Rho Family GTPases Regulate Mammary Epithelium Cell Growth and Metastasis Through Distinguishable Pathways

Abstract

Background: Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, including the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulate diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis, however a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed. Materials and Methods:We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac-and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size. Results: Cell spreading of MTLn3 cells was selectively abrogated by N17Rac1. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the colocalization of vinculin with phosphotyrosine at FCs. While N17Rac1 and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU. Conclusions: These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.

Pro-metastatic signaling by c-Met through RAC-1 and reactive oxygen species (ROS)

“…In both cases, cell manipulation lead to a significant and comparable decrease in cell metastatic capacity. Notably, a similar effect of Rac inhibition on experimental metastasis had already been reported in a similar model utilizing breast cancer cells (Bouzahzah et al, 2001).…”

“…Although the finding that Rac-1 blockade inhibits experimental metastasis is not completely surprising (Bouzahzah et al, 2001), data on metastasis modulation by peroxide-producing (CuZnSOD) or -scavenging (Catalase) enzymes add novel information to the picture, directly implicating ROS and redox signaling in the metastatic process. In this respect, of particular interest is the evidence, presented in Figure 5A, b, of a regain of metastatic capacity by Met-shrna F10 cells overexpressing CuZn SOD, which suggests that peroxide could be able, at least in some circustances, to recapitulate the all pro-metastatic signal downstream of Met.…”

Role of NF-κB signaling in hepatocyte growth factor/scatter factor-mediated cell protection

“…The Rho, Cdc42, and Rac1 expression vectors have been described earlier (Bouzahzah et al, 2001). The Src and DN-Src vectors were also described earlier (Ostareck-Lederer et al, 2002).…”

Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion

“…In humans, the Rho family comprises 20 members that regulate a large variety of different functions, including the organization of the actin cytoskeleton, cell migration, mitogenesis and cell survival (Van Aelst and D'Souza-Schorey, 1997; Burridge and Wennerberg, 2004). Importantly, work from a large number of laboratories has shown that the Rho family members Rac, Cdc42 and Rho are essential for cell transformation (Khosravi-Far et al, 1995;Qiu et al, 1995aQiu et al, , b, 1997Lin et al, 1997), tumor cell invasion (Keely et al, 1997;Shaw et al, 1997) and metastasis (Bouzahzah et al, 2001); reviewed in Sahai E and Marshall (2002).…”