Susanne Sebens Müerköster

Role of NF-κB and Akt/PI3K in the resistance of pancreatic carcinoma cell lines against gemcitabine-induced cell death

Pancreatic cancer is resistant to almost all cytotoxic drugs. Currently, gemcitabine appears to be the only clinically active drug but, because of pre-existing or acquired chemoresistance of most of the tumor cells, it failed to significantly improve the outcome of pancreatic carcinoma patients. The current study examined the relevance of nuclear factor κB (NF-κB) and PI3K/Akt in the resistance of five pancreatic carcinoma cell lines towards gemcitabine. Treatment for 24 h with gemcitabine (0.04–20 μM) led to a strong induction of apoptosis in PT45-P1 and T3M4 cells but not in BxPc-3, Capan-1 and PancTu-1 cells. These resistant cell lines exhibited a high basal NF-κB activity in contrast to the sensitive cell lines. Furthermore, gemcitabine showed a dose-dependent induction of NF-κB. At a dose of 0.04 μM, gemcitabine still induced apoptosis in the sensitive cell lines, but did not induce NF-κB. In addition, NF-κB inhibition by MG132, sulfasalazine or the IκBα super-repressor strongly diminished the resistance against gemcitabine (0.04–20 μM). In contrast to this obvious correlation between basal NF-κB activity and gemcitabine resistance, PI3K/Akt seems not to be involved in gemcitabine resistance of these cell lines. Neither did the basal Akt activity correlate with the sensitivity towards gemcitabine treatment, nor did the inhibition of PI3K/Akt by LY294002 alter gemcitabine-induced apoptosis. These results indicate that constitutive NF-κB activity confers resistance against gemcitabine and that modulation of this activity by pharmacological or genetic approaches may have therapeutical potential when combined with gemcitabine in the treatment of pancreatic carcinoma.

Tumor Stroma Interactions Induce Chemoresistance in Pancreatic Ductal Carcinoma Cells Involving Increased Secretion and Paracrine Effects of Nitric Oxide and Interleukin-1β

Pancreatic ductal carcinoma is characterized by a profound chemoresistance. As we have shown previously, these tumor cells can develop chemoresistance by interleukin (IL)-1β in an autocrine and nuclear factor-κB-dependent fashion. Because pancreatic ductal carcinoma contains many mesenchymal stromal cells, we further investigated how tumor–stroma interactions contribute to chemoresistance by using a transwell coculture model, including murine pancreatic fibroblasts and the chemosensitive human pancreatic carcinoma cell lines T3M4 and PT45-P1. If cultured with fibroblast-conditioned medium or kept in coculture with fibroblasts, both cell lines became much less sensitive toward treatment with etoposide than cells cultured under standard conditions. Furthermore, the secretion of IL-1β in T3M4 and PT45-P1 cells was increased by the fibroblasts, and IL-1β-receptor blockade abolished the resistance-inducing effect during cocultivation. This stimulated IL-1β secretion could be attributed to nitric oxide (NO) released by the fibroblasts as an IL-1β-inducing factor. Although both tumor cells secreted only little NO, which was in line with undetectable inducible nitric oxide synthase (iNOS) expression, fibroblasts exhibited significant iNOS expression and NO secretion that could be further induced by the tumor cells. Incubation of T3M4 and PT45-P1 cells with the NO donor S-Nitroso-N-acetyl-D,l-penicillamine up-regulated IL-1β secretion and conferred resistance toward etoposide-induced apoptosis. Conversely, the resistance-inducing effect of the fibroblasts was significantly abolished, when the specific iNOS inhibitor aminoguanidine was added during coculture. Immunohistochemistry on tissue sections from human pancreatic ductal carcinoma also revealed iNOS expression in stromal cells and IL-1β expression in tumor cells, thus supporting the in vitro findings. These data clearly demonstrate that fibroblasts contribute to the development of chemoresistance in pancreatic carcinoma cells via increased secretion of NO, which in turn leads to an elevated release of IL-1β by the tumor cells. These findings substantiate the implication of tumor–stromal interactions in the chemoresistance of pancreatic carcinoma.

Increased Expression of the E3-Ubiquitin Ligase Receptor Subunit βTRCP1 Relates to Constitutive Nuclear Factor-κB Activation and Chemoresistance in Pancreatic Carcinoma Cells

The permanent activation of the transcription factor nuclear factor-kappaB (NF-kappaB) in pancreatic cancer cells is associated with a profound resistance towards chemotherapy. In the present study, we show that chemoresistant pancreatic cancer cell lines exhibiting constitutive NF-kappaB activity (i.e., PancTu-1, BxPc3, and Capan-1) express significantly elevated levels of the E3-ubiquitin ligase receptor subunit betaTRCP1, compared with pancreatic carcinoma cell lines lacking constitutive NF-kappaB activity and chemoresistance (i.e., PT45-P1 and T3M4). If transfected with betaTRCP1, PT45-P1 cells exhibit an elevated NF-kappaB activity and become less sensitive towards anticancer drug treatment (i.e., etoposide). Conversely, blockade of betaTRCP1 expression in PancTu-1 cells by transfection with a vector-expressed small interfering RNA reduces NF-kappaB activation and chemoresistance. In PancTu-1 cells, betaTRCP1 expression is inhibited, at least in part, by the interleukin-1 (IL-1) receptor(I) antagonist, whereas stimulation of PT45-P1 cells with IL-1beta resulted in an increased expression of betaTRCP1, and transfection of this cell line with betaTRCP1 induced IL-1beta secretion in a NF-kappaB-dependent fashion. Thus, via its close and mutual link to IL-1beta secretion, betaTRCP1 expression might substantially contribute to the persistent, IL-1beta-dependent activation of NF-kappaB in pancreatic carcinoma cells. In support of this, betaTRCP1 expression is detectable at considerable levels in a great number of pancreatic ductal adenocarcinoma specimens, along with an intense staining for activated NF-kappaB. Altogether, our findings of the elevated betaTRCP1 expression in pancreatic carcinoma cells pinpoint to another important mediator of constitutive NF-kappaB activation and thereby of chemoresistance.

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.

Targeting apoptosis pathways in pancreatic cancer

Pancreatic cancer - here in particular pancreatic ductal adenocarcinoma (PDAC) - is still a highly therapy refractory disease. Amongst the mechanisms by which PDAC cells could escape any non-surgical therapy, anti-apoptotic protection seems to be the most relevant one. PDAC cells have acquired resistance to apoptotic stimuli such as death ligands (FasL, TRAIL) or anti-cancer drugs (gemcitabine) by a great number of molecular alterations either disrupting an apoptosis inducing signal or counteracting the execution of apoptosis. Thus, PDAC cells exhibit alterations in the EGFR/MAPK/Ras/raf1-, PI3K/Akt-, TRAIL/TRAF2-, or IKK/NF-κB pathway accompanied by deregulations in the expression of apoptosis regulators such as cIAP, Bcl2, XIAP or survivin. Along with protection against apoptosis, PDAC cells also overexpress histone deacetylases (HDACs) giving rise to epigenetic patterns of chemoresistance and to acetylation of other regulatory proteins, as well. With respect to the multitude of anti-apoptotic pathways, a great number of molecular targets might be of high potential in novel therapy strategies. Thus, natural compounds as well as novel synthetic drugs are considered to be used in single or combined therapy of PDAC. A number of proteasome and HDAC inhibitors or selective inhibitors of IKK, EGFR, Akt and mTOR have been widely explored in preclinical settings and clinical studies. Even though these early studies encouraged an application in a clinical setting, most of the trials have been rather disappointing yet. Thus, new molecular targets and novel concepts of combination therapies need to get access into clinical trials - either in neoadjuvant/adjuvant or in palliative treatments.

CX3CR1 promotes recruitment of human glioma-infiltrating microglia/macrophages (GIMs)

The transmembrane chemokine CX3CL1 and its receptor CX3CR1 are thought to be involved in the trafficking of immune cells during an immune response and in the pathology of various human diseases including cancer. However, little is known about the expression and function of CX3CR1 in human glioma-infiltrating microglia/macrophages (GIMs), representing the major cellular stroma component of highly malignant gliomas. Here, we show that CX3CR1 is overexpressed at both the mRNA and protein level in solid human astrocytomas of different malignancy grades and in glioblastomas. CX3CR1 was localized in ionized calcium-binding adapter molecule 1 (Iba1) and CD11b/c positive GIMs in situ as shown by fluorescence microscopy. In accordance with this, freshly isolated human GIM-enriched fractions separated by CD11b MACS technology displayed high Iba1 and CX3CR1 mRNA expression levels in vitro. Moreover, cultured human GIMs responded to CX3CL1-triggered activation of CX3CR1 with adhesion and migration in vitro. Besides an increase in motility, CX3CL1 also enhanced expression of matrix metalloproteases 2, 9, and 14 in GIM fractions in vitro. These data indicate that the CX3CL1/CX3CR1 system has a crucial tumor-promoting role in human glioblastomas via its impact on glioma-infiltrating immune subsets.

Up-regulation of L1CAM in Pancreatic Duct Cells Is Transforming Growth Factor β1– and Slug-Dependent: Role in Malignant Transformation of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is thought to originate from ductal structures, exhibiting strong desmoplastic reaction with stromal pancreatic myofibroblasts (PMF), which are supposed to drive PDAC tumorigenesis. Previously, we observed high expression of the adhesion molecule L1CAM (CD171) in PDAC cells accounting for chemoresistance. Thus, this study aimed to investigate whether PMFs are involved in the induction of tumoral L1CAM and whether this contributes to malignant transformation of pancreatic ductal cells and PDAC tumorigenesis. Immunohistochemistry of tissues from chronic pancreatitis specimens revealed considerable L1CAM expression in ductal structures surrounded by dense fibrotic tissue, whereas no L1CAM staining was seen in normal pancreatic tissues. Using the human pancreatic duct cell line H6c7, we show that coculture with PMFs led to a transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of L1CAM expression. Similarly, L1CAM expression increased in monocultured H6c7 cells after administration of exogenous TGF-beta1. Both TGF-beta1- and PMF-induced L1CAM expression were independent of Smad proteins but required c-Jun NH(2)-terminal kinase activation leading to the induction of the transcription factor Slug. Moreover, Slug interacted with the L1CAM promoter, and its knockdown abrogated the TGF-beta1- and PMF-induced L1CAM expression. As a result of L1CAM expression, H6c7 cells acquired a chemoresistant and migratory phenotype. This mechanism of TGF-beta1-induced L1CAM expression and the resulting phenotype could be verified in the TGF-beta1-responsive PDAC cell lines Colo357 and Panc1. Our data provide new insights into the mechanisms of tumoral L1CAM induction and how PMFs contribute to malignant transformation of pancreatic duct cells early in PDAC tumorigenesis.

CD95 and TRAF2 promote invasiveness of pancreatic cancer cells

Pancreatic adenocarcinoma represents a tumor type with extremely poor prognosis. High apoptosis resistance and a strong invasive and early metastatic potential contribute to its highly malignant phenotype. Here we identified the death receptor adaptor molecule TRAF2 as a key player in pancreatic cancer pathophysiology. Using immunohistochemistry and Western blot analysis we found TRAF2 overexpressed in 34 of 36 pancreatic tumor samples as well as in pancreatic tumor cell lines resistant to CD95‐mediated apoptosis. The high TRAF2 protein level was not related to chromosomal changes, as monitored by FISH analysis. Instead, the NF‐κB‐and MEK‐signaling pathways were involved. Introduction of a TRAF2 expression vector in CD95‐sensitive Colo357 cells resulted in (i) resistance to CD95‐induced apoptosis; (ii) increased constitutive NF‐κB and AP‐1 activity; and (iii) higher basal secretion of matrix metalloproteinases (MMPs), urokinase‐type plasminogen activator (uPA), and IL‐8, leading to increased invasiveness. High apoptosis resistance and uPA secretion could be reverted by TRAF2‐specific siRNA. Stimulation of TRAF2‐overexpressing cells with CD95 ligand led to induction of NF‐κB and AP‐1, enhanced IL‐8‐ and uPA‐secretion, and a further increased invasiveness. Thus, TRAF2 overexpression does not only block apoptosis induction by CD95 but also converts this death receptor into a mediator of invasiveness.

Role of myofibroblasts in innate chemoresistance of pancreatic carcinoma—Epigenetic downregulation of caspases

We recently reported on continuous tumor–stroma interactions essentially contributing to chemoresistance of pancreatic ductal adenocarinoma (PDAC) cells. As demonstrated here, long‐term coculture with pancreatic myofibroblasts representing the main stromal compartment of PDAC resulted in a chemoresistant phenotype in the pancreatic ductal epithelial cell line H6c7 as well as in the chemosensitive PDAC cell line T3M4. This involved a reduced expression of caspases and the caspase inducing transcription factor STAT1, both caused by diminished gene transcription. The DNA‐methylation inhibitor 5‐azadeoxycytidine enhanced caspase and STAT1 expression in cocultured H6c7 and T3M4 cells along with an increased chemosensitivity, indicating a role for CpG DNA‐hypermethylation in the downregulation of these crucial apoptosis mediators. Cocultured H6c7 and T3M4 cells exhibited elevated nuclear levels of DNA‐methyltransferase‐1 (DNMT1). Silencing of DNMT1 expression by siRNA increased expression of caspases and STAT1 and restored chemosensitivity. In SCID mice, tumors arising from coinoculated T3M4 cells and myofibroblasts (co‐tumors) responded less towards chemotherapy than mono‐tumors, exhibiting decreased apoptosis, no remission and reduced expression of caspases and STAT1. These data underscore the role of myofibroblasts in chemoresistance of PDAC and point to the importance of caspases as central target structures of epigenetic regulation in this scenario. Furthermore, an activated microenvironment might apparently promote the manifestation of chemoresistance already in premalignant precursor cells at early stages of PDAC tumorigenesis.

Enhanced L1CAM expression on pancreatic tumor endothelium mediates selective tumor cell transmigration

L1 cell adhesion molecule (L1CAM) is a transmembrane cell adhesion molecule initially defined as a promigratory molecule in the developing nervous system that appears to be also expressed in some endothelial cells. However, little is known about the functional role of L1CAM on endothelial cells. We observed that L1CAM expression was selectively enhanced on endothelium associated with pancreatic adenocarcinoma in situ and on cultured pancreatic tumor-derived endothelial cells in vitro. L1CAM expression of endothelial cells could be augmented by incubation with immunomodulatory cytokines such as tumor necrosis factor alpha, interferon gamma, or transforming growth factor beta 1. Antibodies to L1CAM and the respective ligand neuropilin-1 blocked tube formation and stromal cell-derived factor 1β induced transmigration of tumor endothelial cells in vitro. L1CAM expression on tumor-derived-endothelial cells enhanced Panc1 carcinoma cell adhesion to endothelial cell monolayers and transendothelial migration. Our data demonstrate a functional role of L1CAM expression on tumor endothelium that could favor metastasis and angiogenesis during tumor progression.