Rap1-GTP-interacting Adaptor Molecule (RIAM) Protein Controls Invasion and Growth of Melanoma Cells

Abstract

The Mig-10/RIAM/lamellipodin (MRL) family member Rap1-GTP-interacting adaptor molecule (RIAM) interacts with active Rap1, a small GTPase that is frequently activated in tumors such as melanoma and prostate cancer. We show here that RIAM is expressed in metastatic human melanoma cells and that both RIAM and Rap1 are required for BLM melanoma cell invasion. RIAM silencing in melanoma cells led to inhibition of tumor growth and to delayed metastasis in a severe combined immunodeficiency xenograft model. Defective invasion of RIAM-silenced melanoma cells arose from impairment in persistent cell migration directionality, which was associated with deficient activation of a Vav2-RhoA-ROCK-myosin light chain pathway. Expression of constitutively active Vav2 and RhoA in cells depleted for RIAM partially rescued their invasion, indicating that Vav2 and RhoA mediate RIAM function. These results suggest that inhibition of cell invasion in RIAM-silenced melanoma cells is likely based on altered cell contractility and cell polarization. Furthermore, we show that RIAM depletion reduces ␤1 integrin-dependent melanoma cell adhesion, which correlates with decreased activation of both Erk1/2 MAPK and phosphatidylinositol 3-kinase, two central molecules controlling cell growth and cell survival. In addition to causing inhibition of cell proliferation, RIAM silencing led to higher susceptibility to cell apoptosis. Together, these data suggest that defective activation of these kinases in RIAM-silenced cells could account for inhibition of melanoma cell growth and that RIAM might contribute to the dissemination of melanoma cells.Rap1 belongs to the Ras family of small GTPases, which are molecular switches that couple extracellular signals with a variety of cellular responses, such as cell proliferation and survival, cell adhesion, and cell polarity (1-3). Rap1 activation is regulated by guanine-nucleotide exchange factors, which stimulate exchange of bound GDP by GTP, and by GTPase-activating proteins, which promote GTP hydrolysis (4). The GTP-associated form of Rap1 is responsible for interaction with its effectors, which then transmit the signaling necessary to generate cell responses.Rap1-GTP-interacting adaptor molecule (RIAM) 3 is an effector protein for Rap1 that belongs to the MRL (Mig-10/ RIAM/Lamellipodin) family of adaptor proteins (5). RIAM contains an N-terminal coiled-coil region, a central Ras association, and pleckstrin homology domains, a C-terminal region rich in prolines, as well as several FPPP motifs with the potential of interacting with ENA-VASP proteins. RIAM is broadly expressed, and it was first characterized as a molecule that promotes Rap1-dependent ␤1 and ␤2 integrin activation on T lymphocytes (5). Subsequent work demonstrated that Rap1-RIAM association favors talin targeting to the plasma membrane through direct talin interaction with an N-terminal segment of RIAM, a process that contributed to integrin activation (6, 7). Furthermore, RIAM co-localizes with talin in lamellipodia (6), representing a mecha...

Focal adhesion disassembly is regulated by a RIAM to MEK-1 pathway

“…, a potent integrin activator, led to similar increased levels of b1 integrin activation and binding of soluble fibronectin (Hernández-Varas et al, 2011). Cell treatment with Mn 2+ resulted in significant increase in paxillin-containing peripheral FA number in mock cells, whereas Mn 2+ did not correct the altered FA distribution of RIAM-depleted cells and did not further enhance their FA number (Fig.…”

“…Alterations in adhesion complex distribution were not only seen in BLM-knockdown cells, but RIAM depletion in MV3 melanoma cells (Hernández-Varas et al, 2011) also led to formation of adhesion complexes that covered the central area (supplementary material Fig. S1D).…”

“…We previously showed that RIAM depletion only moderately affects b1 integrin activation in BLM cells, and does not significantly alter binding of soluble fibronectin, as assessed by flow cytometry (Hernández-Varas et al, 2011). Furthermore, incubation of mock and D11 cells with Mn…”

“…To investigate FA distribution in RIAM-knockdown cells, paxillin staining was analysed in BLM melanoma cells stably interfered for RIAM expression (D11 and F8-Luc transfectants), as well as in cells transiently transfected with siRNA for RIAM (Hernández-Varas et al, 2011). Interestingly, whereas mock cells displayed a predominantly peripheral distribution of paxillincontaining FAs either on fibronectin or type I collagen, adhesions accumulated centrally on the ventral cell surface in RIAMdepleted D11 cells ( Fig.…”

“…We recently reported that RIAM depletion in human melanoma cells leads to impairment in persistent cell migration directionality, causing deficient cell invasion (Hernández-Varas et al, 2011). Furthermore, melanoma cells depleted of RIAM display significant inhibition of lung metastasis in a mouse xenograft model.…”