A: Signaling

A1: Role of pro-inflammatory genes for the induction of gastric MALT lymphomas

2009-06-03T11:02:19Z

Our research group was one of the first that showed an association of Helicobacter pylori (H.p.) infection with the development of gastric MALT lymphoma (Bayerdorffer et al., Lancet 1995; Neubauer et al., J Natl Cancer Inst 1997; Wündisch et al., JCO 2005). We have shown that the genetic profile of H.p.-induced early gastric MALT lymphoma distinctly differed from tissue of chronic gastritis. Up-regulation of Phospholipase C γ2 (PLCγ2) in gastric MALT lymphoma, which leads to activation of NF-kB, was one of the most interesting finding (Huynh et al. Leukemia Lymphoma 2008).

Different genetic host factors are associated with increased risk of developing gastric MALT lymphomas. These are e.g. polymorphism in IL-1, CTLA4, GST-T1 and TNF-alpha (Rollinson et al., Blood 2003; Wu et al., Haematologica 2005; Wu et al., Int J Cancer 2004). All of these host factors result in deregulated inflammation after H.p. infection.

Mice with gain-of-function mutation in PLC γ2 show autoimmunity and inflammatory disease (Yu et al., Immunity 2005). In this project we want to infect these mice with Helicobacter felis. As control groups serve wt mice and in further studies PLCγ2-knockout mice. Our hypothesis is that Balb/c mice with gain-of-function in PLCγ2 develop earlier and in a higher frequency MALT lymphomas compared to control groups.

References:

Bayerdörffer E, Neubauer A, Rudolph B, Thiede C, Lehn N, Eidt S, Stolte M. (1995).
Regression of primary gastric lymphoma of mucosa-associated lymphoid tissue type after cure of Helicobacter pylori infection. MALT Lymphoma Study Group. Lancet 345(8965):1591-4.

Neubauer A, Thiede C, Morgner A, Alpen B, Ritter M, Neubauer B, Wündisch T, Ehninger G, Stolte M, Bayerdörffer E. (1997). Cure of Helicobacter pylori infection and duration of remission of low-grade gastric mucosa-associated lymphoid tissue lymphoma. J Natl Cancer Inst 17;89(18):1350-5.

Wündisch T, Thiede C, Morgner A, Dempfle A, Günther A, Liu H, Ye H, Du MQ, Kim TD, Bayerdörffer E, Stolte M, Neubauer A. (2005). Long-term follow-up of gastric MALT lymphoma after Helicobacter pylori eradication. J Clin Oncol. 23(31):8018-24.

Huynh MQ, Wacker HH, Wündisch T, Sohlbach K, Daniel Kim T, Krause M, Stabla K, Roth P, Fischbach W, Stolte M, Neubauer A. (2008). Expression profiling reveals specific gene expression signatures in gastric MALT lymphomas. Leuk Lymphoma. 49(5):974-83.

Rollinson S, Levene AP, Mensah FK, Roddam PL, Allan JM, Diss TC, Roman E, Jack A, MacLennan K, Dixon MF, Morgan GJ. (2003). Gastric marginal zone lymphoma is associated with polymorphisms in genes involved in inflammatory response and antioxidative capacity. Blood. 102(3):1007-11.

Wu KL, van Wieringen W, Vellenga E, Zweegman S, Lokhorst HM, Sonneveld P. (2005).
Analysis of the efficacy and toxicity of bortezomib for treatment of relapsed or refractory multiple myeloma in community practice. Haematologica 90(7):996-7.

Wu MS, Chen LT, Shun CT, Huang SP, Chiu HM, Wang HP, Lin MT, Cheng AL, Lin JT. (2004). Promoter polymorphisms of tumor necrosis factor-alpha are associated with risk of gastric mucosa-associated lymphoid tissue lymphoma. Int J Cancer 110(5):695-700.

Yu P, Constien R, Dear N, Katan M, Hanke P, Bunney TD, Kunder S, Quintanilla-Martinez L, Huffstadt U, Schröder A, Jones NP, Peters T, Fuchs H, de Angelis MH, Nehls M, Grosse J, Wabnitz P, Meyer TP, Yasuda K, Schiemann M, Schneider-Fresenius C, Jagla W, Russ A, Popp A, Josephs M, Marquardt A, Laufs J, Schmittwolf C, Wagner H, Pfeffer K, Mudde GC. (2005). Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry. Immunity 22(4):451-65.

A2: Role of Nerve growth factor (NGF) for tumor growth and angiogenesis in a mouse model of lung carcinoma

2009-06-03T11:02:59Z

Nerve growth factor (NFG) is a member of the neurotrophin growth factor family. These proteins were first discovered as essential growth and survival factors in the nervous system but recent findings also reveal multiple biological functions in other cell systems. So NGF has been described to support activation and survival of various immune cells and has proangiogenic activities in endothelial cells. Moreover NGF also support proliferation and repair mechanisms of tissue epithelia and NGF has been recently implicated as a prooncogenic factor in various carcinomas.
In carcinomas an intense expression of NGF and its receptor TrkA has been shown to predict high tumorgenity and poor prognosis. In the current project we want to investigate the role of this factor in the progression of bronchial carcinoma. The pathogenic changes involved in lung cancer include hyperplasia and dysplasia of the bronchial epithelium and development of invasive growing carcinoma at a late stage. At this stage tumor growth is accompanied by increased local vascularity and tumor angiogenesis is thought to mainly contribute to cancer pathogenesis.
In previous studies we could show that NGF is an autocrine growth factor for murine airway epithelial and epithelial NGF is upregulated during wounding of these cells. Increased NGF expression by epithelial cells mediates epithelial proliferation and wound closure in an autocine fashion but may also act in a paracrine manner to support epithelial growth. In this regard we were able to demonstrate a direct proangiogenic activity of NGF on cultured endothelial cells as well as during NGF overexpression in the mouse lung.
Therefore we speculate that NGF also mediates tumor progression in bronchial carcinomas by promoting tumor growth and angiogenesis. This is investigated during in vitro studies of murine lung carcinoma cells as well as in a mouse model of lung carcinoma. In tumor cell lines autocrine NGF expression will be knocked down and tumor proliferation as well as angiogenesis will be compared using NGF+/+ versus NGF-/- cells. Moreover we want to investigate the role of tumor NGF synthesis on lung infiltration of immune cells. It is well known that tumor-associated immune cells can inhibit but also support tumor growth and especially mast cells and a subpopulation of macrophages have been implicated to support tumor angiogenesis. While in allergic reactions the epithelial expression of NGF enhances immune cell infiltration in the lung also tumor-derived NGF may influence a local immune response. Therefore we will compare number and phenotype of tumor-associated immune cells after inoculation of NGF+/+ vs. NGF-/- tumor cells.

A3: Role of neuropeptides and their receptors for tumorigenesis, tumorprogression and immune evasion

2009-06-03T11:03:13Z

Many neuropeptides, in particular VIP (Vasoactive Intestinal Peptide) and PACAP (pituitary adenylate-cyclase activating peptide), members of the secretin/glucagon/VIP peptide family have - in addition to their intrinsic functions in the CNS – important modulatory roles in the immune system and interact with many cytokines and other immune modulating molecules to govern immune responses. Such pleiotropic neuropeptides can also act peripherally as growth factors and influence tumor growth. PACAP like most other neuropeptides exerts its functions through G-protein-coupled receptors which are expressed by cells of the immune system and neurons. Three PACAP-specific receptors are known, PAC1, VPAC1 and VPAC2, which are linked to different intracellular signalling pathways. Furthermore, a variety of PAC1 splice variants of were identified. PACAP could be shown to act as an anti-inflammatory/immune suppressive modulator in addition to its antiapoptotic, neuroprotective properties in the CNS (DiCicco-Bloom et al. 2000, Nicot et al. 2001,Vaudry et al. 2000, 2002, 2003). Recently, it was reported (Nemetz et al. and Yazutaka et al. 2008) that PACAP antagonizes cancer development in experimentally induced chronic colitis. The assumption that PACAP and its receptors could be targets for a new innovative neuroimmune regulatory therapy stands to reason.

References:

Azuma YT, Hagi K, Shintani N, Kuwamura M, Nakajima H, Hashimoto H, Baba A, Takeuchi T. “PACAP provides colonic protection against dextran sodium sulfate induced colitis.” J Cell Physiol. 2008 Jul;216(1):111-9.

DiCicco-Bloom E, Deutsch PJ, Maltzman J, Zhang J, Pintar JE, Zheng J, Friedman WF, Zhou X, Zaremba T. “Autocrine expression and ontogenetic functions of the PACAP ligand/receptor system during sympathetic development.” Dev Biol. 2000 Mar 15;219(2):197-213.

Nemetz N, Abad C, Lawson G, Nobuta H, Chhith S, Duong L, Tse G, Braun J, Waschek JA. “Induction of colitis and rapid development of colorectal tumors in mice deficient in the neuropeptide PACAP.” Int J Cancer. 2008 Apr 15;122(8):1803-9.

Nicot A, DiCicco-Bloom E. “Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression.” Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4758-63.

Ravni A, Vaudry D, Gerdin MJ, Eiden MV, Falluel-Morel A, Gonzalez BJ, Vaudry H, Eiden LE. „A cAMP-dependent, protein kinase A-independent signaling pathway mediating neuritogenesis through Egr1 in PC12 cells.” Mol Pharmacol. 2008 Jun;73(6):1688-708. Epub 2008 Mar 24.

Vaudry D, Gonzalez BJ, Basille M, Pamantung TF, Fontaine M, Fournier A, Vaudry H. “ The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32.” Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):13390-5.

Vaudry D, Pamantung TF, Basille M, Rousselle C, Fournier A, Vaudry H, Beauvillain JC, Gonzalez BJ. “ PACAP protects cerebellar granule neurons against oxidative stress-induced apoptosis.” Eur J Neurosci. 2002 May;15(9):1451-60.

Vaudry D, Falluel-Morel A, Basille M, Pamantung TF, Fontaine M, Fournier A, Vaudry H, Gonzalez BJ. “Pituitary adenylate cyclase-activating polypeptide prevents C2-ceramide-induced apoptosis of cerebellar granule cells.” J Neurosci Res. 2003 May 1;72(3):303-16.

A4: Interaction of inflammatory stroma and tumor cells in pancreatic cancer

2009-06-03T11:03:23Z

Pancreatic cancer is characterized by the presence of a profound desmoplastic reaction and inflammatory infiltrates. Among other, tumor-associated macrophages (TAMs) are known to influence tumor progression via a cytokine-mediated interaction with tumor cells. Previously, we identified the transcription factor Cut-like-1 (CUTL1, also known as CUX1) as crucial modulator of tumor cell motility and invasion. Interestingly, CUTL1 is highly expressed in both tumor cells and adjacent tumor-associated macrophages. Initial co-culture experiments suggest that CUTL1 expressed in TAMs stimulates proliferation and migration of neighbouring pancreatic cancer cells. Based on these observations, the aim of the project is to elucidate the effect of TAMs and signalling pathways including CUTL1 on the proliferation and invasion of pancreatic cancer cells in vitro and in vivo. This includes:

I) characterization of the upstream regulation of CUTL1 by cytokines and growth factors involved in the differentiation of TAMs,

II) identification of the CUTL1-induced cytokine-profile mediating its effects on tumor cells,

III) the in vivo evaluation of CUTL1 effects in TAMs in a xenograft mouse model,

IV) evaluation of the effects of TAM´s and their therapeutic modulation in genetic mouse models of pancreatic cancer.

A6: Role for neuropeptides in melanoma specific immunity vs. tolerance induction

2009-06-17T07:49:11Z

Previous studies have shown that the tumor progression of malignant melanoma is apparently induced by a tolerogenic milieu caused by the tumor itself, which also causes the induction of regulatory T-cells that inhibit effector cells. Furthermore, by the induction of T reg anti-inflammatory cytokines like IL-10 and TGF-ß were discussed as well as neuropeptides like CGRP, VIP and PACAP. This probably occurs through the induction of tolerogenic dendritic cells as antigen-presenting cells (reduction of co-stimulating signals like CD80 and CD86). On the other side in case of vitiligo, it could be found that T cell-mediated or antibody-mediated autoimmunity against melanocyte antigens may lead to a loss of pigment. In this project, the frequency of tumor antigen-specific effector and regulatory T cells from blood and tissue should be studied in patients with melanoma and vitiligo. Skin biopsies of patients should be investigated in terms of the composition of the inflammatory infiltrates and the expression of pro- and anti-inflammatory cytokines under particular consideration of neuropeptides. In this context pro-inflammatory and regulatory T cells should also be isolated from melanoma tissue and vitiligo biopsies i.e. by employing laser dissection and examined ex vivo with respect to the expression of pro- and anti-inflammatory cytokines and chemokines as well as neuropeptides CGRP, VIP and PACAP.

References

Erfurt, C., ..... Schultz E.S. (2007). Tumor-reactive CD4+ T cell responses to the melanoma-associated chondroitin sulphate proteoglycan in melanoma patients and healthy individuals in the absence of autoimmunity. J Immunol. 178:7703-7709.

Hertl, M., Eming, R., Veldman, C. (2006). T cell control in autoimmune bullous skin disorders. J Clin Invest. 116:1159-1166.

Veldman, ….. and Hertl, M. (2006). Inhibition of the transcription factor Foxp3 converts desmoglein 3-specific type 1 regulatory T cells into T helper 2-like cells. J Immunol. 176:3215-3222.

TP5: Oncosome-mediated reprogramming of host cells

2010-12-07T09:31:48Z

Microvesicles, which can also become shedded off from non-apoptotic plasma membrane blebs, are small membrane-enclosed structures. They are now considered as important players in the intercellular communication that participate in many pathophysiological processes such as chronical inflammation and cancer. Many metastatic tumor cells display elevated production of microvesicles, often associated with poor prognosis and more advanced disease. However, neither the intrinsic mechanisms leading to their overproduction in tumors nor the nature of their action on tumor-associated host or immune cells have been defined so far.

We previously identified the human formin Dia1 (DIAPH1) as one important actin regulator necessary for bleb-associated cancer cell invasion through 3D collagen through regulation of myosin function and contractility at the cell cortex (Kitzing et al., 2007 and 2010). However, we have not investigated how bleb-induced microvesicles may affect inflammatory or immune responses in vivo. A central goal is to analyze the effects of such bleb shedding on cancer-associated fibroblasts (CAFs) as well as on immune cell reprogramming and motility.