Rac Affects Invasion of Human Renal Cell Carcinomas by Up-regulating Tissue Inhibitor of Metalloproteinases (TIMP)-1 and TIMP-2 Expression

Abstract

Rho-like GTPases, including Cdc42, Rac1, and RhoA, regulate distinct actin cytoskeleton changes required for adhesion, migration, and invasion of cells. Tiam1 specifically activates Rac, and earlier studies have demonstrated that Tiam1-Rac signaling affects migration and invasion in a cell type-and cell substrate-specific manner. In the present study, we examined the role of Tiam1-Rac signaling in migration and invasion of human renal cell carcinomas. Stable overexpression of Tiam1 or constitutively active V12-Rac1 in a human renal cell carcinoma cell line (clearCa-28) strongly inhibited cell migration by promoting E-cadherin-mediated cell-cell adhesion. Blocking E-cadherin-mediated adhesion by E-cadherin-specific HAV peptides allowed cells to migrate, but was not sufficient to antagonize Tiam1-and V12-Rac1-induced inhibition of Matrigel invasion, suggesting that Rac may influence invasion also through other mechanisms. Indeed, Tiam1-mediated Rac activation induced transcriptional up-regulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) and post-transcriptional up-regulation of TIMP-2, whereas secretion and activity levels of their counterparts, matrix metalloproteinase-9 and matrix metalloproteinase-2, respectively, were not affected. Application of recombinant TIMP-1 and TIMP-2 proteins significantly inhibited invasion of mock-transfected clearCa-28 cells, supporting a role of TIMPs in Rac-mediated inhibition of invasion. To our knowledge, this is the first evidence that increased Rac signaling may inhibit invasion of epithelial tumor cells by up-regulation of TIMP-1 and TIMP-2.Tumor invasion is a complex biological process, during which tumor cells detach from the primary tumor and infiltrate the surrounding tissue. This process requires loss of cell contacts between tumor cells, active cell migration, adhesion to the extracellular matrix (ECM) 1 and proteolytic degradation of the ECM (1, 2). On the molecular level, a number of different molecules, including cadherins, integrins, proteases, and growth factors, have been implicated in the regulation of tumor cell invasiveness (2). Family members of the Rho-like GTPases, including Cdc42, Rac1, and RhoA, mediate distinct actin cytoskeleton changes required for cell adhesion, migration, and invasion (3-5). Their effects both on migration and invasion of epithelial cells seem to be at least in part cell type-and cell substrate-specific. Sustained activation of Rac by overexpression of the Rac-specific activator, Tiam1, or overexpression of constitutively active V12-Rac1 induces invasion and metastasis of murine T-lymphoma cells (6, 7). In contrast, activation of Rac promotes E-cadherin-mediated cell-cell adhesion and, therefore, inhibits migration and invasion of epithelial cells (8 -11). However, in other studies, Rac promotes migration and invasion of epithelial cells, as shown for T47D, colon epithelial, and Madin-Darby canine kidney (MDCK) cells (12-15). These seemingly opposing effects of Rac on both migration and invasion of epithelial cells were r...

Prognostic relevance of increased Rac GTPase expression in prostate carcinomas

“…Interestingly, the effects of Rac1 on migration and invasion in vitro may be either stimulating or inhibitory, depending on the cell-substrate used, the fact as to whether or not the formation of E-cadherin-mediated cell-cell adhesions is prevented, and the cell type studied (Sander et al 1998, Braga et al 1999, Engers et al 2001, Price & Collard 2001. Given these heterogeneous effects of Rac1 on migration and invasion of epithelial cells in vitro, the effect of increased Rac expression in a given tumor in vivo on DFS might be positive or negative.…”

“…So far, three highly homologous Rac proteins have been described in mammals: the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3 (for review see: Wennerberg & Der 2004). Rac1 is a major regulator of the actin cytoskeleton (Ridley et al 1992), which affects cell cycle progression (Olson et al 1995), cell adhesion (Hordijk et al 1997, Kuroda et al 1998, migration (Keely et al 1997, Sander et al 1998, Engers et al 2001, invasion (Hordijk et al 1997, Keely et al 1997, Engers et al 2001, 2006b, and metastasis (Shan et al 2005) of tumor cells. Moreover, Rac1 has been implicated in oncogenic transformation of cells.…”

Prognostic relevance of Tiam1 protein expression in prostate carcinomas

“…In addition to its role in oncogenic transformation, Tiam1 has also been implicated in the regulation of migration, invasion, and metastasis of tumour cells (Habets et al, 1994;Hordijk et al, 1997;Adam et al, 2001;Engers et al, 2001). Interestingly, the effects of Tiam1 on migration and invasion in vitro may be either stimulating or inhibitory, depending on the cell-substrate used, the fact as to whether or not the formation of E-cadherin-mediated cell -cell adhesions is prevented, and the cell type studied (Sander et al, 1998;Engers et al, 2001;Price and Collard, 2001;Minard et al, 2004). Given these heterogeneous effects of Tiam1 on migration and invasion of epithelial cells in vitro, the effect of increased Tiam1 expression in a given tumour in vivo on DFS might be positive or negative.…”

“…As a negative control, representative sections were subjected to the same immunostaining procedure except for the fact that the primary antibody was omitted. As a positive control, paraffinembedded tumour spheroids of Tiam1-transfected renal carcinoma cells (Engers et al, 2001) were used. The specificity of the Tiam1 antibody used in this study has previously been demonstrated (Habets et al, 1994;Malliri et al, 2002).…”

“…The guanine nucleotide exchange factor, Tiam1, specifically activates the Rho-like GTPase Rac (Michiels et al, 1995) and Tiam1-Rac signaling affects cell migration (Hordijk et al, 1997;Sander et al, 1998), invasion (Michiels et al, 1995;Keely et al, 1997;Engers et al, 2001), and metastasis (Habets et al, 1994) of tumour cells. Moreover, Tiam1 and Rac have been implicated in oncogenic transformation of cells.…”