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miR-200 enhances mouse breast cancer cell colonization to form distant metastases.

Dykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, Martinvalet D, Song E, Lim B, Lieberman J - PLoS ONE (2009)

Bottom Line: The most metastatic 4T1 cells acquired epithelial properties (high expression of E-cadherin and cytokeratin-18) compared to the less metastatic cells.These results suggest that for some tumors, tumor colonization at metastatic sites might be enhanced by MET.Therefore the epithelial nature of a tumor does not predict metastatic outcome.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Immune Disease Institute, Harvard Medical School, Boston, Massachusetts, United States of America. ddykxhoorn@med.miami.edu

ABSTRACT

Background: The development of metastases involves the dissociation of cells from the primary tumor to penetrate the basement membrane, invade and then exit the vasculature to seed, and colonize distant tissues. The last step, establishment of macroscopic tumors at distant sites, is the least well understood. Four isogenic mouse breast cancer cell lines (67NR, 168FARN, 4TO7, and 4T1) that differ in their ability to metastasize when implanted into the mammary fat pad are used to model the steps of metastasis. Only 4T1 forms macroscopic lung and liver metastases. Because some miRNAs are dysregulated in cancer and affect cellular transformation, tumor formation, and metastasis, we examined whether changes in miRNA expression might explain the differences in metastasis of these cells.

Methodology/principal findings: miRNA expression was analyzed by miRNA microarray and quantitative RT-PCR in isogenic mouse breast cancer cells with distinct metastatic capabilities. 4T1 cells that form macroscopic metastases had elevated expression of miR-200 family miRNAs compared to related cells that invade distant tissues, but are unable to colonize. Moreover, over-expressing miR-200 in 4TO7 cells enabled them to metastasize to lung and liver. These findings are surprising since the miR-200 family was previously shown to promote epithelial characteristics by inhibiting the transcriptional repressor Zeb2 and thereby enhancing E-cadherin expression. We confirmed these findings in these cells. The most metastatic 4T1 cells acquired epithelial properties (high expression of E-cadherin and cytokeratin-18) compared to the less metastatic cells.

Conclusions/significance: Expression of miR-200, which promotes a mesenchymal to epithelial cell transition (MET) by inhibiting Zeb2 expression, unexpectedly enhances macroscopic metastases in mouse breast cancer cell lines. These results suggest that for some tumors, tumor colonization at metastatic sites might be enhanced by MET. Therefore the epithelial nature of a tumor does not predict metastatic outcome.

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Protein expression of Zeb2 protein negatively correlates and E-cadherin positively correlates with miR-200 expression.(A) Zeb2 protein, analyzed by immunoblot relative to α-tubulin as a loading control, is significantly lower in 4T1 cells. (B) Zeb2 mRNA, analyzed by qRT-PCR and normalized to Gapdh, is higher in 67NR cells but similarly expressed in the other cell lines. Snail mRNA is somewhat lower in 4T1 cells than the other cell lines. (C–E) E-cadherin protein (C) and mRNA (D) expression is only detected in 4T1 cells, while N-cadherin protein (C) and mRNA (E) is restricted to 67NR cells. Vimentin protein (C) is expressed in all 4 cell lines, but expression is greater in 67NR cells, while vimentin mRNA is expressed at similar levels in all 4 cell lines (F). Cytokeratin-18 (CK-18) mRNA is expressed in 4TO7 and 4T1 cells, while Epidermal Growth Factor Receptor (EGFR) is limited to 4T1 cells (F). Protein was analyzed relative to α-tubulin by immunoblot and mRNA was quantified by qRT-PCR relative to Gapdh. Levels of protein and mRNA for both cadherins changed in parallel. The qRT-PCR results represent the mean and standard deviation from three independent experiments (*p<0.01, **p<0.001).
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pone-0007181-g002: Protein expression of Zeb2 protein negatively correlates and E-cadherin positively correlates with miR-200 expression.(A) Zeb2 protein, analyzed by immunoblot relative to α-tubulin as a loading control, is significantly lower in 4T1 cells. (B) Zeb2 mRNA, analyzed by qRT-PCR and normalized to Gapdh, is higher in 67NR cells but similarly expressed in the other cell lines. Snail mRNA is somewhat lower in 4T1 cells than the other cell lines. (C–E) E-cadherin protein (C) and mRNA (D) expression is only detected in 4T1 cells, while N-cadherin protein (C) and mRNA (E) is restricted to 67NR cells. Vimentin protein (C) is expressed in all 4 cell lines, but expression is greater in 67NR cells, while vimentin mRNA is expressed at similar levels in all 4 cell lines (F). Cytokeratin-18 (CK-18) mRNA is expressed in 4TO7 and 4T1 cells, while Epidermal Growth Factor Receptor (EGFR) is limited to 4T1 cells (F). Protein was analyzed relative to α-tubulin by immunoblot and mRNA was quantified by qRT-PCR relative to Gapdh. Levels of protein and mRNA for both cadherins changed in parallel. The qRT-PCR results represent the mean and standard deviation from three independent experiments (*p<0.01, **p<0.001).

Mentions: The TargetScan5.0 algorithm identified the zinc finger E-box binding homeobox 2 (Zeb2/SIP1/ZFXH1B) gene as the highest likelihood target gene of the miR-200 family with 6 potential miR-429/miR-200b/miR-200c binding sites and an additional 3 potential miR-141/miR-200a binding sites in its 3′UTR. Zeb2 protein was strongly expressed in 67NR, 168FARN and 4TO7 cells, but suppressed in 4T1 cells (Figure 2A). Zeb2 mRNA levels were significantly higher in 67NR cells than in the other cell lines, which had similar levels (Figure 2B). The low expression of Zeb2 protein in 4T1 cells relative to 168FARN and 4TO7 cells is consistent with inhibition of Zeb2 translation by miR-200. However, other post-transcriptional mechanisms (including other miRNAs) might explain the lack of difference in Zeb2 protein between 67NR and 168FARN and 4TO7 cells. Consistent with the known repressive role of Zeb2 on E-cadherin transcription, 4T1 cells, which have low endogenous levels of Zeb2, have high E-cadherin mRNA and protein (Figure 2C and 2D). Surprisingly, N-cadherin (Cdh2) mRNA and protein, a mesenchymal marker often reciprocally expressed with E-cadherin, was only detected in non-metastatic 67NR cells (Figure 2C and 2E). Immunoblot analysis showed that vimentin was most highly expressed in 67NR cells, but was comparable in the other 3 cell lines (Figure 2C). Vimentin mRNA was similar in all 4 cell lines. Expression of the epithelial cell-associated intermediate filament cytokeratin 18 (CK-18) mRNA was limited to 4TO7 and 4T1 cells and was higher in 4T1 cells [39] (Figure 2F). In addition, expression of Epidermal Growth Factor Receptor (EGFR) mRNA was restricted to 4T1 cells (Figure 2F). These data suggest that contrary to the EMT hypothesis, the nonmetastatic 67NR cells have a mesenchymal phenotype, while the metastatic cell lines have features of both mesenchymal and epithelial cells. Paradoxically, the most metastatic 4T1 cells have more epithelial characteristics based on enhanced Cdh1, CK-18 and EGFR expression, than the less metastatic cells.


miR-200 enhances mouse breast cancer cell colonization to form distant metastases.

Dykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, Martinvalet D, Song E, Lim B, Lieberman J - PLoS ONE (2009)

Protein expression of Zeb2 protein negatively correlates and E-cadherin positively correlates with miR-200 expression.(A) Zeb2 protein, analyzed by immunoblot relative to α-tubulin as a loading control, is significantly lower in 4T1 cells. (B) Zeb2 mRNA, analyzed by qRT-PCR and normalized to Gapdh, is higher in 67NR cells but similarly expressed in the other cell lines. Snail mRNA is somewhat lower in 4T1 cells than the other cell lines. (C–E) E-cadherin protein (C) and mRNA (D) expression is only detected in 4T1 cells, while N-cadherin protein (C) and mRNA (E) is restricted to 67NR cells. Vimentin protein (C) is expressed in all 4 cell lines, but expression is greater in 67NR cells, while vimentin mRNA is expressed at similar levels in all 4 cell lines (F). Cytokeratin-18 (CK-18) mRNA is expressed in 4TO7 and 4T1 cells, while Epidermal Growth Factor Receptor (EGFR) is limited to 4T1 cells (F). Protein was analyzed relative to α-tubulin by immunoblot and mRNA was quantified by qRT-PCR relative to Gapdh. Levels of protein and mRNA for both cadherins changed in parallel. The qRT-PCR results represent the mean and standard deviation from three independent experiments (*p<0.01, **p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2749331&req=5

pone-0007181-g002: Protein expression of Zeb2 protein negatively correlates and E-cadherin positively correlates with miR-200 expression.(A) Zeb2 protein, analyzed by immunoblot relative to α-tubulin as a loading control, is significantly lower in 4T1 cells. (B) Zeb2 mRNA, analyzed by qRT-PCR and normalized to Gapdh, is higher in 67NR cells but similarly expressed in the other cell lines. Snail mRNA is somewhat lower in 4T1 cells than the other cell lines. (C–E) E-cadherin protein (C) and mRNA (D) expression is only detected in 4T1 cells, while N-cadherin protein (C) and mRNA (E) is restricted to 67NR cells. Vimentin protein (C) is expressed in all 4 cell lines, but expression is greater in 67NR cells, while vimentin mRNA is expressed at similar levels in all 4 cell lines (F). Cytokeratin-18 (CK-18) mRNA is expressed in 4TO7 and 4T1 cells, while Epidermal Growth Factor Receptor (EGFR) is limited to 4T1 cells (F). Protein was analyzed relative to α-tubulin by immunoblot and mRNA was quantified by qRT-PCR relative to Gapdh. Levels of protein and mRNA for both cadherins changed in parallel. The qRT-PCR results represent the mean and standard deviation from three independent experiments (*p<0.01, **p<0.001).
Mentions: The TargetScan5.0 algorithm identified the zinc finger E-box binding homeobox 2 (Zeb2/SIP1/ZFXH1B) gene as the highest likelihood target gene of the miR-200 family with 6 potential miR-429/miR-200b/miR-200c binding sites and an additional 3 potential miR-141/miR-200a binding sites in its 3′UTR. Zeb2 protein was strongly expressed in 67NR, 168FARN and 4TO7 cells, but suppressed in 4T1 cells (Figure 2A). Zeb2 mRNA levels were significantly higher in 67NR cells than in the other cell lines, which had similar levels (Figure 2B). The low expression of Zeb2 protein in 4T1 cells relative to 168FARN and 4TO7 cells is consistent with inhibition of Zeb2 translation by miR-200. However, other post-transcriptional mechanisms (including other miRNAs) might explain the lack of difference in Zeb2 protein between 67NR and 168FARN and 4TO7 cells. Consistent with the known repressive role of Zeb2 on E-cadherin transcription, 4T1 cells, which have low endogenous levels of Zeb2, have high E-cadherin mRNA and protein (Figure 2C and 2D). Surprisingly, N-cadherin (Cdh2) mRNA and protein, a mesenchymal marker often reciprocally expressed with E-cadherin, was only detected in non-metastatic 67NR cells (Figure 2C and 2E). Immunoblot analysis showed that vimentin was most highly expressed in 67NR cells, but was comparable in the other 3 cell lines (Figure 2C). Vimentin mRNA was similar in all 4 cell lines. Expression of the epithelial cell-associated intermediate filament cytokeratin 18 (CK-18) mRNA was limited to 4TO7 and 4T1 cells and was higher in 4T1 cells [39] (Figure 2F). In addition, expression of Epidermal Growth Factor Receptor (EGFR) mRNA was restricted to 4T1 cells (Figure 2F). These data suggest that contrary to the EMT hypothesis, the nonmetastatic 67NR cells have a mesenchymal phenotype, while the metastatic cell lines have features of both mesenchymal and epithelial cells. Paradoxically, the most metastatic 4T1 cells have more epithelial characteristics based on enhanced Cdh1, CK-18 and EGFR expression, than the less metastatic cells.

Bottom Line: The most metastatic 4T1 cells acquired epithelial properties (high expression of E-cadherin and cytokeratin-18) compared to the less metastatic cells.These results suggest that for some tumors, tumor colonization at metastatic sites might be enhanced by MET.Therefore the epithelial nature of a tumor does not predict metastatic outcome.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Immune Disease Institute, Harvard Medical School, Boston, Massachusetts, United States of America. ddykxhoorn@med.miami.edu

ABSTRACT

Background: The development of metastases involves the dissociation of cells from the primary tumor to penetrate the basement membrane, invade and then exit the vasculature to seed, and colonize distant tissues. The last step, establishment of macroscopic tumors at distant sites, is the least well understood. Four isogenic mouse breast cancer cell lines (67NR, 168FARN, 4TO7, and 4T1) that differ in their ability to metastasize when implanted into the mammary fat pad are used to model the steps of metastasis. Only 4T1 forms macroscopic lung and liver metastases. Because some miRNAs are dysregulated in cancer and affect cellular transformation, tumor formation, and metastasis, we examined whether changes in miRNA expression might explain the differences in metastasis of these cells.

Methodology/principal findings: miRNA expression was analyzed by miRNA microarray and quantitative RT-PCR in isogenic mouse breast cancer cells with distinct metastatic capabilities. 4T1 cells that form macroscopic metastases had elevated expression of miR-200 family miRNAs compared to related cells that invade distant tissues, but are unable to colonize. Moreover, over-expressing miR-200 in 4TO7 cells enabled them to metastasize to lung and liver. These findings are surprising since the miR-200 family was previously shown to promote epithelial characteristics by inhibiting the transcriptional repressor Zeb2 and thereby enhancing E-cadherin expression. We confirmed these findings in these cells. The most metastatic 4T1 cells acquired epithelial properties (high expression of E-cadherin and cytokeratin-18) compared to the less metastatic cells.

Conclusions/significance: Expression of miR-200, which promotes a mesenchymal to epithelial cell transition (MET) by inhibiting Zeb2 expression, unexpectedly enhances macroscopic metastases in mouse breast cancer cell lines. These results suggest that for some tumors, tumor colonization at metastatic sites might be enhanced by MET. Therefore the epithelial nature of a tumor does not predict metastatic outcome.

Show MeSH
Related in: MedlinePlus