The identification of osteopontin as a metastasis-related gene product in a rodent mammary tumour model.

The rat mammary epithelial cell line, Rama 37, yields benign, non-metastasizing adenomatous tumours in syngeneic Furth-Wistar rats. Transfection of this stably diploid cell line with genomic DNA fragments from a human metastasizing breast cancer cell line yields cells which, when injected subcutaneously in syngeneic rats, give rise to secondary tumours in a number of the animals. From one such secondary lung tumour, a cell line was established designated Ca2-5-LT1. This cell line, when introduced into the syngeneic rat host, also showed the ability to metastasise. To determine key changes in gene expression that occur during the progression from Rama 37, the benign tumour-inducing cell line, to the metastatic derivative Ca2-5-LT1, a general method of subtractive hybridization has been employed. This procedure in conjunction with Northern blotting and nucleic acid sequencing has been used to identify mRNAs expressed differentially between the metastatic and nonmetastatic cell lines described above. So far, of the subtracted cDNAs that have been identified which represent differentially expressed mRNAs, a large proportion of these cDNAs corresponded to the mRNA for rat osteopontin (OPN). The mRNA for OPN was expressed at a ninefold higher level in the metastatic Ca2-5-LT1 cell line when compared to the nonmetastatic parental Rama 37 cell line. Rama 37 cells transfected with DNA from a human benign cell line failed to show elevated levels of OPN mRNA. Following transfection of Rama 37 cells with an expression-construct producing elevated levels of OPN, the newly-transfected cells, when introduced into the rat host, developed metastases in 55% of the animals that produced primary tumours. These experiments show that increasing the expression of OPN in a previously benign cell tine is sufficient to produce a metastatic phenotype in this particular rat mammary model.