ARHGAP8 encodes a GTPase activating protein (GAP) for RHO, RAC, and CDC42 in vitro, although the target(s) of its GAP activity have not yet been defined in vivo. ARHGAP8 is structurally related to ARHGAP1 and they both contain a protein- and lipid-interacting BCH domain at their N-termini, a central proline-rich SH3 binding motif, and a C-terminal GAP domain.1,2 The gene encoding human ARHGAP8 resides on chromosome 22q13.31, within a region commonly deleted in breast cancer.3We analyzed the ARHGAP8 gene sequence and expression in human and mouse breast tumors. While somatic mutations were not found, mRNA levels were dramatically down-regulated in approximately 1/3 of human ductal carcinomas analyzed (n=31), and in invasive breast carcinoma cell lines displaying epithelial-to-mesenchymal transition (EMT) including MDA-MB-231, SUM159, Hs578T, and BT549. Unlike the situation in melanoma cells, a histone deacetylase inhibitor was more potent than demethylating agents in inducing expression of ARHGAP8 in EMT-positive human breast carcinoma cell lines suggesting silencing of ARHGAP8 expression may not occur through methylation in breast cancer. Expression of mouse Arhgap8 was dramatically down-regulated in a transgenic mouse model of EMT in breast cancer.4 Finally, Arhgap8 was induced along with other epithelial markers such as E-cadherin in 4T1.2 mouse breast cancer cells stably expressing miR-200b, a microRNA that promotes the epithelial phenotype.5 Induction of EMT through short- or long-term treatment of NMuMG normal mouse mammary epithelial cells with TGF-beta1 reduced Arhgap8 expression levels by 80%, whereas expression of the gene encoding E-cadherin was completely silenced. The functional role of ARHGAP8 loss in breast cancer progression is now being evaluated through overexpression or silencing in human breast cancer cell lines. Stable shRNA-mediated knockdown of ARHGAP8 was achieved in the estrogen receptor positive lines MCF7 and BT474 using the pLV3 lentiviral vector. Doxycycline-inducible re-expression in MDA-MB-231 and SUM159 cells is also being pursued.