Dynamic cell adhesion and migration form an integral part of tumour cell invasion, therefore an understanding of the molecular effectors involved is essential in order to develop more effective therapies targeting tumour progression. This study has investigated the role of phospholipase C gamma1 (PLCγ1), a key signal transduction enzyme, in such processes.
PLCγ1 is activated downstream of a variety of extracellular stimuli and has previously been implicated in the regulation of motility pathways in a variety of cell types. In this study, PC3LN3 prostate cancer cell lines with inducible shRNA-mediated knockdown (KD) of PLCγ1 were generated in order to monitor the effect on cell behaviours relevant to tumour invasion. Subsequent microarray analyses have identified changes occurring at the gene expression level that may underlie the observed phenotypes.
Two independent approaches were used to monitor adhesion of PLCγ1 KD cells (i) direct counting of floating and attached cell populations post-seeding (ii) a novel assay designed to measure the force required to detach cells from their underlying substrata. Both techniques revealed that PLCγ1 KD reduces the adhesive properties of cells. Concurrent with this defect, PLCγ1 KD cells displayed a 20-25% reduction in growth rate compared to their wild-type controls.
Using a transwell assay, PLCγ1 KD cells displayed reduced chemotactic migration towards 5% FCS ranging from 27-52% of controls. In addition, PLCγ1 KD cells were unable to polarise, elongate and extend dynamic membrane protrusions in a similar manner to controls when in contact with the basement membrane extract MatrigelTM. Visualization of the actin cytoskeleton using Texas-red phalloidin revealed that actin remains uniformly distributed around the periphery of the PLCγ1 KD cells whereas wild-type cells display concentrated foci of actin staining within the cell tips.
cDNA expression analysis was performed on PLCγ1 KD versus control PC3LN3 cells cultured on both plastic and MatrigelTM. Expression changes were predominantly found in genes previously implicated in the regulation of cell motility and cytoskeletal dynamics, in particular members of the integrin family (integrins α2 and α6) and Rho GTPases (Cdc42, Rac2). Of particular interest was the down-regulation of Rap guanine nucleotide exchange factor 1 (Rap GEF1) as Rap GEF1 null mouse embryonic fibroblasts display an adhesion defect similar to that noted for the PLCγ1 KD cells.
Preliminary follow-up studies have indicated that transient KD of Rap GEF1 in PC3LN3 cells significantly reduces adhesion of cells to their underlying substrata. In addition, exogenous expression of RapGEF1 in PLCγ1 KD cells is able to rescue the adhesion defect displayed by these cells. These findings strongly implicate Rap GEF1 as a key player in effecting responses central to tumour cell invasion downstream of PLCγ1.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA