Reduction of Cripto-1 Expression by a Hammerhead-Shaped RNA Molecule Results from Inhibition of Translation Rather Than mRNA Cleavage

Abstract

Cripto-1 (CR-1) is a transforming growth factor which has been associated with breast, colon, and pancreatic cancer. Overexpression of CR-1 in non-tumorigenic mouse mammary epithelial cells and fibroblasts results in an increase in anchorage-dependent and -independent growthin vitro.Reduction of CR-1 expression in human colon carcinoma or embryonal teratoma cells results in a decrease in growthin vitro.In an effort to better define the role of CR-1 in breast cancer, we have developed an underexpression vector for CR-1 to reduce CR-1 levels in a tumorigenic mouse mammary epithelial cell line (-SA). This vector specifically targets the expression of the murine homolog of CR-1 in murine cancer lines and utilizes a hammerhead ribozyme-like structure directed toward the extreme 5′ end of the Cripto-1 mRNA. We dramatically reduced expression of CR-1 through the expression of this RNA. This is the first use of a ribozyme-like molecule to alter Cripto-1 expression. This ribozyme-shaped molecule appears to act principally through a block in translation. A possible mechanism for this block is described, and its implications for modifying expression of other bioactive proteins are discussed.