Open Access Highly Accessed Research article

Ezrin phosphorylation on tyrosine 477 regulates invasion and metastasis of breast cancer cells

Hannah Mak1, Alexandra Naba24, Sonal Varma3, Colleen Schick1, Andrew Day3, Sandip K SenGupta3, Monique Arpin2 and Bruce E Elliott1*

Author Affiliations

1 Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, ON, K7L 3N6, Canada

2 Laboratoire de Morphogenèse et Signalisation Cellulaires, UMR (Unité Mixte de Recherche) 144 CNRS (Centre National de la Recherche Scientifique), Institut Curie, Paris, France

3 Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, K7L3N6, Canada

4 Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

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BMC Cancer 2012, 12:82  doi:10.1186/1471-2407-12-82

Published: 7 March 2012

Additional files

Additional file 1:

Figure S1. Effect of Y477F ezrin on growth of AC2M2 cells in 3D Matrigel cultures. Panels A and B) AC2M2 cell clones expressing pCB6 empty vector or Y477F ezrin (clones A43 and C13) were cultured in 3D Matrigel. Representative phase contrast images of 9 day cultures of clones pCB6 (A) and C13 (B) photographed with a 4x objective are shown. Panel C) AC2M2 cell clones described above were cultured in 96 well plates (104 cells/100 μl/well) with 20% Matrigel, supplemented with Phenol Red-free complete DMEM medium. After 3 days, an MTT assay was performed according to the manufacturer's instructions. Values represent mean O.D. (Absorbance at 570 nm) of 4 wells +/- SD. No significant difference in growth was detected as determined by one way ANOVA (p = 0.319). Panel D) The number of colonies per well in 9 day cultures described in A and B was counted visually using phase contrast microscopy, and plotted as the mean of three wells +/- SD. The colony forming ability (% colonies per 7.5 x 103 cells plated) for each group is indicated. A marginal increase in colony forming ability was apparent in A43 and C13 (perhaps due to some clustering of more diffuse pCB6 colonies), but this difference was not significant as determined by one way ANOVA.

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Additional file 2:

Figure S2. Effect of the expression of Y477F ezrin on primary tumor growth: Mice injected in the mammary fat pad with AC2M2 cell clones (pCB6, A43, C13) in Figure 2 were monitored every two days, and palpable tumors were measured using Vernier calipers. Primary tumors were excised after 21-23 days, and mice were allowed to live for 40 days (20 days post-resection of the primary tumor). Panel A) Equal protein amounts of primary tumor tissue lysates were subjected to SDS-PAGE. Western blotting was performed using antibodies against VSVG, ezrin and γ-tubulin. Mean optical density ratios of ezrin vs γtubulin bands for the three tumor groups (0.52, 0.55 and 0. 64) showed no significant difference, most likely due to host tissue contribution to the total ezrin pool. Panel B) Primary tumor volumes in each group were plotted as a function of days post engraftment. The mean +/- one standard deviation (bars) is indicated for each group (pCB6, A43, C13) at each time point. The natural log of the tumor volumes as measured on days 15, 17, 20, and 23 was compared among the groups by a linear mixed effect model as estimated by restricted maximum likelihood using the SAS MIXED procedure (SAS Institute Inc., 2008). A first order autoregressive correlation structure was used to account for within mouse dependence [39]. No significance among groups on all four days was detected, using a global F-test (overall p = 0.57).

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Additional file 3:

Figure S3. Histopathology of local invasion and metastasis of pCB6 tumors. Representative examples of local invasion and distant metastasis from mammary tumors expressing empty pCB6 vector in experiments from Figures 4 and 5 are shown. Primary tumors were excised after 21-23 days, and animals were allowed to survive for a total of 40 days. Primary tumors (Panels A-C), excised previously, and additional organs (Panels D-H) were retrieved by detailed autopsy and were processed for histopathological analyses. FFPE processed tissues were sectioned (5 μm), and stained with hematoxylin and eosin. Images show the diverse invasive and metastatic characteristics of the pCB6 control tumors, including a suggestion of perineural invasion (Panels A,B), direct invasion into the fatpad (Panel C), seeding of the small intestine mesentery and serosal invasion (Panel D), pancreatic invasion (Panel E), seeding of the splenic capsule (Panel F), and lung metastases (Panels G and H). Label with "T" indicates primary tumor, "N" indicates nerve encased by tumor cells, "M" indicates mucosa, "S" indicates serosa, "P" indicates pancreatic acini, "SC" indicates splenic capsule, and "met" indicates metastatic nodule. Image magnifications are indicated in lower right corner of each image.

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