Protein Profiles Associated with Anoikis Resistance of Metastatic MDA-MB-231 Breast Cancer Cells

  • Akekawatchai, Chareeporn (Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University) ;
  • Roytrakul, Sittiruk (National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency) ;
  • Kittisenachai, Suthathip (National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency) ;
  • Isarankura-Na-Ayudhya, Patcharee (Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University) ;
  • Jitrapakdee, Sarawut (Department of Biochemistry, Faculty of Sciences, Mahidol University)
  • Published : 2016.03.07


Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.


Anoikis resistance;MDA-MB-231;MCF-7;proteomics;GeLC-MS/MS;STITCH


Supported by : Thailand Research Fund (TRF), Commission on Higher Education (CHE)


  1. Abalsamo L, Spadaro F, Bozzuto G, et al (2012). Inhibition of phosphatidylcholine-specific phospholipase C results in loss of mesenchymal traits in metastatic breast cancer cells. Breast Cancer Res, 14, 50.
  2. Akekawatchai C, Holland JD, Kochetkova M, et al (2005). Transactivation of CXCR4 by the insulin-like growth factor-1 receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells. J Biol Chem, 280, 39701-8.
  3. Bharadwaj S, Thanawala R, Bon G, et al (2005). Resensitization of breast cancer cells to anoikis by tropomyosin-1: role of Rho kinase-dependent cytoskeleton and adhesion. Oncogene, 24, 8291-303.
  4. Buchheit CL, Weigel KJ, Schafer ZT (2014). Cancer cell survival during detachment from the ECM: multiple barriers to tumour progression. Nat Rev Cancer, 14, 632-41.
  5. Chesor M, Roytrakul S, Graidist P, et al (2014). Proteomics analysis of siRNA-mediated silencing of Wilms' tumor 1 in the MDA-MB-468 breast cancer cell line. Oncol Rep, 31, 1754-60.
  6. Cui W, Zhang S, Shan C, et al (2013). microRNA-133a regulates the cell cycle and proliferation of breast cancer cells by targeting epidermal growth factor receptor through the EGFR/Akt signaling pathway. FEBS J, 280, 3962-74.
  7. Culty M, Shizari M, Thompson EW, et al (1994). Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: correlation with invasive potential. J Cell Physiol, 160, 275-86.
  8. Eckert LB, Repasky GA, Ulku AS, et al (2004). Involvement of Ras activation in human breast cancer cell signaling, invasion, and anoikis. Cancer Res, 64, 4585-92.
  9. Fukazawa H, Uehara Y (2000). U0126 reverses Ki-ras-mediated transformation by blocking both mitogen-activated protein kinase and p70 S6 kinase pathways. Cancer Res, 60, 2104-7.
  10. Glinskii AB, Smith BA, Jiang P, et al (2003). Viable circulating metastatic cells produced in orthotopic but not ectopic prostate cancer models. Cancer Res, 63, 4239-43.
  11. Henkels KM, Boivin GP, Dudley ES, et al (2013). Phospholipase D (PLD) drives cell invasion, tumor growth and metastasis in a human breast cancer xenograph model. Oncogene, 32, 5551-62.
  12. Holliday DL, Speirs V (2011). Choosing the right cell line for breast cancer research. Breast Cancer Res, 13, 215.
  13. Hooshmand S, Ghaderi A, Yusoff K, et al (2014). Differentially expressed proteins in ER+ MCF7 and ER- MDA- MB-231 human breast cancer cells by RhoGDI-alpha silencing and overexpression. Asian Pac J Cancer Prev, 15, 3311-7.
  14. Kim JB, Yu JH, Ko E, et al (2010). The alkaloid Berberine inhibits the growth of Anoikis-resistant MCF-7 and MDAMB-231 breast cancer cell lines by inducing cell cycle arrest. Phytomedicine, 17, 436-40.
  15. Kim YN, Koo KH, Sung JY, et al (2012). Anoikis resistance: an essential prerequisite for tumor metastasis. Int J Cell Biol, 2012, 306879.
  16. Kuhn M, Szklarczyk D, Pletscher-Frankild S, et al (2014). STITCH 4: integration of protein-chemical interactions with user data. Nucleic Acids Res, 42, 401-7.
  17. Kumar B, Kumar A, Ghosh S, et al (2012). Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells. Biochem Biophys Res Commun, 417, 903-9.
  18. Lai TC, Chou HC, Chen YW, et al (2010). Secretomic and proteomic analysis of potential breast cancer markers by two-dimensional differential gel electrophoresis. J Proteom Res, 9, 1302-22.
  19. Laothumthut T, Jantarat J, Paemanee A, et al (2015). Shotgun proteomics analysis of proliferating STRO-1-positive human dental pulp cell after exposure to nacreous water-soluble matrix. Clin Oral Invest, 19, 261-70.
  20. Mahadev K, Raval G, Bharadwaj S, et al (2002). Suppression of the transformed phenotype of breast cancer by tropomyosin-1. Exp Cell Res, 279, 40-51.
  21. Malin D, Strekalova E, Petrovic V, et al (2015). ERK-regulated alphaB-crystallin induction by matrix detachment inhibits anoikis and promotes lung metastasis in vivo. Oncogene, 34, 5626-34.
  22. Nagaraja GM, Othman M, Fox BP, et al (2006). Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics. Oncogene, 25, 2328-38.
  23. Niu M, Klingler-Hoffmann M, Brazzatti JA, et al (2013). Comparative proteomic analysis implicates eEF2 as a novel target of PI3Kgamma in the MDA-MB-231 metastatic breast cancer cell line. Proteome Sci, 11, 4.
  24. Paoli P, Giannoni E, Chiarugi P (2013). Anoikis molecular pathways and its role in cancer progression. Biochim Biophys Acta, 1833, 3481-98.
  25. Park JB, Lee CS, Jang JH, et al (2012). Phospholipase signalling networks in cancer. Nat Rev Cancer, 12, 782-92.
  26. Pledgie-Tracy A, Sobolewski MD, Davidson NE (2007). Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines. Mol Cancer Ther, 6, 1013-21.
  27. Rizzo MA, Shome K, Vasudevan C, et al (1999). Phospholipase D and its product, phosphatidic acid, mediate agonistdependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway. J Biol Chem, 274, 1131-9.
  28. Roskoski R, Jr. (2014). The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res, 79, 34-74.
  29. Thompson EW, Paik S, Brunner N, et al (1992). Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines. J Cell Physiol, 150, 534-44.
  30. Zheng Y, Rodrik V, Toschi A, et al (2006). Phospholipase D couples survival and migration signals in stress response of human cancer cells. J Biol Chem, 281, 15862-8.
  31. Zhong M, Shen Y, Zheng Y, et al (2003). Phospholipase D prevents apoptosis in v-Src-transformed rat fibroblasts and MDA-MB-231 breast cancer cells. Biochem Biophys Res Commun, 302, 615-9.

Cited by

  1. Bioinformatics-based identification of miR-542-5p as a predictive biomarker in breast cancer therapy vol.155, pp.1, 2018,
  2. KIAA0100 Modulates Cancer Cell Aggression Behavior of MDA-MB-231 through Microtubule and Heat Shock Proteins vol.10, pp.6, 2018,