Despite improved treatments, more than 40,000 women died from breast cancer in the United States in 2006. Thus, there is an urgent need to continue the search for genes that are involved in breast tumorigenesis and metastasis. We have characterized the previously unknown breast tumor suppressor properties of RIN1. RIN1 is a RAS effector that regulates epithelial cell functions. We determined that RIN1 expression is transcriptionally silenced at a high frequency in breast cancer cell lines and human breast tumors. We characterized two mechanisms that silence RIN1. First, the transcriptional repressor SNAI1 negatively regulates RIN1 expression. In addition, treatment with TGFβ, an inducer of SNAI1 and promoter of tumor metastasis, caused a reduction in RIN1 expression in normal mammary epithelial cells and tumor cells. Second, DNA methylation was found in the promoter and first exon of the RIN1 gene, suggesting methylation as another silencing mechanism. Furthermore, we found that ectopic restoration of RIN1 inhibited the growth of tumor cells in anchorage independent assays ( in vitro) and reduced the growth of mammary tumors in nude mice ( in vivo), consistent with a tumor suppressor function.

The RIN1 gene lies in a tight cluster with the BRMS1 (Breast Tumor Metastasis Suppressor 1) and B3GNT1 (β-1,3-N-acetylglucosaminyltransferase 1) genes. This alignment is highly conserved in mammals. We have shown that this three gene locus (B3GNT1, BRMS1, RIN1) displays coordinated silencing in multiple breast tumor cell lines and a tissue sample. We found that SNAI1 knockdown restored B3GNT1, BRMS1 and RIN1 expression. Furthermore, treatment with TGFβ, caused a reduction in B3GNT1, BRMS1 and RIN1 expression in normal mammary epithelial cells and tumor cells. Finally, B3GNT1, RIN1 (this work) and BRMS1 (published elsewhere) each independently act as negative regulators of cell migration. The discovery of a tumor suppressor gene cluster (B3GNT1-BRMS1-RIN1 ) represents a unique opportunity to understand how the silencing of this locus may cooperatively lead to tumor formation and metastatic spread.

The identification of RIN1 as a tumor suppressor, and the investigation of it's role as part of breast tumor suppressor locus, should facilitate the identification of new targets for breast tumor therapeutics as well as provide insight into epithelial plasticity during mammary tissue development.