Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells

Stimulation of growth factor signaling has been implicated;in the developme nt of invasive phenotypes and the activation of pal-activated kinase (Pak1) in human breast; cancer cells (Adam, L., Vadlamudi, R., Kondapaka, S. B., Chernoff, J., Mendelsohn, J,, and Kumar, Ri (1998) J. Biol. Chem. 273, 2823 8-28246; Adam, L., Vadlamudi, R,, Mandal, M,, Chernoff, J., and Kumar, R. ( 2000) J. Biol. Chem. 275, 12041-12050). To study the role of: Pak1 in the r egulation of motility and growth of breast:epithelial cells, we developed h uman epithelial MCF-7 clones that overexpressed the kinase-active T423E Pak 1 mutant under an inducible tetracycline promoter or that stably expressed the kinase-active H83L,H86L Pak1 mutant, which is deficient in small GTPase binding sites, The expression of both T423E and H83L,H86L Pak1 mutants in breast epithelial cells was accompanied:by increased cell motility without any apparent effect On the growth rate of cells, The T423E Pak1 mutant was primarily localized to filopodia, and the H83L,H86L Pak1 mutant was primari ly localized to ruffles. Cells expressing T423E Pak1 exhibited a regulatabl e stimulation of mitogen-activated protein kinase and Jun N-terminal kinase activities. The expression of kinase-active Pak1 mutants significantly sti mulated anchorage-independent growth of cells in soft agar in a preferentia l mitogen-activated protein kinase-sensitive manner.: In addition, regulata ble expression of kinase-active Pak1-resulted in an abnormal organization o f mitotic spindles characterized by appearance of multiple spindle orientat ions. We also provide evidence to suggest a close correlation between the s tatus of Pak1 kinase activity and base-line invasiveness of human breast ca ncer cells and breast tumor grades. This study is the first demonstration o f Pak1 regulation of anchorage-independent growth, potential Pak1 regulatio n of invasiveness, and abnormal organization of mitotic spindles of human e pithelial breast cancer cells.