Epithelial membrane protein-2 promotes endometrial tumor formation through activation of FAK and Src - PubMed (original) (raw)

Epithelial membrane protein-2 promotes endometrial tumor formation through activation of FAK and Src

Maoyong Fu et al. PLoS One. 2011.

Retraction in

Abstract

Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2), a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK)/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Tumor volume is increased in HEC-1A/EMP2 tumors.

(A) HEC-1A/EMP2, HEC-1A/V, and HEC-1A/RIBO cells were injected s.c. into nude mice. Tumor volume was determined using calipers. HEC-1A/EMP2 tumors were larger than tumors injected with HEC-1a/V and HEC-1a/RIBO cells. Values are averages (±SEM, n = 6). Comparison by ANOVA, p<0.05 (B) Representative nude Balb/c mouse displaying HEC-1A/EMP2 and HEC-1A/V subcutaneous tumors. (C) Representative Balb/c nude mouse displaying HEC-1A/V and HEC-1A/RIBO subcutaneous tumors. At day 30, HEC-1A/EMP2 (D), HEC-1A/V (E), and HEC-1A/RIBO (F) tumors were excised, fixed, and stained by hematoxylin and eosin or EMP2. H/E Magnification, 40X; EMP2 Staining Magnification, 400X. Scale bar = 10 µM.

Figure 2

Figure 2. EMP2 expression and FAK/Src signaling promote wound healing.

(A) HEC-1A/EMP2, HEC-1A/V, and HEC-1A/RIBO cells were analyzed quantitatively for total cell numbers from 0–7 days or (B) BrdU incorporation after 2 or 24 hours. No significant differences in cellular proliferation or total cell numbers were observed between the three cell lines. The experiment was repeated at least 3 times with similar results. (C) HEC-1A/EMP2, HEC-1A/V, or HEC-1A/RIBO cells were grown in a monolayer and then a “wound” created. After 24 h, wound closure was measured. Experiments were performed at least three times, and the results averaged. Comparison by Student's t test, * p = 0.03; ** p = 0.05. (D) The same experiment was also performed on Ishikawa/EMP2 and Ishikawa/V. Comparison by Student's t test, * p = 0.02. (E) HEC-1A cells were grown to reach a confluent monolayer. Cells were incubated with the PP2, Dasatinib, Erlotinib, AKTi VIII, or a vehicle control, and a wound created. After 36 h, plates were imaged, and the percentage of wound closure was calculated. Values are averages (±SEM, n = 3). Comparison by Student's t test, * p<0.05. (F) The same experiment was also performed on Ishikawa cells. Values are averages (±SEM, n = 3). Comparison by Student's t test, * p<0.05.

Figure 3

Figure 3. EMP2 promotes activated FAK and Src expression.

Expression of EMP2, 576/577 P-FAK, and 416 P-Src were assessed in (A) HEC-1A/EMP2, HEC-1A/V and HEC-1A/RIBO cells or (B) Ishikawa/EMP2 and Ishikawa/V cells. Semi-quantitative analysis of 576/577 P-FAK after correction for total FAK in both HEC-1A (C) and Ishikawa cells (D) from three independent experiments, respectively. β-actin expression was used as an additional loading control. Comparison by Student's t test, * p<0.05. EMP2 and β-actin panels of Figure 3A are excluded from this article's CC-BY license. See the accompanying retraction notice for more information.

Figure 4

Figure 4. Total FAK and EMP2 associate with each other.

(A) Cellular images of HEC-1A cells. Cells were stained for EMP2 (FITC) and total FAK (Rhodamine) expression and imaged using confocal microscopy. EMP2 and FAK colocalize (yellow) in the cytoplasm and on the membrane of HEC-1A cells. Scale bar, 20 µM. (B) In order to assess if EMP2 and FAK immunoprecipitate together, HEC-1A cells were lysed in 1% NP-40 and an interaction assessed using immunoprecipitation/SDS-PAGE analysis. Both α-FAK antibodies and EMP2 antisera pulled down EMP2, FAK, and 576/577 p-FAK. Normal rabbit antisera served as the negative isotype control. Experiments were repeated three times with similar results; a representative image is displayed.

Figure 5

Figure 5. EMP2 and activated FAK colocalize.

(A) Cellular images of HEC-1A/EMP2 cells stained for confocal microscopy using EMP2 antisera (FITC) and FAK (activated at tyrosine 397; Rhodamine). (B) Data from at least four separate samples were quantitated using Pascal software to calculate pixel intensity, and the resultant data was evaluated by Student's unpaired _t_-test. Overexpression of EMP2 demonstrated a two-fold increase in colocalization with p-FAK compared to vector control cells. Less than 5% colocalization was observed in HEC-1A/RIBO cells. Student's t test, * p = 0.01; ** p = 0.0001. Scale bar, 25 µM. (C) Ishikawa/EMP2 cells were stained using EMP2 antisera (FITC) and FAK (activated at tyrosine 397; Rhodamine) and analyzed by confocal microscopy. (D) Data from at least four separate samples were quantitated and analyzed as above. Student's t test, * p = 0.01. Scale bar, 25 µM.

Figure 6

Figure 6. EMP2 forms a complex with FAK and Src in DIG lipid raft membrane domains.

(A) EMP2 expression in HEC-1A lipid raft membrane domains was verified by Brij-58 insolubility. Cells were lysed in 1% Brij-58 and centrifuged in a sucrose density gradient. Nine fractions (500 µl each) were collected from the top of the gradient and tested for GM1 by a cholera toxin-HRP dot blot and for EMP2 (∼_M_r 20 kDa) by using SDS-PAGE and Western blot analysis. (B) To verify the cholesterol dependence of EMP2 expression within the lipid raft. HEC-1A cells were preincubated in the presence (+) or absence (–) of MβCB. Cells were lysed in 1% Triton X-100, gradient fractionated, and EMP2 expression detected by Western blot analysis. (C) HEC-1A/EMP2, HEC-1A/V, and HEC-1A/RIBO cells were lysed in 1% Triton X-100 and centrifuged as above. Samples were probed by Western blot analysis for EMP2, activated FAK, activated Src, total FAK, total Src, Flotillin-2, and EEA1. Experiments were performed independently three times with similar results.

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