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An Intracellular Signal Pathway That Regulates Cancer Cell Adhesion in Response to Extracellular Forces

Surgical Service, John D. Dingell VA Medical Center and Departments of Surgery, Anesthesiology, Anatomy, and Cell Biology, Wayne State University Detroit, Michigan

Requests for reprints: Marc D. Basson, Surgical Service, John D. Dingell VA Medical Center, 4646 John R. Street, Detroit, MI 48201-1932. Phone: 313-576-3598; Fax: 313-576-1002; E-mail: marc.basson{at}va.gov.

	Abstract

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Increasing evidence suggests that tumor cells can regulate their own adhesion via intracellular signals that modulate integrin binding affinity. Although the full pathway has not yet been elucidated, the effects of pressure seem likely to require cytoskeletal mechanosensing, Src, phosphatidylinositol 3-kinase, focal adhesion kinase, and Akt-1 activation. Ultimately, activated focal adhesion kinase accumulates at the membrane in association with β₁-integrin heterodimers and may modulate integrin binding affinity. This pathway may be a promising target for manipulation to inhibit metastatic cancer cell adhesion. [Cancer Res 2008;68(1):2–4]

	Background

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The adhesion of metastasizing cancer cells is often viewed as a passive process on the part of the cancer cells themselves. Cells swirling through the lymphatic or venous circulation or around a body cavity or surgical site contact endothelial or other cells or extracellular matrix proteins, adhering if the correct membrane receptors are deployed at the right place to bind to adhesion sites and if the energy of cell movement is not so great that it overcomes binding affinity. However, work from our laboratory (1–3) and others (4, 5) suggests that cancer cells may regulate their own adhesion to matrix proteins, endothelial cells, or surgical wounds by intracellular signals that regulate the binding affinity of matrix receptors including β₁-integrin heterodimers. At least one pathway by which this occurs seems to involve focal adhesion kinase (FAK) and Src activation (2) and paxillin (6, 7) and has been shown to be active in breast (8), head and neck (6), and colon (1) cancer cell lines, as well as in primary human colon cancer cells isolated directly from surgical specimens (2). These signals mediating increased adhesion are stimulated by increased extracellular pressure (2) as well as by laminar or nonlaminar shear stress (4, 5, 9).

Although the entire pathway has not yet been elucidated, previous work suggested that the signal events involved occurred primarily within the focal adhesion complex (where FAK and Src bind to the adapter protein paxillin), could be activated by the stimulation of pressure or shear acting on the cell, and could then modulate β₁-integrin binding affinity. An intact cytoskeleton seems to be required for FAK activation and the ultimate effect on adhesion (10), so the cytoskeleton may act as a tensegrity style sensor for external forces that activate this pathway. Src activation by extracellular pressure is not affected by cytoskeletal manipulation, however, and what causes Src to be activated when extracellular pressure is increased is as yet unclear.

Elements of the phosphatidylinositol 3-kinase (PI3K) pathway are known to be overexpressed in some malignancies and to influence cell adhesion and motility in other settings. Thus, the search for further elements in the pathway that mediates pressure-stimulated cancer cell adhesion led us to investigate whether signal elements of the PI3K pathway might play a role in mediating the effects of pressure on cancer cell adhesion. Further studies also extended our understanding of these signals away from focal adhesions and into the cytosol.

	Key Findings

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These additional studies led to several important additional conclusions in our recent study. These are briefly enumerated here, although the interested reader is referred back to the original publication for more details (11).

First, PI3K and Akt are also activated by extracellular pressure and required for the stimulation of adhesion by extracellular pressure. The downstream S6 kinase is not involved, however, because rapamycin does not block the effect. This was particularly interesting because rapamycin does block the effect of increased extracellular pressure on macrophage phagocytosis (12). We have previously described other differences between macrophage and cancer cell signaling in response to extracellular pressure.

Second, Src activation seems to be required for PI3K activation, which is in turn required for the activation of FAK and Akt. Indeed, the p85 subunit of PI3K directly associates with FAK in increased proportions in response to increased extracellular pressure, based on coprecipitation studies. This changed our view of the more proximal part of this signal pathway because we had previously postulated that FAK-397 autophosphorylation within focal adhesions was an early event in the pressure-induced pathway, which is required for subsequent FAK-Src interaction and FAK-576/577 phosphorylation by Src (2). In contrast, this observation suggested that FAK-397 phosphorylation in cancer cells responding to extracellular pressure actually requires prior activation of PI3K.

Third, the proportion of cellular phosphorylated (and presumably active) Akt localized within the membrane/cytoskeletal fraction then increases, along with the proportion of cellular phosphorylated (activated) FAK in the same fraction. This is an important finding that may be explained by alternate models. The pressure stimulus may cause one or both of these important kinases to translocate to the membrane either before activation or after activation. Alternatively, the apparent shift in localization of these activated kinases may reflect an inhibition of movement back into the cytosol after activation at the cell membrane. The shifts in Akt and FAK do seem to be linked in some fashion. An Akt inhibitor prevented the shift into the membrane/cytoskeletal fraction of FAK as well as the shift of Akt itself; conversely, FAK seemed to be required for Akt activation because small interfering RNA (siRNA) reduction of FAK blocked Akt activation. A nonphosphorylatable FAK mutant also seemed to shift into the membrane/cytoskeletal fraction with pressure. Although this does not tell us whether FAK activation occurs at the membrane or within the cytosol, it does suggest that the shift in FAK localization in response to extracellular pressure does not require FAK activity. Distinguishing among these competing possibilities will require further study.

However, regardless of the details of these shifts in FAK and Akt activation and localization, the end result of these signals, and our fourth significant finding, is that increased extracellular pressure results in increased association of FAK with β₁-integrin heterodimers in a manner sensitive to blockade of Src or PI3K. Such FAK-integrin association could directly modulate integrin binding affinity and explain the ultimate increase in adhesiveness observed in response to increased extracellular pressure. Previous observations confirm that the expression of integrins on the cell surface is not altered in response to extracellular pressure (2).

Fifth and finally, only Akt-1 seems to participate in this pathway. siRNA reduction of Akt-1 blocks the effect of pressure on cell adhesion, but siRNA reduction of Akt-2 does not. This was true both for SW620 colon cancer cells in which Akt-1 predominates and for Caco-2 colon cancer cells in which Akt-1 and Akt-2 expression levels seem to be similar. One caution about these results was that we were only able to achieve a relatively modest 50% to 60% reduction of each Akt isoform by siRNA transfection. Thus, these studies left unanswered the question of whether a more complete reduction in Akt-2 might have been able to inhibit the pressure effect. More recent unpublished observations have further supported our hypothesis that Akt-2 is not involved, however (13).

	Implications

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The concept that physical forces such as pressure, shear, and deformation can modulate cell biology without ligand-receptor interactions is not new (Fig. 1 ). Many cell types exhibit mechanosensitive behavior, including intestinal epithelial cells (14). Cancer cells may experience physical forces in a variety of settings. Cancers growing rapidly against a constraining stroma may exhibit increased interstitial pressures (15, 16), whereas metastasizing tumor cells experience pressure and shear during passage through the lymphatics or vasculature. Perioperatively, cancer cells are commonly shed from tumors into the surgical site or the venous circulation during procedures, and the presence of free tumor cells on the outside of the surgical specimen correlates adversely with prognosis for colon cancers (17). Surgeons apply force to tumors during dissection and insufflate the peritoneal cavity to 15 mm Hg above ambient during laparoscopic surgery. Irrigation of the surgical site may also generate pressure and shear stress. Finally, patients exhibit similar increases in intra-abdominal pressure for 2 to 3 days after surgery as bowel wall swelling because of third spacing increases the volume of the intra-abdominal viscera, so intra-abdominal cancer cells may experience increased extracellular pressures for days after surgery. Conversely, the adhesiveness of the cancer cells themselves correlates inversely with prognosis (18).

View larger version (47K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Physical forces such as shear and pressure stimulate the adhesion of cancer cells in suspension to extracellular matrix proteins (ECM) or nonmalignant host cells in vitro or in vivo. The signal pathway that mediates this effect seems to involve Src and PI3K interaction to activate FAK in a complex manner that also requires an intact cytoskeleton, including paxillin, and Akt-1 activation. These events increase the proportion of phosphorylated FAK (p-FAK) that associates with β1-integrin heterodimers, increasing integrin binding affinity for substrate. This pathway may be one mechanism by which cancer cell adhesiveness is internally regulated.

Although this work has focused on mechanotransduced stimuli to date, growth factors or cytokines that act on various elements of this pathway could also modulate cancer cell adhesiveness. Interestingly, the proliferation of colon cancer cells is also stimulated by increased extracellular pressure over hours, albeit via a different signal mechanism (19). These signals might, in the future, offer new targets for intervention to inhibit metastasis, either in the perioperative period or in the setting of unresectable primary tumors. As the proponents of antiangiogenesis have pointed out, an unresectable primary cancer may not be a life-threatening event in many cases if metastasis can be blocked.

The observation of Akt isoform specificity may be interesting in this regard. Although many of the initial studies of Akt function did not distinguish among Akt isoforms, there are actually three Akt isoforms. Akt-1 and Akt-2 are found in all cells, whereas Akt-3 is chiefly found in the brain, heart, and kidney. There is increasing interest in differentiating the functions of the various Akt isoforms. Although opposing roles have been suggested for Akt-2 and Akt-1 in modulating cell motility (20), the current study suggests that Akt-1 is the sole pressure-responsive element that mediates the effect of pressure on cancer cell adhesion. Akt-1 has also this year been reported to selectively regulate integrin activation in endothelial cells and fibroblasts (21).

It is interesting that the stimulation of macrophage phagocytosis by extracellular pressure seems to depend instead on Akt-2 (12). For some other aspects of cell biology, Akt-2 may have other unique functions or the Akt isoforms may have interchangeable functions. The isoform specificity of the pressure-stimulated adhesion effect suggests that Akt-1 may be an interesting target of opportunity on which to act to inhibit cancer cell adhesion with less toxic inhibition of other effects in which Akt-1 may be replaced by Akt-2.

Like any good study, these observations pose more questions than they answer. These include the nature of the proximal mechanosensor by which the cancer cell perceives the physical force; the mechanism responsible for the Akt isoform specificity; whether FAK and Akt are translocating toward the membrane or inhibited from moving away from it and where within the cell their phosphorylation occurs; and the manner in which integrin binding affinity is altered. Such questions await further research, as does the question of whether growth factors or cytokines modulate cancer cell adhesion by acting on this pathway. However, taken together with previous work from our laboratory and others, this study suggests that internal signaling events within tumor cells modulate tumor cell adhesiveness. These signals may prove important targets for therapeutic intervention to inhibit metastasis in the future.

	Acknowledgments

 
Grant support: NIH grant RO1DK06771 and a VA Merit Research Award.

Received 8/ 3/07. Accepted 10/ 8/07.

	References

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1. Basson MD, Yu CF, Herden-Kirchoff O, et al. Effects of increased ambient pressure on colon cancer cell adhesion. J Cell Biochem 2000;78:47–61.[CrossRef][Medline]
2. Thamilselvan V, Basson MD. Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling. Gastroenterology 2004;126:8–18.[CrossRef][Medline]
3. van der Voort van Zyp J, Thamilselvan V, Walsh M, Polin L, Basson MD. Extracellular pressure stimulates colon cancer cell adhesion in vitro and to surgical wounds by Src (sarcoma protein) activation. Am J Surg 2004;188:467–73.[CrossRef][Medline]
4. von Sengbusch A, Gassmann P, Fisch KM, Enns A, Nicolson GL, Haier J. Focal adhesion kinase regulates metastatic adhesion of carcinoma cells within liver sinusoids. Am J Pathol 2005;166:585–96.[Abstract/Free Full Text]
5. Lawler K, Meade G, O'Sullivan G, Kenny D. Shear stress modulates the interaction of platelet-secreted matrix proteins with tumor cells through the integrin vβ3. Am J Physiol 2004;287:C1320–7.[CrossRef]
6. Conway WC, Van der Voort van Zyp J, Thamilselvan V, Walsh MF, Crowe DL, Basson MD. Paxillin modulates squamous cancer cell adhesion and is important in pressure-augmented adhesion. J Cell Biochem 2006;98:1507–16.[CrossRef][Medline]
7. van Zyp JV, Conway WC, Craig DH, van Zyp NV, Thamilselvan V, Basson MD. Extracellular pressure stimulates tumor cell adhesion in vitro by paxillin activation. Cancer Biol Ther 2006;5:1169–78.[Medline]
8. Downey C, Alwan K, Thamilselvan V, et al. Pressure stimulates breast cancer cell adhesion independently of cell cycle and apoptosis regulatory protein (CARP)-1 regulation of focal adhesion kinase. Am J Surg 2006;192:631–5.[CrossRef][Medline]
9. Thamilselvan V, Patel A, van der Voort van Zyp J, Basson MD. Colon cancer cell adhesion in response to Src kinase activation and actin-cytoskeleton by non-laminar shear stress. J Cell Biochem 2004;92:361–71.[CrossRef][Medline]
10. Thamilselvan V, Basson MD. The role of the cytoskeleton in differentially regulating pressure-mediated effects on malignant colonocyte focal adhesion signaling and cell adhesion. Carcinogenesis 2005;26:1687–97.[Abstract/Free Full Text]
11. Thamilselvan V, Craig DH, Basson MD. FAK association with multiple signal proteins mediates pressure-induced colon cancer cell adhesion via a Src-dependent PI3K/Akt pathway. FASEB J 2007;21:1730–41.[Abstract/Free Full Text]
12. Shiratsuchi H, Basson MD. Akt2, but not Akt1 or Akt3 mediates pressure-stimulated serum-opsonized latex bead phagocytosis through activating mTOR and p70 S6 kinase. J Cell Biochem 2007;102:353–67.[CrossRef][Medline]
13. Wang S, Basson MD. Identification of functional domains in AKT responsible for distinct roles of AKT isoforms in pressure-stimulated cancer cell adhesion. Exp Cell Res. In press.
14. Basson MD. Paradigms for mechanical signal transduction in the intestinal epithelium. Category: molecular, cell, and developmental biology. Digestion 2003;68:217–25.[Medline]
15. Diresta GR, Nathan SS, Manoso MW, et al. Cell proliferation of cultured human cancer cells are affected by the elevated tumor pressures that exist in vivo. Ann Biomed Eng 2005;33:1270–80.[CrossRef][Medline]
16. Taghian AG, Abi-Raad R, Assaad SI, et al. Paclitaxel decreases the interstitial fluid pressure and improves oxygenation in breast cancers in patients treated with neoadjuvant chemotherapy: clinical implications. J Clin Oncol 2005;23:1951–61.[Abstract/Free Full Text]
17. Baskaranathan S, Philips J, McCredden P, Solomon MJ. Free colorectal cancer cells on the peritoneal surface: correlation with pathologic variables and survival. Dis Colon Rectum 2004;47:2076–9.[CrossRef][Medline]
18. Benoliel AM, Pirro N, Marin V, et al. Correlation between invasiveness of colorectal tumor cells and adhesive potential under flow. Anticancer Res 2003;23:4891–6.[Medline]
19. Walsh MF, Woo RK, Gomez R, Basson MD. Extracellular pressure stimulates colon cancer cell proliferation via a mechanism requiring PKC and tyrosine kinase signals. Cell Prolif 2004;37:427–41.[CrossRef][Medline]
20. Zhou GL, Tucker DF, Bae SS, Bhatheja K, Birnbaum MJ, Field J. Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration. J Biol Chem 2006;281:36443–53.[Abstract/Free Full Text]
21. Somanath PR, Kandel ES, Hay N, Byzova TV. Akt1 signaling regulates integrin activation, matrix recognition, and fibronectin assembly. J Biol Chem 2007;282:22964–76.[Abstract/Free Full Text]

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Basson, M. D. Search for Related Content PubMed PubMed Citation Articles by Basson, M. D. Related Collections Tumor Biology Tumor Biology: Invasion and Metastasis