Tenascin (TN) is a large hexameric glycoprotein which is expressed transiently in the extracellular matrix at times of tissue development, proliferation and reorganisation. It is overexpressed in the stroma of several types of malignant tumours. The expression of TN in ovarian cancer has not previously been investigated. The aims of the project were therefore to (i) define the expression of TN in ovarian cancer, (ii) examine factors controlling its production and (iii) explore the functionality of the protein.
Initial studies characterised the expression of TN in a series of malignant, benign and borderline ovarian tumours. TN protein was examined by immunohistochemical staining of frozen sections of tumours. Malignant and borderline tumours showed a significantly greater incidence and intensity of stromal staining as compared to benign tumours. Omental metastases showed a pattern of protein distribution strikingly similar to their primary counterpart. These data suggest that TN is overexpressed in malignant ovarian tumours.
TN exists in a number of different isoforms, which are produced by alternative splicing of the RNA transcript. The pattern of different isoforms expressed was investigated using RT-PCR and hybridisation to specific oligonucleotides. The smallest TN splice variant was found in all tumours examined while the appearance of larger molecular weight transcripts was predominantly limited to malignant tumours
In order to determine which cell types were capable of producing TN, in vitro, fibroblast cells, cultured from ascitic fluids of ovarian cancer patients, and established ovarian carcinoma cell lines were studied. TN levels in conditioned media were measured by ELISA. "Basal" levels of TN secretion were determined by culturing cells in serum-free media; under these conditions the ovarian fibroblasts secreted levels of TN over 100 fold greater than the epithelial cells. RT-PCR data showed that epithelial and fibroblast cell lines express TN RNA and display multiple RNA splice forms.
To examine whether a paracrine interaction between the fibroblast and epithelial cells can influence TN production, the cells were co-cultured in compartments separated by a filter, which allowed diffusion of soluble factors. The co-cultured populations of cells produced significantly more TN than either cell type alone. The effects of a number of potential modulating factors, on secretion of TN, have been investigated. Several factors (IGF II, TGFB, progesterone and EGF) stimulate TN secretion by fibroblasts while other factors (gonadotrophins and interferon) inhibit TN secretion in the same cell type. Of the factors studied TGF-B provided the greatest induction of TN in fibroblasts. None of these factors induced the PE01 epithelial cell line to produce measurable levels of TN.
Adhesion and migration assays were used to examine how ovarian carcinoma cell lines interacted with TN, as compared with the ECM proteins fibronectin and collagen IV. The assays demonstrated that TN promoted cell adhesion, spreading and migration in the SKOV-3, 59M, PE01 and PE04 ovarian carcinoma cell lines, however, fibronectin and collagen IV appeared to be preferable substrates. Immuno-staining and analysis by flow cytometry of these cell lines demonstrated that all expressed the integrin a2B1which can bind TN, the SKOV-3 cell line also expressed the integrin aVB3.
These studies have demonstrated that TN is overexpressed in malignant ovarian tumours, and paracrine growth factors, such as TGFB, can induce the synthesis of TN in ovarian fibroblasts. Tumour cells can adhere to, and migrate on TN. These data would be consistent with TN playing a role in the invasion and metastasis of ovarian cancer.