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Breast Cancer Res. 2017 Jul 27;19(1):86. doi: 10.1186/s13058-017-0880-z.

Epithelial requirement for in vitro proliferation and xenograft growth and metastasis of MDA-MB-468 human breast cancer cells: oncogenic rather than tumor-suppressive role of E-cadherin.

Hugo HJ1,2,3,4, Gunasinghe NPAD5, Hollier BG6,7, Tanaka T8, Blick T5,9,10,8, Toh A9,10,8, Hill P11, Gilles C12, Waltham M5,9,10, Thompson EW5,9,10,8,13.

Author information

1
Invasion and Metastasis Unit, St. Vincent's Institute, Melbourne, VIC, Australia. honor.hugo@qut.edu.au.
2
Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia. honor.hugo@qut.edu.au.
3
School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia. honor.hugo@qut.edu.au.
4
Translational Research Institute, Woolloongabba, QLD, Australia. honor.hugo@qut.edu.au.
5
Invasion and Metastasis Unit, St. Vincent's Institute, Melbourne, VIC, Australia.
6
Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia.
7
Australian Prostate Cancer Research Centre-Queensland, Brisbane, Australia.
8
Translational Research Institute, Woolloongabba, QLD, Australia.
9
Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia.
10
School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia.
11
Department of Pathology, University of Melbourne, Melbourne, VIC, Australia.
12
Interdisciplinary Cluster for Applied Genoproteomics (GIGA)-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium.
13
Department of Surgery, University of Melbourne, St. Vincent's Hospital, Melbourne, VIC, Australia.

Abstract

BACKGROUND:

Epithelial-to-mesenchymal transition (EMT) is associated with downregulated E-cadherin and frequently with decreased proliferation. Proliferation may be restored in secondary metastases by mesenchymal-to-epithelial transition (MET). We tested whether E-cadherin maintains epithelial proliferation in MDA-MB-468 breast cancer cells, facilitating metastatic colonization in severe combined immunodeficiency (SCID) mice.

METHODS:

EMT/MET markers were assessed in xenograft tumors by immunohistochemistry. Stable E-cadherin manipulation was effected by transfection and verified by Western blotting, immunocytochemistry, and quantitative polymerase chain reaction (qPCR). Effects of E-cadherin manipulation on proliferation and chemomigration were assessed in vitro by performing sulforhodamine B assays and Transwell assays, respectively. Invasion was assessed by Matrigel outgrowth; growth in vivo was assessed in SCID mice; and EMT status was assessed by qPCR. Hypoxic response of E-cadherin knockdown cell lines was assessed by qPCR after hypoxic culture. Repeated measures analysis of variance (ANOVA), one- and two-way ANOVA with posttests, and paired Student's t tests were performed to determine significance (p < 0.05).

RESULTS:

EMT occurred at the necrotic interface of MDA-MB-468 xenografts in regions of hypoxia. Extratumoral deposits (vascular and lymphatic inclusions, local and axillary nodes, and lung metastases) strongly expressed E-cadherin. MDA-MB-468 cells overexpressing E-cadherin were more proliferative and less migratory in vitro, whereas E-cadherin knockdown (short hairpin CDH1 [shCDH1]) cells were more migratory and invasive, less proliferative, and took longer to form tumors. shCDH1-MDA-MB-468 xenografts did not contain the hypoxia-induced necrotic areas observed in wild-type (WT) and shSCR-MDA-MB-468 tumors, but they did not exhibit an impaired hypoxic response in vitro. Although vimentin expression was not stimulated by E-cadherin knockdown in 2D or 3D cultures, xenografts of these cells were globally vimentin-positive rather than exhibiting regional EMT, and they expressed higher SNA1 than their in vitro counterparts. E-cadherin suppression caused a trend toward reduced lung metastasis, whereas E-cadherin overexpression resulted in the reverse trend, consistent with the increased proliferation rate and predominantly epithelial phenotype of MDA-MB-468 cells outside the primary xenograft. This was also originally observed in WT xenografts. Furthermore, we found that patients with breast cancer that expressed E-cadherin were more likely to have metastases.

CONCLUSIONS:

E-cadherin expression promotes growth of primary breast tumors and conceivably the formation of metastases, supporting a role for MET in metastasis. E-cadherin needs to be reevaluated as a tumor suppressor.

KEYWORDS:

Breast cancer; E-cadherin; Epithelial-mesenchymal plasticity; Epithelial-to-mesenchymal transition; Metastasis; Proliferation

PMID:
28750639
PMCID:
PMC5530912
DOI:
10.1186/s13058-017-0880-z
[Indexed for MEDLINE]
Free PMC Article

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