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. 2014 Mar;105(3):281-9.
doi: 10.1111/cas.12336. Epub 2014 Jan 29.

Mesenchymal-transitioned cancer cells instigate the invasion of epithelial cancer cells through secretion of WNT3 and WNT5B

Shinichiro Kato  1 , Yoshihiro Hayakawa, Hiroaki Sakurai, Ikuo Saiki, Satoru Yokoyama
Affiliations

Mesenchymal-transitioned cancer cells instigate the invasion of epithelial cancer cells through secretion of WNT3 and WNT5B

Shinichiro Kato et al. Cancer Sci. 2014 Mar.

Abstract

Although the heterogeneities of epithelial and mesenchymal-transitioned cancer cells are often observed within the tumor microenvironment, the biological significance of the interaction between epithelial cancer cells and mesenchymal-transitioned cancer cells is not yet understood. In this study, we show that the mesenchymal-transitioned cancer cells instigate the invasive ability and metastatic potential of the neighboring epithelial cancer cells in vitro and in vivo. We identify WNT3 and WNT5B as critical factors secreted from Transforming growth factor-induced mesenchymal cancer cells for instigating the epithelial cancer cell invasion along with the induction of secondary EMT phenotype. These results shed light on the significance of cancer heterogeneity and the interaction between epithelial and mesenchymal-transitioned cancer cells within the tumor microenvironment in promoting metastatic disease through the WNT-dependent mechanism.

Keywords: Cross-talk; epithelial-to-mesenchymal transition; heterogeneity; metastasis WNT.

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Figures

Figure 1
Figure 1
Epithelial cancer cells acquire metastatic potential upon co-culture with mesenchymal-transitioned cancer cells. (a) A549 (left) or Panc-1 (right) cells were subjected to Matrigel invasion assay after 24-h co-culture of epithelial cancer cells (E-cells) with mesenchymal-transitioned cancer cells (M-cells) at the indicated cell numbers. Total invaded cells were counted after H&E staining. Data represented as the mean ± SD of four independent experiments. (b) Labeled E-cells (E-A549/Luc2; left panel or E-Panc/EGFP; right panel) were subjected to Matrigel invasion assay after 24-h-co-culture with M-cells. Invasive abilities were determined by measuring luciferase activity (A549) or counting invaded EGFP+ cells (Panc-1), respectively. Invasion ratio was calculated by the division of invaded E-cells by total E-cells applied. Data represented as the mean ± SD of triplicate experiment. *P < 0.05, **P < 0.01 versus E-cells alone group by Dunnett's test. (c) Epithelial A549 cells overexpressing Luc2 gene (E-A549/Luc2) were co-cultured with either epithelial A549 (E-A549) cells or mesenchymal-transitioned A549 (M-A549) cells and i.v. inoculated into mice. Mice were killed 24 h after the tumor inoculation and lungs were subjected to bioluminescent imaging to determine total flux (photon/s) for lung metastasis quantification. The representative ex vivo images are shown. Data represented as the mean ± SEM (n = 5).
Figure 2
Figure 2
Cell–cell contact independent induction of secondary epithelial-to-mesenchymal transition (EMT) phenotype and invasiveness in neighboring epithelial cancer cells by mesenchymal-transitioned cancer cells. (a, b) A549 cells were co-cultured directly (a) or separately in transwell cell culture chamber (b) at the indicated cell numbers for 24 h and EMT-related protein expression were determined by western blotting. No cells (−), E-cells (E) or M-cells (M) were seeded in upper compartment of transwell chamber. In the separated co-culture, the total protein from E-cells in lower compartment was examined. (c, d) Epithelial A549 cells were treated with conditioned mediums (CM) from E-/M-A549 cells (c) or E-/M-Panc cells (d) for 48 h and subjected to Matrigel invasion assay or western blotting. Total invaded cells were counted after H&E staining. Data represented as the mean ± SD of triplicate experiment. **P < 0.01 versus E-CM group by two-tailed Student's t test.
Figure 3
Figure 3
Secretory WNT3 and WNT5B from mesenchymal-transitioned cancer cells induce secondary epithelial-to-mesenchymal transition (EMT) phenotype in epithelial cancer cells. (a) Commonly upregulated genes encoding soluble protein in the top 5% in GSE17708 (Panc-1) and GSE23952 (A549) datasets were shown as Benn diagram. (b) Epithelial or mesenchymal-transitioned A549 or Panc-1 cells were subjected to western blotting to determine the expression of indicated proteins. (c) Conditioned mediums from E-cells or M-cells were subjected to ELISA for detecting WNT3 or WNT5B.
Figure 4
Figure 4
Critical requirement of secretory WNT3 and WNT5B from mesenchymal-transitioned cancer cells for inducing secondary epithelial-to-mesenchymal transition (EMT) phenotype of epithelical cancer cells. (a, b) E-A549 (left) and E-Panc (right) cells were treated with WNT-depleted CM derived from E-cells or M-cells either by siRNAs against WNT3/WNT5B (a) or by 10 μM WNT secretion inhibitor (IWP-2) (b) for 48 h. The cells were subjected to Matrigel invasion assay and western blotting. **P < 0.01 by one-way anova with the Bonferroni correction. (c) E-A549 or E-Panc cells were treated with WNT-depleted CM derived from E-cells or M-cells by the indicated dose of IWP-2 for 48 h and the expression of proteins were determined by western blotting.
Figure 5
Figure 5
Secretory WNT-dependent metastasis instigation of epithelial cancer cells by mesenchymal-transitioned cancer cells. E-A549 cells were co-cultured with either control (E-A549) cells or mesenchymal-transitioned A549 (M-A549) cells in the presence or absence of IWP-2 for 48 h and i.v. inoculated into mice. Mice were killed 24 h after the tumor inoculation and lungs were subjected to bioluminescent imaging to determine total flux (photon/s) for lung metastasis quantification. The representative ex vivo images are shown. Data represented as the mean ± SEM (n = 3–5).
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