Frauke Bataille

Expression of Dickkopf genes is strongly reduced in malignant melanoma

The Dickkopf (DKK) genes were originally identified as factors inducing head formation in Xenopus. The genes code for inhibitors that are involved in Wnt signaling. We speculate that loss of DKK expression plays a role in development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of DKK, especially DKK-3 transcription. We found that DKK-1, -2 and -3 were downregulated or lost in all cell lines and in most of the tumor samples analysed. Reduced DKK-3 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription–polymerase chain reaction RT–PCR analysis. Functional assays with stable DKK-3 transfected cell lines revealed that DKK-3 expression increased cell-cell adhesion and decreased cell migration. Further, downregulation of fibronectin, snail-1 and re-expression of E-cadherin was found in the DKK-3 expressing cell clones supporting a role of DKK-3 in tumor progression. Our studies thus indicate that loss of DKK-3 expression may contribute to melanoma progression.

Reduction in Raf Kinase Inhibitor Protein Expression Is Associated with Increased Ras-Extracellular Signal-Regulated Kinase Signaling in Melanoma Cell Lines

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.

Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma

Liver cirrhosis is the main risk factor for the development of hepatocellular carcinoma (HCC). Activated hepatic stellate cells (HSC) are the effector cells of hepatic fibrosis and also infiltrate the HCC stroma where they might play a critical role in HCC progression. Here we aimed to analyze the effects of activated HSC on the proliferation and growth of HCC cell lines in vitro and in vivo. Conditioned media (CM) collected from HSC significantly induced proliferation and migration of HCC cells cultured in monolayers. In a 3‐dimensional spheroid coculture system, HSC promoted HCC growth and diminished the extent of central necrosis. In accordance, in vivo simultaneous implantation of HSC and HCC cells into nude mice promoted tumor growth and invasiveness, and inhibited necrosis formation. As potential mechanism of the tumorigenic effects of HSC we identified activation of NFkappaB and extracellular‐regulated kinase (ERK) in HCC cells, two signaling cascades that play a crucial role in HCC progression. In summary, our data indicate that stromal HSC promotes HCC progression and suggest the HSC–HCC interaction as an interesting tumor differentiation‐independent target for therapy of this highly aggressive cancer. (Cancer Sci 2009; 100: 646–653)

Functional implication of BMP4 expression on angiogenesis in malignant melanoma

Analyses of malignant melanomas revealed a strong expression of bone morphogenic proteins (BMPs) and their autocrine effect in promoting cell invasion and migration. Here, we report a paracrine effect of BMPs on the vascular network. Both BMP2 and BMP4 induced tube formation as well as the migratory efficiency of microvascular endothelial cells. Melanoma cells with reduced BMP activity attracted less endothelial cells in invasion assays than control cells. Furthermore, reduction of BMPs in melanoma cells had a strong effect on vasculogenic mimicry. Tube formation on matrigel was analysed for melanoma cells as well as in co-cultures of endothelial and melanoma cells. Melanoma cells with reduced BMP activity were not capable of forming cord-like structures by themselves and additionally inhibited tube formation of the endothelial cells. Genes involved in angiogenesis turned out to be strongly downregulated in these cell clones. Tumors derived from cells with impaired BMP activity showed reduced tumor growth or large necrotic areas owing to lack of angiogenesis in in vivo analyses.

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.

Reduced expression of Hugl-1, the human homologue of Drosophila tumour suppressor gene lgl, contributes to progression of colorectal cancer.

The human gene, human giant larvae (Hugl-1/Llg1/Lgl1) has significant homology to the Drosophila tumour suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that binds Myosin II and is involved in maintaining cell polarity and epithelial integrity. The human protein, Hugl-1 contains several conserved functional domains found in Lgl, suggesting that these proteins may have closely related functions. Whether loss of Hugl expression plays a role in human tumorigenesis has so far not been extensively investigated. Thus, we evaluated tumour tissues from 94 patients undergoing surgery for colorectal cancer (CRC) for loss of Hugl-1 transcription and compared our findings with the clinical data from each of these patients. We found that Hugl-1 was lost in 75% of tumour samples and these losses were associated with advanced stage and particularly with lymph node metastases. Reduced Hugl-1 expression during the adenoma-carcinoma sequence occurring as early as in colorectal adenomas was detected by both immunohistochemical and reverse transcription–polymerase chain reaction analysis. Functional assays with ecdysone-inducible cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Our studies thus indicate that downregulation of Hugl-1 contributes to CRC progression.

Anti-Inflammatory Role of Sympathetic Nerves in Chronic Intestinal Inflammation

Background: Substance P (SP) is a pro-inflammatory neuropeptide in colitis, whereas sympathetic neurotransmitters are anti-inflammatory at high concentrations. Aim and methods: In all layers of the colon, nerve fibre densities of SP+ and sympathetic nerve fibres were investigated (22 Crohn’s disease, six diverticulitis, and 22 controls). In addition, the nerve fibre repellent factor semaphorin 3C (SEMA3C) was studied. The functional role of the sympathetic nervous system was tested in dextran sodium sulfate (DSS) and Il10−/− colitis. Results: In all layers, Crohn’s disease patients demonstrated a loss of sympathetic nerve fibres. Sprouting of SP+ nerve fibres was particularly observed in the mucosa and muscular layer in Crohn’s disease. SEMA3C was detected in epithelial cells, and there was a marked increase of SEMA3C-positive crypts in the mucosa of Crohn’s disease patients compared to controls. In Crohn’s disease, the number of SEMA3C-positive crypts was negatively related to the density of mucosal sympathetic nerve fibres. Sympathectomy reduced acute DSS colitis but increased chronic DSS colitis. Sympathectomy also increased chronic colitis in Il10−/− mice. Conclusions: This study demonstrated a loss of sympathetic and an increase of SP+ nerve fibres in Crohn’s disease. SEMA3C, a sympathetic nerve repellent factor, is highly expressed in the epithelium of Crohn’s disease patients. In chronic experimental colitis, the sympathetic nervous system confers an anti-inflammatory influence. Thus, the loss of sympathetic nerve fibres in the chronic phase of the disease is most probably a pro-inflammatory signal, which might be related to repulsion of these fibres by SEMA3C and other repellents.

Analysis of intestinal haem-oxygenase-1 (HO-1) in clinical and experimental colitis.

Haem‐oxygenase‐1 (HO‐1) has been shown to exert anti‐inflammatory, anti‐apoptotic and anti‐proliferative effects. We investigated HO‐1 expression in patients with inflammatory bowel disease (IBD) and could demonstrate a scattered expression of HO‐1 in the intestinal epithelium of severely inflamed colonic mucosa of patients with IBD compared to control specimens such as diverticulitis, suggesting dysregulated expression in IBD. To further analyse potential mechanisms of HO‐1 induction in the intestine we employed an in vitro epithelial cell apoptosis model and an experimental colitis model. In vitro induction of HO‐1 by the HO‐1 inducer cobalt protoporphyrin (CoPP) resulted in a dose‐dependent down‐regulation of caspase‐3 activation in HT‐29 cells, indicating an anti‐apoptotic function of HO‐1 in the intestine. In vivo, preventive HO‐1 induction by CoPP in acute dextran sodium sulphate (DSS)‐induced colitis led to a significant down‐regulation of colonic inflammation (P < 0·01) with a concomitant reduction in interferon (IFN)‐γ − but unaffected interleukin (IL)‐10‐secretion by isolated mesenteric lymph nodes (P < 0·01). Additionally, TUNEL staining of colonic sections demonstrated fewer apoptotic epithelial cells in the colon of CoPP treated animals. No beneficial effects were observed if HO‐1 was induced by CoPP after the onset of acute colitis or in chronic DSS‐induced colitis. In conclusion, the data suggest a protective role of HO‐1 if it is induced before the onset of inflammation. However, as shown by the lack of effects in established acute or in chronic colitis, the induction of HO‐1 may not be a promising approach for the treatment of IBD.

Gene expression profiling of colorectal cancer and metastases divides tumours according to their clinicopathological stage

Gene expression profiling of matched colorectal carcinomas and metastases could reveal key molecular events involved in tumour progression and metastasis. Expression profiles have been created from 25 colorectal carcinomas (CRCs, pT1–4), corresponding normal colonic mucosa, and 14 liver metastases using cDNA arrays containing 1176 cancer‐related genes (Clontech). Hierarchical clustering clearly distinguished carcinomas from non‐cancerous tissues, separated tumours into high‐stage (pT4 and extensive lymph node or distant metastases) and low‐stage (≤pT3) groups, and correlated with the histopathological classification in 87% (33/38 cases). Most primary tumours and matched liver metastases clustered on terminal branches of the dendrogram. Statistical analysis (Mann–Whitney U‐test) revealed 40 tumour‐specific genes (29 up‐regulated, 11 down‐regulated) which allowed identification of malignant tissue samples by cluster analysis. A specific expression signature in matching metastases was not found, but a set of 23 classifier genes with statistically significant expression patterns in high‐ and low‐stage tumours was identified. These genes may represent important targets in colorectal carcinogenesis and might provide useful clinicopathological tools in the management of colorectal cancer. Copyright

Expression of Hugl-1 is strongly reduced in malignant melanoma

The human gene Hugl-1 (Llgl/Lgl1) has significant homology to the Drosophila tumor suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that is involved in maintaining cell polarity and epithelial integrity. We speculate that Hugl-1 might play a role in epithelial–mesenchymal transition (EMT) and that loss of Hugl-1 expression plays a role in the development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of Hugl-1 transcription. We found that Hugl-1 was downregulated or lost in all cell lines and in most of the tumor samples analysed, and that these losses were associated with advanced stage of the disease. Reduced Hugl-1 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription–polymerase chain reaction (RT–PCR) analysis. Functional assays with stable Hugl-1-transfected cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Further, downregulation of MMP2 and MMP14 (MT1-MMP) and re-expression of E-cadherin was found in the Hugl-1-expressing cell clones supporting a role of Hugl-1 in EMT. Our studies thus indicate that loss of Hugl-1 expression contributes to melanoma progression.