Adam L, Vadlamudi R, Mandal M, Chernoff J, Kumar R
The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Biol Chem. 2000 Apr 21;275(16):12041-50. doi: 10.1074/jbc.275.16.12041.
Stimulation of growth factor signaling has been implicated in the development of invasive phenotype and p21-activated kinase (PAK1) activation in human breast epithelial cancer cells. To further explore the roles of PAK1 in the invasive behavior of breast cancer cells, in the present study we investigated the influence of inhibition of PAK1 activity on the reorganization of cytoskeleton components that control motility and invasiveness of cells, using a highly invasive breast cancer MDA-MB435 as a model system. Our results demonstrate that overexpression of a kinase dead K299R PAK1 mutant leads to suppression of motile phenotypes as well as invasiveness of cells both in the absence or presence of exogenous heregulin-beta1. In addition, these phenotypic changes were accompanied by a blockade of disassembly of focal adhesion points, stabilization of stress fibers, and enhanced cell spreading and were dependent on the presence of the kinase dead domain but independent of the presence of the Rac/cdc42 intact (Cdc42/Rac interactive binding) domain of PAK1. We also demonstrated that in K299R PAK1-expressing cells, F-actin filaments were stabilized by persistent co-localization with the actin-binding proteins tropomyosin and caldesmon. Extension of these studies to invasive breast cancer MDA-MB231 cells illustrated that conditional expression of kinase-defective K299R PAK1 was also accompanied by persistent cell spreading, multiple focal adhesion points, and reduced invasiveness. Furthermore, inhibition of PAK1 activity in breast cancer cells was associated with a reduction in c-Jun N-terminal kinase activity, inhibition of DNA binding activity of transcription factor AP-1, and suppression of in vivo transcription driven by AP-1 promoter (known to be involved in breast cancer invasion). These findings suggest that PAK1 downstream pathways have a role in the development and maintenance of invasive phenotypes in breast cancer cells.
生长因子信号的刺激与人类乳腺上皮癌细胞侵袭性表型的发展以及p21激活激酶（PAK1）的激活有关。为了进一步探究PAK1在乳腺癌细胞侵袭行为中的作用，在本研究中，我们以高侵袭性乳腺癌MDA-MB435作为模型系统，研究了抑制PAK1活性对控制细胞运动性和侵袭性的细胞骨架成分重组的影响。我们的结果表明，激酶失活的K299R PAK1突变体的过表达导致细胞运动表型以及侵袭性受到抑制，无论是否存在外源性神经调节蛋白-β1。此外，这些表型变化伴随着粘着斑的解体受阻、应力纤维的稳定以及细胞铺展增强，并且依赖于激酶失活结构域的存在，但不依赖于PAK1完整的Rac/cdc42（Cdc42/Rac相互作用结合）结构域的存在。我们还证明，在表达K299R PAK1的细胞中，F-肌动蛋白丝通过与肌动蛋白结合蛋白原肌球蛋白和钙调蛋白持续共定位而得以稳定。将这些研究扩展到侵袭性乳腺癌MDA-MB231细胞表明，激酶缺陷型K299R PAK1的条件性表达也伴随着持续的细胞铺展、多个粘着斑以及侵袭性降低。此外，乳腺癌细胞中PAK1活性的抑制与c-Jun N端激酶活性降低、转录因子AP-1的DNA结合活性受到抑制以及AP-1启动子驱动的体内转录受到抑制有关（已知AP-1启动子参与乳腺癌侵袭）。这些发现表明，PAK1下游通路在乳腺癌细胞侵袭性表型的发展和维持中发挥作用。
