Cellular Oncology

, Volume 42, Issue 1, pp 67–80 | Cite as

S100P and Ezrin promote trans-endothelial migration of triple negative breast cancer cells

  • Kyoko Kikuchi
  • Keely May McNamara
  • Yasuhiro Miki
  • Erina Iwabuchi
  • Ayako Kanai
  • Minoru Miyashita
  • Takanori Ishida
  • Hironobu SasanoEmail author
Original Paper



Triple negative breast cancer (TNBC) patients generally have an adverse clinical outcome because their tumors often recur and metastasize to distant sites in the first 3 years after surgery. Therefore, it has become pivotal to identify potential factors associated with metastasis. Here, we focused on the effects of S100P and Ezrin on the trans-endothelial migration (TEM) of TNBC cells, as they have both been suggested to play a role in this process in other malignancies.


The expression of S100P and Ezrin was examined by immunohistochemistry in 58 primary TNBC samples. The mRNA and protein levels of S100P and Ezrin were assessed in breast cancer-derived cell lines using qRT-PCR and Western blotting, respectively. Proliferation and migration assays were performed using TNBC-derived MFM-223 and SUM-185-PE cells transfected with S100P and Ezrin siRNAs. Two different timeframes were employed for TEM assays using TNBC-derived cells and human umbilical vein endothelial-derived cells, respectively. Correlations between the status of EzrinThr-567 expression and various clinicopathological features were analyzed by immunohistochemistry.


We found that S100P and Ezrin double negative TNBC cases were significantly associated with a better disease-free survival. We also found that single and double siRNA-mediated knockdown of S100P and Ezrin in TNBC-derived cells significantly inhibited their TEM and destabilized the intercellular junctions of endothelial cells. In addition, we found that EzrinThr-567 immunoreactivity significantly correlated with vascular invasion in TNBC patients.


From our data we conclude that S100P, Ezrin and EzrinThr-567 are involved in the trans-endothelial migration of TNBC cells and that they may serve as potential targets in TNBC patients.


Triple negative breast carcinoma S100P Ezrin Trans-endothelial migration 



We would like to acknowledge the support and assistance of the members of the Department of Pathology, Tohoku University School of Medicine. We appreciate Mr. H. Hiranuma (SCRUM Inc., Tokyo, Japan) for his technical assistance regarding xCELLigence. We also acknowledge the technical support of the Biomedical Research Unit of Tohoku University Hospital.

Compliance with ethical standards

This study was approved by Ethics Committee at Tohoku University School of Medicine. Informed consent was obtained from all patients.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13402_2018_408_MOESM1_ESM.doc (58 kb)
ESM 1 (DOC 57 kb)
13402_2018_408_MOESM2_ESM.pdf (59 kb)
Supplementary Fig. 1 S100P expression induces proliferation of TNBC cells. The viability of TNBC cells transfected with single and double knockdown S100P and Ezrin siRNAs was determined using a CCK-8 assay. The data are presented as mean ± standard deviation. S + E: S100P and Ezrin, *p < 0.05. (PDF 58 kb)


  1. 1.
    K.R. Bauer, M. Brown, R.D. Cress, C.A. Parise, V. Caggiano, Descriptive analysis of estrogen receptor (ER)- negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype. Cancer 109, 1721–1728 (2007)CrossRefGoogle Scholar
  2. 2.
    M.D. Laurentiis, D. Cianniello, R. Caputo, B. Stanzione, G. Arpino, S. Cinieri, V. Lorusso, S.D. Placido, Treatment of triple negative breast cancer (TNBC): Current options and future perspectives. Cancer Treat Rev 36, S80–S86 (2010)CrossRefPubMedGoogle Scholar
  3. 3.
    R. Rouzier, C.M. Perou, W.F. Symmans, N. Ibrahim, M. Cristofanilli, K. Anderson, K.R. Hess, J. Stec, M. Ayers, P. Wagner, P. Morandi, C. Fan, I. Rabiul, J.S. Ross, G.N. Hortobagyi, L. Pusztai, Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res 11, 5678–5685 (2005)CrossRefGoogle Scholar
  4. 4.
    C. Liedtke, C. Mazouni, K.R. Hess, F. Andre, A. Tordai, J.A. Mejia, W.F. Symmans, A.M. Gonzalez-Angulo, B. Hennessy, M. Green, M. Cristofanilli, G.N. Hortobagyi, L. Pusztai, Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. J Clin Oncol 26, 1275–1281 (2008)CrossRefGoogle Scholar
  5. 5.
    R. Dent, M. Trudeau, K.I. Pritchard, W.M. Hanna, H.K. Kahn, C.A. Sawka, L.A. Lickley, E. Rawlinson, P. Sun, S.A. Narod, Triple-negative breast cancer; clinical features and patterns of recurrence. Clin Cancer Res 13, 4429–4435 (2007)CrossRefPubMedGoogle Scholar
  6. 6.
    M. Yousefi, R. Nosrati, A. Salmaninejad, S. Dehghani, A. Shahryari, A. Saberi, Organ-specific metastasis of breast cancer: Molecular and cellular mechanisms underlying lung metastasis. Cell Oncol 41, 123–140 (2018)CrossRefGoogle Scholar
  7. 7.
    R. Sharma, R. Sharma, T.P. Khaket, C. Dutta, B. Chakraborty, T.K. Mukherjee, Breast cancer metastasis: Putative therapeutic role of vascular cell adhesion molecule-1. Cell Oncol 40, 199–208 (2017)CrossRefGoogle Scholar
  8. 8.
    T. Arumugam, C.D. Logsdon, S100P: A novel therapeutic target for cancer. Amino Acids 41, 893–899 (2011)CrossRefPubMedGoogle Scholar
  9. 9.
    F. Prica, T. Radon, Y. Cheng, T. Crnogorac-Jurcevic, The life and works of S100P - from conception to cancer. Am J Cancer Res 6, 562–576 (2016)PubMedPubMedCentralGoogle Scholar
  10. 10.
    A. Maciejczyk, A. Łacko, M. Ekiert, E. Jagoda, T. Wysocka, R. Matkowski, A. Hałoń, B. Györffy, H. Lage, P. Surowiak, Elevated nuclear S100P expression is associated with poor survival in early breast cancer patients. Histol Histopathol 28, 513–524 (2013)PubMedGoogle Scholar
  11. 11.
    L. Chung, L. Phillips, M.Z. Lin, K. Moore, D.J. Marsh, F.M. Boyle, R.C. Baxter, A novel truncated form of S100P predicts disease-free survival in patients with lymph node positive breast cancer. Cancer Lett 368, 64–70 (2015)CrossRefPubMedGoogle Scholar
  12. 12.
    S. Zhang, Z. Wang, W. Liu, R. Lei, J. Shan, L. Li, X. Wang, Distinct prognostic values of S100 mRNA expression in breast cancer. Sci Rep 7, 39786 (2017)CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    T. Becker, V. Gerke, E. Kube, K. Weber, S100P, a novel Ca2+-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties. Eur J Biochem 207, 541–547 (1992)CrossRefPubMedGoogle Scholar
  14. 14.
    S. Parkkila, P. Pan, A. Ward, A. Gibadulinova, I. Oveckova, S. Pastorekova, J. Pastorek, A.R. Martinez, H.O. Helin, J. Isola, The calcium-binding protein S100P in normal and malignant human tissues. BMC Clin Pathol 8, 2 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    M. Komatsu, T. Yoshimura, T. Matsuo, K. Kiyotani, Y. Miyoshi, T. Tanihashi, K. Rokutani, R. Yamaguchi, A. Saito, S. Imoto, S. Miyano, Y. Nakamura, M. Sasa, M. Shimada, T. Katagiri, Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis. Int J Oncol 42, 478–506 (2013)CrossRefPubMedGoogle Scholar
  16. 16.
    M. Maierthaler, M. Kriegsmann, C. Peng, S. Jauch, A. Szabo, M. Wallwiener, J. Rom, C. Sohn, A. Schneeweiss, H. Sinn, R. Yang, B. Burwinkel, S100P and HYAL2 as prognostic markers for patients with triple-negative breast cancer. Exp Mol Pathol 99, 180–187 (2015)CrossRefPubMedGoogle Scholar
  17. 17.
    Q. Li, M. Wu, H. Wang, G. Xu, T. Zhu, Y. Zhang, P. Liu, A. Song, C. Gang, Z. Han, J. Zhou, L. Meng, Y. Lu, S. Wang, D. Ma, Ezrin silencing by small hairpin RNA reverses metastatic behaviors of human breast cancer cells. Cancer Lett 261, 55–63 (2008)CrossRefPubMedGoogle Scholar
  18. 18.
    S.D. Choi, Ezrin is an essential marker for metastasis of gynecologic cancer. J Korean Soc Menopause 18, 81–93 (2012)CrossRefGoogle Scholar
  19. 19.
    J. Clucas, F. Valderrama, ERM proteins in cancer progression. J Cell Sci 127, 267–275 (2014)CrossRefPubMedGoogle Scholar
  20. 20.
    T. Matsui, M. Maeda, Y. Doi, S. Yonemura, M. Amano, K. Kaibuchi, S. Tsukita, S. Tsukita, Rho-kinase phosphorylates COOH-terminal threonines of Ezrin/Radixin/Moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol 140, 647–657 (1998)CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    A. Gautreau, D. Louvard, M. Arpin, Morphogenic effects of Ezrin require a phosphorylation-induced transition from oligomers to monomers at the plasma membrane. J Cell Biol 150, 193–203 (2000)CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    J. Li, K. Wei, H. Yu, D. Jin, G. Wang, B. Yu, Prognostic value of Ezrin in various cancers: A systematic review and updated meta-analysis. Sci Rep 5, 17903 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    M. Bartova, J. Hlavaty, Y. Tan, C. Singer, K. Pohlodek, J. Luha, I. Walter, Expression of Ezrin and Moesin in primary breast carcinoma and matched lymph node metastases. Clin Exp Metastasis. 34, 333–344 (2017)CrossRefPubMedGoogle Scholar
  24. 24.
    T. Jin, J. Jin, X. Li, S. Zhang, Y.H. Choi, Y. Piao, X. Shen, Z. Lin, Prognostic implications of Ezrin and phosphorylated Ezrin expression in non-small cell lung cancer. BMC Cancer 14, 191 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    J. He, G. Ma, J. Qian, Y. Zhu, M. Liang, N. Yao, Q. Ding, L. Chen, X. Liu, T. Xia, S. Wang, Interaction between Ezrin and Cortactin in promoting epithelial to mesenchymal transition in breast cancer cells. Med Sci Monit 23, 1583–1596 (2017)CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    M. Koltzscher, C. Neumann, S. Konig, V. Gerke, Ca2+-dependent binding and activation of dormant Ezrin by dimeric S100P. Mol Biol Cell 14, 2372–2384 (2003)CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    H. Zhang, G. Wang, Y. Ding, Z. Wang, R. Barraclough, P.S. Rudland, D.G. Fernig, Z. Rao, The crystal structure at 2A resolution of the Ca2+-binding protein S100P. J Mol Biol 325, 785–794 (2003)CrossRefPubMedGoogle Scholar
  28. 28.
    A. Gibadulinova, V. Tothova, J. Pastorek, S. Pastorekova, Transcriptional regulation and functional implication of S100P in cancer. Amino Acids 41, 885–892 (2011)CrossRefPubMedGoogle Scholar
  29. 29.
    S. Diederichs, E. Bulk, B. Steffen, P. Ji, L. Tickenbrock, K. Lang, K.S. Zanker, R. Metzger, P.M. Schneider, V. Gerke, M. Thomas, W.E. Berdel, H. Serve, C. Muller-Tidow, S100 family members and trypsinogens are predictors of distant metastasis and survival in early-stage non-small cell lung cancer. Cancer Res 64, 5564–5569 (2004)CrossRefPubMedGoogle Scholar
  30. 30.
    J. Austermann, A.R. Nazmi, C. Muller-Tidow, V. Gerke, Characterization of the Ca2+-regulated Ezrin-S100P interaction and its role in tumor cell migration. J Biochem 283, 29331–29340 (2008)Google Scholar
  31. 31.
    S. Barry, C. Chelala, K. Lines, M. Sunamura, A. Wang, F.M. Marelli-Berg, C. Brennan, N.R. Lemoine, T. Crnogorac-Jurcevic, S100P is a metastasis-associated gene that facilitates transendothelial migration of pancreatic cancer cells. Clin Exp Metastasis 30, 251–264 (2013)CrossRefPubMedGoogle Scholar
  32. 32.
    C. Artus, F. Glacial, K. Ganeshamoorthy, N. Ziegler, M. Godet, T. Guilbert, S. Liebner, P. Couraud, The Wnt/planar cell polarity signaling pathway contributes to the integrity of tight junctions in brain endothelial cells. J Cerebr Blood F Met 34, 433–440 (2014)CrossRefGoogle Scholar
  33. 33.
    N. Maishi, Y. Ohba, K. Akiyama, N. Ohga, J. Hamada, H. Nagao-Kitamoto, M.T. Alam, K. Yamamoto, T. Kawamoto, N. Inoue, A. Taketomi, M. Shindoh, Y. Hida, K. Hida, Tumour endothelial cells in high metastatic tumours promote metastasis via epigenetic dysregulation of biglycan. Sci Reports 6, 28039 (2016)CrossRefGoogle Scholar
  34. 34.
    J.M. Iglesias, I. Beloqui, F. Garcia-Garcia, O. Leis, A. Vazquez- Martin, A. Eguiara, S. Cufi, A. Pavon, J.A. Menendez, J. Dopazo, A.G. Martin, Mammosphere formation in breast carcinoma cell lines depends upon expression of E-cadherin. PLoS One e77281, 8 (2013)Google Scholar
  35. 35.
    D. Sarrio, S.M. Marıa, A. Dotor, F. Calero, D. Hardisson, J. Palacios, Abnormal ezrin localization is associated with clinicopathological features in invasive breast carcinomas. Breast cancer Res Tr 98, 71–79 (2006)CrossRefGoogle Scholar
  36. 36.
    A.P.T. Schor, F.M. Carvalho, C. Kemp, I.D.C.G. Silva, J. Russo, S100P calcium-binding protein expression is associated with high-risk proliferative lesions of the breast. Oncol Rep 15, 3–6 (2006)PubMedGoogle Scholar
  37. 37.
    L. Guo, S. Chen, H. Jiang, J. Huang, W. Jin, S. Yao, The expression of S100P increases and promotes cellular proliferation by increasing nuclear translocation of β-catenin in endometrial cancer. Int J Clin Exp Pathol 7, 2102–2112 (2014)PubMedPubMedCentralGoogle Scholar
  38. 38.
    Y. Liu, C. Wang, X. Shan, J. Wu, H. Liu, H. Liu, J. Zhang, W. Xu, Z. Sha, J. He, J. Fan, S100P is associated with proliferation and migration in nasopharyngeal carcinoma. Oncology Lett 14, 525–532 (2017)CrossRefGoogle Scholar
  39. 39.
    S. Dakhel, L. Padilla, J. Adan, M. Masa, J.M. Martinez, L. Roque, T. Coll, R. Hervas, C. Calvis, R. Messeguer, F. Mitjans, J.L. Hernandez, S100P antibody-mediated therapy as a new promising strategy for the treatment of pancreatic cancer. Oncogene 3, e92 (2014)CrossRefGoogle Scholar
  40. 40.
    J.K. Kim, K.H. Jung, J.H. Noh, J.W. Eun, H.J. Bae, H.J. Xie, Y.M. Ahn, J.C. Ryu, W.S. Park, J.Y. Lee, S.W. Nam, Targeted disruption of S100P suppresses tumor cell growth by down-regulation of cyclin D1 and CDK2 in human hepatocellular carcinoma. Int J Oncol 35, 1257–1264 (2009)PubMedGoogle Scholar
  41. 41.
    X. Wang, T. Tian, M. Zhao, Y. Lou, J. Qian, Z. Liu, H. Chen, Z. Cui, High expression of S100P is associated with unfavorable prognosis and tumor progression in patients with epithelial ovarian cancer. Am J Cancer Res 5, 2409–2421 (2015)PubMedPubMedCentralGoogle Scholar
  42. 42.
    G.D. Basu, D.O. Azorsa, J.A. Kiefer, A.M. Rojas, S. Tuzmen, M.T. Barrett, J.M. Trent, O. Kallioniemi, S. Mousses, Functional evidence implicating S100P in prostate cancer progression. Int J Cancer 123, 330–339 (2008)CrossRefPubMedGoogle Scholar
  43. 43.
    A. Chandramouli, M.E. Mercado-Pimentel, A. Hutchinson, A. Gibadulinová, E.R. Olson, S. Dickinson, R. Shañas, J. Davenport, J. Owens, A.K. Bhattacharyya, J.W. Regan, S. Pastorekova, T. Arumugam, C.D. Logsdon, M.A. Nelson, The induction of S100p expression by the Prostaglandin E2 (PGE2 )/EP4 receptor signaling pathway in colon cancer cells. Cancer Biol Ther 10, 1056–1066 (2010)CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    M.E. Mercado-Pimentel, B.C. Onyeagucha, Q. Li, A.C. Pimentel, J. Jandova, M.A. Nelson, The S100P/RAGE signaling pathway regulates expression of microRNA-21 in colon cancer cells. FEBS Lett 589, 2388–2393 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Z. Wu, T. Boonmars, I. Nagano, S. Boonjaraspinyo, P. Srinontong, P. Ratasuwan, K. Narong, P.S. Nielsen, Y. Maekawa, Significance of S100P as a biomarker in diagnosis, prognosis and therapy of opisthorchiasis-associated cholangiocarcinoma. Int J Cancer 138, 396–408 (2015)CrossRefPubMedGoogle Scholar
  46. 46.
    C. Zhou, Q. Zhong, L.V. Rhodes, I. Townley, M.R. Bratton, Q. Zhang, E.C. Martin, S. Elliott, B.M. Collins-Burow, M.E. Burow, G. Wang, Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration. Breast Cancer Res 14, R45 (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    T. Arumugam, D.M. Simeone, A.M. Schmidt, C.D. Logsdon, S100P stimulates cell proliferation and survival via receptor for activated glycation end products (RAGE). J Biol Chem 279, 5059–5065 (2004)CrossRefPubMedGoogle Scholar
  48. 48.
    Y. Chen, D. Wang, Z. Guo, J. Zhao, B. Wu, H. Deng, T. Zhou, H. Xiang, F. Gao, X. Yu, J. Liao, T. Ward, P. Xia, C. Emenari, X. Ding, W. Thompson, K. Ma, J. Zhu, F. Aikhionbare, K. Dou, S.Y. Cheng, X. Yao, Rho kinase phosphorylation promotes Ezrin-mediated metastasis in hepatocellular carcinoma. Cancer Res 71, 1721–1730 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    S. Giampieri, C. Manning, S. Hooper, L. Jones, C.S. Hill, E. Sahai, Localized and reversible TGFβ signalling switches breast cancer cells from cohesive to single cell motility. Nat Cell Biol 11, 1287–1296 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Y.L. Hsu, J.Y. Hung, Y.Y. Liang, Y.S. Lin, M.J. Tsai, S.H. Chou, C.Y. Lu, P.L. Kuo, S100P interacts with integrin α7 and increases cancer cell migration and invasion in lung cancer. Oncotarget 6, 29585–29598 (2015)PubMedPubMedCentralGoogle Scholar
  51. 51.
    L.L. Cao, J.W. Xie, Y. Lin, C.H. Zheng, P. Li, J.B. Wang, J.X. Lin, J. Lu, Q.Y. Chen, C.M. Huang, miR-183 inhibits invasion of gastric cancer by targeting Ezrin. Int J Clin Exp Pathol 7, 5582–5594 (2014)PubMedPubMedCentralGoogle Scholar
  52. 52.
    Y. Li, Z. Lin, B. Chen, S. Chen, Z. Jiang, T. Zhou, Z. Hou, Y. Wang, Ezrin/NF-kB activation regulates epithelial- mesenchymal transition induced by EGF and promotes metastasis of colorectal cancer. Biomed Pharmacother 92, 140–148 (2017)CrossRefPubMedGoogle Scholar
  53. 53.
    S. Hamada, K. Satoh, M. Hirota, W. Fujibuchi, A. Kanno, J. Umino, H. Ito, A. Satoh, K. Kikuta, K. Kume, A. Masamune, T. Shimosegawa, Expression of the calcium-binding protein S100P is regulated by bone morphogenetic protein in pancreatic duct epithelial cell lines. Cancer Sci 100, 103–110 (2008)CrossRefPubMedGoogle Scholar

Copyright information

© International Society for Cellular Oncology 2018

Authors and Affiliations

  • Kyoko Kikuchi
    • 1
  • Keely May McNamara
    • 1
  • Yasuhiro Miki
    • 2
  • Erina Iwabuchi
    • 1
  • Ayako Kanai
    • 1
    • 3
  • Minoru Miyashita
    • 3
  • Takanori Ishida
    • 3
  • Hironobu Sasano
    • 1
    Email author
  1. 1.Department of PathologyTohoku University Graduate School of MedicineMiyagiJapan
  2. 2.Department of Disaster Obstetrics and Gynecology, International Research Institute of Disaster Science (IRIDeS)Tohoku UniversityMiyagiJapan
  3. 3.Department of Breast and Endocrine Surgical OncologyTohoku University Graduate School of MedicineMiyagiJapan

Personalised recommendations