Abstract
S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 μM; IIB, K(d) = 8 μM; IIC, K(d) = 1.0 μM). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.
Highlights
Certain S100 proteins induce cell migration and metastasis but the molecular mechanism is not clear
Regulation of Levels of Intracellular S100P Affects Cell Migration—When the rat mammary 37 (Rama 37) cells were transfected with an expression vector for S100P, the resultant transfectants, R37-S100P-1 and R37-S100P-2, and two pooled clones, pool-1 and pool-2 [7], expressed 6 –14-fold of S100P mRNA and 5–12-fold of S100P protein over the pooled empty vector-transfected Rama 37 cells (R37-vector), causing an increase in cell migration of 30 –70% (Fig. 1, A and B) (ANOVA test, p Ͻ 0.01)
Either when S100P was induced before (Fig. 3C, induced) or after (Fig. 3F) NMIIC was knocked down, cell migration was significantly increased by 55–121% (Student’s t test, p Յ 0.033), which is similar to cells transfected with control siRNA, suggesting that NMIIC fails to play a major role in regulating S100P-enhanced cell migration
Summary
Certain S100 proteins induce cell migration and metastasis but the molecular mechanism is not clear. In different cellular systems, when expression of NMIIA is knocked down, cell migration is increased, but when NMIIB is knocked down, cell migration is reduced [23] These observations suggest that S100A4 and perhaps other S100 proteins may interact differentially and regulate these different NMII isoforms to achieve an overall increase in cell migration, but how the overall increase is achieved is unknown. To test how this is brought about, S100P has been used as a representative of the S100 proteins, because its metastasis-promoting property has been well characterized in vivo [7] and S100P-inducible cell lines have been successfully established from a rat breast tumor cell line and HeLa ovarian cancer cells. These changes suggest that the weakening of the anchoring forces generated through NMIIA to FAS will allow intact NMIIB filaments to drive cell migration
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