E- and P-selectin determine peritoneal spread of ovarian cancer in a xenograft model

Ovarian cancer is the sixth most common cancer and seventh most common cause of cancer death in women world-wide. It is a heterogeneous, rapidly progressive, highly lethal disease of low prevalence.

However, recent progress in the understanding of the peritoneal spread of pancreatic cancer indicated that selectin carbohydrate ligand interactions play a major role in the intraabdominal spread of this neoplasm. To cover this, we hypothesised that E- and P-selectins are also of importance in the formation of intraperitoneal metastases in ovarian cancer, because endothelial cells and peritoneal mesothelium are both derived from the third germinal layer and thus share many morphological and functional similarities.

Therefore two ovarian carcinoma cell lines (SKOV3 and OVCAR3) were xenografted into the peritoneum of E- and P-selectin-deficient scid mice and selectin wild-type controls. Sections of primary tumors and peritoneal carcinomatosis were used for RNA isolation and further on for Affymetrix cDNA analysis. For xenografted SKOV3 as well as OVCAR3 we found a significantly reduced overall survival in selectin wild-type controls compared to the E- and P-selectin-deficient scid mice. Our data obtained that peritoneal selectins play a crucial role in the interaction of the ovarian tumor cells during the peritoneal spread.

But in addition intraperitoneal spread was not completely abrogated by selectin deficiency hence we investigated further molecules like beta4 integrin, we established stable integrin beta4 downregulation in SKOV3 cells via shRNA. The cells were xenografted into the peritoneum of E- and P-selectin-deficient scid mice and selectin wild-type controls and currently we examine the consequences of integrin beta4 downregulation on the biology of human ovarian cancer cells.

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The Role of the Transcription factor Fra-2 in human breast cancer

Fos-related antigen 2 (Fra-2) is a member of the Fos family of AP-1 transcription factors which are often up-regulated in clinical samples of breast cancer. Previous preliminary clinical studies and initial experiment investigations using human breast cancer cell lines suggested that Fra-2 might be involved in the regulation of tumor invasion and metastasis in breast cancer.

In order to analyze the impact of Fra-2 on the aggressive behaviour of breast cancer cells, we established stable Fra-2 over expressing transfectants of highly invasive MDA-MB231 human breast cancer cells. Over expression of Fra-2 resulted in an increase of invasiveness and by microarray analysis, we found that Fra-2 over expression is associated with deregulation of various adhesion molecules, i.e. down-regulation of molecules involved in cell-cell contacts within tissues (Cx43, DSC2, ALCAM), and up-regulation of others involved in adhesion to ECM during migration and/or attachment to endothelial cells during extravasation (ICAM1, L1-CAM, etc.).

The highly increased adhesion to E-selectin, an important cell adhesion molecules expressed by activated endothelial cells, supports the hypothesis that Fra-2 promotes metastasis of breast cancer cells by changing the expression pattern of cell adhesion molecules. This hypotheses will be currently check in an in vitro xenograft model.

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Role of cell adhesion molecules in spontaneous metastasis of neuroblastoma

Neuroblastoma is with six to eight percent the second most common solid tumor in childhood. It is an embryonic tumor which arises from immature cells of the sympathetic nervous system. Clinical presentation of neuroblastoma is very heterogeneous. Thus, metastatic neuroblastoma of young children (< eighteen months), the so-called stage 4s, can spontaneously regress or differentiate whereas fast-growing aggressive neuroblastomas are difficult to treat.

Prognosis is dependent on tumor spread, detection of distant metastases and malignancy of the tumor. Microscopic and molecular genetic evaluation such as amplification of the oncogene MYCN are considered as criteria of malignancy. Prognosis for older children (stage 4) with metastatic neuroblastoma remains still poor. Unfortunately, nearly half of these patients present with metastatic disease at time of diagnosis. Common metastatic sites are cortical bone, bone marrow and liver, less often brain and lung.

As occurence of distant metastases is one important prognostic factor in neuroblastoma, it is important to understand the complex mechanism of the metastatic cascade in order to develop new therapeutic approaches.

Cell adhesion molecules (CAMs) play an important role in the development of distant metastases. CAMs mediating homotypic cell cell adhesion between tumor cells have to be downregulated so that tumor cells can dissociate from the primary tumor and invade into the blood vessels. However, CAMs which mediate cell adhesion to endothelial cells have to be upregulated at the site of distant metastases because adhesion to endothelium is required for extravasation of cells. Integrins are one of the major families of cell adhesion receptors. Integrins are important for leukocyte extravasation at inflammation as well as for neoplastic transformation and formation of metastases. Integrins (ITG) can bind to extracellular matrix components as well as heterotypically to other cells. Furthermore they are involved in several biological processes and can influence survival and migration of cells.

The aim of our studies is to analyze the role of integrins and other CAMs e.g. of the immunoglobulin superfamiliy on neuroblastoma metastasis. Influence of these molecules on cell proliferation, migration, invasion and adhesive properties are studied in vitro. Tumor growth and metastasis are investigated in a xenograft model.

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Intraperitoneal carcinomatosis in pancreatic cancer

Despite decades of research carcinoma still prove fatal for patients in many cases. More than 90% of carcinoma related deaths are caused by metastatic spread of the primary tumor. Tumor cells can reach the site of future metastases via lymphatic vessels (lymphogenous metastasis) or blood vessels (hematogenous metastasis). Additionally, a particular form of metastasis exists in the peritoneal cavaties: Intraperitoneal carcinomatosis. Once peritoneal carcinomatosis occurs, it is generally regarded as the terminal stage of the disease and considerably contributes to the demise of the patients. Peritoneal carcinomatosis is a typical, especially postoperative, complication for ovarian, gastric and pancreatic cancer and it is fair to say that a curable therapy still does not exist. New insights into the mechanisms of peritoneal tumor spread are thus urgently needed, especially regarding the role of the peritoneal epithelium consisting of mesothelial cells.

Recent studies have shown that cell adhesion molecules normally regulating leukocyte migration to sites of infection play a pivotal role in hematogenous metastasis. The first step in the leukocyte adhesion cascade is mediated by E- and P-selectin which initiate leukocyte adhesion to endothelial cells. As endo- and mesothelial cells of the peritoneal cavity both express E- and P-selectin, a mechanism for peritoneal carcinomatosis similar to hematogenous metastasis was almost self-evident, especially as endo- and mesothelial cells are of the same embryological origin. Using a xenograft model with human pancreatic carcinoma cells, we could indeed demonstrate that E- and P-selectin obviously play an essential role in peritoneal tumor spread.

As selectin deficiency drastically reduced carcinomatosis in the model, however did not completely abolish it, we decided to study involvement of other molecules normally regulating the leukocyte adhesion cascade. Direct after the selectins, a second group of molecules - the integrins - mediates further steps in leukocyte adhesion. In intraperitoneal carcinomatoses that developed despite the lack of selectins, we found an upregulation of integrins, indicating integrin involvement. We thus modified human pancreatic carcinoma cells using RNAi to generate sub-lines with drastically reduced integrin expression. In first experiments, we found that tumor growth is significantly reduced for the modified tumor cells.

At the moment we are developing a xenograft model to study the effect of selectin and integrin blockers on peritoneal carcinomatosis.

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