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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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Over-expression of miR-200 in 4TO7 cells converts fibroblastic cells to an epithelial morphology.(A) Phase contrast microscopy of 4TO7 cells that were either mock treated or transfected with the miRNA control (ctl), miR-200b, or miR-200c mimic. The white bar represents 10 µm. (B) E-cadherin expression visualized by fluorescence microscopy of parental 4T1 and 4TO7 cells (top) and 4TO7 cells that were transfected with miR-200b and/or miR-200c or control mimics (bottom). 4TO7 cells treated with either of the miR-200 mimics adopted an epithelial-like morphology and expressed high levels of E-cadherin, similar to the highly metastatic 4T1 cells. E-cadherin (Cdh1) concentration at the cell-cell junction is shown in the magnified image. Cell nuclei were stained with DAPI.
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pone-0007181-g004: Over-expression of miR-200 in 4TO7 cells converts fibroblastic cells to an epithelial morphology.(A) Phase contrast microscopy of 4TO7 cells that were either mock treated or transfected with the miRNA control (ctl), miR-200b, or miR-200c mimic. The white bar represents 10 µm. (B) E-cadherin expression visualized by fluorescence microscopy of parental 4T1 and 4TO7 cells (top) and 4TO7 cells that were transfected with miR-200b and/or miR-200c or control mimics (bottom). 4TO7 cells treated with either of the miR-200 mimics adopted an epithelial-like morphology and expressed high levels of E-cadherin, similar to the highly metastatic 4T1 cells. E-cadherin (Cdh1) concentration at the cell-cell junction is shown in the magnified image. Cell nuclei were stained with DAPI.

Mentions: The effect of exogenous miR-200 expression on E-cadherin expression and cell morphology of 4TO7 cells was also analyzed by fluorescence microscopy (Figure 4). In support of the immunoblot and qRT-PCR data, E-cadherin was readily detected in 4T1 cells, but not in 4TO7 cells. In line with this, 4TO7 cells looked more like fibroblasts growing as single cells than cultured 4T1 cells, which had epithelial morphology and adhered to their neighbors. E-cadherin in 4T1 cells concentrated at intercellular junctions. Transfection of miR-200b and/or miR-200c in 4TO7 cells increased E-cadherin expression, which also concentrated at cell junctions and shifted 4TO7 morphology from spindle-shaped cells to cobblestone-forming epithelial 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)

Over-expression of miR-200 in 4TO7 cells converts fibroblastic cells to an epithelial morphology.(A) Phase contrast microscopy of 4TO7 cells that were either mock treated or transfected with the miRNA control (ctl), miR-200b, or miR-200c mimic. The white bar represents 10 µm. (B) E-cadherin expression visualized by fluorescence microscopy of parental 4T1 and 4TO7 cells (top) and 4TO7 cells that were transfected with miR-200b and/or miR-200c or control mimics (bottom). 4TO7 cells treated with either of the miR-200 mimics adopted an epithelial-like morphology and expressed high levels of E-cadherin, similar to the highly metastatic 4T1 cells. E-cadherin (Cdh1) concentration at the cell-cell junction is shown in the magnified image. Cell nuclei were stained with DAPI.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007181-g004: Over-expression of miR-200 in 4TO7 cells converts fibroblastic cells to an epithelial morphology.(A) Phase contrast microscopy of 4TO7 cells that were either mock treated or transfected with the miRNA control (ctl), miR-200b, or miR-200c mimic. The white bar represents 10 µm. (B) E-cadherin expression visualized by fluorescence microscopy of parental 4T1 and 4TO7 cells (top) and 4TO7 cells that were transfected with miR-200b and/or miR-200c or control mimics (bottom). 4TO7 cells treated with either of the miR-200 mimics adopted an epithelial-like morphology and expressed high levels of E-cadherin, similar to the highly metastatic 4T1 cells. E-cadherin (Cdh1) concentration at the cell-cell junction is shown in the magnified image. Cell nuclei were stained with DAPI.
Mentions: The effect of exogenous miR-200 expression on E-cadherin expression and cell morphology of 4TO7 cells was also analyzed by fluorescence microscopy (Figure 4). In support of the immunoblot and qRT-PCR data, E-cadherin was readily detected in 4T1 cells, but not in 4TO7 cells. In line with this, 4TO7 cells looked more like fibroblasts growing as single cells than cultured 4T1 cells, which had epithelial morphology and adhered to their neighbors. E-cadherin in 4T1 cells concentrated at intercellular junctions. Transfection of miR-200b and/or miR-200c in 4TO7 cells increased E-cadherin expression, which also concentrated at cell junctions and shifted 4TO7 morphology from spindle-shaped cells to cobblestone-forming epithelial 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