Antisense Downregulation of a Mouse Mammary Tumor Virus Activated Protooncogene in Mouse Mammary Tumor Cells Reverses the Malignant Phenotype

Abstract

Activation of the protooncogeneWnt-1 by insertion of the mouse mammary tumor virus (MMTV) is known to cause mammary tumors in mice.Wnt-1 expression in mammary glands has been postulated to confer direct local growth stimulation of mammary epithelial cells leading to their acquisition of a preneoplastic state.Wnt-1 expression also induces morphological alterations in cultured normal mammary cells. However, it has not been determined whether or not transformed mammary cells require continuousWnt-1 expression for their ability to form tumorsin vivo.To address this question, we constructed antisense and senseWnt-1 expression vectors containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (GRE5). This promoter is at least 50-fold more inducible by dexamethasone than the promoter contained in the long terminal repeats of MMTV. The vectors were introduced into a mouse mammary tumor cell line (R/Sa-MT) that expresses high levels of endogenousWnt-1 mRNA and forms rapidly growing tumors when transplanted into syngeneic hosts. Of the 12 stably transfected cell lines established (9 with antisense and 3 with sense constructs), 2 antisense cell lines (R/Sa-MT/antisense) and 1 sense cell line (R/Sa-MT/sense) were examined for inducibility by dexamethasone of antisense and senseWnt-1 RNAs, changes in endogenousWnt-1 RNA expression, and changes in cell morphology. The growth patterns of the cellsin vitroandin vivowere also examined. Our results show that (1) the levels of the expression of endogenousWnt-1 mRNA and protein were reduced significantly (>80%) in those cells (R/Sa-MT/antisense) that expressed antisenseWnt-1 RNA at high levels following exposure to dexamethasone, compared to the R/Sa-MT/sense and R/Sa-MT control cells and (2) transplantation of the R/Sa-MT/antisense cells produced smaller tumors (≃0.2 cm in 16 weeks) compared to the tumors (≃2.0 cm in 8 weeks) that were produced by the R/Sa-MT/sense and R/Sa-MT cells. We therefore suggest thatWnt-1 expression is required not only for the transformation of normal mammary cells into tumor cells, but also for the maintenance of their tumorigenicity.