Frauke Grohmann

Usage of the NF-κB inhibitor sulfasalazine as sensitizing agent in combined chemotherapy of pancreatic cancer

Sulfasalazine is commonly used as an anti inflammatory agent and is known as a potent inhibitor of NF‐κB. Some pancreatic carcinomas are characterized by a constitutively elevated NF‐κB activity accounting for chemoresistance. To elucidate whether blockade of NF‐κB activity with sulfasalazine is suitable for overcoming this chemoresistance in vivo, we employed a mouse model with subcutaneously xenotransplanted human Capan‐1 pancreatic carcinoma cells. Fourteen days upon tumor inoculation, animals were randomized in 6 groups, receiving no treatment, treatment with gemcitabine or with etoposide, either alone or in combination with sulfasalazine, or with sulfasalazine alone. Two therapy regimens were given with a 7‐day interval in between. Upon treatment with etoposide or gemcitabine alone, tumor sizes were moderately reduced to 65–68% and 50–65%, respectively, as compared to untreated tumors. Sulfasalazine alone only decreased temporarily the tumor sizes. Sulfasalazine in combination with gemcitabine showed only partially higher reduction in tumor sizes than gemcitabine alone, whereas the combination with etoposide reduced significantly the tumor sizes in all experiments (down to 20%). TUNEL‐staining showed higher numbers of apoptotic cells in tumors from the combination groups, in particular with etoposide, and proliferation as indicated by Ki67 staining was strongly reduced. Furthermore, combined treatment of sulfasalazine with the cytostatic drugs led to a decreased blood vessel density. Immunohistochemical staining of the activated p65 subunit showed that sulfasalazine treatment abolished the basal NF‐κB activity in tumor xenografts. These data imply that the well established anti‐inflammatory drug sulfasalazine sensitizes pancreatic carcinoma cells to anti cancer drugs, in particular to etoposide in vivo by inhibition of NF‐κB. This combined chemotherapy offers great potential for improved anti‐tumor responses in pancreatic carcinomas.

IEX-1 directly interferes with RelA/p65 dependent transactivation and regulation of apoptosis.

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-kappaB 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-kappaB 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-kappaB inhibition accounts for the reduced expression of the anti-apoptotic NF-kappaB 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-kappaB dependent regulation of apoptotic responses.

Immediate early gene-X1 interferes with 26 S proteasome activity by attenuating expression of the 19 S proteasomal components S5a/Rpn10 and S1/Rpn2

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.

[Autocrine IL-1beta secretion leads to NF-kappabeta-mediated chemoresistance in pancreatic carcinoma cells in vivo].

Zusammenfassung.Hintergrund und Ziel:Das duktale Pankreaskarzinom (PDAC) ist durch einen hochmalignen Phänotyp und eine profunde Chemoresistenz charakterisiert. In der vorliegenden Studie wurde die autokrine Sekretion von Interleukin-(IL)1β durch Pankreaskarzinomzellen in vivo als eine Ursache für einen chemoresistenten Phänotyp untersucht.Material und Methodik:Zellen der humanen PDAC-Zelllinie PancTu1 mit ausgeprägter IL-1β-Sekretion wurden subkutan in weibliche SCID-Mäuse inokuliert. Nach 10 Tagen wurden die Tiere randomisiert und entweder unbehandelt gelassen oder für 14 Tage mit einem IL-1β-RI-Antikörper, mit Etoposid oder einer Kombination aus beidem behandelt. Anschließend wurden die Tumorgrößen bestimmt und die Tumoren immunhistologisch auf Apoptose, Vaskularisierung sowie die Aktivität des Transkriptionsfaktors NF-κB untersucht.Ergebnisse:Die Kombination aus IL-1β-RI-Antikörper und Etoposid führte im Vergleich zu den entsprechenden Monotherapien bzw. keiner Behandlung zu einem deutlich verringerten Wachstum der PancTu1-Tumoren. Entsprechend konnte in Tumoren der Kombinationsbehandlung eine signifikant erhöhte Anzahl an apoptotischen Zellen detektiert werden. Nach der Behandlung mit dem IL-1β-RI-Antikörper wiesen die Tumoren eine deutlich verminderte Präsenz von aktiviertem NF-κB im Vergleich zu den Kontrolltumoren auf. Ferner war in den Tumoren nach Kombinationstherapie die Vaskularisierung (CD31-positive Zellen) weniger stark ausgeprägt.Schlussfolgerung:Die autokrine Sekretion von IL-1β ist an der Entstehung konstitutiver NF-κB-Aktivität und einer dadurch vermittelten Chemoresistenz in Pankreaskarzinomzellen beteiligt.Abstract.Background and Purpose:The pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly malignant phenotype and a profound chemoresistance. Thus, options for an effective treatment of this disease are still quite poor. In this study, it was investigated whether the autocrine secretion of interleukin-(IL-)1β is related to a chemoresistant phenotype of PDAC cells in vivo.Material and Methods:Human PancTu1 PDAC cells were inoculated subcutaneously into female SCID mice. After 10 days of outgrowth, animals were randomized and left untreated or treated with an IL-1β-RI antibody, etoposide, or a combination of both. After treatment for 14 days, tumor sizes were determined and each tumor was analyzed immunohistochemically for apoptosis (TUNEL), activated NF-κB (p65), and vascularization (CD31 staining).Results:The combination of IL-1β-RI antibody and etoposide led to a significantly reduced outgrowth of PancTu1 tumors in comparison to the monotherapies or no treatment. Accordingly, the number of apoptotic cells was significantly elevated in tumors of the combination group. After treatment with the IL-1β-RI antibody, less activated NF-κB was present in tumors compared to the control group. Moreover, tumors of the combination group showed a clearly reduced vascularization.Conclusion:The autocrine secretion of IL-1β contributes to a constitutively increased NF-κB activity in PDAC cells along with a chemoresistant phenotype.

Role of CCL20 mediated immune cell recruitment in NF-κB mediated TRAIL resistance of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers. From a clinical view, the transcription factor NF-κB is of particular importance, since this pathway confers apoptosis resistance and limits drug efficacy. Whereas the role of the most abundant NF-κB subunit p65/RelA in therapeutic resistance is well documented, only little knowledge of the RelA downstream targets and their functional relevance in TRAIL mediated apoptosis in PDAC is available. In the present study TRAIL resistant and sensitive PDAC cell lines were analyzed for differentially expressed RelA target genes, to define RelA downstream targets mediating TRAIL resistance. The most upregulated target gene was then further functionally characterized. Unbiased genome-wide expression analysis demonstrated that the chemokine CCL20 represents the strongest TRAIL inducible direct RelA target gene in resistant PDAC cells. Unexpectedly, targeting CCL20 by siRNA, blocking antibodies or by downregulation of the sole CCL20 receptor CCR6 had no effect on PDAC cell death or cancer cell migration, arguing against an autocrine role of CCL20 in PDAC. However, by using an ex vivo indirect co-culture system we were able to show that CCL20 acts paracrine to recruit immune cells. Importantly, CCL20-recruited immune cells further increase TRAIL resistance of CCL20-producing PDAC cells. In conclusion, our data show a functional role of a RelA-CCL20 pathway in PDAC TRAIL resistance. We demonstrate how the therapy-induced cross-talk of cancer cells with immune cells affects treatment responses, knowledge needed to tailor novel bi-specific treatments, which target tumor cell as well as immune cells.

TRAIL/NF-κB/CX3CL1 Mediated Onco-Immuno Crosstalk Leading to TRAIL Resistance of Pancreatic Cancer Cell Lines

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.

Abstract 2273: c-Rel is a critical mediator of NF-κB-dependent apoptosis resistance of pancreatic cancer cells against TRAIL

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 Hasler, Sebastian Zeissig, Stefan Schreiber, Philip Rosenstiel, Heiner Schafer, 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

Autokrine IL-1β-Sekretion führt zu erhöhter NF-κB-Aktivität und zu Chemoresistenz in Pankreaskarzinomzellen in vivo

Zusammenfassung.Hintergrund und Ziel:Das duktale Pankreaskarzinom (PDAC) ist durch einen hochmalignen Phänotyp und eine profunde Chemoresistenz charakterisiert. In der vorliegenden Studie wurde die autokrine Sekretion von Interleukin-(IL)1β durch Pankreaskarzinomzellen in vivo als eine Ursache für einen chemoresistenten Phänotyp untersucht.Material und Methodik:Zellen der humanen PDAC-Zelllinie PancTu1 mit ausgeprägter IL-1β-Sekretion wurden subkutan in weibliche SCID-Mäuse inokuliert. Nach 10 Tagen wurden die Tiere randomisiert und entweder unbehandelt gelassen oder für 14 Tage mit einem IL-1β-RI-Antikörper, mit Etoposid oder einer Kombination aus beidem behandelt. Anschließend wurden die Tumorgrößen bestimmt und die Tumoren immunhistologisch auf Apoptose, Vaskularisierung sowie die Aktivität des Transkriptionsfaktors NF-κB untersucht.Ergebnisse:Die Kombination aus IL-1β-RI-Antikörper und Etoposid führte im Vergleich zu den entsprechenden Monotherapien bzw. keiner Behandlung zu einem deutlich verringerten Wachstum der PancTu1-Tumoren. Entsprechend konnte in Tumoren der Kombinationsbehandlung eine signifikant erhöhte Anzahl an apoptotischen Zellen detektiert werden. Nach der Behandlung mit dem IL-1β-RI-Antikörper wiesen die Tumoren eine deutlich verminderte Präsenz von aktiviertem NF-κB im Vergleich zu den Kontrolltumoren auf. Ferner war in den Tumoren nach Kombinationstherapie die Vaskularisierung (CD31-positive Zellen) weniger stark ausgeprägt.Schlussfolgerung:Die autokrine Sekretion von IL-1β ist an der Entstehung konstitutiver NF-κB-Aktivität und einer dadurch vermittelten Chemoresistenz in Pankreaskarzinomzellen beteiligt.Abstract.Background and Purpose:The pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly malignant phenotype and a profound chemoresistance. Thus, options for an effective treatment of this disease are still quite poor. In this study, it was investigated whether the autocrine secretion of interleukin-(IL-)1β is related to a chemoresistant phenotype of PDAC cells in vivo.Material and Methods:Human PancTu1 PDAC cells were inoculated subcutaneously into female SCID mice. After 10 days of outgrowth, animals were randomized and left untreated or treated with an IL-1β-RI antibody, etoposide, or a combination of both. After treatment for 14 days, tumor sizes were determined and each tumor was analyzed immunohistochemically for apoptosis (TUNEL), activated NF-κB (p65), and vascularization (CD31 staining).Results:The combination of IL-1β-RI antibody and etoposide led to a significantly reduced outgrowth of PancTu1 tumors in comparison to the monotherapies or no treatment. Accordingly, the number of apoptotic cells was significantly elevated in tumors of the combination group. After treatment with the IL-1β-RI antibody, less activated NF-κB was present in tumors compared to the control group. Moreover, tumors of the combination group showed a clearly reduced vascularization.Conclusion:The autocrine secretion of IL-1β contributes to a constitutively increased NF-κB activity in PDAC cells along with a chemoresistant phenotype.

Autokrine IL-1-Sekretion fhrt zu erhhter NF-?B-Aktivitt und zu Chemoresistenz in Pankreaskarzinomzellen in vivo@@@Autocrine IL-1 Secretion Leads to NF-?-Mediated Chemoresistance in Pancreatic Carcinoma Cells in Vivo

Zusammenfassung.Hintergrund und Ziel:Das duktale Pankreaskarzinom (PDAC) ist durch einen hochmalignen Phänotyp und eine profunde Chemoresistenz charakterisiert. In der vorliegenden Studie wurde die autokrine Sekretion von Interleukin-(IL)1β durch Pankreaskarzinomzellen in vivo als eine Ursache für einen chemoresistenten Phänotyp untersucht.Material und Methodik:Zellen der humanen PDAC-Zelllinie PancTu1 mit ausgeprägter IL-1β-Sekretion wurden subkutan in weibliche SCID-Mäuse inokuliert. Nach 10 Tagen wurden die Tiere randomisiert und entweder unbehandelt gelassen oder für 14 Tage mit einem IL-1β-RI-Antikörper, mit Etoposid oder einer Kombination aus beidem behandelt. Anschließend wurden die Tumorgrößen bestimmt und die Tumoren immunhistologisch auf Apoptose, Vaskularisierung sowie die Aktivität des Transkriptionsfaktors NF-κB untersucht.Ergebnisse:Die Kombination aus IL-1β-RI-Antikörper und Etoposid führte im Vergleich zu den entsprechenden Monotherapien bzw. keiner Behandlung zu einem deutlich verringerten Wachstum der PancTu1-Tumoren. Entsprechend konnte in Tumoren der Kombinationsbehandlung eine signifikant erhöhte Anzahl an apoptotischen Zellen detektiert werden. Nach der Behandlung mit dem IL-1β-RI-Antikörper wiesen die Tumoren eine deutlich verminderte Präsenz von aktiviertem NF-κB im Vergleich zu den Kontrolltumoren auf. Ferner war in den Tumoren nach Kombinationstherapie die Vaskularisierung (CD31-positive Zellen) weniger stark ausgeprägt.Schlussfolgerung:Die autokrine Sekretion von IL-1β ist an der Entstehung konstitutiver NF-κB-Aktivität und einer dadurch vermittelten Chemoresistenz in Pankreaskarzinomzellen beteiligt.Abstract.Background and Purpose:The pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly malignant phenotype and a profound chemoresistance. Thus, options for an effective treatment of this disease are still quite poor. In this study, it was investigated whether the autocrine secretion of interleukin-(IL-)1β is related to a chemoresistant phenotype of PDAC cells in vivo.Material and Methods:Human PancTu1 PDAC cells were inoculated subcutaneously into female SCID mice. After 10 days of outgrowth, animals were randomized and left untreated or treated with an IL-1β-RI antibody, etoposide, or a combination of both. After treatment for 14 days, tumor sizes were determined and each tumor was analyzed immunohistochemically for apoptosis (TUNEL), activated NF-κB (p65), and vascularization (CD31 staining).Results:The combination of IL-1β-RI antibody and etoposide led to a significantly reduced outgrowth of PancTu1 tumors in comparison to the monotherapies or no treatment. Accordingly, the number of apoptotic cells was significantly elevated in tumors of the combination group. After treatment with the IL-1β-RI antibody, less activated NF-κB was present in tumors compared to the control group. Moreover, tumors of the combination group showed a clearly reduced vascularization.Conclusion:The autocrine secretion of IL-1β contributes to a constitutively increased NF-κB activity in PDAC cells along with a chemoresistant phenotype.