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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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Related in: MedlinePlus

miR-424 and miR-200c/141 act independently and additively to affect cell plasticity.DU145-LN4 cells were treated with control pre-miR combined with control antimiR, premiR-424 combined with antimiR control, antimiR-200c/141 combined with premiR control, or antimiR-200c/141 combined with premiR-424. (A) Whole cell lysates were collected at 7 and 14 days. Immunoblots showed reduced E-cadherin and EpCAM expression, and increased vimentin levels in premiR-424 or antimiR-200c/141 treated cells. Combining these treatments produced additive inhibition of epithelial markers. The same membrane was stripped and reprobed for each subsequent marker. Images were cropped to save space; original images are shown in Supplemental Figure 5. (B) In parallel with cell lysate collection, treated cells were examined using phase contrast microscopy and E-cadherin immunocytochemistry. Cell scattering was more distinct after premiR-424 treatment at 7 days, compared to antimiR-141/200c. At 14 days we observed additive effects of miR-424 expression and miR-141/200c inhibition, with greater cell scatter and loss of E-cadherin staining.
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f7: miR-424 and miR-200c/141 act independently and additively to affect cell plasticity.DU145-LN4 cells were treated with control pre-miR combined with control antimiR, premiR-424 combined with antimiR control, antimiR-200c/141 combined with premiR control, or antimiR-200c/141 combined with premiR-424. (A) Whole cell lysates were collected at 7 and 14 days. Immunoblots showed reduced E-cadherin and EpCAM expression, and increased vimentin levels in premiR-424 or antimiR-200c/141 treated cells. Combining these treatments produced additive inhibition of epithelial markers. The same membrane was stripped and reprobed for each subsequent marker. Images were cropped to save space; original images are shown in Supplemental Figure 5. (B) In parallel with cell lysate collection, treated cells were examined using phase contrast microscopy and E-cadherin immunocytochemistry. Cell scattering was more distinct after premiR-424 treatment at 7 days, compared to antimiR-141/200c. At 14 days we observed additive effects of miR-424 expression and miR-141/200c inhibition, with greater cell scatter and loss of E-cadherin staining.

Mentions: We sought to determine whether manipulation of the miR-424 and miR-200 pathways could act synergistically to regulate tumor cell EMT. In our model, the epithelial phenotype was represented by high miR-200 and low miR-424 levels. We transfected epithelial DU145-LN4 cells with anti-miR200c/141 and premiR-424, alone or in combination. By Western blot analysis, both miR-424 overexpression and miR-200 inhibition reduced E-cadherin and EpCAM expression and increased vimentin levels, indicating an EMT (Figure 7A). Inhibition of miR-200c and miR-141 had a more dramatic effect on EpCAM and vimentin level, compared to premiR-424 expression. These markers are known to be targets of the miR-200 family; EpCAM has been reported to be a target of miR-200c35, while vimentin is induced by ZEB36. The EMT marker expression changes were supported by phase microscopy and immunocytochemical staining for E-cadherin (Figure 7B), which indicated that at 7 days cell scattering was induced more effectively by miR-424 overexpression, compared to miR-200 inhibition. By combining miR-200c/141 inhibition and miR-424 overexpression, a greater reduction in epithelial marker expression was observed in DU145-LN4 cells after both 7 and 14 days, compared to either treatment alone (Figure 7A and B). However, vimentin was more effectively induced by miR-200c/141 inhibition alone, compared to miR-200c/141 inhibition combined with miR-424 overexpression. Our data suggest that miR-200c/141 and miR-424 influence cell plasticity through additive, rather than synergistic effects.


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)

miR-424 and miR-200c/141 act independently and additively to affect cell plasticity.DU145-LN4 cells were treated with control pre-miR combined with control antimiR, premiR-424 combined with antimiR control, antimiR-200c/141 combined with premiR control, or antimiR-200c/141 combined with premiR-424. (A) Whole cell lysates were collected at 7 and 14 days. Immunoblots showed reduced E-cadherin and EpCAM expression, and increased vimentin levels in premiR-424 or antimiR-200c/141 treated cells. Combining these treatments produced additive inhibition of epithelial markers. The same membrane was stripped and reprobed for each subsequent marker. Images were cropped to save space; original images are shown in Supplemental Figure 5. (B) In parallel with cell lysate collection, treated cells were examined using phase contrast microscopy and E-cadherin immunocytochemistry. Cell scattering was more distinct after premiR-424 treatment at 7 days, compared to antimiR-141/200c. At 14 days we observed additive effects of miR-424 expression and miR-141/200c inhibition, with greater cell scatter and loss of E-cadherin staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f7: miR-424 and miR-200c/141 act independently and additively to affect cell plasticity.DU145-LN4 cells were treated with control pre-miR combined with control antimiR, premiR-424 combined with antimiR control, antimiR-200c/141 combined with premiR control, or antimiR-200c/141 combined with premiR-424. (A) Whole cell lysates were collected at 7 and 14 days. Immunoblots showed reduced E-cadherin and EpCAM expression, and increased vimentin levels in premiR-424 or antimiR-200c/141 treated cells. Combining these treatments produced additive inhibition of epithelial markers. The same membrane was stripped and reprobed for each subsequent marker. Images were cropped to save space; original images are shown in Supplemental Figure 5. (B) In parallel with cell lysate collection, treated cells were examined using phase contrast microscopy and E-cadherin immunocytochemistry. Cell scattering was more distinct after premiR-424 treatment at 7 days, compared to antimiR-141/200c. At 14 days we observed additive effects of miR-424 expression and miR-141/200c inhibition, with greater cell scatter and loss of E-cadherin staining.
Mentions: We sought to determine whether manipulation of the miR-424 and miR-200 pathways could act synergistically to regulate tumor cell EMT. In our model, the epithelial phenotype was represented by high miR-200 and low miR-424 levels. We transfected epithelial DU145-LN4 cells with anti-miR200c/141 and premiR-424, alone or in combination. By Western blot analysis, both miR-424 overexpression and miR-200 inhibition reduced E-cadherin and EpCAM expression and increased vimentin levels, indicating an EMT (Figure 7A). Inhibition of miR-200c and miR-141 had a more dramatic effect on EpCAM and vimentin level, compared to premiR-424 expression. These markers are known to be targets of the miR-200 family; EpCAM has been reported to be a target of miR-200c35, while vimentin is induced by ZEB36. The EMT marker expression changes were supported by phase microscopy and immunocytochemical staining for E-cadherin (Figure 7B), which indicated that at 7 days cell scattering was induced more effectively by miR-424 overexpression, compared to miR-200 inhibition. By combining miR-200c/141 inhibition and miR-424 overexpression, a greater reduction in epithelial marker expression was observed in DU145-LN4 cells after both 7 and 14 days, compared to either treatment alone (Figure 7A and B). However, vimentin was more effectively induced by miR-200c/141 inhibition alone, compared to miR-200c/141 inhibition combined with miR-424 overexpression. Our data suggest that miR-200c/141 and miR-424 influence cell plasticity through additive, rather than synergistic effects.

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