ABC  Vol.2 No.1 , February 2012
Studies on Focal Adhesion Kinase in human breast cancer cell MDA-MB-231
ABSTRACT
AIM: 1) To study the participation of Focal Adhesion Kinase (FAK) in regulation of Breast Cancer cell migration in relation with MMP-9 and other signaling proteins. 2) To study the effect of some natural products on FAK. METHODS: Cell culture, Western Blot, Immunoprecipitation, Immunocytochemistry, Zymography, SiRNA transfection, RT-PCR, Real-Time PCR. RESULTS: For our study on FAK, we selected invasive Breast Cancer cell line MDA-MB-231 and treated the cells with Fibronectin (FN). Treatment of FN was found to increase FAK expression, phosphorylation (Tyr 397). FAK was found to be involved in re- gulation of breast cancer cell migration and MMP-9 expression, activity. Fi-bronectin increases association of FAK with integrin α5β1, Paxillin, Actin, ERK, PI3K and localization at Focal Adhesion sites. FAK was found to be involved in modulation of ERK and PI3K phosphorylation. Moreover, FAK signal was found to be transduced through ERK and PI3K, which modulate MMP-9 and thereby cell migration. CONCLUSION: FAK expression, phosphorylation and processing are induced in response to Cell-ECM interactions. Integrin α5β1 is involved in FN induced FAK phosphorylation. FAK is a potent regulator of MMP-9 expression and activity. FAK is involved in regulation of ERK and PI3K phosphorylation. ERK and PI3K are involved in FAK regulated MMP-9 expression & activity. FAK regulates MMP-9 expression and activity and thereby migration of human breast cancer cell. By the regulation of FAK, cell attachment and migration may be regulated by Curcumin, ATRA or EGCG treatment. It may be concluded that invasive potential of breast cancer cells may be modulated by regulation of FAK.

Cite this paper
Ganguly, K. , Sen, T. , Pal, S. , Biswas, J. and Chatterjee, A. (2012) Studies on Focal Adhesion Kinase in human breast cancer cell MDA-MB-231. Advances in Biological Chemistry, 2, 29-42. doi: 10.4236/abc.2012.21004.
References
[1]   Schaller, M.D. (2001) Biochemical signals and biological res-ponses elicited by the focal adhesion kinase. Biochi- mica Bio-physica Acta, 1540, 1-21. doi:10.1016/S0167-4889(01)00123-9

[2]   Mitra, S.K., Hanson, D.A. and Schlaepfer, D.D. (2005) Focal adhesion kinase: In command and control of cell motility. Nature Reviews Molecular Cell Biology, 6, 56- 68. doi:10.1038/nrm1549

[3]   Ilic, D., Damsky, C.H. and Yamamoto, T. (1997) Focal adhesion kinase: At the crossroads of signal transduction. Journal of Cell Science, 110, 401-407.

[4]   Luo, M. and Guan, J.L. (2010) Focal adhesion kinase: A prominent determinant in breast cancer initiation; progression and metastasis. Cancer Letters, 289, 127-139. doi:10.1016/j.canlet.2009.07.005

[5]   Zhao, J. and Guan, J.L. (2009) Signal transduction by focal adhesion kinase in cancer. Cancer Metastasis Reviews, 28, 35-49. doi:10.1007/s10555-008-9165-4

[6]   Clark, E.A. and Brugge, J.S. (1995) Integrins and signal transduction pathways: the road taken. Sciences, 268, 233- 239. doi:10.1126/science.7716514

[7]   Lee, Y.C., Cheng, T.H., Lee, J.S., Chen, J.H., Liao, Y.C., Fong, Y., Wu, C.H. and Shih, Y.W. (2011) Nobiletin; a citrus flavonoid; suppresses invasion and migration involving FAK/PI3K/Akt and small GTPase signals in human gastric adenocarcinoma AGS cells. Molecular and Cellular Biochemistry, 347, 103-115. doi:10.1007/s11010-010-0618-z

[8]   Chen, Y.J., Wei, Y.Y., Chen, H.T., Fong, Y.C., Hsu, C.J., Tsai, C.H., Hsu, H.C., Liu, S.H. and Tang, C.H. (2009) Osteopontin increases migration and MMP-9 up-regulation via alphavbeta3 integrin; FAK; ERK; and NF-kappaB-dependent pathway in human chondrosarcoma cells. Journal of Cell Physiology, 221, 98-108. doi:10.1002/jcp.21835

[9]   Meng, X.N., Jin, Y., Yu, Y., Bai, J., Liu, G.Y., Zhu, J., Zhao, Y.Z., Wang, Z., Chen, F., Lee, K.Y. and Fu, S.B. (2009) Characterisation of Fibronectin-mediated FAK signalling pathways in lung cancer cell migration and invasion. British Journal of Cancer, 101, 327-334. doi:10.1038/sj.bjc.6605154

[10]   Ilic, D., Kovacic, B., Johkura, K., Schlaepfer, D.D., Toma- sevic, N., Han, Q., Kim, J.B., Howerton, K., Baumbusch, C., Ogiwara, N., Streblow, D.N., Nelson, J.A., Dazin, P., Shino, Y., Sasaki, K. and Damsky, C.H. (2004) FAK promotes organization of fibronectin matrix and fibrillar adhesions. Journal of Cell Science, 117, 177-187. doi:10.1242/jcs.00845

[11]   Springman, E.B., Angleton, E.L., Birkedal-Hansen, H. and Van Wart, H.E. (1990) Multiple modes of activation of latent human fibroblast collagenase: Evidence for the role of a Cys73 active-site zinc complex in latency and a “cysteine switch” mechanism for activation. Proceedings of the National Academy of Sciences USA, 87, 364-368. doi:10.1073/pnas.87.1.364

[12]   Han, Y.P., Yan, C., Zhou, L., Qin, L. and Tsukamoto, H. (2007) A matrix metalloproteinase-9 activation cascade by hepatic stellate cells in trans-differentiation in the three-dimensional extracellular matrix. Journal of Bio-logical Chemistry, 282, 12928-12939. doi:10.1074/jbc.M700554200

[13]   Han, Y.P., Nien, Y.D. and Garner, W.L. (2002) Tumor necrosis factor-alpha-induced pro-teolytic activation of pro- matrix metalloproteinase-9 by human skin is controlled by down-regulating tissue inhibitor of metal-loproteinase- 1 and mediated by tissue-associated chymotryp-sin-like proteinase. Journal of Biological Chemistry, 277, 27319- 27327. doi:10.1074/jbc.M202842200

[14]   Ramos-DeSimone, N., Hahn-Dantona, E., Sipley, J., Nagase, H., French, D.L. and Quigley, J.P. (1999) Activation of matrix metalloproteinase-9 (MMP-9) via a converging plasmin/stromelysin-1 cascade en-hances tumor cell invasion. Journal of Biological Chemistry, 274, 13066-13076. doi:10.1074/jbc.274.19.13066

[15]   Toth, M., Chvyrkova, I., Bernardo, M.M., Hernandez- Barrantes, S. and Fridman, R. (2003) Pro-MMP-9 activation by the MT1-MMP/MMP-2 axis and MMP-3: Role of TIMP-2 and plasma membranes. Bio-chemical and Biophysical Research Communications, 308, 386-395. doi:10.1016/S0006-291X(03)01405-0

[16]   Segarra, M., Vilar-dell, C., Matsumoto, K., Esparza, J., Lozano, E., Serra-Pages, C., Urbano-Marquez, A., Yama- da, K.M. and Cid, M.C. (2005) Dual function of focal adhesion kinase in regulating inte-grin-induced MMP-2 and MMP-9 release by human T lymphoid cells. FASEB Journal, 19, 1875-1877.

[17]   Anderson, D.G., Li, X. and Balian, G.A. (2005) Fibro- nectin fragment alters the metabolism by rabbit intervertebral disc cells in vitro. Spine, 30, 1242-1246. doi:10.1097/01.brs.0000164097.47091.4c

[18]   Chana, R.S., Martin, J., Rahman, E.U. and Wheeler, D.C. (2003) Monocyte adhesion to mesangial matrix modulates cytokine and metallo-proteinase production. Kidney International, 63, 889-898. doi:10.1046/j.1523-1755.2003.00828.x

[19]   Thant, A.A., Nawa, A., Kikkawa, F., Ichigotani, Y., Zhang, Y., Sein, T.T., Amin, A.R. and Hamaguchi, M. (2000) Fibronectin activates matrix metalloproteinase-9 secretion via the MEK1-MAPK and the PI3K-Akt pathways in ovarian cancer cells. Clinical & Expe-rimental Metastasis, 18, 423-428. doi:10.1023/A:1010921730952

[20]   Han, S., Ritzenthaler, J.D., Sitaraman, S.V. and Roman, J. (2006) Fibronectin increases matrix metalloproteinase 9 expression through activation of c-Fos via extracellular-regulated kinase and phosphatidylinositol 3-kinase pathways in human lung carcinoma cells. Journal of Biological Chemistry, 281, 29614-29624. doi:10.1074/jbc.M604013200

[21]   Shibata, K., Kikkawa, F., Nawa, A., Thant, A.A., Naruse, K., Mizutani, S. and Hamaguchi, M. (1998) Both focal adhesion kinase and c-Ras are required for the enhanced matrix metalloproteinase 9 secretion by fibronectin in ova- rian cancer cells. Cancer Research, 58, 900-903.

[22]   Sein, T.T., Thant, A.A., Hiraiwa, Y., Amin, A.R., Sohara, Y., Liu, Y., Matsuda, S., Yamamoto, T. and Hamaguchi, M. (2000) A role for FAK in the concanavalin a-depen- dent secretion of matrix metalloproteinase-2 and -9. Oncogene, 19, 5539-5542.

[23]   Xia, H., Nho, R.S., Judy, K., Jill, K. and Henke, C.A. (2004) FAK is upstream of PI-3-kinase/Akt in regulating fibroblast survival in response to contraction of type I collagen matrices via a β1 integrin-viability signaling pathway. Journal of Biological Chemistry, 279, 33024- 33034. doi:10.1074/jbc.M313265200

[24]   Shi, J., Zeng, X., Zhou, M. and Chen, Q. (2009) Activation of ERK-FAK signaling pathway and enhancement of cell migration involved in the early interaction between oral keratinocytes and candida albicans. Mycopathologia, 167, 1-7. doi:10.1007/s11046-008-9142-z

[25]   Das, S., Banerji, A., Frei, E. and Chatterjee, A. (2008) Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium. Life Sciences, 82, 467- 476. doi:10.1016/j.lfs.2007.12.013

[26]   Maity, G., Sen, T. and Chatterjee, A. (2011) Laminin induces matrix metalloproteinase-9 expression and activation in human cervical cancer cell line (SiHa). Journal of Cancer Research Clinical Oncology, 137, 347-357. doi:10.1007/s00432-010-0892-x

[27]   Sen, T., Moulik, S., Dutta, A., Roy Choudhury, P., Ba- nerji, A., Das, S., Roy, M. and Chatterjee, A. (2009) Multifunctional effect of epigallocate-chin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7. Life Sciences, 84, 194-204. doi:10.1016/j.lfs.2008.11.018

[28]   Maity, G., Fahreen, S., Ba-nerji, A., Roychoudhury, P., Sen, T., Dutta, A. and Chatterjee, A. (2010) Fibronectin-inte- grin mediated signaling in human cervical cancer cells (SiHa). Molecular and Cellular Biochemi-stry, 336, 65- 74. doi:10.1007/s11010-009-0256-5

[29]   Sen, T., Dutta, A. and Chatterjee, A. (2010) EGCG down- regulates Ge-latinase-B (MMP-9) by involvement of FAK/ ERK, NF-kB and AP-I in the human breast cancer cell line MDA-MB-231. Anti Cancer Drugs, 21, 632-644. doi:10.1097/CAD.0b013e32833a4385

[30]   Dutta, A., Sen, T. and Chatterjee A. (2010) All-trans retinoic acid (ATRA) down-regulates MMP-9 by modulating its regulatory molecules. Cell Adhesion & Migration, 4, 409-418.

[31]   Lin, H.J., Su, C.C., Lu, H.F., Yang, J.S., Hsu, S.C., Ip, S.W., Wu, J.J., Li, Y.C., Ho, C.C., Wu, C.C. and Chung, J.G. (2010) Curcumin blocks mi-gration and invasion of mouse-rat hybrid retina ganglion cells (N18) through the inhibition of MMP-2, -9, FAK, Rho A and Rock-1 gene expression. Oncology Reports, 23, 665-670.

[32]   Miyazaki, T., Kato, H., Nakajima, M., Sohda, M., Fukai, Y., Masuda, N., Manda, R., Fukuchi, M., Tsukada, K. and Kuwano, H. (2003) FAK overexpression is correlated with tumour invasiveness and lymph node metasta sis in oesophageal squamous cell carcinoma. British Journal of Cancer, 89, 140-145. doi:10.1038/sj.bjc.6601050

[33]   Lark, A.L., Livasy, C.A., Dressler, L., Moore, D.T., Millikan, R.C., Geradts, J., Iacocca, M., Cowan, D., Little, D., Craven, R.J. and Cance, W. (2005) High focal adhesion kinase expression in invasive breast carcinomas is associated with an aggressive phenotype. Modern Pathology, 18, 1289-1294. doi:10.1038/modpathol.3800424

[34]   Cance, W.G., Harris, J.E., Iacocca, M.V., Roche, E., Yang, X., Chang, J., Simkins, S. and Xu, L. (2000) Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive pheno-types. Clinical Cancer Research, 6, 2417-2423.

[35]   Beierle, E.A., Massoll, N.A., Hartwich, J., Kurenova, E.V., Golubovs-kaya, V.M., Cance, W.G., McGrady, P. and London, W.B. (2008) Focal adhesion kinase expression in human neuroblastoma: Immunohistochemical and real- time PCR analyses. Clinical Cancer Research, 14, 3299- 305. doi:10.1158/1078-0432.CCR-07-1511

[36]   Ioachim, E., Char-chanti, A., Briasoulis, E., Karavasilis, V., Tsanou, H., Arvanitis, D.L., Agnantis, N.J. and Pavlidis, N. (2002) Immunohisto-chemical expression of extracellular matrix components tenascin; fibronectin; collagen type IV and laminin in breast cancer: Their prognostic value and role in tumour invasion and progression. European Journal Cancer, 38, 2362-2370. doi:10.1016/S0959-8049(02)00210-1

[37]   Reiske, H.R., Zhao, J., Han, D.C., Cooper, L.A. and Guan, J.L. (2000) Analysis of FAK-associated signaling pathways in the regulation of cell cycle progression. FEBS Letters, 486, 275-280. doi:10.1016/S0014-5793(00)02295-X

[38]   Sieg, D.J., Hauck, C.R. and Schlaepfer, D.D. (1999) Required role of focal adhe-sion kinase (FAK) for integrin- stimulated cell migration. Journal of Cell Sciences, 112, 2677-2691.

[39]   Bang, O.S., Kim, E.J., Chung, J.G., Lee, S.R., Park, T.K. and Kang, S.S. (2000) Association of focal adhesion kinase with fibronectin and paxillin is required for precartilage condensation of chick mesenchymal cells. Biochemical and Biophysical Research Communications, 278, 522-529. doi:10.1006/bbrc.2000.3831

[40]   Maity, G., Roychoudhury, P., Sen, T., Ganguly, K.K., Sil, H. and Chatterjee, A. (2011) Culture of human breast cancer cell line (MDA-MB-231) on fibro-nectin-coated surface induces pro-matrix metalloproteinase-9 expression and activity. Tumour Biology, 32, 129-138. doi:10.1007/s13277-010-0106-9

[41]   Chen, H.C., Appeddu, P.A., Isoda, H. and Guan, J.L. (1996) Phosphorylation of tyro-sine 397 in focal adhesion kinase is required for binding phos-phatidylinositol 3-ki- nase. Journal of Biological Chemistry, 271, 26329-26334. doi:10.1074/jbc.271.42.26329

[42]   Schlaepfer, D.D. and Hunter, T. (1996) Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyro- sine kinases. Molecular and Cellular Biology, 16, 5623- 5633.

[43]   Crowe, D.L. and Ohannessian, A. (2004) Recruitment of focal adhesion kinase and paxillin to beta1 integrin promotes cancer cell migration via mitogen activated protein kinase activation. BMC Cancer, 4, 18. doi:10.1186/1471-2407-4-18

[44]   Serrels, B., Serrels, A., Brunton, V.G., Holt, M., McLean, G.W., Gray, C.H., Jones, G.E. and Frame, M.C. (2007) Focal adhesion kinase controls actin assembly via a FERM- mediated interaction with the Arp2/3 complex. Nature Cell Biology, 9, 1046-1056. doi:10.1038/ncb1626

[45]   Reiske, H.R., Kao, S.C., Cary, L.A., Guan, J.L., Lai, J.F. and Chen, H.C. (1999) Requirement of phosphatidyli- nositol 3-kinase in focal adhesion ki-nase-promoted cell migration. Journal of Biological Chemistry, 274, 12361- 12366. doi:10.1074/jbc.274.18.12361

[46]   Siu, L.L, Burris, H.A., Mileshkin, L.R., et al. (2008) A phase I clinical, pharmacokinetic (PK) and pharmacodynamic (PD) evaluation of PF-00562271 targeting focal adhesion kinase (FAK) in patients (pts) with advanced so- lid tumors. Journal of Clinical Oncology, 26, 3534.

[47]   Leu, T.H., Su, S.L, Chuang, Y.C. and Maa, M.C. (2003) Direct inhibitory effect of curcumin on Src and focal adhesion kinase activity. Biochemical Pharmacology, 66, 2323-2331. doi:10.1016/j.bcp.2003.08.017

 
 
Top