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. 2005 Feb;166(2):585-96.
doi: 10.1016/S0002-9440(10)62280-8.

Focal adhesion kinase regulates metastatic adhesion of carcinoma cells within liver sinusoids

Anke von Sengbusch  1 , Peter Gassmann, Katja M Fisch, Andreas Enns, Garth L Nicolson, Jörg Haier
Affiliations

Focal adhesion kinase regulates metastatic adhesion of carcinoma cells within liver sinusoids

Anke von Sengbusch et al. Am J Pathol. 2005 Feb.

Abstract

Organ-specific tumor cell adhesion to extracellular matrix (ECM) components and cell migration into host organs often involve integrin-mediated cellular processes that can be modified by environmental conditions acting on metastasizing tumor cells, such as shear forces within the blood circulation. Since the focal adhesion kinase (FAK) appears to be essential for the regulation of the integrin-mediated adhesive and migratory properties of tumor cells, its role in early steps of the metastatic cascade was investigated using in vitro and in vivo approaches. Human colon and hepatocellular carcinoma cells were used to study adhesive properties under static conditions and in a parallel plate laminar flow chamber in vitro. In addition, intravital fluorescence microscopy was used to investigate early interactions between circulating tumor cells and the microvasculature of potential target organs in vivo. Shear forces caused by hydrodynamic fluid flow induced Tyr-hyperphosphorylation of FAK in cell monolayers. Reduced expression of FAK or its endogenous inhibition by FAK-related non-kinase (FRNK) interfered with early adhesion events to extracellular matrix components under flow conditions. In contrast, tumor cell adhesion to endothelial cells under these conditions was not affected. Furthermore, down-regulation of FAK inhibited metastatic cell adhesion in vivo within the liver sinusoids. In summary, FAK appears to be involved in early events of integrin-mediated adhesion of circulating carcinoma cells under fluid flow in vitro and in vivo. This kinase may take part in the establishment of definitive adhesive interactions that enable adherent tumor cells to resist fluid shear forces, resulting in an organ-specific formation of distant metastases.

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Figures

Figure 1
Figure 1
Stimulation of Tyr-phosphorylation by hydrodynamic shear forces. HT-29 cells grown on glass slides were subjected to different rates of WSS in a laminar flow chamber. Tyr-phosphorylated FAK were measured after 60 minutes of flow exposure. Experiments were performed three times and one representative example is shown.
Figure 2
Figure 2
Inhibition of FAK function or expression. A: HT-29 cells were incubated with 0.5–1 μmol/L FAK-AS2 or FAK-AS control oligonucleotides using Lipofectin (10 μl/ml) in serum-free DME-F12 medium for 36 hours. Expression of FAK was determined using Western blotting. In antisense-treated cells expression of FAK was significantly reduced to less than 30% compared to untreated cells (P < 0.05 from four experiments), whereas mismatch-control treatment had only slight effects (88%) on FAK protein amounts. In contrast, the focal adhesion protein paxillin was not affected. B: Transient transfection of Hep-G2 cells resulted in overexpression of FRNK after 24 to 72 hours as determined by Western blot using monoclonal antibodies against the C-terminal region of FAK. Endogeneous FRNK has not been detected in these cells. C: The 46 kd-FRNK fragment was found using pcDNA3.1 plasmids, where the EGFP-FRNK plasmid produced a 73-kd fragment. Control cells were transfected with genuine plasmids.
Figure 3
Figure 3
Static cell adhesion and spreading of FRNK-transfected cells. A: Hep-G2 cells transfected with EGFP-plasmid (//) or pcDNA3.1-plasmid () containing FRNK or in its genuine form (mock) were allowed to adhere to collagen IV in microtiter plate assays for 30 minutes. Significant differences in the numbers of adherent cells were not found between FRNK- and control-transfected cells. Values are shown as mean ± SD from quadruplicate experiments. Comparable results were obtained using collagen I or colon carcinoma cells (not shown). B: Hep-G2 cells adherent to collagen IV for 30 minutes were fluorescence-stained for FAK (epitope within FRNK-fragment, B1) and F-actin (phalloidin, B2) in a double-labeling technique. The cell expressing FRNK remained round without cell spreading and reorganization of the actin cytoskeleton. In addition, FRNK-transfected cells did not show adhesion-specific phosphorylation of FAK (B4), whereas mock-transfected cells demonstrated typical focal adhesions with phosphorylated Tyr397 residues of FAK (B3). This inhibition was confirmed by Western blot (not shown).
Figure 4
Figure 4
Effect of reduced FAK expression on dynamic cell adhesion to ECM. HT-29 cells treated with FAK antisense or mismatch-control oligonucleotides were used for adhesion assays in a laminar flow chamber. BSA was used as nonspecific adhesion substrate. Values are shown as mean ± SD from five different experiments. Numbers of adherent cells were compared to dynamic adhesion of untreated cells to C I using Student’s t-test (#, P < 0.05). Similar results were obtained using C IV as adhesive substrate.
Figure 5
Figure 5
FRNK negatively regulates dynamic cell adhesion to collagen. Hep-G2 cells transfected with genuine EGFP-vector or EGFP-FRNK were used for dynamic adhesion assays on C I. Values are shown as mean ± SD from six different experiments. WSAT was not significantly affected (4.7 ± 0.6 dyn/cm2 versus 4.1 ± 0.3 dyn/cm2), but DAR (7.8 ± 6.8 cells; P < 0.005) and ASR (71 ± 7% versus 55 ± 15%; 0.05) were significantly reduced in FRNK-transfected cells. Similar results were obtained using C IV, but total numbers of cells were smaller, if this ECM component was used (not shown).
Figure 6
Figure 6
FAK is not required for tumor cell adhesion to endothelial cells. Hep-G2 cells transfected with genuine EGFP-vector or EGFP-FRNK were used for (A) static or (B) dynamic adhesion assays on HUVEC in vitro. Values are shown as mean ± SD from five different experiments. FRNK did not affect dynamic adhesion, but cell adhesion under static conditions was significant higher in FRNK-transfected cells (P < 0.005).
Figure 7
Figure 7
In vivo cell adhesion within the hepatic microcirculation. Hep-G2 cells transfected with (A) genuine EGFP-vector or (B) EGFP-FRNK were used for the intravital observation of circulating tumor cells within the liver sinusoids. Numbers of adherent cells, migrated cells and the total number of arrested cells (adherent + migrated) are shown as mean ± SD from seven animals in each group. Expression of FRNK almost completely inhibited adhesive interactions of circulating Hep-G2 cells within the liver.
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