Skip to main content

Advertisement

SpringerLink
Log in

An altered fibronectin matrix induces anoikis of human squamous cell carcinoma cells by suppressing integrin alpha v levels and phosphorylation of FAK and ERK

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Fibronectin regulates many cellular processes, including migration, proliferation, differentiation, and survival. Previously, we showed that squamous cell carcinoma (SCC) cell aggregates escape suspension-induced, p53-mediated anoikis by engaging in fibronectin-mediated survival signals through focal adhesion kinase (FAK). Here we report that an altered matrix, consisting of a mutated, nonfunctional high-affinity heparin-binding domain and the V region of fibronectin (V+H−), induced anoikis in human SCC cells; this response was blocked by inhibitors of caspase-8 and caspase-3. Anoikis was mediated by downregulation of integrin alpha v in a panel of SCC cells and was shown to be proteasome-dependent. Overexpression of integrin alpha v or FAK inhibited the increase in caspase-3 activation and apoptosis, whereas suppression of alpha v or FAK triggered a further significant increase in apoptosis, indicating that the apoptosis was mediated by suppression of integrin alpha v levels and dephosphorylation of FAK. Treatment with V+H− decreased the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, and direct activation of ERK by constitutively active MEK1, an ERK kinase, increased ERK1 and ERK2 phosphorylation and inhibited the increase in apoptosis induced by V+H−. ERK acted downstream from alpha v and FAK signals, since alpha v and FAK overexpression inhibited both the decrease in ERK phosphorylation and the increase in anoikis triggered by V+H−. These findings provide evidence that mutations in the high-affinity heparin-binding domain in association with the V region of fibronectin, or altered fibronectin matrices, induce anoikis in human SCC cells by modulating integrin alpha v-mediated phosphorylation of FAK and ERK.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

ECM:

Extracellular matrix

FAK:

Focal adhesion kinase

MAPK:

Mitogen-activated protein kinase

V+ :

V region of fibronectin

H+ :

Heparin-binding region of fibronectin

H− :

Mutated, nonfunctional heparin-binding region of fibronectin

References

  1. Gilcrease MZ (2007) Integrin signaling in epithelial cells. Cancer Lett 247:1–25

    Article  PubMed  CAS  Google Scholar 

  2. Pytela R, Pierschbacher MD, Ruoslahti E (1985) Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 40:191–198

    Article  PubMed  CAS  Google Scholar 

  3. Busk M, Pytela R, Sheppard D (1992) Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein. J Biol Chem 267:5790–5796

    PubMed  CAS  Google Scholar 

  4. Charo IF, Nannizzi L, Smith JW, Cheresh DA (1990) The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin. J Cell Biol 111:2795–2800

    Article  PubMed  CAS  Google Scholar 

  5. Frisch SM, Ruoslahti E (1997) Integrins and anoikis. Curr Opin Cell Biol 9:701–706

    Article  PubMed  CAS  Google Scholar 

  6. Mould AP, Komoriya A, Yamada KM, Humphries MJ (1991) The CS5 peptide is a second site in the IIICS region of fibronectin recognized by the integrin alpha 4 beta 1. Inhibition of alpha 4 beta 1 function by RGD peptide homologues. J Biol Chem 266:3579–3585

    PubMed  CAS  Google Scholar 

  7. Mould AP, Humphries MJ (1991) Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin. EMBO J 10:4089–4095

    PubMed  CAS  Google Scholar 

  8. Mitra SK, Hanson DA, Schlaepfer DD (2005) Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 6:56–68

    Article  PubMed  CAS  Google Scholar 

  9. Sieg DJ, Hauck CR, Ilic D et al (2000) FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2:249–256

    Article  PubMed  CAS  Google Scholar 

  10. Schaller MD, Otey CA, Hildebrand JD, Parsons JT (1995) Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains. J Cell Biol 130:1181–1187

    Article  PubMed  CAS  Google Scholar 

  11. Guan JL, Shalloway D (1992) Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature 358:690–692

    Article  PubMed  CAS  Google Scholar 

  12. Huynh QN, Wang S, Tafolla E, Gansky SA, Kapila S, Armitage GC, Kapila YL (2002) Specific fibronectin fragments as markers of periodontal disease status. J Periodontol 73:1101–1110

    Article  PubMed  CAS  Google Scholar 

  13. Ffrench-Constant C, Van de Water L, Dvorak HF, Hynes RO (1989) Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat. J Cell Biol 109:903–914

    Article  PubMed  CAS  Google Scholar 

  14. Trefzer U, Hofmann M, Reinke S, Guo YJ, Audring H, Spagnoli G, Sterry W (2006) Concordant loss of melanoma differentiation antigens in synchronous and asynchronous melanoma metastases: implications for immunotherapy. Melanoma Res 16:137–145

    Article  PubMed  CAS  Google Scholar 

  15. Frisch SM, Francis H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124:6619–6626

    Article  Google Scholar 

  16. Wahl SM, Allen JB, Hines KL et al (1994) Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment. J Clin Invest 94:655–662

    Article  PubMed  CAS  Google Scholar 

  17. Natal C, Oses-Prieto JA, Pelacho B et al (2006) Regulation of apoptosis by peptides of fibronectin in human monocytes. Apoptosis 11:209–219

    Article  PubMed  CAS  Google Scholar 

  18. Dai R, Iwama A, Wang S, Kapila YL (2005) Disease-associated fibronectin matrix fragments trigger anoikis of human primary ligament cells: p53 and c-myc are suppressed. Apoptosis 10:503–512

    Article  PubMed  CAS  Google Scholar 

  19. Streuli CH, Gilmore AP (1999) Adhesion-mediated signaling in the regulation of mammary epithelial cell survival. J Mammary Gland Biol Neoplasia 4:183–191

    Article  PubMed  CAS  Google Scholar 

  20. Shanmugathasan M, Jothy S (2000) Apoptosis, anoikis and their relevance to the pathobiology of colon cancer. Pathol Int 50:273–279

    Article  PubMed  CAS  Google Scholar 

  21. Diaz-Montero CM, McIntyre BW (2003) PI3-K/Akt-mediated anoikis resistance of human osteosarcoma cells requires Src activation. Eur J Cancer 39:2395–2402

    Article  PubMed  CAS  Google Scholar 

  22. Wei L, Yang Y, Yu Q (2001) Tyrosine kinase-dependent, phosphatidylinositol 3′-kinase, and mitogen-activated protein kinase-independent signaling pathways prevent lung adenocarcinoma cells from anoikis. Cancer Res 61:2439–2444

    PubMed  CAS  Google Scholar 

  23. Thomas GJ, Jones J, Speight PM (1997) Integrins and oral cancer. Oral Oncol 33:381–388

    PubMed  CAS  Google Scholar 

  24. Silverman S Jr (2001) Demographics and occurrence of oral and pharyngeal cancers. The outcomes, the trends, the challenge. J Am Dent Assoc 132:7S–11S

    PubMed  Google Scholar 

  25. Xue H, Atakilit A, Zhu W, Li X, Ramos DM, Pytela R (2001) Role of the alpha(v)beta6 integrin in human oral squamous cell carcinoma growth in vivo and in vitro. Biochem Biophys Res Commun 288:610–618

    Article  PubMed  CAS  Google Scholar 

  26. Koivisto L, Grenman R, Heino J, Larjava H (2000) Integrins alpha5beta1, alphavbeta1, and alphavbeta6 collaborate in squamous carcinoma cell spreading and migration on fibronectin. Exp Cell Res 255:10–17

    Article  PubMed  CAS  Google Scholar 

  27. Thomas GJ, Hart IR, Speight PM, Marshall JF (2002) Binding of TGF-beta1 latency-associated peptide (LAP) to alpha(v) beta6 integrin modulates behaviour of squamous carcinoma cells. Br J Cancer 87:859–867

    Article  PubMed  CAS  Google Scholar 

  28. Ylipalosaari M, Thomas GJ, Nystrom M et al (2005) Alpha v beta 6 integrin down-regulates the MMP-13 expression in oral squamous cell carcinoma cells. Exp Cell Res 309:273–283

    Article  PubMed  CAS  Google Scholar 

  29. Vacca A, Ria R, Presta M et al (2001) Alpha(v)beta(3) integrin engagement modulates cell adhesion, proliferation, and protease secretion in human lymphoid tumor cells. Exp Hematol 29:993–1003

    Article  PubMed  CAS  Google Scholar 

  30. Mitjans F, Sander D, Adan J et al (1995) An anti-alpha v-integrin antibody that blocks integrin function inhibits the development of a human melanoma in nude mice. J Cell Sci 108:2825–2838

    PubMed  CAS  Google Scholar 

  31. Zhang Z, Vuori K, Reed JC, Ruoslahti E (1995) The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 92:6161–6165

    Article  PubMed  CAS  Google Scholar 

  32. O’Brien V, Frisch SM, Juliano RL (1996) Expression of the integrin alpha 5 subunit in HT29 colon carcinoma cells suppresses apoptosis triggered by serum deprivation. Exp Cell Res 224:208–213

    Article  PubMed  CAS  Google Scholar 

  33. Zhang Y, Lu H, Dazin P, Kapila Y (2004) Squamous cell carcinoma cell aggregates escape suspension-induced, p53-mediated anoikis: fibronectin and integrin alphav mediate survival signals through focal adhesion kinase. J Biol Chem 279:48342–48349

    Article  PubMed  CAS  Google Scholar 

  34. Kapila YL, Niu J, Johnson PW (1997) The high affinity heparin-binding domain and the V region of fibronectin mediate invasion of human oral squamous cell carcinoma cells in vitro. J Biol Chem 272:18932–18938

    Article  PubMed  CAS  Google Scholar 

  35. Akiyama SK, Olden K, Yamada KM (1995) Fibronectin and integrins in invasion and metastasis. Cancer Met Rev 14:173–189

    Article  CAS  Google Scholar 

  36. Yokota T, Oritani K, Mitsui H et al (1998) Growth-supporting activities of fibronectin on hematopoietic stem/progenitor cells in vitro and in vivo: structural requirement for fibronectin activities of CS1 and cell-binding domains. Blood 91:3263–3272

    PubMed  CAS  Google Scholar 

  37. Kapila YL, Wang S, Dazin P, Tafolla E, Mass MJ (2002) The heparin-binding domain and V region of fibronectin regulate apoptosis by suppression of p53 and c-myc in human primary cells. J Biol Chem 277:8482–8491

    Article  PubMed  CAS  Google Scholar 

  38. Clark RA, An JQ, Greiling D, Khan A, Schwarzbauer JE (2003) Fibroblast migration on fibronectin requires three distinct functional domains. J Invest Dermatol 121:695–705

    Article  PubMed  CAS  Google Scholar 

  39. Peterson JA, Sheibani N, David G, Garcia-Pardo A, Peters DM (2005) Heparin II domain of fibronectin uses alpha4beta1 integrin to control focal adhesion and stress fiber formation, independent of syndecan-4. J Biol Chem 280:6915–6922

    Article  PubMed  CAS  Google Scholar 

  40. Owens LV, Xu L, Craven RJ et al (1995) Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 55:2752–2755

    PubMed  CAS  Google Scholar 

  41. Carey TE (1994) Head and neck tumor lines. In: Hay R, Gazdar A, Park J-G (eds) Atlas of human tumor cell lines. Academic Press Inc., Harcourt Brace Jovanovich Publishers, Orlando, pp 79–120

    Google Scholar 

  42. Kapila YL, Wang S, Johnson PW (1999) Mutations in the heparin binding domain of fibronectin in cooperation with the V region induce decreases in pp125(FAK) levels plus proteoglycan-mediated apoptosis via caspases. J Biol Chem 274:30906–30913

    Article  PubMed  CAS  Google Scholar 

  43. Barkalow FJ, Schwarzbauer JE (1991) Localization of the major heparin-binding site in fibronectin. J Biol Chem 266:7812–7818

    PubMed  CAS  Google Scholar 

  44. Hamel W, Dazin P, Israel M (1996) Adaptation of a simple flow cytometric assay to identify different stages during apoptosis. Cytometry 25:173–181

    Article  PubMed  CAS  Google Scholar 

  45. Lallier T, Bronner-Fraser M (1993) Inhibition of neural crest cell attachment by integrin antisense oligonucleotides. Science 259:692–695

    Article  PubMed  CAS  Google Scholar 

  46. Townsend PA, Villanova I, Uhlmann E (2000) An antisense oligonucleotide targeting the alphaV integrin gene inhibits adhesion and induces apoptosis in breast cancer cells. Eur J Cancer 36:397–409

    Article  PubMed  CAS  Google Scholar 

  47. Akhtar S, Kole R, Juliano RL (1991) Stability of antisense DNA oligodeoxynucleotide analogs in cellular extracts and sera. Life Sci 49:1793–1801

    Article  PubMed  CAS  Google Scholar 

  48. Zhang Y, Lu H, Dazing P, Kapila Y (2004) Functional differences between integrin alpha4 and integrins alpha5/alphav in modulating the motility of human oral squamous carcinoma cells in response to the V region and heparin-binding domain of fibronectin. Exp Cell Res 295:48–58

    Article  PubMed  CAS  Google Scholar 

  49. Lee BH, Ruoslahti E (2005) Alpha5beta1 integrin stimulates Bcl-2 expression and cell survival through Akt, focal adhesion kinase, and Ca2+/calmodulin-dependent protein kinase IV. J Cell Biochem 95:1214–1223

    Article  PubMed  CAS  Google Scholar 

  50. Giancotti FG, Ruoslahti E (1999) Integrin signaling. Science 285:1028–1032

    Article  PubMed  CAS  Google Scholar 

  51. Micheloni A, De Luca N, Tadini G, Zambruno G, D’Alessio M (2004) Intracellular degradation of beta4 integrin in lethal junctional epidermolysis bullosa with pyloric atresia. Br J Dermatol 151:796–802

    Article  PubMed  CAS  Google Scholar 

  52. Parsons JT (2003) Focal adhesion kinase: the first ten years. J Cell Sci 116:1409–1416

    Article  PubMed  CAS  Google Scholar 

  53. Schaller MD (2004) FAK and paxillin: regulators of N-cadherin adhesion and inhibitors of cell migration? J Cell Biol 166:157–159

    Article  PubMed  CAS  Google Scholar 

  54. Kapila YL, Lancero H, Johnson PW (1998) Specific fibronectin fragments as markers of periodontal disease status. J Periodontal 69:1008–1019

    CAS  Google Scholar 

  55. Golubovskaya VM, Gross S, Kaur AS et al (2003) Simultaneous inhibition of focal adhesion kinase and SRC enhances detachment and apoptosis in colon cancer cell lines. Mol Cancer Res 1:755–764

    PubMed  CAS  Google Scholar 

  56. Kurenova E, Xu LH, Yang X et al (2004) Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein. Mol Cell Biol 24:4361–4371

    Article  PubMed  CAS  Google Scholar 

  57. Golubovskaya V, Beviglia L, Xu LH, Earp HS III, Craven R, Cance WG (2002) Dual inhibition of focal adhesion kinase and epidermal growth factor receptor pathways cooperatively induces death receptor-mediated apoptosis in human breast cancer cells. J Biol Chem 277:38978–38987

    Article  PubMed  CAS  Google Scholar 

  58. Hungerford JE, Compton MT, Matter ML, Hoffstrom BG, Otey CA (1996) Inhibition of pp125FAK in cultured fibroblasts results in apoptosis. J Cell Biol 135:1383–1390

    Article  PubMed  CAS  Google Scholar 

  59. Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY (1996) Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 134:793–799

    Article  PubMed  CAS  Google Scholar 

  60. Beviglia L, Golubovskaya V, Xu L, Yang X, Craven RJ, Cance WG (2003) Focal adhesion kinase N-terminus in breast carcinoma cells induces rounding, detachment and apoptosis. Biochem J 373:201–210

    Article  PubMed  CAS  Google Scholar 

  61. Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE (2004) Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: a novel therapeutic strategy in pancreatic cancer. Surgery 135:555–562

    Article  PubMed  CAS  Google Scholar 

  62. Miyazaki T, Kato H, Nakajima MH et al (2003) FAK overexpression is correlated with tumour invasiveness and lymph node metastasis in oesophageal squamous cell carcinoma. Br J Cancer 89:140–145

    Article  PubMed  CAS  Google Scholar 

  63. Mitra SK, Lim ST, Chi A, Schlaepfer DD (2006) Intrinsic focal adhesion kinase activity controls orthotopic breast carcinoma metastasis via the regulation of urokinase plasminogen activator expression in a syngeneic tumor model. Oncogene 25:4429–4440

    Article  PubMed  CAS  Google Scholar 

  64. Scibelli A, Tafuri S, Ferrante MC et al (2003) Ochratoxin A affects COS cell adhesion and signaling. Toxicol Appl Pharmacol 192:222–230

    Article  PubMed  CAS  Google Scholar 

  65. Reginato MJ, Mills KR, Paulus JK et al (2003) Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis. Nat Cell Biol 5:733–740

    Article  PubMed  CAS  Google Scholar 

  66. Zhang J, Zhi H, Zhou C et al (2005) Up-regulation of fibronectin in oesophageal squamous cell carcinoma is associated with activation of the Erk pathway. J Pathol 207:402–409

    Article  PubMed  CAS  Google Scholar 

  67. Mishima K, Inoue K, Hayashi Y (2002) Overexpression of extracellular-signal regulated kinases on oral squamous cell carcinoma. Oral Oncol 38:468–474

    Article  PubMed  CAS  Google Scholar 

  68. Schlaepfer DD, Hunter E (1997) Focal adhesion kinase overexpression enhances ras-dependent integrin signaling to ERK2/mitogen-activated protein kinase through interactions with and activation of c-Src. J Biol Chem 272:13189–13195

    Article  PubMed  CAS  Google Scholar 

  69. Renshaw MW, Price LS, Schwartz MA (1999) Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase. J Cell Biol 147:611–618

    Article  PubMed  CAS  Google Scholar 

  70. Zhang L, Bewick M, Lafrenie RM (2002) Role of Raf-1 and FAK in cell density-dependent regulation of integrin-dependent activation of MAP kinase. Carcinogenesis 23:1251–1258

    Article  PubMed  CAS  Google Scholar 

  71. Mitra SK, Mikolon D, Molina JE et al (2006) Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in tumors. Oncogene 25:5969–5984

    Article  PubMed  CAS  Google Scholar 

  72. Zhou X, Murphy FR, Gehdu N, Zhang J, Iredale JP, Benyon RC (2004) Engagement of alphavbeta3 integrin regulates proliferation and apoptosis of hepatic stellate cells. J Biol Chem 279:23996–24006

    Article  PubMed  CAS  Google Scholar 

  73. Xue D-L, Meyers R, Homandberg GA (1992) Fibronectin fragments in osteoarthritic synovial fluid. J Rheumatol 19:1448–1452

    Google Scholar 

  74. Liu Y, Huang B, Yuan Y et al (2007) Inhibition of hepatocarcinoma and tumor metastasis to liver by gene therapy with recombinant CBD-HepII polypeptide of fibronectin. Int J Cancer 121:184–192

    Article  PubMed  CAS  Google Scholar 

  75. Birchler MT, Milisavlijevic D, Pfaltz M et al (2003) Expression of the extra domain B of fibronectin, a marker of angiogenesis, in head and neck tumors. Laryngoscope 113:1231–1237

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Tina Chunyuan Chen for assistance with protein purification, Drs. Steve Hanks, David Cheresh, Kunliang Guan, Nisha J. D’Silva, Thomas E. Carey, Paul W. Johnson and Ken Yamada for gifts of reagents, and Stephen Ordway for editorial assistance. This work was supported by Grant R01 DE014429 from the National Institutes of Health (to Y.K.).

Author information

Authors and Affiliations

  1. Department of Periodontics and Oral Medicine, University of Michigan, School of Dentistry, 1011 N. University Ave, Room 5223, Ann Arbor, MI, 48109-1078, USA

    Pachiyappan Kamarajan & Yvonne L. Kapila

Authors
  1. Pachiyappan Kamarajan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yvonne L. Kapila
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yvonne L. Kapila.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kamarajan, P., Kapila, Y.L. An altered fibronectin matrix induces anoikis of human squamous cell carcinoma cells by suppressing integrin alpha v levels and phosphorylation of FAK and ERK. Apoptosis 12, 2221–2231 (2007). https://doi.org/10.1007/s10495-007-0138-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-007-0138-9

Keywords

Search

Navigation