Extracellular pressure stimulates adhesion of sarcoma cells via activation of focal adhesion kinase and Akt

Abstract

Background: The effect of extracellular pressure on adhesion and adhesiogenic focal adhesion kinase (FAK) and Akt signaling in sarcomas was investigated.

Methods: Human sarcoma cells (HT-1080 fibrosarcoma, KHOS-240S osteosarcoma, and A-673 rhabdomyosarcoma) were subjected to increased pressure followed by adhesion assay. Two cell lines were pretreated with the FAK inhibitor 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) or Akt IV inhibitor, followed by Western analysis for activated FAK and Akt. Parallel studies were conducted in cells from a resected human fibrous histiosarcoma.

Results: Pressure increased adhesion in all 3 sarcoma lines and primary histosarcoma cells by 7% to 18% (n = 6; P < .01 each). Pressure activated FAK and Akt (n = 5; P < .01). Inhibiting FAK or Akt inhibited FAK or Akt phosphorylation and the stimulation of adhesion by increased pressure (n = 5 each; P < .01 each).

Conclusions: Pressure increases sarcoma cell adhesiveness via Akt and FAK. Perioperative manipulation or forces in lymphatic or circulatory systems may potentiate local recurrence or distant metastasis.

Fig. 1

Fibrous histiosarcoma, HT-1080, and A-673cells were pre-treated with 10μM Akt IV inhibitor or 20μM Y15 (FAK Inh) before adhesion assay. Y15 or Akt IV inhibitor prevented pressure-associated increased adhesion in all three (n=5 each). FAK inhibitor reduced basal adhesion in all three cells and Akt IV inhibitor reduced basal adhesion in A-673 and fibrous histiosarcoma cells. **p<0.01 vs. DMSO ambient pressure; ##p<0.01 vs. DMSO ambient pressure; NS – not significant.

Fig. 2

Densitometric analysis of pressure (shaded bars) effects on (A) Akt and (B) FAK phosphorylation in HT-1080 cells vs. ambient pressure (open bars), with typical blots from one of nine similar studies. Increasing ambient pressure by 15 mm Hg (DMSO-closed bar) stimulated Akt and FAK phosphorylation vs. ambient pressure controls (DMSO-open bar). Akt IV inhibitor or Y15 (FAK inhibitor) prevented pressure-induced Akt and FAK phosphorylation vs. cells under ambient pressure. *p<0.05 vs. DMSO ambient pressure, #p<0.05 vs. DMSO ambient pressure, NS – not significant.

Fig. 3

Densitometric analysis of the effects of pressure (shaded bars) on (A) Akt and (B) FAK phosphorylation in primary fibrous histiosarcoma cells vs. ambient pressure (open bars), with typical blots from one of four studies. Increasing ambient pressure by 15 mm Hg (DMSO-closed bar) stimulated Akt and FAK phosphorylation vs. ambient pressure controls (DMSO-open bar). Akt IV inhibitor or Y15 (FAK inhibitor) prevented pressure-induced Akt and FAK phosphorylation vs. cells under ambient pressure. *p<0.05 vs. DMSO ambient pressure, #p<0.05 vs. DMSO ambient pressure, NS – not significant.