Hans-Jürgen Schlitt

Mammalian target of rapamycin is activated in human gastric cancer and serves as a target for therapy in an experimental model

The mammalian target of rapamycin (mTOR) has become an interesting target for cancer therapy through its influence on oncogenic signals, which involve phosphatidylinositol‐3‐kinase and hypoxia‐inducible factor‐1α (HIF‐1α). Since mTOR is an upstream regulator of HIF‐1α, a key mediator of gastric cancer growth and angiogenesis, we investigated mTOR activation in human gastric adenocarcinoma specimens and determined whether rapamycin could inhibit gastric cancer growth in mice. Expression of phospho‐mTOR was assessed by immunohistochemical analyses of human tissues. For in vitro studies, human gastric cancer cell lines were used to determine S6K1, 4E‐BP‐1 and HIF‐1α activation and cancer cell motility upon rapamycin treatment. Effects of rapamycin on tumor growth and angiogenesis in vivo were assessed in both a subcutaneous tumor model and in an experimental model with orthotopically grown tumors. Mice received either rapamycin (0.5 mg/kg/day or 1.5 mg/kg/day) or diluent per intra‐peritoneal injections. In addition, antiangiogenic effects were monitored in vivo using a dorsal‐skin‐fold chamber model. Immunohistochemical analyses showed strong expression of phospho‐mTOR in 60% of intestinal‐ and 64% of diffuse‐type human gastric adenocarcinomas. In vitro, rapamycin‐treatment effectively blocked S6K1, 4E‐BP‐1 and HIF‐1α activation, and significantly impaired tumor cell migration. In vivo, rapamycin‐treatment led to significant inhibition of subcutaneous tumor growth, decreased CD31‐positive vessel area and reduced tumor cell proliferation. Similar significant results were obtained in an orthotopic model of gastric cancer. In the dorsal‐skin‐fold chamber model, rapamycin‐treatment significantly inhibited tumor vascularization in vivo. In conclusion, mTOR is frequently activated in human gastric cancer and represents a promising new molecular target for therapy.

IL-13 Signaling via IL-13Rα2 Induces Major Downstream Fibrogenic Factors Mediating Fibrosis in Chronic TNBS Colitis

BACKGROUND & AIMSnPrevious studies have shown that fibrosis developing in chronic experimental colitis is driven by interleukin (IL)-13 signaling via IL-13R alpha(2) and the production of transforming growth factor (TGF)-beta1. In the present study, we sought to determine the fibrogenic downstream events set in motion by such signaling.nnnMETHODSnExperimental colitis with late-onset intestinal fibrosis was induced by weekly intrarectal administration of trinitrobenzene sulfonic acid (TNBS) to BALB/c mice. Blockade of IL-13 signaling via IL-13R alpha(2) and TGF-beta1 signaling was achieved by the administration of small interfering RNA or decoy oligonucleotides that target promoter sequences of signaling components of these receptors. Effects of blockade were determined by enzyme-linked immunosorbent assay or Western blotting detecting specific key fibrogenic factors and by measurement of collagen production.nnnRESULTSnInitially, we showed that abrogation of IL-13 activity via blockade of IL-13R alpha(2) and TGF-beta1 signaling results in severe inhibition of expression of colonic insulin-like growth factor (IGF)-I and early growth response gene (Egr)-1, factors known to initiate and sustain fibrosis. We then showed that Egr-1 was necessary early in the fibrotic process for caspase-mediated apoptosis of myofibroblasts and the production of urokinase plasminogen activator, a protein that enhances TGF-beta1 activation. Finally, we showed that IGF-I (together with TGF-beta1) acts later in the process to stimulate myofibroblasts to deposit collagen in the colon.nnnCONCLUSIONSnThese studies establish that IL-13 signaling via the IL-13R alpha(2) is a key initiation point for a complex fibrotic program in the colon consisting of TGF-beta1 activation, IGF-I and Egr-1 expression, myofibroblast apoptosis, and myofibroblast production of collagen.

Targeting FGFR/PDGFR/VEGFR impairs tumor growth, angiogenesis and metastasis by effects on tumor cells, endothelial cells and pericytes in pancreatic cancer

Activation of receptor tyrosine kinases, such as fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), and VEGF receptor (VEGFR), has been implicated in tumor progression and metastasis in human pancreatic cancer. In this study, we investigated the effects of TKI258, a tyrosine kinase inhibitor to FGFR, PDGFR, and VEGFR on pancreatic cancer cell lines (HPAF-II, BxPC-3, MiaPaCa2, and L3.6pl), endothelial cells, and vascular smooth muscle cells (VSMC). Results showed that treatment with TKI258 impaired activation of signaling intermediates in pancreatic cancer cells, endothelial cells, and VSMCs, even upon stimulation with FGF-1, FGF-2, VEGF-A, and PDGF-B. Furthermore, blockade of FGFR/PDGFR/VEGFR reduced survivin expression and improved activity of gemcitabine in MiaPaCa2 pancreatic cancer cells. In addition, motility of cancer cells, endothelial cells, and VSMCs was reduced upon treatment with TKI258. In vivo, therapy with TKI258 led to dose-dependent inhibition of subcutaneous (HPAF-II) and orthotopic (L3.6pl) tumor growth. Immunohistochemical analysis revealed effects on tumor cell proliferation [bromodeoxyuridine (BrdUrd)] and tumor vascularization (CD31). Moreover, lymph node metastases were significantly reduced in the orthotopic tumor model when treatment was initiated early with TKI258 (30 mg/kg/d). In established tumors, TKI258 (30 mg/kg/d) led to significant growth delay and improved survival in subcutaneous and orthotopic models, respectively. These data provide evidence that targeting FGFR/PDFGR/VEGFR with TKI258 may be effective in human pancreatic cancer and warrants further clinical evaluation. Mol Cancer Ther; 10(11); 2157–67. ©2011 AACR.

Restoration of Tumor Immunosurveillance via Targeting of Interleukin-13 Receptor-{alpha}2

In previous studies, we described a counter-immunosurveillance mechanism initiated by tumor-activated, interleukin-13 (IL-13)-producing natural killer T cells that signal Gr-1(+) cells to produce transforming growth factor-beta(1) (TGF-beta(1)), a cytokine that suppresses the activity of tumor-inhibiting cytolytic CD8(+) T cells. Here, we show that in two tumor models (the CT-26 metastatic colon cancer and the 15-12RM fibrosarcoma regressor models), this counter-surveillance mechanism requires the expression of a novel IL-13 receptor, IL-13R alpha(2), on Gr-1(intermediate) cells, because down-regulation of IL-13R alpha(2) expression or the activator protein-1 signal generated by the receptor via in vivo administration of specific small interfering RNA or decoy oligonucleotides leads to loss of TGF-beta(1) production. Furthermore, acting on prior studies showing that IL-13R alpha(2) expression is induced (in part) by tumor necrosis factor-alpha (TNF-alpha), we show that receptor expression and TGF-beta(1) production is inhibited by administration of a TNF-alpha-neutralizing substance, TNF-alpha R-Fc (etanercept). Taking advantage of this latter fact, we then show in the CT-26 model that counter-immunosurveillance can be inhibited, anti-CT-26-specific CD8(+) cytolytic activity can be restored, and CT-26 metastatic tumor nodules can be greatly decreased by administration of TNF-alpha R-Fc. Corroborative data were obtained using the 15-12RM fibrosarcoma model. These studies point to the prevention of metastatic cancer with an available agent with already known clinically acceptable adverse effects and toxicity.

Tumor development in murine ulcerative colitis depends on MyD88 signaling of colonic F4/80 + CD11b high Gr1 low macrophages

Patients with prolonged ulcerative colitis (UC) frequently develop colorectal adenocarcinoma for reasons that are not fully clear. To analyze inflammation-associated colonic tumorigenesis, we developed a chronic form of oxazolone-induced colitis in mice that, similar to UC, was distinguished by the presence of IL-13-producing NKT cells. In this model, the induction of tumors using azoxymethane was accompanied by the coappearance of F4/80+CD11b(high)Gr1(low) M2 macrophages, cells that undergo polarization by IL-13 and are absent in tumors that lack high level IL-13 production. Importantly, this subset of macrophages was a source of tumor-promoting factors, including IL-6. Similar to dextran sodium sulfate-induced colitis, F4/80+CD11b(high)Gr1(intermediate) macrophages were present in the mouse model of chronic oxazolone-induced colitis and may influence tumor development through production of TGF-β1, a cytokine that inhibits tumor immunosurveillance. Finally, while robust chronic oxazolone-induced colitis developed in myeloid differentiation primary response gene 88-deficient (Myd88-/-) mice, these mice did not support tumor development. The inhibition of tumor development in Myd88-/- mice correlated with cessation of IL-6 and TGF-β1 production by M2 and F4/80+CD11b(high)Gr1(intermediate) macrophages, respectively, and was reversed by exogenous IL-6. These data show that an UC-like inflammation may facilitate tumor development by providing a milieu favoring development of MyD88-dependent tumor-supporting macrophages.

Urinary Biomarkers TIMP-2 and IGFBP7 Early Predict Acute Kidney Injury after Major Surgery

Objective To assess the ability of the urinary biomarkers IGFBP7 (insulin-like growth factor-binding protein 7) and TIMP-2 (tissue inhibitor of metalloproteinase 2) to early predict acute kidney injury (AKI) in high-risk surgical patients. Introduction Postoperative AKI is associated with an increase in short and long-term mortality. Using IGFBP7 and TIMP-2 for early detection of cellular kidney injury, thus allowing the early initiation of renal protection measures, may represent a new concept of evaluating renal function. Methods In this prospective study, urinary [TIMP-2]×[IGFBP7] was measured in surgical patients at high risk for AKI. A predefined cut-off value of [TIMP-2]×[IGFBP7] >0.3 was used for assessing diagnostic accuracy. Perioperative characteristics were evaluated, and ROC analyses as well as logistic regression models of risk assessment were calculated with and without a [TIMP-2]×[IGFBP7] test. Results 107 patients were included in the study, of whom 45 (42%) developed AKI. The highest median values of biomarker were detected in septic, transplant and patients after hepatic surgery (1.24 vs 0.45 vs 0.47 ng/l2/1000). The area under receiving operating characteristic curve (AUC) for the risk of any AKI was 0.85, for early use of RRT 0.83 and for 28-day mortality 0.77. In a multivariable model with established perioperative risk factors, the [TIMP-2]×[IGFBP7] test was the strongest predictor of AKI and significantly improved the risk assessment (p<0.001). Conclusions Urinary [TIMP-2]×[IGFBP7] test sufficiently detect patients with risk of AKI after major non-cardiac surgery. Due to its rapid responsiveness it extends the time frame for intervention to prevent development of AKI.

Transforming growth factor-beta 2 gene silencing with trabedersen (AP 12009) in pancreatic cancer

Pancreatic cancer is one of the most aggressive human cancers with a 5‐year survival rate of <5%. Overexpression of transforming growth factor‐beta 2 (TGF‐β2) in pancreatic malignancies is suggested to be a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation. Trabedersen (AP 12009) is a phosphorothioate antisense oligodeoxynucleotide specific for human TGF‐β2 mRNA and was successfully tested in a randomized, active‐controlled phase IIb clinical study in patients with high‐grade glioma. Here, we report on the antitumor activity of trabedersen in human pancreatic cancer cells and in an orthotopic xenograft mouse model of human metastatic pancreatic cancer. Trabedersen reduced TGF‐β2 secretion in human pancreatic cell lines with an IC50 in the low μM range without transfection reagent, clearly inhibited cell proliferation, and completely blocked migration of pancreatic cancer cells. Additionally, trabedersen reversed TGF‐β2‐mediated immunosuppression of pancreatic cancer cells targeted by lymphokine activated killer (LAK) cells, resulting in considerably increased LAK cell‐mediated cytotoxicity. Moreover, in an orthotopic mouse model of metastatic pancreatic cancer, intraperitoneal (i.p.) treatment with trabedersen significantly reduced tumor growth, lymph node metastasis and angiogenesis. These promising results warrant further clinical development of trabedersen. Cancer Sci 2011; 102: 1193–1200)

Cytokines mediating the induction of chronic colitis and colitis-associated fibrosis

To investigate the immunopathogenesis of inflammation-associated fibrosis we analyzed the chronic colitis and late-developing fibrosis occurring in BALB/c mice administered weekly doses of intrarectal trinitrobenzene sulfonic acid (TNBS). We showed first in this model that an initial T helper type 1 response involving interleukin (IL)-12p70 and interferon-γ subsides after 3 weeks to be supplanted by an IL-23/IL-25 response beginning after 4–5 weeks. This evolution is followed by gradually increasing production of IL-17 and cytokines ordinarily seen in a T helper type 2 response, particularly IL-13, which reaches a plateau at 8–9 weeks. We then show that IL-13 production results in the induction of an IL-13 receptor formerly thought to function only as a decoy receptor, IL-13Rα2, and this receptor is critical to the production of tumor growth factor (TGF)-β1 and the onset of fibrosis. Thus, if IL-13 signaling through this receptor is blocked by administration of soluble IL-13Rα2-Fc, or by administration of IL-13Rα2–specific siRNA, TGF-β1 is not produced and fibrosis does not occur. These studies show that in chronic TNBS colitis, fibrosis is dependent on the development of an IL-13 response that acts through a novel cell-surface-expressed IL-13 receptor to induce TGF-β1.

Deletion of Foxp3+ regulatory T cells in genetically targeted mice supports development of intestinal inflammation.

BackgroundMice lacking Foxp3+ regulatory T (Treg) cells develop severe tissue inflammation in lung, skin, and liver with premature death, whereas the intestine remains uninflamed. This study aims to demonstrate the importance of Foxp3+ Treg for the activation of T cells and the development of intestinal inflammation.MethodsFoxp3-GFP-DTR (human diphtheria toxin receptor) C57BL/6 mice allow elimination of Foxp3+ Treg by treatment with Dx (diphtheria toxin). The influence of Foxp3+ Treg on intestinal inflammation was tested using the CD4+ T-cell transfer colitis model in Rag−/− C57BL/6 mice and the acute DSS-colitis model.ResultsContinuous depletion of Foxp3+ Treg in Foxp3-GFP-DTR mice led to dramatic weight loss and death of mice by day 28. After 10 days of depletion of Foxp3+ Treg, isolated CD4+ T-cells were activated and produced extensive amounts of IFN-γ, IL-13, and IL-17A. Transfer of total CD4+ T-cells isolated from Foxp3-GFP-DTR mice did not result in any changes of intestinal homeostasis in Rag−/− C57BL/6 mice. However, administration of DTx between days 14 and 18 after T-cell reconstitution, lead to elimination of Foxp3+ Treg and to immediate weight loss due to intestinal inflammation. This pro-inflammatory effect of Foxp3+ Treg depletion consecutively increased inflammatory cytokine production. Further, the depletion of Foxp3+ Treg from Foxp3-GFP-DTR mice increased the severity of acute dSS-colitis accompanied by 80% lethality of Treg-depleted mice. CD4+ effector T-cells from Foxp3+ Treg-depleted mice produced significantly more pro-inflammatory cytokines.ConclusionIntermittent depletion of Foxp3+ Treg aggravates intestinal inflammatory responses demonstrating the importance of Foxp3+ Treg for the balance at the mucosal surface of the intestine.

Interleukin 21 controls tumour growth and tumour immunosurveillance in colitis-associated tumorigenesis in mice

Background and aims Colitis-associated tumorigenesis is a balance between proliferation of tumour cells and tumour immunosurveillance. The role of T-helper-cell-derived cytokines in tumour growth is not fully understood. In this study the authors investigated the influence of interleukin (IL) 21 on intestinal tumorigenesis. Methods Chronic colitis was induced in IL-21−/− and littermate control wild-type mice with three cycles of 1.5% dextran sulphate sodium (DSS) over 7u2005days followed by 7u2005days of drinking water. Mice received an azoxymethane injection on day 0 of DSS-colitis to induce tumorigenesis. Immunohistochemistry was performed on inflamed and tumour-bearing areas of colons. Cytokine expression of isolated colonic CD4 T cells was determined by ELISA. Cytotoxic capacity of isolated colonic CD8 T cells targeting tumour cells was evaluated by flow cytometry and quantitative cytotoxicity assay. Apoptosis of tumour cells was determined by TUNEL assay of colonic sections. Results Increasing expression of IL-21 was observed in chronic colitis, which showed functional importance, since IL-21 deficiency prevented chronic DSS-colitis development. Further, in the absence of IL-21, significantly fewer tumour nodules were detected, despite a similar extent of intestinal inflammation. In wild-type mice, 8.6±1.9 tumour nodules were found compared with 1.0±1.2 in IL-21-deficient mice. In tumour-bearing IL-21-deficient mice, intestinal inflammation was restored and partly dependent on interferon (IFN)-γ, whereas the inflammation in wild-type mice showed high IL-17A concentrations. In these rare tumours in IL-21-deficient mice, tumour cell proliferation (Ki-67) was decreased, while cell apoptosis was increased, compared with wild-type mice. Increased IFNγ expression in tumour-bearing IL-21-deficient mice led to increased tumour immunosurveillance mediated by cytotoxic CD8CD103 T cells targeting E-cadherin+ colonic tumour cells and therefore limited tumour growth. Conclusion These results indicate that IL-21 orchestrates colitis-associated tumorigenesis, leading to the hypothesis that high IFNγ and low IL-17A expression reduces tumour cell proliferation and increases tumour immunosurveillance.