating with higher amounts of activated B cells in the spleen of those mice. An analysis of the humoral immune response against a T-cell-dependent antigen, in that case ovalbumin, reveals increased Ovalbumin-specific antibodies before the immunization. Whereas, after 14 days of immunization the primary immune response against ovalbumin of mice deficient for the p300 acetyltransferase activity in B cells is similar to control mice. In contrast, mice show an impaired memory response. Notably, in addition to an increased amount of activated B cells, p300 acetyltransferase-deficient mice show a significant augmented number of activated T cells independently of ovalbumin. Since the mice expressing the B-cell-specific acetyltransferase-deficient p300 are displaying B cell activation after administration of the antigen, the proliferation of the B cells is analyzed after activation of different signaling pathways ex vivo. Only the activation of the BCR signaling pathway via addition of α-IgM antibodies leads to a reduced proliferation of the B cells. Notably, the reduced amount of proliferating cells is not due to an increased apoptosis rate suggesting a blockage of the proliferation by the acetyltransferase activity of p300. In addition, an observed deregulation of the BCR signaling pathway in microarray analysis in those B cells is consistent with that suggestion. In order to check whether the presence of an acetyltransferase activity deficient form of p300 had an incidence on the overall acetylation status, biochemical analyses of the histone acetylation levels didn’t show any difference in the global or promoter-specific histone H3K18 acetylation in B cells with reduced p300 acetyltransferase activity. Rather, a reduced acetylation of proteins larger than histones is detectable suggesting that other proteins might be the critical substrates of the p300 acetyltransferase activity in vivo. For further analyses, I established a B cell line via homologous recombination expressing the mutant p300AS. This cell line which shows a reduced acetyltransferase activity in vitro will provide a helpful tool for the identification of the substrates which are dependent on the p300 acetyltransferase activity through proteomics or other analyses.