Molecular Mechanism for oral cancer invasion

侵袭足是侵袭性肿瘤细胞向细胞外基质的膜突起。在体外纤维连接蛋白降解/侵袭实验中，这些结构与基质降解和恶性肿瘤细胞侵袭性的位置有关。在这项研究中，我们首先利用激光扫描共聚焦显微镜系统在垂直面上证明了RPM17951人黑色素瘤细胞的侵袭足延伸到基质基质中。我们证实了该物种富含肌动蛋白细丝(F-肌动蛋白)，并且在垂直和水平方向上都能清楚地看到F-肌动蛋白染色。Rho家族小G蛋白由Rho、Rac和CDC42亚家族组成，通过肌动蛋白细胞骨架的重组参与细胞的各种功能，如细胞形状的改变、黏附和运动。然后，我们研究了Rho、Rac和Cdc42在同一细胞系侵袭性中的作用。在体外纤维连接蛋白降解/侵袭实验中，cdc42的显性活性突变体促进点状降解，而rac的显性活性突变体促进弥漫型降解。此外，具有F-肌动蛋白结合活性的CDC42的GDP/GTP交换蛋白Frabin可以促进点状和弥漫型降解。然而，Rho的显性活性突变体不影响纤维连接蛋白的降解。此外，抑制磷脂酰肌醇-3激酶(P13K)可破坏依赖RAC和CDC42的肌动蛋白结构，并阻止纤维连接蛋白的降解。这些结果表明，CDC42和Rac通过Frabin-CDc42/Rac-PI3K信号通路协同作用于纤维连接蛋白的降解和侵袭。

Invadopodia are membrane protrusions into the extracellular matrix by aggressive tumor cells. These structures are associated with sites of matrix degradation and invasiveness of malignant tumor cells in an in vitro fibronectin degradation/invasion assay. In this study, we first demonstrated that invadopodia of RPM17951 human melanoma cells extended into the matrix substratum on a vertical view using a laser scanning confocal microscope system. We confirmed that invadopodia were rich in actin filaments (F-actin) and visualized clearly with F-actin staining on a vertical view as well as on a horizontal view. The Rho family small G proteins, consisting of the Rho, Rac, and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, adhesion, and motility, through reorganization of the actin cytoskeleton. We then studied the roles of Rho, Rac, and Cdc42 in invasiveness of the same cell line. In the in vitro fibronectin degradation/invasion assay, a dominant active mutant of Cdc42 enhanced dot-like degradation, whereas a dominant active mutant of Rac enhanced diffuse-type degradation. Furthermore, frabin, a GDP/GTP exchange protein for Cdc42 with F-actin-binding activity, enhanced both dot-like and diffuse-type degradation. However, a dominant active mutant of Rho did not affect the fibronectin degradation. Moreover, inhibition of phosphatidylinositol-3 kinase (P13K) disrupted the Rac and Cdc42-dependent actin structures and blocked the fibronectin degradation. These results suggest that Cdc42 and Rac play important roles in fibronectin degradation and invasiveness in a coordinate manner through the frabin-Cdc42/Rac-PI3K signaling pathway.