Silencing of D4-GDI Inhibits Growth and Invasive Behavior in MDA-MB-231 Cells by Activation of Rac-dependent p38 and JNK Signaling

The Rho GDP dissociation inhibitor D4-GDI is overexpressed in some human breast cancer cell lines (Zhang, Y., and Zhang, B. (2006) Cancer Res. 66, 5592–5598). Here, we show that silencing of D4-GDI by RNA interference abrogates tumor growth and lung metastasis of otherwise highly invasive MDA-MB-231 breast cancer cells. Under anchorage-independent culture conditions, D4-GDI-depleted cells undergo rapid apoptosis (anoikis), which is known to hinder metastasis. We also found that D4-GDI associates with Rac1 and Rac3 in breast cancer cells, but not with other Rho GTPases tested (Cdc42, RhoA, RhoC, and TC10). Silencing of D4-GDI results in constitutive Rac1 activation and translocation from the cytosol to cellular membrane compartments and in sustained activation of p38 and JNK kinases. Rac1 blockade inhibits p38/JNK kinase activities and the spontaneous anoikis of D4-GDI knockdown cells. These results suggest that D4-GDI regulates cell function by interacting primarily with Rac GTPases and may play an integral role in breast cancer tumorigenesis. D4-GDI could prove to be a potential new target for therapeutic intervention.Human breast cancer is a heterogeneous disease with diverse metastatic behavior and treatment responses (1). Attempts to classify this disease into clinically relevant subtypes have yielded multiple sets of gene expression signatures of noninvasive and invasive breast cancers (2–6). However, only a few genes overlap among the results from different laboratories, and most of the genes are not yet characterized as functional mediators of breast cancer progression. The molecular basis of breast tumorigenesis remains to be fully understood.Rho GTPases, including Rac1, Rac3, Cdc42, and RhoA, are pivotal regulators of cell morphology, gene expression, cell proliferation, and apoptosis (7). The aberrant signaling through these molecules has been implicated in many aspects of tumorigenesis, including uncontrolled cell growth and metastatic phenotypes (8–12). In particular, Rac1 and its isoforms are key regulators of malignant transformation and invasive behavior of cancer cells (13–17). This is achieved at least partially by their ability to control cell growth under anchorage-independent conditions and resistance to anoikis, apoptosis induced by loss of adhesion (18–20).As molecular switches, Rac/Rho GTPases cycle between inactive GDP-bound and active GTP-bound states (21). Their biological activity is tightly controlled by the Rho-GDP dissociation inhibitors (RhoGDIs),² including RhoGDI (RhoGDI-1 or RhoGDI-α), D4-GDI (RhoGDI-2 or RhoGDI-β), and RhoGDI-3 (RhoGDI-γ). These proteins are thought to form stable complexes with individual Rho GTPases, thus keeping them in the cytosol. Upon growth factor stimulation, the GTPases are directed to effector sites, such as the plasma membrane, for activation (21–23). Thus, the expression levels of RhoGDIs relative to Rho GTPases must be precisely controlled to achieve normal cell function. RhoGDI binds most Rho GTPases in most types of cells (22). However, the Rho protein(s) regulated by D4-GDI in vivo are not clearly defined.RhoGDIs are differentially expressed in human cancers, and this may contribute to the deregulation of Rho-dependent pathways in cancer cells. For instance, D4-GDI is widely expressed in hematopoietic tissues (24, 25) and is selectively down-regulated in Hodgkin lymphoma cells compared with non-Hodgkin lymphoma (26). Moreover, D4-GDI is reduced as a function of disease progression in bladder cancer (27–29). In contrast, D4-GDI is overexpressed in ovarian (30), colon (31), and breast (32) cancer cell lines as well as primary breast tumors (33–35). Notably, elevated D4-GDI expression correlates with the in vitro invasiveness of ovarian (30) and breast (32) cancer cells. In the latter, targeted disruption of D4-GDI prevents cells from invading through Matrigel (32), supporting the hypothesis that D4-GDI may be a promoter of tumorigenesis and metastasis in breast cancer.In this study, we explored the roles of D4-GDI in breast tumor growth and metastasis by manipulating its protein expression in MDA-MB-231 cells, a highly invasive breast cancer cell line that expresses high levels of D4-GDI (32). Targeted disruption of D4-GDI abolishes tumor growth and experimental metastasis of MDA-MB-231 cells both in vitro and in vivo. We also show that D4-GDI regulates breast cancer cell growth through a signaling pathway that involves Rac GTPases and p38/JNK kinases. Thus, our results support a functional link between D4-GDI expression and enhanced breast cancer cell growth and invasion.