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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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Stable miR-141-200c over-expression or Zeb2 silencing in 4TO7 cells promoted adoption of an epithelial-like morphology.(A) miR-200c expression is increased in 4TO7 cells stably expressing the miR-141-200c cluster from a retroviral vector. miR-200c levels, normalized to U6 snRNA expression, are shown relative to expression in 4T1 cells. Mean and standard deviation of 3 independent experiments are shown (*, p = 0.02, relative to 4T1 cells). (B) Stable over-expression of the miR-141-200c cluster leads to an increase in E-cadherin expression, as measured by immunoblot relative to α-tubulin. (C) The stable miR-141-200c-expressing 4TO7 cells adopt an epithelial morphology compared to the control transfected cells or the parental cells when examined by phase contrast microscopy. 4TO7 cells stably expressing a Zeb2 shRNA had reduced Zeb2 and concomitantly increased E-cadherin protein by immunoblot (D), and altered morphology from a fibroblastic to an epithelial morphology (E). 4T1 cells are shown for comparison. The white bars represent 10 µm.
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pone-0007181-g005: Stable miR-141-200c over-expression or Zeb2 silencing in 4TO7 cells promoted adoption of an epithelial-like morphology.(A) miR-200c expression is increased in 4TO7 cells stably expressing the miR-141-200c cluster from a retroviral vector. miR-200c levels, normalized to U6 snRNA expression, are shown relative to expression in 4T1 cells. Mean and standard deviation of 3 independent experiments are shown (*, p = 0.02, relative to 4T1 cells). (B) Stable over-expression of the miR-141-200c cluster leads to an increase in E-cadherin expression, as measured by immunoblot relative to α-tubulin. (C) The stable miR-141-200c-expressing 4TO7 cells adopt an epithelial morphology compared to the control transfected cells or the parental cells when examined by phase contrast microscopy. 4TO7 cells stably expressing a Zeb2 shRNA had reduced Zeb2 and concomitantly increased E-cadherin protein by immunoblot (D), and altered morphology from a fibroblastic to an epithelial morphology (E). 4T1 cells are shown for comparison. The white bars represent 10 µm.

Mentions: To evaluate the effect of miR-200 and Zeb2 on tumor formation and metastasis, we next engineered retroviruses encoding the miR-141-200c cluster mature miRNAs or control virus expressing firefly luciferase shRNA or Zeb2 shRNA within the miR-30 stem. Infection with these retroviruses allowed us to produce 4TO7 cell lines that stably expressed the miR141-200c cluster or had stably knocked-down Zeb2. Stable expression of the miR-200c cluster in 4TO7 cells increased miR-200c expression to ∼3-fold higher than that of 4T1 cells as measured by qRT-PCR (Figure 5A). As expected, the miR-200c cluster-transfected cells expressed E-cadherin protein, which was undetected in the control virus-treated cells (Figure 5B). They also adopted an epithelial-like morphology (Figure 5C). This same phenotype was recapitulated in 4TO7 cells that had stably silenced Zeb2 expression (Figure 5D and 5E)). 4TO7 cells over-expressing miR-200 or knocked down for Zeb2 morphologically resembled 4T1 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)

Stable miR-141-200c over-expression or Zeb2 silencing in 4TO7 cells promoted adoption of an epithelial-like morphology.(A) miR-200c expression is increased in 4TO7 cells stably expressing the miR-141-200c cluster from a retroviral vector. miR-200c levels, normalized to U6 snRNA expression, are shown relative to expression in 4T1 cells. Mean and standard deviation of 3 independent experiments are shown (*, p = 0.02, relative to 4T1 cells). (B) Stable over-expression of the miR-141-200c cluster leads to an increase in E-cadherin expression, as measured by immunoblot relative to α-tubulin. (C) The stable miR-141-200c-expressing 4TO7 cells adopt an epithelial morphology compared to the control transfected cells or the parental cells when examined by phase contrast microscopy. 4TO7 cells stably expressing a Zeb2 shRNA had reduced Zeb2 and concomitantly increased E-cadherin protein by immunoblot (D), and altered morphology from a fibroblastic to an epithelial morphology (E). 4T1 cells are shown for comparison. The white bars represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749331&req=5

pone-0007181-g005: Stable miR-141-200c over-expression or Zeb2 silencing in 4TO7 cells promoted adoption of an epithelial-like morphology.(A) miR-200c expression is increased in 4TO7 cells stably expressing the miR-141-200c cluster from a retroviral vector. miR-200c levels, normalized to U6 snRNA expression, are shown relative to expression in 4T1 cells. Mean and standard deviation of 3 independent experiments are shown (*, p = 0.02, relative to 4T1 cells). (B) Stable over-expression of the miR-141-200c cluster leads to an increase in E-cadherin expression, as measured by immunoblot relative to α-tubulin. (C) The stable miR-141-200c-expressing 4TO7 cells adopt an epithelial morphology compared to the control transfected cells or the parental cells when examined by phase contrast microscopy. 4TO7 cells stably expressing a Zeb2 shRNA had reduced Zeb2 and concomitantly increased E-cadherin protein by immunoblot (D), and altered morphology from a fibroblastic to an epithelial morphology (E). 4T1 cells are shown for comparison. The white bars represent 10 µm.
Mentions: To evaluate the effect of miR-200 and Zeb2 on tumor formation and metastasis, we next engineered retroviruses encoding the miR-141-200c cluster mature miRNAs or control virus expressing firefly luciferase shRNA or Zeb2 shRNA within the miR-30 stem. Infection with these retroviruses allowed us to produce 4TO7 cell lines that stably expressed the miR141-200c cluster or had stably knocked-down Zeb2. Stable expression of the miR-200c cluster in 4TO7 cells increased miR-200c expression to ∼3-fold higher than that of 4T1 cells as measured by qRT-PCR (Figure 5A). As expected, the miR-200c cluster-transfected cells expressed E-cadherin protein, which was undetected in the control virus-treated cells (Figure 5B). They also adopted an epithelial-like morphology (Figure 5C). This same phenotype was recapitulated in 4TO7 cells that had stably silenced Zeb2 expression (Figure 5D and 5E)). 4TO7 cells over-expressing miR-200 or knocked down for Zeb2 morphologically resembled 4T1 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