Our long term objective is to identify those stages in cancer metastasis in which urokinase type plasminogen activator (uPA) and its specific receptor (uPAR), play a determining role. This would provide opportunity for testing whether inhibition of expression of these proteins, or their interaction with each other, can curtail the malignant behavior of cancer cells, and thus become therapeutically advantageous. This goal required an in vivo model of metastasis in which each step could be individually accessed and quantitated, a goal that has been accomplished. The model consists of a chick embryo and a human carcinoma of the oral cavity, a type of cancer in which the epithelial tumor cells, and not stromal cells, are responsible for the production of both uPA and uPAR. Quantitation of primary tumor growth, overall level of metastasis, local invasion of connective tissue, invasion of blood vessels (intravasation) and exit from the blood vessels (extravasation) is now possible. Using the novel assay for intravasation, based in a polymerase chain reaction (PCR) of human alu- sequences as a target of amplification, we will determine the requirement for completion of this process and ways to block it. Another important aspect of the proposed work will explore the link we have discovered between reduced level of uPAR and induction of tumor dormancy. Tumor cells, in which uPAR is reduced by antisense-expressing vectors, grow well in culture, but enter a state of protracted dormancy in vivo. We have preliminary data that dormancy is induced through a reduction in the rate of proliferation, and not through excessive death rate, and that integrins and epidermal growth factor receptors are involved in the mechanism responsible for the induction. We will further assess their role and examine the connection between these factors and uPAR, as well as identify properties responsible for interruption of dormancy and test whether once established, metastases can be forced into dormancy by reduction in uPAR levels. Genes responsible for dormancy and for interruption of dormancy will be identified through subtractive hybridization. The possibility that blocking of uPAR expression may induce a state of dormancy in metastases will be tested with the aid of vectors producing antisense of uPAR-RNA in a regulated (inducible promoters) fashion. The initial experiments will be performed in chick embryos, and whenever possible or necessary, confirmed in nude mice.
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