Myeloid STAT3 promotes formation of colitis-associated colorectal cancer in mice

Myeloid cells lacking STAT3 promote antitumor responses of NK and T cells but it is unknown if this crosstalk affects development of autochthonous tumors. We deleted STAT3 in murine myeloid cells (STAT3(Δm)) and examined the effect on the development of autochthonous colorectal cancers (CRCs). Formation of Azoxymethane/Dextransulfate (AOM/DSS)-induced CRCs was strongly suppressed in STAT3(Δm) mice. Gene expression profiling showed strong activation of T cells in the stroma of STAT3(Δm) CRCs. Moreover, STAT3(Δm) host mice were better able to control the growth of transplanted MC38 colorectal tumor cells which are known to be killed in a T cell-dependent manner. These data suggest that myeloid cells lacking STAT3 control formation of CRCs mainly via cross activation of T cells. Interestingly, the few CRCs that formed in STAT3(Δm) mice displayed enhanced stromalization but appeared normal in size indicating that they have acquired ways to escape enhanced tumor surveillance. We found that CRCs in STAT3(Δm) mice consistently activate STAT3 signaling which is implicated in immune evasion and might be a target to prevent tumor relapse

STAT3 is a negative regulator of KRAS-induced lung tumorigenesis

Lungenkrebs ist eine der aggressivsten und tödlichsten Krebserkrankungen. Da die Behandlungsmethoden vor allem in Kirsten-rat sarcoma (KRAS)-mutierten Lungenkarzinom äußerst beschränkt sind, werden neue Behandlungsmöglichkeiten gesucht. Ziel ist es Kooperationspartner oder Signalkaskaden, die durch die KRAS Mutation ausgelöst werden zu blockieren. Diese Arbeit beschäftigt sich mit der Funktion von Signal Transducer and Activator of Transcription 3 (STAT3) im KRAS-mutierten Lungenkarzinom. STAT3 ist vorwiegend für seine pro-onkogene Rolle in Leukämie und kolorektalen Karzinom bekannt wurde aber auch bereits als tumor-suppressiv beschrieben. In vitro- Studien im Lungenkarzinom suggerieren eine pro-onkogene Funktion von STAT3, dennoch fehlen notwenige in vivo Versuche um diese Bedeutung zu bestätigen. Mit Hilfe eines transgenen Mausmodells wird in dieser Arbeit die Funktion von STAT3 während der Lungenkrebsentstehung in vivo erforscht. Die spezifische Aktivierung der KrasG12D Mutation in Lungen-epithelzellen führt zu Lungenkarzinomen, die dem menschlichen Krankheitsbild ähneln. Interessanterweise führte die gleichzeitige genetische Deletion von Stat3 (homo oder heterozygot) in KrasG12D-mutierten, entstehenden Tumorzellen zu einer drastischen Verschlechterung der Überlebensrate der Mäuse im Vergleich zu Kontrolltieren mit endogener Stat3 Expression. STAT3-Knockdown in einer humanen Lungenkrebs-Zelllinie führten ebenso zu einem signifikanten Anstieg in der Wachstumskurve im Xenograft Mausmodel. Diese Modelle bestätigten, dass STAT3 als haploid-insuffizienter Tumor-Suppressor im KRAS-induzierten Lungenkarzinom fungiert. Die Analyse von Patientendaten zeigte, dass die STAT3 Expression in KRAS-mutierten Tumoren signifikant de-reguliert ist im Vergleich zu KRAS-wildtyp Lungenkarzinomen. Desweitern ist die STAT3 Expression in hochgradigen Tumoren signifikant negativ reguliert im Vergleich zu geringgradigen Tumoren. Die Überlebensrate von Lungenkarzinom Patienten (mit oder ohne Raucher-geschichte) zeigte, dass Tumore mit geringerer STAT3 Expression einen deutlich signifikanten Nachteil hatten im Vergleich zu Patienten mit erhöhter STAT3 Expression. Diese Ergebnisse zeigten, dass die de-Regulierung von STAT3 in Patienten eine negative Auswirkung auf den Fortschritt der Erkrankung und die Überlebensrate haben kann. Mechanistisch konnten wir beweisen, dass in Abwesenheit von STAT3 das pro-inflammatorische chemokine (C-X-C motif) ligand 1 (CXCL1, im Menschen als IL-8 bekannt) verstärkt exprimiert wird und daher vermehrt Makrophagen und Granulozyten den Tumor infiltrieren. Außerdem führt eine erhöhte CXCL1/IL-8 Expression zu einer Aktivierung von Endothelzellen, wodurch die Blutversorgung des Tumors verbessert wird. Sowohl eine verbesserte Durchblutung des Tumors als auch die Infiltrierung von Immun-Zellen begünstigen das Tumorwachstum. Durch Blockade dieses Signalweges wird Tumorentstehung, Tumorwachstum und Blutversorgung in Stat3-defizienten Tumoren in vivo unterbunden. Anhand von in vivo und in vitro Modellen zeigen wir, dass STAT3 aktiv mit der p65-Untereinheit von nuklear Faktor kappa B (NFkB) eine Bindung eingeht und es im Zytoplasma zurückhält, was zu einer negativ-Regulation von CXCL-1/ IL-8 führt. Zusammenfassend können wir in dieser Arbeit zeigen, dass STAT3 in KRAS-mutiertem Lungenkarzinom eine wichtige Rolle zur Anti-tumor Bekämpfung durch Inhibition von NFkB induzierter CXCL1/IL-8 Expression beiträgt.Lung cancer belongs to the most aggressive and deadliest diseases. Treatment possibilities, especially for Kirsten-rat sarcoma (KRAS)-mutant lung adenocarcinoma (AC), are limited, since KRAS does not represent a druggable target. Research focus has shifted towards targeting downstream signaling molecules and cooperative partners, including signal transducer and activator of transcription-3 (STAT3). STAT3 is known for its pro-oncogenic function in leukemia and colorectal cancer, although recent data implicated tumor-suppressive function. In lung AC, various in vitro studies suggested an oncogenic role of STAT3; however, in vivo data were missing. With the help of a transgenic mouse model we aimed to elucidate STAT3's functionality during KrasG12D-driven lung cancer development in vivo. Our findings were validated using a human to mouse xenograft model, human lung cancer databases and with a human lung AC cell line in vitro. Hetero- or homozygous deletion of Stat3 in KrasG12D- mutant lung tumors resulted in a drastic survival disadvantage compared to mice with Stat3-competent tumors. Small hairpin-mediated knockdown of STAT3 in xenografted human lung AC cells enhanced tumor growth. Moreover, Stat3-deficient tumors expressed increased levels of the pro-inflammatory chemokine (C-X-C motif) ligand 1 (CXCL1, the human orthologue is IL-8), resulting in infiltration of pro-tumorigenic granulocytes and macrophages and enhanced tumor-angiogenesis. Blockade of CXCL1 signaling cascade reduced tumor formation, growth and vascularization of Stat3-deficient tumors in vivo. Based on in vivo and in vitro models we demonstrate that activated STAT3 retained nuclear factor kappa B (NFkB)-subunit p65 in the cytoplasm to repress CXCL1 expression. Clinically we could show that STAT3 expression is significantly reduced in KRAS mutant compared to KRAS wildtype AC cases. ^Furthermore, high-grade tumors display significantly reduced STAT3 expression levels compared to low-grade cases. Survival analysis of lung AC cases with smoking history demonstrated that patients with low STAT3 expression have a worse prognosis than patients with higher STAT3 expression within the tumors. These results further elucidate that STAT3 de-regulation in patients has a negative impact on tumor progression as well as survival outcome. Altogether, our data demonstrate a tumor suppressive function of STAT3 in KRAS-driven lung cancer through inhibition of NFkB-induced CXCL1/IL-8 expression. This is of particular importance since treatment with STAT3 inhibitors may worsen tumor progression and outcome in a subset of patients, precisely with KRAS mutations. Hence, we recommend that lung AC patients should be stratified according to their driver mutations before treatment with STAT3 inhibitors is considered.submitted by Beatrice GrabnerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, Med. Univ., Diss., 2015OeBB(VLID)271261

Trondheim cityscape. Preservation of the city identity

The concept of the Cultural Heritage Protection in Climate Change (CHePiCC) School focuses on built cultural heritage as integral part of cultural landscapes and vice versa, both seriously affected by climate change and related natural catastrophes. Climate change itself is the connecting element and affects both cultural landscapes and cultural heritage. Higher Education (HE) students are given the chance to learn about applicable maintenance, preparedness and preservation measures by developing tailored measures for a given site and by implementing and actually testing their ideas. The concept focuses on the effects of climate change in a certain climate zone, and develops possibilities to teach sustainable, eco-friendly and cost-efficient preparedness measures for built cultural heritage and maintenance measures for cultural landscapes. The whole concept is hybrid, student-centred, following a strict hands-on approach and is research based. The entire concept of the School can easily be transferred to other frameworks

Vitamin D treatment attenuates cardiac FGF23/FGFR4 signaling and hypertrophy in uremic rats

Vitamin D deficiency and excess of circulating fibroblast growth factor 23 (FGF23) contribute to cardiovascular mortality in patients with chronic kidney disease (CKD). FGF23 activates FGF receptor 4 and (FGFR4) calcineurin/nuclear factor of activated T cells (NFAT) signaling in cardiac myocytes, thereby causing left ventricular hypertrophy (LVH). Here, we determined if 1,25-dihydroxyvitamin D (calcitriol) inhibits FGF23-induced cardiac signaling and LVH. 5/6 nephrectomized (5/6 Nx) rats were treated with different doses of calcitriol for 4 or 10 weeks and cardiac expression of FGF23/FGFR4 and activation of calcineurin/NFAT as well as LVH were analyzed. FGFR4 activation and hypertrophic cell growth were studied in cultured cardiac myocytes that were co-treated with FGF23 and calcitriol. In 5/6Nx rats with LVH, we detected elevated FGF23 expression in bone and myocardium, increased cardiac expression of FGFR4 and elevated cardiac activation of calcineurin/NFAT signaling. Cardiac expression levels of FGF23 and FGFR4 significantly correlated with the presence of LVH in uremic rats. Treatment with calcitriol reduced LVH as well as cardiac FGFR4 expression and calcineurin/NFAT activation. Bone and cardiac FGF23 expression were further stimulated by calcitriol in a dose-dependent manner, but levels of intact cardiac FGF23 protein were suppressed by high-dose calcitriol. In cultured cardiac myocytes, co-treatment with calcitriol blocked FGF23-induced activation of FGFR4 and hypertrophic cell growth. Our data suggest that in CKD, cardioprotective effects of calcitriol stem from its inhibitory actions on the cardiac FGF23/FGFR4 system, and based on their counterbalancing effects on cardiac myocytes, high FGF23 and low calcitriol synergistically contribute to cardiac hypertrophy

Heterologous protein production using euchromatin-containing expression vectors in mammalian cells

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.(VLID)458983

A Mouse Model to Assess STAT3 and STAT5A/B Combined Inhibition in Health and Disease Conditions

Genetically-engineered mouse models (GEMMs) lacking diseased-associated gene(s) globally or in a tissue-specific manner represent an attractive tool with which to assess the efficacy and toxicity of targeted pharmacological inhibitors. Stat3 and Stat5a/b transcription factors have been implicated in several pathophysiological conditions, and pharmacological inhibition of both transcription factors has been proposed to treat certain diseases, such as malignancies. To model combined inhibition of Stat3 and Stat5a/b we have developed a GEMM harboring a flox Stat3-Stat5a/b allele (Stat5/3loxP/loxP mice) and generated mice lacking Stat3 and Stat5a/b in hepatocytes (Stat5/3Δhep/Δhep). Stat5/3Δhep/Δhep mice exhibited a marked reduction of STAT3, STAT5A and STAT5B proteins in the liver and developed steatosis, a phenotype that resembles mice lacking Stat5a/b in hepatocytes. In addition, embryonic deletion of Stat3 and Stat5a/b (Stat5/3Δ/Δ mice) resulted in lethality, similar to Stat3Δ/Δ mice. This data illustrates that Stat5/3loxP/loxP mice are functional and can be used as a valuable tool to model the combined inhibition of Stat3 and Stat5a/b in tumorigenesis and other diseases.We thank Hans-Christian Theussl for performing blastocyst injections, Francis Stewart for providing the pUBC/EM7-hygromicin and pSC101-BAD-gbaA plasmids.Funding: Open Access Funding by the Austrian Science Fund (FWF): P 25599-B19 (EC) and the Austrian Federal Ministry of Science and Research GENAU grant ‘Austromouse’ (EC and RE). RE was supported by the Austrian Science Fund (FWF) Doktoratskolleg-plus grant “Inflammation and Immunity”, the FWF grants P25925-B20,P26908-B20 and P29222-B28.S

AKT3 drives adenoid cystic carcinoma development in salivary glands

Salivary gland cancer is an aggressive and painful cancer, but a rare tumor type accounting for only 0.5% of cancer cases. Tumors of the salivary gland exhibit heterogeneous histologic and genetic features and they are subdivided into different subtypes, with adenoid cystic carcinomas (ACC) being one of the most abundant. Treatment of ACC patients is afflicted by high recurrence rates, the high potential of the tumors to metastasize, as well as the poor response of ACC to chemotherapy. A prerequisite for the development of targeted therapies is insightful genetic information for driver core cancer pathways. Here, we developed a transgenic mouse model toward establishment of a preclinical model. There is currently no available mouse model for adenoid cystic carcinomas as a rare disease entity to serve as a test system to block salivary gland tumors with targeted therapy. Based on tumor genomic data of ACC patients, a key role for the activation of the PI3KAKTmTOR pathway was suggested in tumors of secretory glands. Therefore, we investigated the role of Akt3 expression in tumorigenesis and report that Akt3 overexpression results in ACC of salivary glands with 100% penetrance, while abrogation of transgenic Akt3 expression could revert the phenotype. In summary, our findings validate a novel mouse model to study ACC and highlight the druggable potential of AKT3 in the treatment of salivary gland patients.(VLID)480659