Rac1 and Rac3 isoform activation is involved in the invasive and metastatic phenotype of human breast cancer cells

INTRODUCTION: The metastatic progression of cancer is a direct result of the disregulation of numerous cellular signaling pathways, including those associated with adhesion, migration, and invasion. Members of the Rac family of small GTPases are known to act as regulators of actin cytoskeletal structures and strongly influence the cellular processes of integrin-mediated adhesion and migration. Even though hyperactivated Rac proteins have been shown to influence metastatic processes, these proteins have never been directly linked to metastatic progression. METHODS: To investigate a role for Rac and Cdc42 in metastatic breast cancer cell invasion and migration, relative endogenous Rac or Cdc42 activity was determined in a panel of metastatic variants of the MDA-MB-435 metastatic human breast cancer cell line using a p21-binding domain-PAK pull down assay. To investigate the migratory and invasive potential of the Rac isoforms in human breast cancer, namely Rac1 and the subsequently cloned Rac3, we stably expressed either dominant active Rac1 or dominant active Rac3 into the least metastatic cell variant. Dominant negative Rac1 or dominant negative Rac3 were stably expressed in the most metastatic cell variant. Cell lines expressing mutant Rac1 or Rac3 were analyzed using in vitro adhesion, migration and invasion assays. RESULTS: We show that increased activation of Rac proteins directly correlates with increasing metastatic potential in a panel of cell variants derived from a single metastatic breast cancer cell line (MDA-MB-435). The same correlation could not be found with activated Cdc42. Expression of a dominant active Rac1 or a dominant active Rac3 resulted in a more invasive and motile phenotype. Moreover, expression of either dominant negative Rac1 or dominant negative Rac3 into the most metastatic cell variant resulted in decreased invasive and motile properties. CONCLUSION: This study correlates endogenous Rac activity with high metastatic potential and implicates Rac in the regulation of cell migration and invasion in metastatic breast cancer cells. Taken together, these results suggest a role for both the Rac1 and Rac3 GTPases in human breast cancer progression

Inhibition of Rho GTPases, Rac, or Cdc42 in migration of the high metastatic MDA-MB-435α6HG6 cell variant

<p><b>Copyright information:</b></p><p>Taken from "Rac1 and Rac3 isoform activation is involved in the invasive and metastatic phenotype of human breast cancer cells"</p><p>Breast Cancer Research 2005;7(6):R965-R974.</p><p>Published online 30 Sep 2005</p><p>PMCID:PMC1410764.</p><p>Copyright © 2005 Baugher et al.; licensee BioMed Central Ltd.</p> MDA-MB-435α6HG6 cells were treated with vehicle or 2 ng/ml toxin B for 24 h and subjected to a basement membrane haptotaxis assay. Cells migrating to the underside of the membrane were stained with propidium iodide and counted under 400× Magnification. MDA-MB-435α6HG6 cells transiently expressing vector alone or myc-Cdc42(T17N) were subjected to a basement membrane haptotaxis assay. Cells migrating to the underside of the membrane were stained with propidium iodide and counted under 400× Magnification. Equal loading was confirmed by a total actin blot, ectopic myc-Cdc42(T17N) expression confirmed by western blotting with anti-Cdc42 or anti-myc. MDA-MB-435α6HG6 cells transiently expressing vector alone, myc-Rac1(T17N), or myc-Rac3(T17N) were subjected to a basement membrane haptotaxis assay. Bars represent ±SEM; equal loading was confirmed by total actin blot. Myc-Rac1(T17N) and myc-Rac3(T17N) expression were confirmed by western blotting with anti-Rac or anti-myc. Data are expressed as mean ±SEM of three independent experiments. A star denotes statistical significance from control ( < 0.05, calculated from paired t-tests)

Combined Resveratrol, Quercetin, and Catechin Treatment Reduces Breast Tumor Growth in a Nude Mouse Model1

Grape polyphenols can act as antioxidants, antiangiogenics, and selective estrogen receptor (ER) modifiers and are therefore especially relevant for gynecological cancers such as breast cancer. The major polyphenols of red wine (resveratrol, quercetin, and catechin) have been individually shown to have anticancer properties. However, their combinatorial effect on metastatic breast cancers has not been investigated in vivo. We tested the effect of low dietary concentrations of resveratrol, quercetin, and catechin on breast cancer progression in vitro by analyzing cell proliferation and cell cycle progression. The effects of these compounds on fluorescently tagged breast tumor growth in nude mice were assessed using in situ fluorescence image analysis. Individual polyphenols at 0.5 µM neither decreased breast cancer cell proliferation nor affected cell cycle progression in vitro. However, a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 µM each significantly reduced cell proliferation and blocked cell cycle progression in vitro. Furthermore, using in situ image analysis, we determined that combined dietary polyphenols at 0.5, 5, or 25 mg/kg reduced primary tumor growth of breast cancer xenografts in a nude mouse model. Peak inhibition was observed at 5 mg/kg. These results indicate that grape polyphenols may inhibit breast cancer progression