Molecular Mechanism for oral cancer invasion

侵袭伪足是侵袭性肿瘤细胞向细胞外基质中的膜突起。在体外纤连蛋白降解/侵袭测定中，这些结构与恶性肿瘤细胞的基质降解和侵袭性的位点相关。在这项研究中，我们首先证明了RPM 17951人黑色素瘤细胞的侵袭伪足延伸到基质的垂直视图上使用激光扫描共聚焦显微镜系统。我们证实，侵入伪足富含肌动蛋白丝（F-actin），并在垂直视图和水平视图上用F-actin染色清晰可见。Rho家族小G蛋白由Rho、Rac和Cdc 42亚家族组成，通过肌动蛋白细胞骨架的重组参与各种细胞功能，如细胞形状改变、粘附和运动。然后，我们研究了Rho，Rac和Cdc 42在同一细胞系的侵袭性中的作用。在体外纤连蛋白降解/侵袭试验中，Cdc 42的显性活性突变体增强了点样降解，而Rac的显性活性突变体增强了扩散型降解。此外，frabin，GDP/GTP交换蛋白Cdc 42与F-肌动蛋白结合活性，增强点样和扩散型降解。然而，Rho的显性活性突变体不影响纤连蛋白的降解。此外，抑制磷脂酰肌醇-3激酶（P13 K）破坏了Rac和Cdc 42依赖的肌动蛋白结构，并阻止纤连蛋白降解。这些结果表明Cdc 42和Rac通过frabin-Cdc 42/Rac-PI 3 K信号通路以协调方式在纤维连接蛋白降解和侵袭中起重要作用。

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.