Abstract One of the earliest steps in oncogenic transformation of hMECs involves loss of senescence and continuous proliferation, a process referred to as immortalization. Delineation of biochemical pathways that mediate hMEC immortalization is therefore likely to provide answers to key questions about early breast cancer. RhoA GTPases are central players in many aspects of basic cell biology, There is little knowledge about the role of Rho in early breast cancer. Our recent findings have revealed the potential role of RhoA GTPase in early oncogenic transformation in breast cancer. We demonstrated that overexpression of constitutively-active RhoA, a Rho effector domain mutant, and the wild-type RhoA(WTRhoA), but not a dominant-negative Rho mutant, induces the immortalization of normal hMECs. This is highly significant as in breast and other cancers WTRhoA overexpression is seen but not mutation. Furthermore, we observed immortalization with a RhoA mutant T37A, that fails to interact with well known Rho effectors, Rho kinase, mDia1 and PKN, thus pointing towards a novel pathway that does not involve actin cytoskeleton organization, which may be a late event. In order to identify the molecular mechanism of RhoA-induced immortalization, we performed microarray expression profiling of RhoA-immortalized vs. parental cells and found alterations in expression of several genes (ELF3, ZNF217, S100P were overexpressed; DAB2, CLCA2, TFPI2, ALOX15B were downregulated) previously implicated in immortalization of mammary epithelial cells, and breast cancer progression. Our western blot and RT-PCR results showed that similar to immortal cells, the expression of these genes were altered in breast cancer cell lines. these results demonstrate that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked to cellular transformation and breast cancer. There is increasing evidence that breast cancer originates in and is maintained by cancer stem cell (CSC) Which has stem cell properties, including self-renewal, tumorigenicity and multi-lineage differentiation capacity. A major hurdle in addressing how normal stem cell become CSC is the lack of in vitro human stem/progenitor cells that are immortal and can be deliberately manipulated to ask questions related to origins of CSC. Our current work showed that RhoA immortal cells express luminal, myoepithelial, basal, and stem cell markers (K8/18, E-cadherin, ESA, K5, K6, K14, p63, CD29, CD49f). Indicate that these cells may have stem/progenitor cell properties. It is important to further examine the role of RhoA in normal and cancer mammary stem/progenitor cell self-renewal and differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3839.