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Breast Cancer Research
Published in: Breast Cancer Research 6/2013

Open Access 01-12-2013 | Research article

Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model

Authors: Yvette Drabsch, Shuning He, Long Zhang, B Ewa Snaar-Jagalska, Peter ten Dijke

Published in: Breast Cancer Research | Issue 6/2013

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Abstract

Introduction

The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells.

Methods

We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy.

Results

Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells.

Conclusions

The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.
Appendix
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Literature
1.
Shi Y, Massague J: Mechanisms of TGF-β signaling from cell membrane to the nucleus. Cell. 2003, 113: 685-700. 10.1016/S0092-8674(03)00432-X.CrossRefPubMed
2.
Drabsch Y, ten Dijke P: TGF-β signaling in breast cancer cell invasion and bone metastasis. J Mammary Gland Biol Neoplasia. 2011, 16: 97-108. 10.1007/s10911-011-9217-1.CrossRefPubMedPubMedCentral
3.
Padua D, Massague J: Roles of TGFβ in metastasis. Cell Res. 2009, 19: 89-102. 10.1038/cr.2008.316.CrossRefPubMed
4.
Sakaki-Yumoto M, Katsuno Y, Derynck R: TGF-β family signaling in stem cells. Biochim Biophys Acta. 1830, 2013: 2280-2296.
5.
6.
Stoletov K, Klemke R: Catch of the day: zebrafish as a human cancer model. Oncogene. 2008, 27: 4509-4520. 10.1038/onc.2008.95.CrossRefPubMed
7.
8.
Goessling W, North TE, Zon LI: New waves of discovery: modeling cancer in zebrafish. J Clin Oncol. 2007, 25: 2473-2479. 10.1200/JCO.2006.08.9821.CrossRefPubMed
9.
Zon LI, Peterson RT: In vivo drug discovery in the zebrafish. Nat Rev Drug Discov. 2005, 4: 35-44. 10.1038/nrd1606.CrossRefPubMed
10.
Feng Y, Santoriello C, Mione M, Hurlstone A, Martin P: Live imaging of innate immune cell sensing of transformed cells in zebrafish larvae: parallels between tumor initiation and wound inflammation. PLoS Biol. 2010, 8: e1000562-10.1371/journal.pbio.1000562.CrossRefPubMedPubMedCentral
11.
Stoletov K, Montel V, Lester RD, Gonias SL, Klemke R: High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish. Proc Natl Acad Sci USA. 2007, 104: 17406-17411. 10.1073/pnas.0703446104.CrossRefPubMedPubMedCentral
12.
Lawson ND, Weinstein BM: In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol. 2002, 248: 307-318. 10.1006/dbio.2002.0711.CrossRefPubMed
13.
Renshaw SA, Loynes CA, Trushell DM, Elworthy S, Ingham PW, Whyte MK: A transgenic zebrafish model of neutrophilic inflammation. Blood. 2006, 108: 3976-3978. 10.1182/blood-2006-05-024075.CrossRefPubMed
14.
15.
Ellett F, Lieschke GJ: Zebrafish as a model for vertebrate hematopoiesis. Curr Opin Pharmacol. 2010, 10: 563-570. 10.1016/j.coph.2010.05.004.CrossRefPubMed
16.
Mathias JR, Dodd ME, Walters KB, Rhodes J, Kanki JP, Look AT, Huttenlocher A: Live imaging of chronic inflammation caused by mutation of zebrafish Hai1. J Cell Sci. 2007, 120: 3372-3383. 10.1242/jcs.009159.CrossRefPubMed
17.
He S, Lamers GE, Beenakker JW, Cui C, Ghotra VP, Danen EH, Meijer AH, Spaink HP, Snaar-Jagalska BE: Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model. J Pathol. 2012, 227: 431-445. 10.1002/path.4013.CrossRefPubMedPubMedCentral
18.
Wiercinska E, Naber HP, Pardali E, van der Pluijm G, van Dam H, ten Dijke P: The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system. Breast Cancer Res Treat. 2011, 128: 657-666. 10.1007/s10549-010-1147-x.CrossRefPubMed
19.
Santner SJ, Dawson PJ, Tait L, Soule HD, Eliason J, Mohamed AN, Wolman SR, Heppner GH, Miller FR: Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells. Breast Cancer Res Treat. 2001, 65: 101-110. 10.1023/A:1006461422273.CrossRefPubMed
20.
Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM: Direct binding of Smad3 and Smad4 to critical TGF β-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. Embo J. 1998, 17: 3091-3100. 10.1093/emboj/17.11.3091.CrossRefPubMedPubMedCentral
21.
Haldi M, Ton C, Seng WL, McGrath P: Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish. Angiogenesis. 2006, 9: 139-151. 10.1007/s10456-006-9040-2.CrossRefPubMed
22.
Zhou Y, He MF, Choi FF, He ZH, Song JZ, Qiao CF, Li SL, Xu HX: A high-sensitivity UPLC-MS/MS method for simultaneous determination and confirmation of triptolide in zebrafish embryos. Biomed Chromatogr. 2011, 25: 851-857. 10.1002/bmc.1534.CrossRefPubMed
23.
Lacroix M, Leclercq G: Relevance of breast cancer cell lines as models for breast tumours: an update. Breast Cancer Res Treat. 2004, 83: 249-289. 10.1023/B:BREA.0000014042.54925.cc.CrossRefPubMed
24.
Strickland LB, Dawson PJ: The hyperplasia-to-carcinoma sequence in the breast, Immunohistochemical-histologic correlations. Appl Immunohistochem Mol Morphol. 2000, 8: 98-103. 10.1097/00022744-200006000-00003.PubMed
25.
Tian F, DaCosta BS, Parks WT, Yoo S, Felici A, Tang B, Piek E, Wakefield LM, Roberts AB: Reduction in Smad2/3 signaling enhances tumorigenesis but suppresses metastasis of breast cancer cell lines. Cancer Res. 2003, 63: 8284-8292.PubMed
26.
Kang Y, He W, Tulley S, Gupta GP, Serganova I, Chen CR, Manova-Todorova K, Blasberg R, Gerald WL, Massague J: Breast cancer bone metastasis mediated by the Smad tumor suppressor pathway. Proc Natl Acad Sci USA. 2005, 102: 13909-13914. 10.1073/pnas.0506517102.CrossRefPubMedPubMedCentral
27.
Deckers M, van Dinther M, Buijs J, Que I, Lowik C, van der Pluijm G, ten Dijke P: The tumor suppressor Smad4 is required for transforming growth factor β-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells. Cancer Res. 2006, 66: 2202-2209. 10.1158/0008-5472.CAN-05-3560.CrossRefPubMed
28.
Ghosh-Choudhury N, Woodruff K, Qi W, Celeste A, Abboud SL, Ghosh Choudhury G: Bone morphogenetic protein-2 blocks MDA MB 231 human breast cancer cell proliferation by inhibiting cyclin-dependent kinase-mediated retinoblastoma protein phosphorylation. Biochem Biophys Res Commun. 2000, 272: 705-711. 10.1006/bbrc.2000.2844.CrossRefPubMed
29.
Kohli G, Hu S, Clelland E, Di Muccio T, Rothenstein J, Peng C: Cloning of transforming growth factor-β 1 (TGF-β 1) and its type II receptor from zebrafish ovary and role of TGF-β 1 in oocyte maturation. Endocrinology. 2003, 144: 1931-1941. 10.1210/en.2002-0126.CrossRefPubMed
30.
Dumont N, Bakin AV, Arteaga CL: Autocrine transforming growth factor-β signaling mediates Smad-independent motility in human cancer cells. J Biol Chem. 2003, 278: 3275-3285. 10.1074/jbc.M204623200.CrossRefPubMed
31.
Halder SK, Beauchamp RD, Datta PK: A specific inhibitor of TGF-β receptor kinase, SB-431542, as a potent antitumor agent for human cancers. Neoplasia. 2005, 7: 509-521. 10.1593/neo.04640.CrossRefPubMedPubMedCentral
32.
Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS: SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2002, 62: 65-74. 10.1124/mol.62.1.65.CrossRefPubMed
33.
Taylor KL, Grant NJ, Temperley ND, Patton EE: Small molecule screening in zebrafish: an in vivo approach to identifying new chemical tools and drug leads. Cell Commun Signal. 2010, 8: 11-10.1186/1478-811X-8-11.CrossRefPubMedPubMedCentral
34.
Sukardi H, Chng HT, Chan EC, Gong Z, Lam SH: Zebrafish for drug toxicity screening: bridging the in vitro cell-based models and in vivo mammalian models. Expert Opin Drug Metab Toxicol. 2011, 7: 579-589. 10.1517/17425255.2011.562197.CrossRefPubMed
35.
Sliva D, Rizzo MT, English D: Phosphatidylinositol 3-kinase and NF-kappaB regulate motility of invasive MDA-MB-231 human breast cancer cells by the secretion of urokinase-type plasminogen activator. J Biol Chem. 2002, 277: 3150-3157. 10.1074/jbc.M109579200.CrossRefPubMed
36.
Oshima M, Oshima H, Taketo MM: TGF-β receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis. Dev Biol. 1996, 179: 297-302. 10.1006/dbio.1996.0259.CrossRefPubMed
37.
Larsson J, Goumans MJ, Sjostrand LJ, van Rooijen MA, Ward D, Leveen P, Xu X, ten Dijke P, Mummery CL, Karlsson S: Abnormal angiogenesis but intact hematopoietic potential in TGF-β type I receptor-deficient mice. Embo J. 2001, 20: 1663-1673. 10.1093/emboj/20.7.1663.CrossRefPubMedPubMedCentral
38.
Souchelnytskyi S, Tamaki K, Engstrom U, Wernstedt C, ten Dijke P, Heldin CH: Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-β signaling. J Biol Chem. 1997, 272: 28107-28115. 10.1074/jbc.272.44.28107.CrossRefPubMed
39.
Abdollah S, Macias-Silva M, Tsukazaki T, Hayashi H, Attisano L, Wrana JL: TβRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling. J Biol Chem. 1997, 272: 27678-27685. 10.1074/jbc.272.44.27678.CrossRefPubMed
40.
Sliva D, Harvey K, Mason R, Lloyd F, English D: Effect of phosphatidic acid on human breast cancer cells exposed to doxorubicin. Cancer Invest. 2001, 19: 783-790. 10.1081/CNV-100107739.CrossRefPubMed
41.
Sabeh F, Shimizu-Hirota R, Weiss SJ: Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited. J Cell Biol. 2009, 185: 11-19. 10.1083/jcb.200807195.CrossRefPubMedPubMedCentral
42.
Naber HP, Drabsch Y, Snaar-Jagalska BE, ten Dijke P, van Laar T: Snail and Slug, key regulators of TGF-β-induced EMT, are sufficient for the induction of single-cell invasion. Biochem Biophys Res Commun. 2013, 435: 58-63. 10.1016/j.bbrc.2013.04.037.CrossRefPubMed
43.
Katz E, Verleyen W, Blackmore CG, Edward M, Smith VA, Harrison DJ: An analytical approach differentiates between individual and collective cancer invasion. Anal Cell Pathol (Amst). 2011, 34: 35-48.CrossRef
44.
Marques IJ, Weiss FU, Vlecken DH, Nitsche C, Bakkers J, Lagendijk AK, Partecke LI, Heidecke CD, Lerch MM, Bagowski CP: Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model. BMC Cancer. 2009, 9: 128-10.1186/1471-2407-9-128.CrossRefPubMedPubMedCentral
45.
Weiss FU, Marques IJ, Woltering JM, Vlecken DH, Aghdassi A, Partecke LI, Heidecke CD, Lerch MM, Bagowski CP: Retinoic acid receptor antagonists inhibit miR-10a expression and block metastatic behavior of pancreatic cancer. Gastroenterology. 2009, 137: e2131-e2137.CrossRef
46.
Ghotra VP, He S, de Bont H, van der Ent W, Spaink HP, van de Water B, Snaar-Jagalska BE, Danen EH: Automated whole animal bio-imaging assay for human cancer dissemination. PLoS One. 2012, 7: e31281-10.1371/journal.pone.0031281.CrossRefPubMedPubMedCentral
47.
Lee SL, Rouhi P, Dahl Jensen L, Zhang D, Ji H, Hauptmann G, Ingham P, Cao Y: Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model. Proc Natl Acad Sci USA. 2009, 106: 19485-19490. 10.1073/pnas.0909228106.CrossRefPubMedPubMedCentral
48.
Vlecken DH, Bagowski CP: LIMK1 and LIMK2 are important for metastatic behavior and tumor cell-induced angiogenesis of pancreatic cancer cells. Zebrafish. 2009, 6: 433-439. 10.1089/zeb.2009.0602.CrossRefPubMed
49.
Harfouche R, Basu S, Soni S, Hentschel DM, Mashelkar RA, Sengupta S: Nanoparticle-mediated targeting of phosphatidylinositol-3-kinase signaling inhibits angiogenesis. Angiogenesis. 2009, 12: 325-338. 10.1007/s10456-009-9154-4.CrossRefPubMed
50.
Latifi A, Abubaker K, Castrechini N, Ward AC, Liongue C, Dobill F, Kumar J, Thompson EW, Quinn MA, Findlay JK, Ahmed N: Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell-like profile. J Cell Biochem. 2011, 112: 2850-2864. 10.1002/jcb.23199.CrossRefPubMed
51.
Corkery DP, Dellaire G, Berman JN: Leukaemia xenotransplantation in zebrafish–chemotherapy response assay in vivo. Br J Haematol. 2011, 153: 786-789. 10.1111/j.1365-2141.2011.08661.x.CrossRefPubMed
52.
Nicoli S, Presta M: The zebrafish/tumor xenograft angiogenesis assay. Nat Protoc. 2007, 2: 2918-2923. 10.1038/nprot.2007.412.CrossRefPubMed
53.
Zhang L, Zhou F, Garcia De Vinuesa A, de Kruijf EM, Mesker WE, Hui L, Drabsch Y, Li Y, Bauer A, Rousseau A, Sheppard KA, Mickanin C, Kuppen PJ, Lu CX, Ten Dijke P: TRAF4 promotes TGF-β receptor signaling and drives breast cancer metastasis. Mol Cell. 2013, 51: 559-572. 10.1016/j.molcel.2013.07.014.CrossRefPubMed
54.
Zhang L, Zhou F, Drabsch Y, Gao R, Snaar-Jagalska BE, Mickanin C, Huang H, Sheppard KA, Porter JA, Lu CX, ten Dijke P: USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-β type I receptor. Nat Cell Biol. 2012, 14: 717-726. 10.1038/ncb2522.CrossRefPubMed
55.
Zhang L, Zhou F, Li Y, Drabsch Y, Zhang J, van Dam H, ten Dijke P: Fas-associated factor 1 is a scaffold protein that promotes β-transducin repeat-containing protein (β-TrCP)-mediated β-catenin ubiquitination and degradation. J Biol Chem. 2012, 287: 30701-30710. 10.1074/jbc.M112.353524.CrossRefPubMedPubMedCentral
56.
Johnson JI, Decker S, Zaharevitz D, Rubinstein LV, Venditti JM, Schepartz S, Kalyandrug S, Christian M, Arbuck S, Hollingshead M, Sausville EA: Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br J Cancer. 2001, 84: 1424-1431. 10.1054/bjoc.2001.1796.CrossRefPubMedPubMedCentral
57.
Xiao H, Siddiqui RA, Al-Hassani MH, Sliva D, Kovacs RJ: Phospholipids released from activated platelets improve platelet aggregation and endothelial cell migration. Platelets. 2001, 12: 163-170. 10.1080/09537100120039389.CrossRefPubMed
58.
Rosol TJ, Tannehill-Gregg SH, Corn S, Schneider A, McCauley LK: Animal models of bone metastasis. Cancer Treat Res. 2004, 118: 47-81. 10.1007/978-1-4419-9129-4_3.CrossRefPubMedPubMedCentral
59.
Korpal M, Yan J, Lu X, Xu S, Lerit DA, Kang Y: Imaging transforming growth factor-β signaling dynamics and therapeutic response in breast cancer bone metastasis. Nat Med. 2009, 15: 960-966. 10.1038/nm.1943.CrossRefPubMed
60.
van der Pluijm G, Que I, Sijmons B, Buijs JT, Lowik CW, Wetterwald A, Thalmann GN, Papapoulos SE, Cecchini MG: Interference with the microenvironmental support impairs the de novo formation of bone metastases in vivo. Cancer Res. 2005, 65: 7682-7690.PubMed
61.
El-Abdaimi K, Ste-Marie LG, Papavasiliou V, Dion N, Cardinal PE, Huang D, Kremer R: Pamidronate prevents the development of skeletal metastasis in nude mice transplanted with human breast cancer cells by reducing tumor burden within bone. Int J Oncol. 2003, 22: 883-890.PubMed
Metadata
Title
Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model
Authors
Yvette Drabsch
Shuning He
Long Zhang
B Ewa Snaar-Jagalska
Peter ten Dijke
Publication date
01-12-2013
Publisher
BioMed Central
Published in
Breast Cancer Research / Issue 6/2013
Electronic ISSN: 1465-542X
DOI
https://doi.org/10.1186/bcr3573

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