Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling  Vijayalakshmi Thamilselvan, Marc D. Basson  Gastroenterology  Volume 126, Issue 1, Pages 8-18 (January 2004) DOI: 10.1053/j.gastro.2003.10.078

Figure 1 Fifteen millimeters of mercury increased pressure stimulates colon cancer adhesion to collagen I and endothelial monolayers. (A) Increased pressure (closed bars) stimulated the adhesion to type I collagen of primary human colon cancer cells from 20 consecutive patients compared with ambient pressure controls from the same patients’ tumors (open bars). Data from individual experiments (20 high power fields in each of triplicate wells counted per experiment) are normalized to control and expressed as mean ± SE (∗P < 0.001; n = 20). (B) Figure represents the adhesion of primary human colon cancer cells under increased pressure conditions (closed bars) compared with their respective controls (open bars) without or with divalent cation supplementation. Adding 0.25 mmol/L Mn2+ stimulated primary colon cancer cell adhesion to collagen I (middle open bar, #P < 0.001), and adhesion was further enhanced by increased pressure (middle closed bar, ∗P < 0.05). Adding 10 mmol/L Ca2+ inhibited basal adhesion by 50% (right open bar, @P < 0.001), but pressure remained able to stimulate adhesion even under these conditions (right closed bar) (n = 5, ∗P < 0.05). (C) Pressure significantly increased SW620 colon cancer cell adhesion to rat aortic endothelial cell monolayer compared with ambient pressure controls (∗P < 0.001; n = 3). (D) Pressure increased the binding force between human SW620 colon cancer cells and the matrix. Pressure treatment (closed bar) significantly increased the percentage of cells remaining on collagen-coated plates after attempted detachment by a centrifugal force of 1500 g compared with detachment of cells that had been subjected only to ambient pressure before detachment (open bar) (∗P < 0.001; n = 3). Gastroenterology 2004 126, 8-18DOI: (10.1053/j.gastro.2003.10.078)

Figure 2 Pressure activated FAK and Src but not ERK in suspended cells. (A) Pressure activated FAK Tyr-397 autophosphorylation and Src in SW620 cells. Figure represents Western blots for phosphorylated FAK Tyr-397 or Src-416 and reprobing of each blot for total FAK or Src as appropriate. Addition of 0.25 mmol/L Mn2+ did not affect FAK or Src phosphorylation. Adding 10 mmol/L Ca2+ significantly increased FAK phosphorylation but not that of Src. (Figure shows representative blots from 6 similar experiments.) (A) Src in vitro kinase assay further confirms Src activation by pressure in SW620 cells. The kinase activity of pSrc are assessed by kinase assays by using enolase and [γ-32P]adenosine triphosphate as substrates. The top panel represents the one of 3 similar autoradiographs of phosphorylated enolase. The bottom panel represents densitometric analysis of 3 similar experiments. Results are normalized to control and expressed as mean ± SE of fold increase in Src activation. Open bar represents amibient pressure and closed bar represents increased pressure (n = 3, ∗P < 0.001). (B) Figure represents ERK phosphorylation in adherent (Adher) and nonadherent (Non A) SW620 cells. The top panel represents a blot for phosphorylated ERK, whereas the bottom panel represents reprobing for total ERK as a loading control. Pressure did not activate ERK in nonadherent cells, but pressure significantly increased ERK phosphorylation in adherent cells compared with control cells under ambient pressure or nonadherent cells. (Figure shows representative blots from 3 similar studies.) Gastroenterology 2004 126, 8-18DOI: (10.1053/j.gastro.2003.10.078)

Figure 3 Effect of signal inhibition on Src phosphorylation and SW620 cell adhesion. Closed bars represent 15 mm Hg increased pressure and open bars represent ambient pressure conditions. (A) Src kinase inhibitor PP2 significantly reduced basal Src phosphorylation and prevents pressure-induced Src phosphorylation compared with a DMSO vehicle control and ambient pressure control respectively. Quantitative values are from densitometric scanning of blots of 3 different experiments. Results are expressed as mean ± SE of phosphorylated Src to total Src normalized to control (∗P < 0.001 increased pressure vs. ambient pressure control; #P < 0.001—PP2 vs. DMSO, not significant). (B) PP2 effect on pressure- and cation-modulated cell adhesion was determined by adhesion assay. PP2 significantly blocked basal and pressure induced SW620 cell adhesion on collagen I. (∗P < 0.001 increased pressure vs ambient pressure control; ΨP < 0.001—PP2 vs. DMSO). Mn2+ 0.25 mmol/L significantly increased cell adhesion (#P < 0.001 Mn2+ vs. control) and 10 mmol/L Ca2+ inhibits cell adhesion (@P < 0.001 Ca2+ vs. control). Despite its effect on basal adhesion, PP2 did not prevent the further stimulation of adhesion by 0.25 mmol/L Mn2+ or the further inhibition of adhesion by 10 mmol/L Ca2+. (C, D) Src inhibitor PP2 lowered basal and prevented pressure-induced FAK Tyr-576 phosphorylation and enhances both basal and pressure induced FAK Tyr-397 autophosphorylation. The top panel represents the Western blot for phosphorylated FAK Tyr-576 (C) or FAK Tyr-397 (D) and the reprobe of the corresponding membrane for total FAK as a loading control. The bottom panels represent densitometric analysis of 3 similar experiments (∗P < 0.05—increased pressure vs. ambient pressure control; #P < 0.05, PP2 vs. DMSO, not significant). (E) The Src inhibitor PP2 significantly inhibited basal adhesion and prevented pressure-stimulated primary colon cancer cell adhesion (∗P < 0.05—increased pressure vs. ambient pressure control; #P < 0.05, PP2 vs. DMSO, not significant). Gastroenterology 2004 126, 8-18DOI: (10.1053/j.gastro.2003.10.078)

Figure 4 Inhibition of FAK decreased pressure-induced SW620 cell adhesion and FAK-397, FAK-576, and Src-416 phosphorylation. Closed bars represent 15 mm Hg increased pressure, and open bars represent ambient pressure conditions. (A) Lowering FAK expression by SiRNA transfection significantly decreased basal adhesion compared with untreated, mock-transfected, or scrambled siRNA-transfected controls (ΨP < 0.001; n = 3) and prevented pressure stimulated adhesion when compared with respective ambient pressure control. C represents untreated control, M represents mock transfection, Sc represents scrambled siRNA, and Si represents FAK siRNA. In contrast, despite its effect on basal adhesion, FAK inhibition did not affect the modulation of cell adhesion by Mn2+ or Ca2+. (#P < 0.001 Mn2+ vs. control; @P < 0.001 Ca2+ vs. control). ∗Increased adhesion by increased pressure compared with the respective ambient pressure controls; NS, not significant. Figure represents one of 3 similar experiments. Results are expressed as mean ± SE of adherent cells. (B) FAK inhibition by transient transfection with HA-tagged FRNK inhibited pressure-induced cell adhesion compared with ambient pressure conditions, whereas sham-transfected cells (Sham), cells not expressing FRNK from the same transfections (FRNK Non-transf), cells transfected with HA-tagged wild-type FAK (wtFAK Transf), or cells not expressing HA-tagged wtFAK (wtFAK Non-transf) all display increased adhesion in response to pressure (∗P < 0.05; n = 3). Differences in basal cell numbers under ambient pressure conditions reflect differences in transfection efficiencies rather than the effect of transfection on basal adhesion per se. Results are expressed as mean ± SE of cells counted over 20 HPF in triplicate wells per experiment. (C–E) Reducing FAK expression by SiRNA (Si) significantly inhibited pressure-induced FAK Tyr-397 and FAK Tyr-576 phosphorylation and Src-416 phosphorylation compared with their respective ambient pressure condition without any significant changes in the basal level of phosphorylation at FAK397 (C), FAK576 (D), or Src (E) compared with untreated (C), mock-transfected (M), or scrambled-siRNA (Sc)-transfected controls. Bars represent densitometric analysis of blots of phosphorylated FAK-397(C), FAK-576 (D), Src-416 (E) and their corresponding total protein loading controls. Data are normalized to control. Representative Western blots are shown above the bars (∗P < 0.05, increased pressure vs. ambient pressure control). Gastroenterology 2004 126, 8-18DOI: (10.1053/j.gastro.2003.10.078)

Figure 5 Pressure-stimulated adhesion was blocked by cytoskeleton inhibition but not by MEK or PKC inhibition. (A) Phalloidin significantly blocked pressure-induced SW620 cell adhesion to collagen I. (∗P < 0.05 pressure vs. control, not significant). In contrast, neither the MEK inhibitor PD98059 nor the PKC inhibitor calphostin C prevented pressure-induced SW620 adhesion to collagen I. Effects of a DMSO vehicle control are also shown (n = 3,∗P < 0.05, increased pressure vs. ambient pressure control; #P < 0.05, phalloidin vs. DMSO, not significant). (B) Phalloidin reduced basal adhesion of primary colon cancer cells and prevented pressure-stimulated cell adhesion (n = 5, ∗P < 0.05 increased pressure vs. ambient pressure control; #P < 0.05, phalloidin vs. DMSO, not significant). Gastroenterology 2004 126, 8-18DOI: (10.1053/j.gastro.2003.10.078)

Figure 6 Schematic diagram represents a model consistent with our results. Extracellular pressure engendered by surgical manipulation or laparoscopic insufflation or by venous, lymphatic, intra-abdominal, or luminal pressure may activate an actin-dependent pathway that involves FAK autophosphorylation at Tyr397, FAK-Src association and Src-416 activation, and further phosphorylation of FAK-Tyr576 by Src. Other phosphorylation sites on FAK or Src may also display similar behavior when these kinases are activated. Such signals at the focal adhesion complex may then alter integrin affinity for the extracellular matrix from within the cell. Conversely, extracellular divalent cations are likely to bind to the divalent cation binding site previously identified on the extracellular domains of many integrin subunits, thus modifying integrin binding affinity independently of the intracellular signal events stimulated by pressure. Gastroenterology 2004 126, 8-18DOI: (10.1053/j.gastro.2003.10.078)