Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

At a glance: The ONWARDS insulin icodec trials

A round-up of the ONWARDS phase 3 clinical trials evaluating the novel weekly insulin icodec in people with diabetes.
ADVANCED SEARCH
Log in
Top
Breast Cancer Research
Published in: Breast Cancer Research 1/2019

Open Access 01-12-2019 | Metastasis | Research article

Intravital imaging reveals systemic ezrin inhibition impedes cancer cell migration and lymph node metastasis in breast cancer

Authors: Abdi Ghaffari, Victoria Hoskin, Gulisa Turashvili, Sonal Varma, Jeff Mewburn, Graeme Mullins, Peter A. Greer, Friedemann Kiefer, Andrew G. Day, Yolanda Madarnas, Sandip SenGupta, Bruce E. Elliott

Published in: Breast Cancer Research | Issue 1/2019

Login to get access

Abstract

Background

Limited understanding of the cancer biology of metastatic sites is a major factor contributing to poor outcomes in cancer patients. The regional lymph nodes are the most common site of metastasis in most solid cancers and their involvement is a strong predictor of relapse in breast cancer (BC). We have previously shown that ezrin, a cytoskeletal–membrane linker protein, is associated with lymphovascular invasion and promotes metastatic progression in BC. However, the efficacy of pharmacological inhibition of ezrin in blocking cancer cell migration and metastasis remains unexplored in BC.

Methods

We quantified ezrin expression in a BC tissue microarray (n = 347) to assess its correlation with risk of relapse. Next, we developed a quantitative intravital microscopy (qIVM) approach, using a syngeneic lymphatic reporter mouse tumor model, to investigate the effect of systemic ezrin inhibition on cancer cell migration and metastasis.

Results

We show that ezrin is expressed at significantly higher levels in lymph node metastases compared to matched primary tumors, and that a high tumor ezrin level is associated with increased risk of relapse in BC patients with regional disease. Using qIVM, we observe a subset of cancer cells that retain their invasive and migratory phenotype at the tumor-draining lymph node. We further show that systemic inhibition of ezrin, using a small molecule compound (NSC668394), impedes the migration of cancer cells in vivo. Furthermore, systemic ezrin inhibition leads to reductions in metastatic burden at the distal axillary lymph node and lungs.

Conclusions

Our findings demonstrate that the tumor ezrin level act as an independent biomarker in predicting relapse and provide a rationale for therapeutic targeting of ezrin to reduce the metastatic capacity of cancer cells in high-risk BC patients with elevated ezrin expression.
Appendix
Available only for authorised users
Literature
1.
Li J, Wei K, Yu H, Jin D, Wang G, Yu B. Prognostic Value of Ezrin in Various Cancers: A Systematic Review and Updated Meta-analysis. Sci Rep. 2015;5:17903.PubMedPubMedCentralCrossRef
2.
3.
4.
5.
Ran S, Volk L, Hall K, Flister MJ. Lymphangiogenesis and lymphatic metastasis in breast cancer. Pathophysiology. 2010;17(4):229–51.PubMedCrossRef
6.
Ragaz J, Jackson SM, Le N, Plenderleith IH, Spinelli JJ, Basco VE, Wilson KS, Knowling MA, Coppin CM, Paradis M, et al. Adjuvant radiotherapy and chemotherapy in node-positive premenopausal women with breast cancer. N Engl J Med. 1997;337(14):956–62.PubMedCrossRef
7.
Whelan TJ, Olivotto IA, Levine MN. Regional Nodal Irradiation in Early-Stage Breast Cancer. N Engl J Med. 2015;373(19):1878–9.PubMed
8.
Ghaffari A, Hoskin V, Szeto A, Hum M, Liaghati N, Nakatsu K, LeBrun D, Madarnas Y, Sengupta S, Elliott BE. A novel role for ezrin in breast cancer angio/lymphangiogenesis. Breast Cancer Res. 2014;16(5):438.PubMedPubMedCentralCrossRef
9.
Yu Z, Sun M, Jin F, Xiao Q, He M, Wu H, Ren J, Zhao L, Zhao H, Yao W, et al. Combined expression of ezrin and E-cadherin is associated with lymph node metastasis and poor prognosis in breast cancer. Oncol Rep. 2015;34(1):165–74.PubMedCrossRef
10.
Piao J, Liu S, Xu Y, Wang C, Lin Z, Qin Y, Liu S. Ezrin protein overexpression predicts the poor prognosis of pancreatic ductal adenocarcinomas. Exp Mol Pathol. 2015;98(1):1–6.PubMedCrossRef
11.
Jin T, Jin J, Li X, Zhang S, Choi YH, Piao Y, Shen X, Lin Z. Prognostic implications of ezrin and phosphorylated ezrin expression in non-small cell lung cancer. BMC Cancer. 2014;14:191.PubMedPubMedCentralCrossRef
12.
Bulut G, Hong SH, Chen K, Beauchamp EM, Rahim S, Kosturko GW, Glasgow E, Dakshanamurthy S, Lee HS, Daar I, et al. Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells. Oncogene. 2012;31(3):269–81.PubMedCrossRef
13.
Celik H, Sajwan KP, Selvanathan SP, Marsh BJ, Pai AV, Kont YS, Han J, Minas TZ, Rahim S, Erkizan HV, et al. Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level. Mol Cell Biol. 2015;35(18):3145–62.PubMedPubMedCentral
14.
Paige M, Kosturko G, Bulut G, Miessau M, Rahim S, Toretsky JA, Brown ML, Uren A. Design, synthesis and biological evaluation of ezrin inhibitors targeting metastatic osteosarcoma. Bioorg Med Chem. 2014;22(1):478–87.PubMedCrossRef
15.
Cass JD, Varma S, Day AG, Sangrar W, Rajput AB, Raptis LH, Squire J, Madarnas Y, Sengupta SK, Elliott BE. Automated Quantitative Analysis of p53, Cyclin D1, Ki67 and pERK Expression in Breast Carcinoma Does Not Differ from Expert Pathologist Scoring and Correlates with Clinico-Pathological Characteristics. Cancers (Basel). 2012;4(3):725–42.CrossRef
16.
McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM, Statistics Subcommittee of NCIEWGoCD. REporting recommendations for tumor MARKer prognostic studies (REMARK). Breast Cancer Res Treat. 2006;100(2):229–35.PubMedCrossRef
17.
Sugiura K, Stock CC. Studies in a tumor spectrum. I. Comparison of the action of methylbis (2-chloroethyl)amine and 3-bis(2-chloroethyl)aminomethyl-4-methoxymethyl -5-hydroxy-6-methylpyridine on the growth of a variety of mouse and rat tumors. Cancer. 1952;5(2):382–402.PubMedCrossRef
18.
Ewens A, Mihich E, Ehrke MJ. Distant metastasis from subcutaneously grown E0771 medullary breast adenocarcinoma. Anticancer Res. 2005;25(6B):3905–15.PubMed
19.
Ager EI, Kozin SV, Kirkpatrick ND, Seano G, Kodack DP, Askoxylakis V, Huang Y, Goel S, Snuderl M, Muzikansky A, et al. Blockade of MMP14 activity in murine breast carcinomas: implications for macrophages, vessels, and radiotherapy. J Natl Cancer Inst. 2015;107(4).
20.
Hoskin V, Szeto A, Ghaffari A, Greer PA, Cote GP, Elliott BE. Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion. Mol Biol Cell. 2015;26(19):3464–79.PubMedPubMedCentralCrossRef
21.
Gorelik R, Gautreau A. Quantitative and unbiased analysis of directional persistence in cell migration. Nat Protoc. 2014;9(8):1931–43.PubMedCrossRef
22.
Hagerling R, Pollmann C, Kremer L, Andresen V, Kiefer F. Intravital two-photon microscopy of lymphatic vessel development and function using a transgenic Prox1 promoter-directed mOrange2 reporter mouse. Biochem Soc Trans. 2011;39(6):1674–81.PubMedCrossRef
23.
Wang J, Kubes P. A Reservoir of Mature Cavity Macrophages that Can Rapidly Invade Visceral Organs to Affect Tissue Repair. Cell. 2016;165(3):668–78.PubMedCrossRef
24.
25.
Prag S, Parsons M, Keppler MD, Ameer-Beg SM, Barber P, Hunt J, Beavil AJ, Calvert R, Arpin M, Vojnovic B, et al. Activated ezrin promotes cell migration through recruitment of the GEF Dbl to lipid rafts and preferential downstream activation of Cdc42. Mol Biol Cell. 2007;18(8):2935–48.PubMedPubMedCentralCrossRef
26.
Elliott BE, Qiao H, Louvard D, Arpin M. Co-operative effect of c-Src and ezrin in deregulation of cell-cell contacts and scattering of mammary carcinoma cells. J Cell Biochem. 2004;92(1):16–28.PubMedCrossRef
27.
Naba A, Reverdy C, Louvard D, Arpin M. Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering. EMBO J. 2008;27(1):38–50.PubMedCrossRef
28.
Yu Y, Khan J, Khanna C, Helman L, Meltzer PS, Merlino G. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med. 2004;10(2):175–81.PubMedCrossRef
29.
Mak H, Naba A, Varma S, Schick C, Day A, SenGupta SK, Arpin M, Elliott BE. Ezrin phosphorylation on tyrosine 477 regulates invasion and metastasis of breast cancer cells. BMC Cancer. 2012;12:82.PubMedPubMedCentralCrossRef
30.
Ren L, Hong SH, Cassavaugh J, Osborne T, Chou AJ, Kim SY, Gorlick R, Hewitt SM, Khanna C. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC. Oncogene. 2009;28(6):792–802.PubMedCrossRef
31.
32.
33.
Giampieri S, Manning C, Hooper S, Jones L, Hill CS, Sahai E. Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility. Nat Cell Biol. 2009;11(11):1287–96.PubMedPubMedCentralCrossRef
34.
Das S, Sarrou E, Podgrabinska S, Cassella M, Mungamuri SK, Feirt N, Gordon R, Nagi CS, Wang Y, Entenberg D, et al. Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses. J Exp Med. 2013;210(8):1509–28.PubMedPubMedCentralCrossRef
35.
Pereira ER, Kedrin D, Seano G, Gautier O, Meijer EFJ, Jones D, Chin SM, Kitahara S, Bouta EM, Chang J, et al. Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice. Science. 2018;359(6382):1403–7.PubMedPubMedCentralCrossRef
36.
Brown M, Assen FP, Leithner A, Abe J, Schachner H, Asfour G, Bago-Horvath Z, Stein JV, Uhrin P, Sixt M, et al. Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice. Science. 2018;359(6382):1408–11.PubMedCrossRef
37.
Benson JR, Jatoi I, Keisch M, Esteva FJ, Makris A, Jordan VC. Early breast cancer. Lancet. 2009;373(9673):1463–79.PubMedCrossRef
38.
Voutsadakis IA, Spadafora S. Axillary lymph node management in breast cancer with positive sentinel lymph node biopsy. World J Clin Oncol. 2015;6(1):1–6.PubMedPubMedCentralCrossRef
39.
Husemann Y, Geigl JB, Schubert F, Musiani P, Meyer M, Burghart E, Forni G, Eils R, Fehm T, Riethmuller G, et al. Systemic spread is an early step in breast cancer. Cancer Cell. 2008;13(1):58–68.PubMedCrossRef
40.
Weaver DL, Ashikaga T, Krag DN, Skelly JM, Anderson SJ, Harlow SP, Julian TB, Mamounas EP, Wolmark N. Effect of occult metastases on survival in node-negative breast cancer. N Engl J Med. 2011;364(5):412–21.PubMedPubMedCentralCrossRef
41.
Martin OA, Anderson RL, Narayan K, MacManus MP. Does the mobilization of circulating tumour cells during cancer therapy cause metastasis? Nat Rev Clin Oncol. 2017;14(1):32–44.PubMedCrossRef
42.
Karagiannis GS, Pastoriza JM, Wang Y, Harney AS, Entenberg D, Pignatelli J, Sharma VP, Xue EA, Cheng E, D'Alfonso TM, et al. Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism. Sci Transl Med. 2017;9(397).
43.
Deming PB, Campbell SL, Stone JB, Rivard RL, Mercier AL, Howe AK. Anchoring of protein kinase A by ERM (ezrin-radixin-moesin) proteins is required for proper netrin signaling through DCC (deleted in colorectal cancer). J Biol Chem. 2015;290(9):5783–96.PubMedPubMedCentralCrossRef
Metadata
Title
Intravital imaging reveals systemic ezrin inhibition impedes cancer cell migration and lymph node metastasis in breast cancer
Authors
Abdi Ghaffari
Victoria Hoskin
Gulisa Turashvili
Sonal Varma
Jeff Mewburn
Graeme Mullins
Peter A. Greer
Friedemann Kiefer
Andrew G. Day
Yolanda Madarnas
Sandip SenGupta
Bruce E. Elliott
Publication date
01-12-2019
Publisher
BioMed Central
Published in
Breast Cancer Research / Issue 1/2019
Electronic ISSN: 1465-542X
DOI
https://doi.org/10.1186/s13058-018-1079-7

Other articles of this Issue 1/2019

Breast Cancer Research 1/2019 Go to the issue

Research article

Profiling molecular regulators of recurrence in chemorefractory triple-negative breast cancers

Research article

Genomic signature of parity in the breast of premenopausal women

Research article

A functional role for the cancer disparity-linked genes, CRYβB2 and CRYβB2P1, in the promotion of breast cancer

Research article

Elacestrant (RAD1901) exhibits anti-tumor activity in multiple ER+ breast cancer models resistant to CDK4/6 inhibitors

Research article

Loss of SIM2s inhibits RAD51 binding and leads to unresolved replication stress

Research article

MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors
Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras
Prof. Fabrice Barlesi
Developed by: Springer Medicine
Image Credits