Differential requirement for MEK Partner 1 in DU145 prostate cancer cell migration

Abstract

ERK signaling regulates focal adhesion disassembly during cell movement, and increased ERK signaling frequently contributes to enhanced motility of human tumor cells. We previously found that the ERK scaffold MEK Partner 1 (MP1) is required for focal adhesion disassembly in fibroblasts. Here we test the hypothesis that MP1-dependent ERK signaling regulates motility of DU145 prostate cancer cells. We find that MP1 is required for motility on fibronectin, but not for motility stimulated by serum or EGF. Surprisingly, MP1 appears not to function through its known binding partners MEK1 or PAK1, suggesting the existence of a novel pathway by which MP1 can regulate motility on fibronectin. MP1 may function by regulating the stability or expression of paxillin, a key regulator of motility.

Figure 1

ERK activation is not required for DU145 migration on FN, but is required for migration on FN induced by serum. A) Results of DU145 wound healing on FN. Shown are averaged results from 4 experiments; error bars represent standard error of the mean (S.E.M.). Migration in response to fibronectin alone was not affected by UO126. Serum and EGF stimulated significant (p < 0.0001) increases in migration, that were significantly inhibited (p < 0.0001 for serum, p = 0.0004 for EGF) by UO126 treatment. B) Sample images from wound healing assay; for each condition, six grid squares were imaged at 0 and 10 h post-wounding, and wound area (outlined in white) was measured using ImageJ software (NIH).

Figure 2

MP1 knockdown inhibits migration in DU145 acutely adherent on fibronectin. A) DU145 cells were transfected with Control (non-targeting) or MP1 siRNA. MP1 depletion inhibits DU145 migration on FN in serum-free medium (SF) by ~50% relative to control. Migration is induced by 1% fetal bovine serum (1%) in cells transfected with control or MP1 siRNA, and the induced component of migration is sensitive to MEK1 inhibition by UO126 (UO). Shown are mean data from three independent experiments; error bars represents S.E.M. B) Representative blot showing MP1 knockdown in DU145 cells treated in parallel with cells in wound-healing assay. Two exposures of MEK1 phospho-SS218/222 are shown to emphasize the low level of MEK1 activation in these assays, which nevertheless remains similar between Control and MP1 siRNA transfected cells. SF, Serum-Free medium; S, 1% fetal bovine Serum medium; S/U, 1% fetal bovine Serum medium + 25 μM UO126.

Figure 3

MP1 depletion decreases membrane activity and the number of focal adhesions at the wound edge. A) DU145 cells transfected with control or MP1 siRNA were wounded as described. Approximately 2 hours post-wounding, cells were imaged at 1 second intervals for 5 min for kymograph analysis. For each cell, 6-7 kymographs were generated (shown by numbered lines); 5-7 cells per condition were analyzed. Boxes around the number indicate the representative kymograph shown. B) Control and MP1 siRNA transfected cells plated on coverslips and imaged for a wound-healing assay were fixed and stained for vinculin to mark focal adhesions. Average number and size of FA occurring at the wound edge were quantified using ImageJ (chart). C) Size distribution of all focal adhesions in the cells imaged for B. MP1 depleted cells have fewer focal adhesions at the wound edge.

Figure 4

MP1 knockdown modestly delays PAK autophosphorylation in response to FN and decreases paxillin expression. A) DU145 cells transfected with control or MP1 siRNA were plated on FN for the times indicated. Whole cell lysates were analyzed by immunoblot for PAK phosphorylation of MEK1 (pS298 MEK1) and PAK1-3 autophosphroylation on S141. B) Cells transfected with control or MP1 siRNA were lysed in RIPA (RIPA) or SDS-PAGE (SDS) loading buffer (see Methods) and expression of focal adhesion proteins was assessed by immunoblot. C, Control siRNA; M, MP1 siRNA. Paxillin expression was quantified by densitometry and is expressed as percent of control for each lysis buffer. C) Cells transfected with control, MP1, or p14 siRNA were analyzed for vinculin, paxillin, MP1 and p14 expression.