The emergence of resistance to systemic therapies in pancreatic ductal adenocarcinoma (PDAC) is still a major obstacle in clinical practice. Both, constitutive and inducible NF-κB activity are known as key players in this context. To identify differentially expressed and TRAIL resistance mediating NF-κB target genes, TRAIL sensitive and resistant PDAC cell lines were analyzed by transcriptome assays. In this context, A20 was identified as an NF-κB/RelA inducible target gene. Translational PDAC tissue analysis confirmed the correlation of elevated A20 protein expression with activated RelA expression in PDAC patients. In in vitro experiments, an elevated A20 expression is accompanied by a specific resistance toward TRAIL-mediated apoptosis but not to chemotherapeutic-induced cell death. This TRAIL resistance was attributed to A20´s E3-ligase activity-mediating Zink finger domain. Furthermore, the ubiquitin-binding scaffold protein p62 was identified as indispensable for the TRAIL-mediated apoptosis-inducing pathway affected by A20. The results of this study identify A20 as a possible therapeutic target to affect resistance to TRAIL-induced apoptosis in PDAC cells.

Obesity and obesity-associated diseases represent one of the key health challenges of our time. In this context, aberrant hepatic lipid accumulation is a central pathological aspect of non-alcoholic fatty liver disease (NAFLD). By comparing methylation signatures of liver biopsies before and after bariatric surgery, we recently demonstrated the strong enrichment of differentially methylated heat shock factor 1 (HSF1) binding sites (>400-fold) in the process of liver remodeling, indicating a crucial role of HSF1 in modulating central aspects of NAFLD pathogenesis. Using cellular models of NAFLD, we were able to show that HSF1 is activated during fat accumulation in hepatocytes, mimicking conditions in patients before bariatric surgery. This induction was abolished by starving the cells, mimicking the situation after bariatric surgery. Regarding this connection, carnitine palmitoyltransferase 1 isoform A (CTP1a), a central regulator of lipid beta-oxidation, was identified as a HSF1 target gene by promoter analysis and HSF1 knockdown experiments. Finally, pharmacological activation of HSF1 through celastrol reduced fat accumulation in the cells in a HSF1-dependent manner. In conclusion, we were able to confirm the relevance of HSF1 activity and described a functional HSF1-CPT1a pathway in NAFLD pathogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant neoplasms and registers rising death rates in western countries. Due to its late detection in advanced stages, its extremely aggressive nature and the minimal effectiveness of currently available therapies, PDAC is a challenging problem in the clinical field. One characteristic of PDAC is a distinct desmoplasia consisting of fibroblasts, endothelial and immune cells as well as non-cellular components, contributing to therapy resistance. It is well established that the NF-κB signaling pathway controls inflammation, cancer progression and apoptosis resistance in PDAC. This study attempts to identify NF-κB target genes mediating therapy resistance of humane PDAC cell lines towards death ligand induced apoptosis. By using a genome wide unbiased approach the chemokine CX3CL1 was established as a central NF-κB target gene mediating therapy resistance. While no direct impact of CX3CL1 expression on cancer cell apoptosis was identified in co-culture assays it became apparent that CX3CL1 is acting in a paracrine fashion, leading to an increased recruitment of inflammatory cells. These inflammatory cells in turn mediate apoptosis resistance of PDAC cells. Therefore, our data dissect a bifunctional cross-signaling pathway in PDAC between tumor and immune cells giving rise to therapy resistance.

Background/Aim: FOLFIRINOX as neoadjuvant therapy for locally advanced pancreatic cancer has shown encouraging R0 resection and response rates.We report our experience for the overall response rate, R0 resection rate, progression free survival of FOLFIRINOX and chemoradiation in patients with) borderline resectable, locally advanced pancreatic cancer (LAPC) and LAPC with isolated liver metastasis.Methods: Retrospectively, 15 consecutive patients were given neoadjuvant FOLFIRINOX between 11/2011 and 8/2014.Radiographic review was used to allocate the patients into 3 groups (5 patients in each) based on National Comprehensive Cancer Network Criteria.Endpoints were objective response rates as per Response Evaluation Criteria in Solid Tumors Criteria, R0 resection rate and progression free survival.Results: All patients' characteristics are summarized in Table1.Out of 15, 5 were borderline resectable (Grp 1), 5 were locally advanced (Grp 2), and 5 had locally advanced disease with isolated liver metastases (Grp 3).Following neoadjuvant FOLFIRINOX the overall R0 resection rate was 27% (20% in Grp 1 and 2, 40% in Grp 3).The ORR was 26.7%.Median follow-up was 14 months.The progression free survival (PFS) and overall survival (OS) in surgical and non-surgical patient's seen in graphs 1 and 2. Conclusion: FOLFIRINOX possesses activity in patients with borderline-resectable and LAPC.The use of FOLFIRINOX was associated with conversion to R0 resectability in 40% of our patients, 27% after FOLFIRINOX alone and 13% after additional treatment with radiation.Despite having locally advanced disease with isolated liver metastasis, 40% of patients were able to proceed to R0 resection.There was also a survival benefit in patients getting neoadjuvant therapy with FOLFIRINOX and then resection.Prospective studies are advised to evaluate the true benefit of this treatment and survival rate.

Einleitung: Der Transkriptionsfaktor NF-kB hat entscheidenden Anteil an der Therapieresistenz des duktalen Pankreasadenokarzinom (PDAC). Obwohl sich die Mehrzahl der Arbeiten auf die am häufigsten untersuchte p65/RelA NF-kB-Untereinheit fokussieren, sind sowohl die Mechanismen als auch die hieran beteiligten Zielgene kaum verstanden.

Pankreasadenokarzinome weisen zahlreiche molekulare Veränderungen auf, die die Chemoresistenz gegenüber Zytostatika bedingen. Veränderungen in anti-apoptotischen Signaltransduktionswegen scheinen hierbei eine entscheidende Bedeutung zu haben. So konnte gezeigt werden, dass in einer Reihe von Pankreaskarzinomzellinien eine hohe konstitutive NFkB-Aktivität die Resistenz gegenüber TopoisomeraseII-Inhibitoren vermittelt und in anderen Zellinien Akt/PI3K an der Chemoresistenz beteiligt zu sein scheint. In der vorliegenden Arbeit sollte geklärt werden, ob NFkB und/oder Akt/PI3K Pankreaskarzinomzellinien gegenüber dem in der Therapie des Pankreaskarzinoms am häufigsten eingesetzten Zytostatikum Gemzar schützt. Anschließend sollte die Eignung dieser Mechanismen als molekulares Ziel einer kombinierten Chemotherapie in vitro und in vivo untersucht werden. Mittels FACS-Analysen gelang es uns nachzuweisen, dass Zellinien mit hoher basaler NFkB-Aktivität, im Gegensatz zu Zellinien mit niedriger basaler NFkB-Aktivität gegenüber Gemzar resistent sind. EMSA und Luciferase-Assay zeigten, dass Gemzar in allen Zellinien eine dosisabhängige NFkB-Induktion bewirkt. Die Tatsache, dass durch NFkB-Inhibition die resistenten Zellen auch gegenüber Gemzar-Konzentrationen sensitiviert werden, die nicht in der Lage sind, NFkB zu induzieren, belegt die Theorie, dass hauptsächlich die konstitutive NFkB-Aktivität für die Resistenz gegenüber Gemzar verantwortlich ist. Im Gegensatz hierzu bestand zwischen der Akt-Aktivität und der Gemzar-Resistenz keine Korrelation. Weiterhin hatte eine pharmakologische PI3K/Akt-Inhibition keine Auswirkungen auf die Sensitivität gegenüber diesem Zytostatikum. Die auf diesen Daten basierenden in vivo Untersuchungen in einem subkutanen SCID-Maus Modell bestätigten, dass durch eine NFkB-Inhibition mittels Sulfasalazin eine signifikante Verbesserung der Ansprechrate von Capan-1 oder PancTu-1 Tumoren auf Gemzar erzielt werden kann. Es ist somit festzuhalten, dass durch eine spezifische NFkB-Inhibition Pankreaskarzinom-Zellinien in vitro und in vivo gegenüber Gemzar sensitiviert werden. Dies lässt die Hoffnung zu, dass die Kombination von Zytostatika mit klinisch etablierten NFkB-hemmenden Mitteln eine neue Therapieoption des Pankreaskarzinoms darstellen könnte.

Einleitung: Das duktale Pankreasadenokarzinom (PDAC) repräsentiert eine Tumorentität mit einer der schlechtesten Prognosen. Dem Transkriptionsfaktor NF-κB wird hierbei durch die Regulierung anti-apoptotischer Zielgene eine entscheidende Rolle zugesprochen.

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies with an overall life expectancy of six months despite palliative radio-chemotherapy. The transcription factor NF-κB has been shown to be a critical component of dysregulated transcription factor activity conferring this profound resistance against chemotherapeutic drugs and death receptor induced apoptosis. Despite extensive data on the role of the most abundant NF-κB subunit p65/RelA in PDAC apoptosis control, only little knowledge of the role of the subunit c-Rel in solid cancers exists. In the present study, three pancreatic carcinoma cell lines (Panc1, Patu8988t, MiaPaca2) were analysed for the role of c-Rel in resistance against TRAIL induced apoptosis. TRAIL resistant Panc1 and Patu8988 cells exhibit a strong TRAIL inducible NF-κB activity, whereas TRAIL sensitive MiaPaca2 cells displayed only a small increase in NF-κB binding activity. Transfection with siRNA against the c-Rel subunit of NF-κB sensitized the TRAIL resistant cells in a comparable fashion like siRNA targeting the p65/RelA subunit. Gel shift analysis revealed that together with the p65/RelA subunit, c-Rel is part of the TRAIL inducible NF-κB complex in PDAC. Array analysis results suggested NFATc2 as a c-Rel target gene that is one of the 15 strongest TRAIL inducible genes in apoptosis-resistant Panc1 cells. siRNA targeting c-Rel strongly reduced TRAIL induced NFATc2 activity in TRAIL resistant PDAC cells. Furthermore siRNA targeting NFATc2 sensitized these PDAC cells against TRAIL induced apoptosis. Finally, TRAIL induced expression of COX-2 was strongly reduced through siRNA targeting c-Rel or NFATc2 and pharmacological inhibition of COX-2 with celecoxib strongly increased TRAIL apoptosis. In conclusion, c-Rel is a critical mediator of NF-κB dependent anti-apoptotic signalling in PDAC through activation of NFATc2 and COX-2. Citation Format: Claudia Geismann, Frauke Grohmann, Gabriele Wirths, Susanne Sebens, Anita Dreher, Robert Häsler, Sebastian Zeissig, Stefan Schreiber, Philip Rosenstiel, Heiner Schäfer, Alexander Arlt. c-Rel is a critical mediator of NF-κB-dependent apoptosis resistance of pancreatic cancer cells against TRAIL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2273. doi:10.1158/1538-7445.AM2014-2273

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies with an overall life expectancy of 6 months despite current therapies. NF-κB signalling has been shown to be critical for this profound cell-autonomous resistance against chemotherapeutic drugs and death receptor-induced apoptosis, but little is known about the role of the c-Rel subunit in solid cancer and PDAC apoptosis control. In the present study, by analysis of genome-wide patterns of c-Rel-dependent gene expression, we were able to establish c-Rel as a critical regulator of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in PDAC. TRAIL-resistant cells exhibited a strong TRAIL-inducible NF-κB activity, whereas TRAIL-sensitive cells displayed only a small increase in NF-κB-binding activity. Transfection with siRNA against c-Rel sensitized the TRAIL-resistant cells in a manner comparable to siRNA targeting the p65/RelA subunit. Gel-shift analysis revealed that c-Rel is part of the TRAIL-inducible NF-κB complex in PDAC. Array analysis identified NFATc2 as a c-Rel target gene among the 12 strongest TRAIL-inducible genes in apoptosis-resistant cells. In line, siRNA targeting c-Rel strongly reduced TRAIL-induced NFATc2 activity in TRAIL-resistant PDAC cells. Furthermore, siRNA targeting NFATc2 sensitized these PDAC cells against TRAIL-induced apoptosis. Finally, TRAIL-induced expression of COX-2 was diminished through siRNA targeting c-Rel or NFATc2 and pharmacologic inhibition of COX-2 with celecoxib or siRNA targeting COX-2, enhanced TRAIL apoptosis. In conclusion, we were able to delineate a novel c-Rel-, NFATc2- and COX-2-dependent antiapoptotic signalling pathway in PDAC with broad clinical implications for pharmaceutical intervention strategies.

Einleitung: Der NFkB Signalweg, und hier vor allem die RelA/p65 Untereinheit, ist im Pankreaskarzinom (PDAC) für die ausgeprägte Apoptoseresistenz essentiell. Es wird allerdings kontrovers diskutiert ob eine basale oder eine induzible NFκB Aktivität den Hauptanteil an dieser Apoptose-Resistenz hat und welche Zielgene involviert sind.

After day 12, tumor growth was measured every 3 days (x-axis) and reported as "Tumor volume" on the Y-axis."Untreated" indicates that tumor cells were not transduced.

Einleitung: Das duktale Pankreasadenokarzinom (PDAC) weist nach wie vor eine nahezu infauste Prognose auf und zählt zu den häufigsten Ursachen krebsbedingter Todesfälle. Die ausgeprägte Chemoresistenz gegenüber Chemotherapeutika und Todesliganden stellt ein wesentliches Problem in der Behandlung dieses Malignomsdar. Ein neuartiger Mechanismus der Apoptoseresistenz bei soliden Tumoren konnte kürzlich in der erhöhten Aktivität des Transkriptionsfaktors Nrf2 aufgezeigt werden. Nrf2 war bisher aufgrund seiner protektiven Wirkung gegenüber durch oxidativen/metabolischen Stress verursachten Zell-/DNA-Schädigung in der Chemoprävention von Bedeutung. Unsere Arbeitsgruppe konnten allerdings kürzlich zeigen, dass Nrf2 auch in der Lage ist, Tumorzellen durch Induktion des 26S-Proteasoms und daraus resultierender verstärkter NF-κB Aktivierung vor Apoptose zu schützen.

Das duktale Pankreasadenokarzinom weist nach wie vor eine nahezu infauste Prognose auf und zählt zu den häufigsten Ursachen krebsbedingter Todesfälle. Die ausgeprägte Chemoresistenz stellt ein wesentliches Problem in der Behandlung des Pankreaskarzinoms dar, wobei dem Transkriptionsfaktor NF-κB eine zentrale Bedeutung zukommt. Kürzlich konnte gezeigt werden, dass der Verlust der Expression des early response Gens IEX-1 mit einer deutlich schlechteren Prognose bei dieser Tumorentität einhergeht. Ziel der Studie war daher die molekularen Mechanismen der durch IEX-1 vermittelten Tumorsuppression und eine mögliche Verbindung mit der NF-κB vermittelten Chemoresistenz zu klären.

The early response gene IEX-1 plays a complex role in the regulation of apoptosis. Depending on the cellular context and the apoptotic stimulus, IEX-1 is capable to either enhance or suppress apoptosis. To further dissect the molecular mechanisms involved in the modulation of apoptosis by IEX-1, we analysed the molecular crosstalk between IEX-1 and the NF-κB pathway. Using GST-pulldown assays, a direct interaction of IEX-1 with the C-terminal region of the subunit RelA/p65 harbouring the transactivation domain of the NF-κB transcription factor was shown. This interaction negatively regulates RelA/p65 dependent transactivation as shown by GAL4-and luciferase assay and was confirmed for the endogenous proteins by co-immunoprecipitation experiments. Using deletion constructs, we were able to map the C-terminal region of IEX-1 as the critical determinant of the interaction with RelA/p65. We could further show, that IEX-1 mediated NF-κB inhibition accounts for the reduced expression of the anti-apoptotic NF-κB target genes Bcl-2, Bcl-xL, cIAP1 and cIAP2, thereby sensitizing cells for apoptotic stimuli. Finally, ChIP-assays revealed that IEX-1 associates with the promoter of these genes. Altogether, our findings suggest a critical role of IEX-1 in the NF-κB dependent regulation of apoptotic responses.

The stress response gene IEX-1 (immediate early gene-X-1) is involved in the regulation of cell growth and cellular viability. To some extent, these effects include an interference with the proteasomal turnover of certain regulatory proteins. Here, we show that IEX-1 directly attenuates the activity and formation of the 26 S proteasome in HEK-293 cells (human embryonic kidney cells). We further demonstrate that IEX-1 reduces the overall expression levels of certain protein components of the 19 S proteasomal subunit such as S5a/Rpn10 and S1/Rpn2, whereas the expression of other proteasomal proteins was less or not affected. In contrast with direct apoptotic stimuli, such as the anti-cancer drug etoposide, leading to caspase-dependent degradation of S1 and S5a, the effect of IEX-1 is independent of proteolytic cleavage of these proteins. Furthermore, the decreasing effect of IEX-1 on S5a and S1 expression is still seen in the presence of cycloheximide, but not in the presence of actinomycin D, and quantitative real-time PCR revealed lower mRNA levels of S5a and S1 in IEX-1-overexpressing cells, suggesting an interference of IEX-1 with the gene transcription of S5a and S1. Additionally, luciferase assays confirmed an interference of IEX-1 with the activity of the S5a promoter. These findings indicate a role of IEX-1 in the maintenance and assembly of the 26 S proteasome, obviously involving an altered gene expression of certain proteasomal proteins. Thereby, IEX-1 may essentially modulate signalling pathways related to 26 S proteasome activity and involved in cellular growth control and apoptosis.

Immediate early gene X1 (IEX-1) represents a stress response gene involved in growth control and modulation of apoptosis. Here, we report a detailed analysis of IEX-1 with respect to its intracellular localization. By means of confocal laser scanning microscopy, a green fluorescent protein-IEX-1 fusion protein transfected into HeLa cells, as well as endogenous IEX-1, could be detected in distinct subnuclear structures. This particular subnuclear localization of IEX-1 was not observed with a green fluorescent protein-IEX-1 fusion protein lacking a putative nuclear localization sequence, along with a decreased effect on apoptosis. Double immunofluorescence staining revealed a partial co-localization of endogenous promyelocytic leukemia protein (PML) and IEX-1 in these subnuclear structures. Nuclear localization of IEX-1 is also enhanced upon treatment of cells with leptomycin B, an inhibitor of the nuclear exporter CRM1. These observations indicate that IEX-1 is specifically shuttled to and from the nucleus. Overexpression experiments using PML isoforms III and IV revealed distinct intranuclear interaction of IEX-1 and PML. Coprecipitation experiments showed physical interaction between IEX-1 and PML. The close structural relation of IEX-1-containing nuclear subdomains and PML nuclear bodies suggests a function of IEX-1 related to the multiple functions of these unique subnuclear regions, particularly during stress response and growth control.