Regulation of Microfilament Reorganization and Invasiveness of Breast Cancer Cells by Kinase Dead p21-activated Kinase-1

Abstract

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-␤1. 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.Breast cancer is one of the most common malignancies in the United States, affecting one in nine women. Localized breast cancer prior to metastasis can be cured by surgery. The high mortality rate associated with breast cancer, however, is related to its ability to metastasize beyond the mammary gland and invade distant sites while the primary tumor is small and undetected. Thus, tumor cell migration/invasion is an important factor in formation of solid tumors and is necessary for their spread to distinct organs. The process of malignancy requires, among other steps, changes in growth factor pathways and increased migration. The exposure of cells to growth factors has been shown to cause cytoskeleton reorganization, formation of lamellipodia, membrane ruffling, and altered cell morphology, and accordingly, such exposure is implicated in stimulating cell migration and invasion (1). Most eukaryotic cells possess the capacity to move over or through a substrate, and cell migration plays a key role in both normal physiology and in a disease setting, including invasion and metastasis (2). In many tiss...

Dysregulation of phosphatidylinositol 3‐kinase and downstream effectors in human breast cancer

“…Importantly, our observations are in keeping with recent observations that concern cell motility and invasiveness, which are regulated by Pak via a PI3-K dependent mechanism. 24,25 It has previously been shown that the MAPK family members, p38 and JNK enzymes are downstream of Pak. 26 However, to our knowledge, neither of these protein kinases have been investigated for enzyme activity in breast cancer, in direct comparison with control tissue.…”

Role of NF-κB signaling in hepatocyte growth factor/scatter factor-mediated cell protection

“…The wild-type (wtPTEN) and mutant (phosphatase-defective) (mut-PTEN) expression vectors were provided by Dr MM Georgescu (Rockefeller University, New York, NY, USA) (Georgescu et al, 1999). Expression vectors encoding wild-type (wtPak1) and kinase dead (K299R) dominant-negative (DN-Pak1) p21-associated kinase have been described earlier (Adam et al, 2000). These Pak1 vectors were provided by Dr R Kumar (University of Texas MD Anderson Cancer Center, Houston, TX, USA).…”

Distinct roles for phosphoinositide 3-kinase, mitogen-activated protein kinase and p38 MAPK in mediating cell cycle progression of breast cancer cells

“…The epidermal growth factor (EGF)-family of peptides are ligands for the ErbB receptor family, the prototype ligand being EGF which binds to the epidermal growth factor receptor (EGFR or ErbB1) (Beerli and Hynes, 1996;Burden and Yarden, 1997;Riese and Stern, 1998). The Neu di erentiation factors (HRGs) or Neuregulins (NRGs) represent a family of ligands that bind and activate di erent subsets of ErbB receptors eliciting a plethora of cellular responses (Adam et al, 1998(Adam et al, , 2000Hall, 1998;Hijazi et al, 2000;Mitchison and Cramer, 1996). One such factor, heregulin b1 (HRGb1 henceforth termed HRG), binds with high a nity to ErbB3 and ErbB4 thus activating these receptors, but also induces tyrosine phosphorylation and activation of ErbB2 Plowman et al, 1993;Tzahar et al, 1994).…”