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Regulation of epithelial plasticity by miR-424 and miR-200 in a new prostate cancer metastasis model.

Banyard J, Chung I, Wilson AM, Vetter G, Le Béchec A, Bielenberg DR, Zetter BR - Sci Rep (2013)

Bottom Line: DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin.Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering.We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital, Karp Family Research Laboratories, 300 Longwood Ave., Boston, MA 02115 [2] Department of Surgery, Harvard Medical School, Boston, MA 02115.

ABSTRACT
Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1-LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

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In vivo prostate-cycled DU145 cells do not display the phenotypic and expression changes observed in the metastatic DU145-LN cell lines.DU145 cells were injected into the mouse prostate, the prostate excised and grown in culture to create DU145-PR1. This process was repeated to create DU145-PR2 and DU145-PR3. (A) Photography of DU145-PR3 gross specimens (tumors and sentinel lymph nodes) after 5 weeks following cell line injection orthotopically into the prostate. Scale bar = 1 cm (B) Phase contrast microscopy shows similar cell phenotype of DU145 parental and DU145-PR3 cells in culture. (C) Western blot analysis of DU145-PR3 and parental DU145 cell lysates indicate that expression of E-cadherin, EpCAM, γ-catenin, vimentin and β-catenin were not changed by in vivo cycling through the prostate, in contrast to the expression changes observed in DU145-LN4 cells. Equal loading shown by actin immunoblots. The same membrane was stripped and reprobed for E-cadherin, EpCAM, γ-catenin, vimentin, and actin. A separate blot was probed for β-catenin and actin. Images were cropped to save space; original images are shown in Supplemental Figure 5. Handwritten pen marks denote molecular weight markers.
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f3: In vivo prostate-cycled DU145 cells do not display the phenotypic and expression changes observed in the metastatic DU145-LN cell lines.DU145 cells were injected into the mouse prostate, the prostate excised and grown in culture to create DU145-PR1. This process was repeated to create DU145-PR2 and DU145-PR3. (A) Photography of DU145-PR3 gross specimens (tumors and sentinel lymph nodes) after 5 weeks following cell line injection orthotopically into the prostate. Scale bar = 1 cm (B) Phase contrast microscopy shows similar cell phenotype of DU145 parental and DU145-PR3 cells in culture. (C) Western blot analysis of DU145-PR3 and parental DU145 cell lysates indicate that expression of E-cadherin, EpCAM, γ-catenin, vimentin and β-catenin were not changed by in vivo cycling through the prostate, in contrast to the expression changes observed in DU145-LN4 cells. Equal loading shown by actin immunoblots. The same membrane was stripped and reprobed for E-cadherin, EpCAM, γ-catenin, vimentin, and actin. A separate blot was probed for β-catenin and actin. Images were cropped to save space; original images are shown in Supplemental Figure 5. Handwritten pen marks denote molecular weight markers.

Mentions: As a parallel to our in vivo metastasis selection model, we also performed repeated rounds of orthotopic prostate injection, establishing tumor cell lines from the prostate (Figure 3A), as described in Methods. This scheme created the in vivo prostate-cycled DU145 sublines; DU145-PR1, DU145-PR2 and DU145-PR3. In contrast to the DU145-LN model, repeated in vivo cycling through the prostate alone produced no significant change in the metastatic potential of the cells when reinjected in vivo. This was assessed by cytokeratin 18 staining of excised lymph nodes as described in Methods. No mice bearing DU145-PR3 tumors were positive for metastatic foci (Table 1). In contrast to the MET-like changes observed in the metastatic DU145-LN model, no effect was observed in cell morphology (Figure 3B), and no significant change in expression of E-cadherin, EpCAM, β-catenin, γ-catenin or vimentin was observed in DU145-PR3 by Western blot (Figure 3C). This suggests that the microenvironment at the secondary site has a role in either selecting or differentiating tumor cells that arrive at that site.


Regulation of epithelial plasticity by miR-424 and miR-200 in a new prostate cancer metastasis model.

Banyard J, Chung I, Wilson AM, Vetter G, Le Béchec A, Bielenberg DR, Zetter BR - Sci Rep (2013)

In vivo prostate-cycled DU145 cells do not display the phenotypic and expression changes observed in the metastatic DU145-LN cell lines.DU145 cells were injected into the mouse prostate, the prostate excised and grown in culture to create DU145-PR1. This process was repeated to create DU145-PR2 and DU145-PR3. (A) Photography of DU145-PR3 gross specimens (tumors and sentinel lymph nodes) after 5 weeks following cell line injection orthotopically into the prostate. Scale bar = 1 cm (B) Phase contrast microscopy shows similar cell phenotype of DU145 parental and DU145-PR3 cells in culture. (C) Western blot analysis of DU145-PR3 and parental DU145 cell lysates indicate that expression of E-cadherin, EpCAM, γ-catenin, vimentin and β-catenin were not changed by in vivo cycling through the prostate, in contrast to the expression changes observed in DU145-LN4 cells. Equal loading shown by actin immunoblots. The same membrane was stripped and reprobed for E-cadherin, EpCAM, γ-catenin, vimentin, and actin. A separate blot was probed for β-catenin and actin. Images were cropped to save space; original images are shown in Supplemental Figure 5. Handwritten pen marks denote molecular weight markers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818652&req=5

f3: In vivo prostate-cycled DU145 cells do not display the phenotypic and expression changes observed in the metastatic DU145-LN cell lines.DU145 cells were injected into the mouse prostate, the prostate excised and grown in culture to create DU145-PR1. This process was repeated to create DU145-PR2 and DU145-PR3. (A) Photography of DU145-PR3 gross specimens (tumors and sentinel lymph nodes) after 5 weeks following cell line injection orthotopically into the prostate. Scale bar = 1 cm (B) Phase contrast microscopy shows similar cell phenotype of DU145 parental and DU145-PR3 cells in culture. (C) Western blot analysis of DU145-PR3 and parental DU145 cell lysates indicate that expression of E-cadherin, EpCAM, γ-catenin, vimentin and β-catenin were not changed by in vivo cycling through the prostate, in contrast to the expression changes observed in DU145-LN4 cells. Equal loading shown by actin immunoblots. The same membrane was stripped and reprobed for E-cadherin, EpCAM, γ-catenin, vimentin, and actin. A separate blot was probed for β-catenin and actin. Images were cropped to save space; original images are shown in Supplemental Figure 5. Handwritten pen marks denote molecular weight markers.
Mentions: As a parallel to our in vivo metastasis selection model, we also performed repeated rounds of orthotopic prostate injection, establishing tumor cell lines from the prostate (Figure 3A), as described in Methods. This scheme created the in vivo prostate-cycled DU145 sublines; DU145-PR1, DU145-PR2 and DU145-PR3. In contrast to the DU145-LN model, repeated in vivo cycling through the prostate alone produced no significant change in the metastatic potential of the cells when reinjected in vivo. This was assessed by cytokeratin 18 staining of excised lymph nodes as described in Methods. No mice bearing DU145-PR3 tumors were positive for metastatic foci (Table 1). In contrast to the MET-like changes observed in the metastatic DU145-LN model, no effect was observed in cell morphology (Figure 3B), and no significant change in expression of E-cadherin, EpCAM, β-catenin, γ-catenin or vimentin was observed in DU145-PR3 by Western blot (Figure 3C). This suggests that the microenvironment at the secondary site has a role in either selecting or differentiating tumor cells that arrive at that site.

Bottom Line: DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin.Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering.We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital, Karp Family Research Laboratories, 300 Longwood Ave., Boston, MA 02115 [2] Department of Surgery, Harvard Medical School, Boston, MA 02115.

ABSTRACT
Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1-LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

Show MeSH
Related in: MedlinePlus