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First published online 31 August 2004
doi: 10.1242/jcs.01367

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The RacGEF Tiam1 inhibits migration and invasion of metastatic melanoma via a novel adhesive mechanism

¹ Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
² Department of Dermatology, University of Würzburg, Sanderring 2, 97070 Würzburg, Germany
³ Department of Dermatology, University of Tübingen, Liebermeisterstraße 25, 72076 Tübingen, Germany
⁴ Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

View larger version (69K): [in a new window]   Fig. 1. Tiam1 expression induces an ALCAM-mediated epithelial-like morphology in metastatic melanoma cells. (A) Western blot analysis of parental and Tiam1-expressing melanoma cell lines. Lysates from parental and Tiam1-expressing 1F6 and MV3 cells were immunoblotted with Tiam1 and ALCAM antibodies. ß-Actin was used as loading control to ensure equal loading. (B) Phase-contrast images of melanoma cell lines. (C) Confocal images of melanoma cell lines stained for Tiam1 (green), ALCAM (white) and F-actin (red). Note that, in MV3-Tiam1 cells, ALCAM is strongly clustered at cell-cell contacts, which is accompanied by distinct colocalization of Tiam1, F-actin and ALCAM. Bars, 20 µm.

View larger version (101K): [in a new window]   Fig. 2. Effect of the anti-ALCAM antibody on ALCAM-mediated cell adhesion. Confocal images of the MV3-Tiam1 cell line stained for ALCAM (blue) and F-actin (red). Cells were treated with a monoclonal anti-rabbit IgG (10 µg/ml) as a control (upper panel) or with the anti-ALCAM antibody L50 (10 µg/ml) (lower panel), respectively. Bars, 20 µm.

View larger version (48K): [in a new window]   Fig. 3. Interrelation of Rac and Rho activity in Tiam1-expressing melanoma cells. (A) GTPase pull-down assay of parental cells (1F6, MV3) and Tiam1-expressing cells (1F6-Tiam1, MV3-Tiam1). Cell lysates of the indicated cell lines (3x106 cells for Rac assay, 6x106 cells for Rho assay) were incubated with GST-PAK-CD (upper panel) or GST-C21 (lower panel), and bound GTPases were detected by western blot using antibodies against Rac1 and RhoA. (B) Rac and Rho activities were visualized indirectly by staining F-actin (red). Focal adhesion complexes were visualized using anti-vinculin antibody (white). Tiam1 was stained with pAb and FITC-conjugated secondary antibody (green). Note that increased Rac activation did not lead to reduction of stress fiber or focal adhesion formation, indicating a stable level of Rho activity and that Tiam1 colocalizes with F-actin at the cell-cell contact sites in MV3-Tiam1 cells. Bars, 20 µm.

View larger version (36K): [in a new window]   Fig. 4. Expression and localization of annexin II and -catenin in melanoma cells. (A) Western blot analysis of parental and Tiam1-expressing melanoma cell lines. Lysates from parental and Tiam1-expressing 1F6 and MV3 cells were immunoblotted with annexin II and -catenin antibodies. A smaller protein at 34 kDa is also visualized in MV3-Tiam1 cells (asterisk), which may be a proteolytical product of annexin II. ß-actin was used as an equal loading control. (B) Confocal images of melanoma cell lines stained for ALCAM (blue), annexin II (green) and F-actin (red). (C) Confocal images of melanoma cell lines stained for ALCAM (green), -catenin (blue) and F-actin (red). Bars, 20 µm.

View larger version (19K): [in a new window]   Fig. 5. The impact of Tiam1 expression on MV3 melanoma proliferation. Equal numbers (5x104) of the indicated cell lines were seeded in culture dishes. Proliferation of control cells (1F6, open triangles; MV3, open circles) and Tiam1-expressing cells (1F6-Tiam1, filled triangles; MV3-Tiam1, filled circles) was monitored over a period of 96 hours, starting one day after seeding. Tiam1 expression remarkably enhanced proliferation of the metastatic cells, whereas proliferation of the non-metastatic cells was slightly reduced.

View larger version (98K): [in a new window]   Fig. 6. The impact of Tiam1 expression on MV3 melanoma migration. (A) Wound closure in a 2D cell motility assay. Closure of a scratch wound in a confluent monolayer of cells was monitored over a period of at least 7 hours. Tiam1 expression strongly inhibited migration of the metastatic MV3-Tiam1 cells (filled circles) compared with the parental cell line MV3 (open circles). The migration potential of non-metastatic cells 1F6 (open triangles) and 1F6-Tiam1 (filled triangles) was not significantly altered in the 2D system. A slight inhibition of migration was detectable. (B) Characterization of MV3 and MV3-Tiam1 cells in human skin reconstructs. Upper panel: MV3 melanoma cells form band-like tumor cell aggregates at the epidermal-dermal junction and exhibit invasive tumor growth of tumor cell strands into the deeper dermis (arrows). Lower panel: MV3-Tiam1 melanoma cells display a high proliferation rate (red) and form giant tumor masses at the epidermal-dermal junction and in the upper dermis; stained with Ki 67 (red), x50. (C) Magnified zones (tetragons) highlight the different cell morphology and position in greater detail. (D) Reinforced cell-cell adhesions and impaired migration after expression of Tiam1. Multicellular spheroids of MV3 control cells (left panel) or Tiam1-expressing MV3 cells (right panel) were incorporated into 3D collagen lattices and monitored by time-lapse videomicroscopy. Paths of individual cells and resulting migration speed were obtained from single cell tracking (after 11 hours). Upon long-term observation, stringent cell-cell junctions preventing cell detachment in Tiam1-positive cells were retained for up to 72 hours (not shown). Bar, 0.6 mm..

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