Expression of Wnt5a is downregulated by extracellular matrix and mutated c-Ha-ras in the human mammary epithelial cell line MCF-10A

Wnt genes are involved in tumour growth and regulate cell adhesion. Some (Wnt5a and Wnt7b) are more highly expressed in human breast cancer compared to normal tissues. Wnt5a is involved in the regulation of cell movement in Xenopus and is upregulated in several human cancers. Factors regulating Wnt gene expression in human breast epithelium are poorly understood, but c-erbB2 is amplified in many breast cancers and associated with rapid growth and metastasis, as is high expression of c-Ha-ras. To further understand the regulation of Wnt gene expression, this study investigated the effect of proto-oncogenes c-Ha-ras and c-erbB2, and collagen on Wnt mRNA expression, in a normal spontaneously immortalised human mammary epithelial cell line MCF-10A. Out of nine human Wnt genes investigated, Wnt5a and Wnt7b were expressed in the parental cell line, and neomycin-, c-Ha-ras- and c-erbB2-transfected cell lines. The level of Wnt5a mRNA expression was decreased 40-fold and 3-fold when parental cells were grown on collagen and in collagen, respectively. This downregulation correlated with cell branching. However, Wnt7b was not regulated by collagen. In the presence of activated c-Ha-ras, the level of Wnt5a mRNA expression was markedly decreased (> 200-fold) and cell growth rate was elevated. When treated with p21ras inhibitor, BZA-5B, there was a moderate reversal of Wnt5a mRNA expression (2-fold) with a parallel decrease in cell growth. The data indicate that c-Ha-ras is an upstream inhibitory regulator of Wnt5a, and provide further evidence of an inverse relationship between Wnt5a mRNA expression and cell branching. This demonstrates selectivity of regulation of individual members of the Wnt gene family by the ras pathway. Overexpression of c-erbB2 had no effect on Wnt5a or Wnt7b mRNA expression. Thus, extracellular matrix and ras regulate Wnt5a, providing a mechanism for feedback of morphogenetic movements, which is relevant also to cancer biology