L1CAM-integrin interaction induces constitutive NF-kappaB activation in pancreatic adenocarcinoma cells by enhancing IL-1beta expression.

L1 cell adhesion molecule (L1CAM) overexpression is often associated with bad prognosis in various human carcinomas. Recent studies also suggest a role of L1CAM in pancreatic ductal adenocarcinomas (PDAC). To further address its contribution, we expressed functional domains of L1CAM in PT45-P1 PDAC cells. We found that L1CAM that is full length (L1-FL), but neither the soluble ectodomain (L1ecto) nor the cytoplasmic part (L1cyt), could enhance cell proliferation or tumour growth in mice. Expression of L1-FL resulted in constitutive activation of NF-kappaB, which was abolished by L1CAM knockdown. We showed that the expression of IL-1beta was selectively upregulated by L1-FL, and increased IL-1beta levels were instrumental for sustained NF-kappaB activation. IL-1beta production and NF-kappaB activation were abolished by knockdown of alpha5-integrin and integrin-linked kinase, but insensitive to depletion of L1CAM cleavage proteinases. Supporting these data, PT45-P1 cells transduced with an L1CAM mutant deficient in integrin binding (L1-RGE) did not support the described L1-FL functions. Our results suggest that membranous L1CAM interacts with RGD-binding integrins, leading to sustained NF-kappaB activation by IL-1beta production and autocrine/paracrine signalling. The unravelling of this novel mechanism sheds new light on the important role of L1CAM expression in PDAC cells.

Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2).

An elevated proteasome activity contributes to tumorigenesis, particularly by providing cancer cells with antiapoptotic protection and efficient clearance from irregular proteins. Still, the underlying mechanisms are poorly known. In this study, we report that in colon cancer patients, higher proteasome activity was detected in tumoral tissue compared with surrounding normal tissue, and also that increased levels of proteasomal subunit proteins, such as S5a/PSMD4 and alpha-5/PSMA5, could be detected. Colon tumors showed higher nuclear levels of nuclear factor E2-related factor 2 (Nrf2), a transcription factor supposed to be involved in the control of proteasomal subunit protein expression. The induction or overexpression of Nrf2 led to stronger S5a and alpha-5 expression in the human colon cancer cell lines, Colo320 and Lovo, as well as in NCM460 colonocytes along with higher proteasome activity. The small interfering RNA (siRNA)-mediated Nrf2 knockdown decreased S5a and alpha-5 expression and reduced proteasome activity. Additionally, Nrf2-dependent S5a and alpha-5 expression conferred protection from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, an effect preceded by an increased nuclear factor (NF)-kappaB activation and higher expression of antiapoptotic NF-kappaB target genes. These findings point to an important role of Nrf2 in the gain of proteasome activity, thereby contributing to colorectal carcinogenesis. Nrf2 may therefore serve as a potential target in anticancer therapy.

The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-kappa B inhibition.

Addressing the puzzling role of amidated gastrin(17) (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis, we analysed potential candidate genes involved in G17-dependent NF-kappaB inhibition and apoptosis. The colorectal carcinoma cell line Colo320 overexpressing the wild-type CCK2 receptor (Colo320wt) underwent G17-induced apoptosis along with suppressed NF-kappaB activation and decreased expression of the antiapoptotic NF-kappaB target genes cIAP1 and cIAP2, whereas G17 was without effect on Colo320 cells expressing a CCK2 receptor bearing a loss of function mutation (Colo320mut). Gene microarray analysis revealed an elevated expression of the stress response gene IEX-1 in G17-treated Colo320wt but not Colo320mut cells. Quantitative real-time PCR and conventional RT-PCR confirmed this G17-dependent increase of IEX-1 expression in Colo320wt cells. If these cells were subjected to IEX-1 knockdown by small interfering RNA transfection, the apoptosis-inducing effect of G17 was abolished. Moreover, tumor necrosis factor alpha (TNFalpha)- or 5-FU-induced apoptosis that is greatly enhanced by G17 treatment in Colo320wt cells was prevented if IEX-1 expression was repressed. Under these conditions of blocked IEX-1 expression, the NF-kappaB activity remained unaffected by G17, in particular in Colo320wt cells co-treated with TNFalpha and also the suppressive effect of G17 on cIAP1 and cIAP2 expression was not observed anymore if IEX-1 expression was blocked. Conversely, IEX-1 overexpression in Colo320mut cells caused an increase of basal and TNFalpha- or 5-FU-induced apoptosis, an effect not further triggered by G17 treatment. Using a xenograft tumor model in severe combined immune deficiency mice, we could show that experimental systemic hypergastrinemia induced by the administration of omeprazole led to enhanced apoptosis as well as to a marked increase of IEX-1 expression in Colo320wt tumors, but not in Colo320mut tumors. These observations indicate that the proapoptotic effect of G17 on human colon cancer cells expressing the wild-type CCK2 receptor is mediated by IEX-1, which modulates NF-kappaB-dependent antiapoptotic protection and thereby exerts tumor-suppressive potential.

Dissecting the role of TGF-beta type I receptor/ALK5 in pancreatic ductal adenocarcinoma: Smad activation is crucial for both the tumor suppressive and prometastatic function.

In the present study, we have analysed the effects of transforming growth factor-beta (TGF-beta) signaling on the growth behavior of pancreatic carcinoma cells in vitro and on their tumorigenicity in vivo. Ectopic expression of dominant-negative mutants of the TGF-beta type II receptor or type I receptor/activin receptor-like kinase 5 (ALK5) in TGF-beta-sensitive pancreatic ductal adenocarcinoma PANC-1 cells prevented the TGF-beta-induced activation of transfected Smad-responsive reporter genes and growth arrest. The growth-inhibitory effect was mimicked by stable expression of kinase-active ALK5 (ALK5-T204D), and was dependent on ALK5's ability to activate Smad signaling, as a ALK5-derived mutant with an intact kinase domain but deficient in its ability to activate Smads (RImL45) failed to suppress proliferation in the absence of added TGF-beta. Moreover, this mutant often displayed opposite effects to those of ALK5-TD and blocked various ligand-induced responses in vitro, indicating that it acts in a dominant-negative fashion to inhibit endogenous wild-type receptors. ALK5-TD-, but not RImL45-TD-transduced cells underwent epithelial-to-mesenchymal transition, exhibited a higher ratio of thrombospondin-1 to vascular endothelial growth factor-A expression and upregulated various metastasis-associated genes. Upon orthotopic transplantation of PANC-1 clones into immunodeficient mice, ALK5-TD, but not RImL45-TD, greatly reduced tumor size and induced the formation of liver metastases in otherwise non-metastatic PANC-1 cells. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in pancreatic tumor cells.

Drug-induced expression of the cellular adhesion molecule L1CAM confers anti-apoptotic protection and chemoresistance in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rapid tumor progression, high metastatic potential and profound chemoresistance. We recently reported that induction of a chemoresistant phenotype in the PDAC cell line PT45-P1 by long-term chemotherapy involves an increased interleukin 1 beta (IL1beta)-dependent secretion of nitric oxide (NO) accounting for efficient caspase inhibition. In the present study, we elucidated the involvement of L1CAM, an adhesion molecule previously found in other malignancies, in this NO-dependent chemoresistance. Chemoresistant PT45-P1res cells, but not chemosensitive parental PT45-P1 cells, express high levels of L1CAM in an ILbeta-dependent fashion. PT45-P1res cells subjected to short interfering RNA (siRNA)-mediated L1CAM knock-down exhibited reduced inducible nitric oxide synthase expression and NO secretion, as well as a significant increase of anti-cancer drug-induced caspase activation, an effect reversed by the NO donor S-nitroso-N-acetyl-D,L-penicillamine. Conversely, overexpression of L1CAM in PT45-P1 cells conferred anti-apoptotic protection to anti-cancer drug treatment. Interestingly, L1CAM ectodomain shedding, in example, by ADAM10, as reported for other L1CAM-related activities, seemed to be dispensable for anti-apoptotic protection by L1CAM. Neither the shedded L1CAM ectodomain was detected in chemoresistant L1CAM-expressing PT45-P1 cells nor did the administration of various metalloproteinase inhibitors affect L1CAM-dependent chemoresistance. Immunohistochemical analysis revealed L1CAM expression in 80% of pancreatic cancer specimens, supporting a potential role of L1CAM in the malignancy of this tumor. These findings substantiate our understanding of the molecular mechanisms leading to chemoresistance in PDAC cells and indicate the importance of L1CAM in this scenario.

Acquired chemoresistance in pancreatic carcinoma cells: induced secretion of IL-1beta and NO lead to inactivation of caspases.

Pancreatic cancer exhibits profound chemoresistance resulting either from pre-existing (intrinsic) mechanisms, or from anticancer drug treatment itself (acquired chemoresistance). To identify molecular alterations leading to acquired chemoresistance, the chemosensitive pancreatic carcinoma cell line PT45-P1 was exposed to low-dose treatment with etoposide for 6 weeks. Afterwards, these cells (PT45-P1res) were much more resistant to high-dose treatment with anticancer drugs than parental cells. Among several differentially expressed genes in PT45-P1res cells, IL-1beta was most significantly upregulated, a finding in line with our previous observation that IL-1beta accounts for intrinsic chemoresistance of pancreatic carcinoma cells. Elevated IL-1beta expression in PT45-P1res cells was confirmed by real-time PCR and ELISA, and treatment with the IL-1 receptor antagonist restored drug-induced apoptosis. The increased IL-1beta secretion was accompanied by an elevated formation of nitric oxide (NO) and a NO-dependent inhibition of the etoposide-induced caspase-3/-7/-8/-9 activity. Caspase activation was restored either by the iNOS inhibitor 1400W, the reducing agent dithiothreitol or the IL-1 receptor antagonist, resulting in greater sensitivity towards anticancer drug treatment. Conversely, IL-1beta or the NO-donor SNAP decreased caspase activation and apoptosis in etoposide-treated PT45-P1 cells. These data confirm IL-1beta and NO as determinants of chemoresistance in pancreatic cancer, and indicate that the intrinsic and acquired chemoresistance rely to some extent on common molecular targets beneficial for improved therapeutical strategies.

Superantigen reactive Vbeta6+ T cells induce perforin/granzyme B mediated caspase-independent apoptosis in tumour cells.

The endogenous viral superantigen 7 in DBA/2 mice serves as a target antigen on syngeneic ESb-MP lymphoma cells for allogeneic graft-vs-leukaemia reactive cells. Allogeneic viral superantigen 7 reactive Vbeta6+ T cells are able to transfer graft-vs-leukaemia reactivity and to kill specifically viral superantigen 7+ ESb-MP tumour cells in vitro. Here we elucidate the mechanism of this superantigen specific cell lysis. Already 10 min after co-incubation with in vitro stimulated Vbeta6+ T cells, viral superantigen 7+ ESb-MP tumour cells show an apoptotic phenotype (Annexin V-positivity, DNA-fragmentation). This extremely rapid type of cell death is not mediated by the death inducing ligands CD95L, TRAIL and TNF but by perforin and granzyme B. Surprisingly, neither mitochondria nor any of the known caspases appear to be involved in this type of tumour cell killing. In contrast, nitric oxide, released by activated macrophages and endothelial cells, induces in the same tumour cells another type of apoptosis which is much slower and involves mitochondria and caspase activation. A synergistic effect between the two different effector mechanisms of superantigen reactive donor cytotoxic T lymphocytes and nitric oxide releasing host macrophages and endothelial cells might explain the effective immune rejection of even advanced metastasised cancer in this graft-vs-leukaemia animal model.

The chemotherapeutic drug 5-fluorouracil induces apoptosis in mouse thymocytes in vivo via activation of the CD95(APO-1/Fas) system.

The CD95/CD95 ligand (CD95L) system has been shown to mediate chemotherapeutic drug-induced apoptosis in vitro. However, the contribution of the CD95 pathway to drug-induced apoptosis is controversial. We have shown previously that 5-fluorouracil (5-FU) induces apoptosis in vitro via the activation of the CD95/CD95L system. To study the effects of the chemotherapeutic drug 5-FU and the contribution of the CD95 system to 5-FU-induced apoptosis in vivo, we gave mice an i.p. injection of 5-FU. Apoptotic cell death peaked in thymocytes at 18 h after administration of 5-FU. Total organ weight and cell number in the thymus were reduced by approximately 40%. This cell loss was due to apoptosis, as measured in cell suspensions by measuring hypodiploid DNA content and by terminal deoxynucleotidyl transferase-mediated nick end labeling staining of tissue sections. The number of apoptotic cells correlated with the extent of weight loss and cell attrition of the organs. Furthermore, in the thymi of 5-FU-treated animals, CD95L was strongly up-regulated. Apoptosis of thymocytes was blocked in vivo with neutralizing anti-CD95L antibodies. In addition, cell loss in the thymus was negligible in lpr mice in comparison with wild-type mice. Thus, a significant portion of apoptosis of thymocytes in vivo on treatment with 5-FU is mediated via the CD95/CD95L pathway. Our findings therefore contribute to the understanding how chemotherapeutic drugs exert their effects in vivo.

Breaking tolerance to a tumor-associated viral superantigen as a basis for graft-versus-leukemia reactivity.

A major goal in tumor immunotherapy consists of breaking potential tumor-specific T-cell unresponsiveness (tolerance), which may explain tumor growth in cancer patients. We report that immunological tolerance to a tumor-associated viral superantigen (SAg) is overcome in a mouse lymphoma model by transfer of allogeneic T cells expressing SAg-reactive Vbeta6 T-cell receptor chains. Surprisingly, upon contact with SAg-expressing lymphoma cells, Vbeta6 T cells became activated rather than tolerized (as reported previously). They also developed SAg-specific cytotoxic T-lymphocyte activity and secreted IL-2 and IFN-gamma. The grafted T cells infiltrated liver metastases, formed close contact with SAg-expressing tumor cells, and caused significant graft-vs. -leukemia (GvL) effects. Selection for tumor resistance among the progeny from a cross between SAg-negative donor and SAg- positive recipient strains revealed a strict correlation between loss of the endogenous SAg tolerogen, rescue of Vbeta6 T cells from SAg-mediated deletion, and leukemia resistance. These findings suggest that immune responses to SAg can be exploited to break tolerance and augment immune responses to tumors.

Functional and in situ evidence for nitric oxide production driven by CD40-CD40L interactions in graft-versus-leukemia reactivity.

In a murine tumor model, complete tumor remission is achievable at even advanced metastasized stages by transfer of immune T cells from donor B10.D2 (H-2d, Mls(b)) into tumor-bearing DBA/2 (H-2d, Mls(a)) mice. We showed previously that this graft-versus-leukemia (GvL) effect is dependent on synergistic interactions of transferred CD4+ and CD8+ T cells with host sialoadhesin (SER)-positive macrophages. We now show that the CD40-CD40L (CD154) interaction is involved in the induction of inducible nitric oxide synthase (iNOS) expression during adoptive immunotherapy (ADI). We demonstrate that during ADI, the level of CD40 expression in the liver becomes significantly augmented in comparison to livers of tumor-bearing, untreated animals. CD40 expression is found mostly on SER+ macrophages and to a lesser extent on dendritic cells (DCs). In GvL animals, more SER+ macrophages express iNOS than untreated animals. iNOS expressing cells are found in close proximity to apoptotic cells, at early time points of the therapy in areas of metastasis, and at late stages around portal veins, where CD4+ and CD8+ T lymphocytes form clusters with SER+ macrophages. Blocking of CD40L in vivo at days 5 and 20, when all iNOS+ cells express CD40, leads to significantly reduced CD40 and iNOS expression as well as to a marked inhibition of the therapeutic effect. These data provide functional and in situ evidence that the increased CD40 and iNOS expression observed during ADI contribute to the eradication of liver metastases and to the clearance of donor lymphocytes from the liver.

Sialoadhesin-positive host macrophages play an essential role in graft-versus-leukemia reactivity in mice.

We recently established an effective immune T-cell-mediated graft-versus-leukemia (GVL) murine model system in which complete tumor remissions were achievable even in advanced metastasized cancer. We now describe that this T-cell-mediated therapy is dependent on host macrophages expressing the lymphocyte adhesion molecule sialoadhesin (Sn). Depletion of Kupffer cells in tumor-bearing mice during adoptive immunotherapy (ADI) or the treatment of these animals with anti-Sn monoclonal antibodies led to complete or partial inhibition of the immune T-cell-mediated therapeutic effect. Furthermore, Sn+ host macrophages in livers formed clusters during ADI with donor CD8 T cells. To test for a possible antigen presentation function of these macrophages, we used as an in vitro model the antigen beta-galactosidase for which a dominant major histocompatibility complex (MHC) class I Ld-restricted peptide epitope is known to be recognized by specific CD8 cytotoxic T lymphocytes (CTL). We demonstrate that purified Sn+ macrophages can process exogenous beta-galactosidase and stimulate MHC class I peptide-restricted CTL responses. Thus, Sn+ macrophages, which are significantly increased in the liver after ADI, may process tumor-derived proteins via the MHC class I pathway as well as via the MHC class II pathway, as shown previously, and present respective peptide epitopes to CD8 as well as to CD4 immune T cells, respectively. The synergistic interactions observed before between immune CD4 and CD8 T cells during ADI could thus occur in the observed clusters with Sn+ host macrophages.

Graft-versus-leukemia reactivity involves cluster formation between superantigen-reactive donor T lymphocytes and host macrophages.

T-cell-mediated antitumor effects play an important role clinically in allogeneic graft-versus-leukemia (GvL) reactivity, whereas T-cell-mediated antihost effects are associated with a risk of developing graft-versus-host (GvH) disease. GvL and GvH were compared in an animal tumor model system after the systemic transfer of allogeneic antitumor immune T lymphocytes from B10.D2 [H-2d; minor lymphocyte-stimulating antigen (Mls)b] mice into ESb-MP tumor-bearing or normal DBA/2 (H-2d; Mls(a)) mice. Here we demonstrate that this T-cell-mediated therapy involves the formation of clusters of donor CD4 and CD8 T cells with host macrophages, in particular, with a subpopulation expressing the lymphocyte adhesion molecule sialoadhesin. DBA/2 mice and the derived tumor ESb-MP express viral superantigen 7 (Mls(a)), an endogenous viral superantigen that is absent from B10.D2 mice. To test the contribution of viral superantigen 7-reactive Vbeta6 donor T cells in the GvL-mediated eradication of liver metastases, we performed immunohistological and transmission electron microscopy studies. Vbeta6+ CD4 and CD8 T cells from B10.D2 donors formed tight clusters with host sialoadhesin-positive macrophages, and transmission electron microscopy pictures revealed direct membrane-membrane interactions between T cells and macrophages. Clusters were more abundant and consisted of more cells in tumor-bearing hosts (GvL model) than in non-tumor-bearing hosts (GvH model). In addition, Vbeta6 T cells within the clusters showed a strong proliferation activity, indicating stimulation. Moreover, in an in vitro tumor cytostasis assay, primed as well as nonprimed purified Vbeta6 T cells from donor mice were able to inhibit the proliferation of superantigen-expressing ESb-MP lymphoma cells. This suggests that the transferred superantigen-reactive Vbeta6 T cells contribute to the eradication of metastases. The observed cell clusters might be sites for antigen presentation and the activation of tumor-reactive T cells.

Antagonistic effects of systemic interleukin 2 on immune Tcell-mediated graft-versus-leukemia reactivity.

This study demonstrates that systemic interleukin 2 (IL-2) can decrease the homing of syngeneic immune T cells to the target organ of metastases and accelerate unwanted side effects of allogeneic immune T cells. As a tumor system, we used the well-characterized highly aggressive DBA/2 mouse leukemia ESb and its less aggressive adhesion variant, ESb-MP. Systemic IL-2 treatment was performed with recombinant human interleukin-2 (Proleukin), which was slowly released via an implanted osmotic pump or was modified with polyethylene glycol (PEG-IL-2) to achieve constant plasma levels. Allogeneic B10.D2 antitumor immune spleen cells (ISPL cells) exerted strong graft-versus-leukemia (GvL) reactivity after adoptive transfer into late-stage ESb-MP tumor-bearing DBA/2 mice. Mls(a) superantigen-reactive vbeta6 donor T cells were not eliminated or tolerized by in vivo priming with the tumor cells and were present in active proliferation in liver infiltrates. When exogenous PEG-IL-2 or Proleukin was applied in addition to ISPL cells in such mice, the strong GvL-mediated protective immunity was converted into a fatal graft-versus-host disease. IL-2 treatment alone had no toxic effect and caused a moderate protection effect in the absence of an effect on local tumor growth. Potentiation of GvH reactivity of B10.D2 ISPL by PEG-IL-2 was proven in non-tumor-bearing DBA/2 mice, in which graft-versus-host disease was characterized by: (a) heavy hepatic lymphocytic infiltration, (b) irreversible increase of serum glutamate-oxalacetate-transaminase and glutamate-pyruvate-transaminase levels, (c) weight loss, and (d) death. Antagonistic effects of systemic IL-2 on GvL were observed with syngeneic DBA/2 anti-ESb immune peritoneal effector cells (PECs). There was a detrimental effect of systemic IL-2 on liver target organ infiltration by immune T cells causing, at day 6 after transfer, a drop from 20-30 CD4 or CD8 T cells per liver lobule in the PEC group to <5 in the PEC plus IL-2 group. The results emphasize the importance of a better understanding of IL-2 function in vivo and of its interaction with immune cell function to improve protocols for optimal application in the clinic to achieve maximal GvL effects.