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

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Hugo, HJ; Gunasinghe, NPAD; Hollier, BG; Tanaka, T; Blick, T; Toh, A; Hill, P; Gilles, C; Waltham, M; Thompson, EWDate
2017-07-27Source Title
Breast Cancer ResearchPublisher
BMCAffiliation
Clinical PathologySurgery (St Vincent's)
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Hugo, H. J., Gunasinghe, N. P. A. D., Hollier, B. G., Tanaka, T., Blick, T., Toh, A., Hill, P., Gilles, C., Waltham, M. & Thompson, E. W. (2017). 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. BREAST CANCER RESEARCH, 19 (1), https://doi.org/10.1186/s13058-017-0880-z.Access Status
Open AccessAbstract
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.
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