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Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1.

Takahashi RU, Miyazaki H, Takeshita F, Yamamoto Y, Minoura K, Ono M, Kodaira M, Tamura K, Mori M, Ochiya T - Nat Commun (2015)

Bottom Line: Here, we identified microRNA-27b (miR-27b) as a key regulator for the generation of a side-population in breast cancer cells that showed CSC properties, and also found that the anti-type II diabetes (T2D) drug metformin reduced this side-population via miR-27b-mediated repression of ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1), which is involved in T2D development.ENPP1 induced the generation of the side-population via upregulation of the ABCG2 transporter.ENPP1 was also identified as a substrate of the 26S proteasome, the activity of which is downregulated in CSCs.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Tokyo 104-0045, Japan.

ABSTRACT
Cancer stem cells (CSCs) have been identified in various types of cancer; however, the mechanisms by which cells acquire CSC properties such as drug resistance and tumour seeding ability are not fully understood. Here, we identified microRNA-27b (miR-27b) as a key regulator for the generation of a side-population in breast cancer cells that showed CSC properties, and also found that the anti-type II diabetes (T2D) drug metformin reduced this side-population via miR-27b-mediated repression of ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1), which is involved in T2D development. ENPP1 induced the generation of the side-population via upregulation of the ABCG2 transporter. ENPP1 was also identified as a substrate of the 26S proteasome, the activity of which is downregulated in CSCs. Overall, these results demonstrate that a T2D-associated gene plays an important role in tumour development and that its expression is strictly controlled at the mRNA and protein levels.

No MeSH data available.


Related in: MedlinePlus

Downregulation of miR-27b is associated with the generation of CD44high/CD24low fraction.(a) Flow cytometry analyses of the CD44high/CD24low population and Zs-DR expression in MCF7-luc Zs-DR-27bs and its tumorigenic cells (MCF7-luc-ZT1 cells) in adherent and mammosphere culture conditions. (b) Flow cytometry analyses of the CD44high/CD24low and GFPhigh populations of ZR75-1-luc anti-miR-27b cells treated with docetaxel (DOC).
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f4: Downregulation of miR-27b is associated with the generation of CD44high/CD24low fraction.(a) Flow cytometry analyses of the CD44high/CD24low population and Zs-DR expression in MCF7-luc Zs-DR-27bs and its tumorigenic cells (MCF7-luc-ZT1 cells) in adherent and mammosphere culture conditions. (b) Flow cytometry analyses of the CD44high/CD24low and GFPhigh populations of ZR75-1-luc anti-miR-27b cells treated with docetaxel (DOC).

Mentions: In breast cancer, CD44high/CD24low cells show a higher tumour seeding ability and chemoresistance than CD44low/CD24high cells1239. As MCF7 cells contained the two main populations (CD44low/CD24high and CD44low/CD24low cell fraction), we first examined the expression of miR-27b in these two populations. A qRT-PCR analysis coupled with cell sorting revealed that miR-27b was downregulated in CD24low cell fraction compared with CD24high cell fraction (Supplementary Fig. 7a). We also found that CD44low/CD24high cell fraction was decreased in MCF7-luc anti-miR-27b cells (Supplementary Fig. 7b); therefore we next investigated the role of miR-27b for the generation of CD44high/CD24low cells using MCF7-luc-ZT1 cells (Fig. 1e,f). Because, a flow cytometry analysis revealed that approximately 90% of MCF7-luc-ZT1 cells showed CD24low antigen phenotype (Fig. 4a, middle panel) and mommsphere culture conditions, which are used for the enrichment of CSCs in breast cancer cells32, induced the generation of CD44high/CD24low cell fraction in MCF7-luc-ZT1 cells (Fig. 4a, right panel). Compared with CD44low/CD24high fraction, elevated expression of Zs-DR was observed in CD44high/CD24low fraction (Fig. 4a, right panel). Furthermore, in ZR75-1-luc anti-miR-27b cells, docetaxel treatment significantly induced the increase of CD44high/CD24low cells (Fig. 4b, upper panel). Because the lentivirus vector expressing the miR-27b antisense sequence also expressed green fluorescent protein (GFP), we confirmed the efficiency of knockdown of miR-27b by monitoring GFP expression (Fig. 4b, lower panel). Exposure of ZR75-1-luc anti-miR-27b cells to 5 nM docetaxel induced a clear increase in the number of GFPhigh cells (Fig. 4b, right pannel). These results suggest that miR-27b inhibits the generation of the SP fraction from the main population of cells, and that the SP fraction derived from cells with low miR-27b expression has CSC properties such as chemoresistance and high tumour seeding ability. These findings also indicate that loss or downregulation of miR-27b is essential but not sufficient for the generation of the CD44high/CD24low fraction of luminal-type breast cancer cells.


Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1.

Takahashi RU, Miyazaki H, Takeshita F, Yamamoto Y, Minoura K, Ono M, Kodaira M, Tamura K, Mori M, Ochiya T - Nat Commun (2015)

Downregulation of miR-27b is associated with the generation of CD44high/CD24low fraction.(a) Flow cytometry analyses of the CD44high/CD24low population and Zs-DR expression in MCF7-luc Zs-DR-27bs and its tumorigenic cells (MCF7-luc-ZT1 cells) in adherent and mammosphere culture conditions. (b) Flow cytometry analyses of the CD44high/CD24low and GFPhigh populations of ZR75-1-luc anti-miR-27b cells treated with docetaxel (DOC).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490376&req=5

f4: Downregulation of miR-27b is associated with the generation of CD44high/CD24low fraction.(a) Flow cytometry analyses of the CD44high/CD24low population and Zs-DR expression in MCF7-luc Zs-DR-27bs and its tumorigenic cells (MCF7-luc-ZT1 cells) in adherent and mammosphere culture conditions. (b) Flow cytometry analyses of the CD44high/CD24low and GFPhigh populations of ZR75-1-luc anti-miR-27b cells treated with docetaxel (DOC).
Mentions: In breast cancer, CD44high/CD24low cells show a higher tumour seeding ability and chemoresistance than CD44low/CD24high cells1239. As MCF7 cells contained the two main populations (CD44low/CD24high and CD44low/CD24low cell fraction), we first examined the expression of miR-27b in these two populations. A qRT-PCR analysis coupled with cell sorting revealed that miR-27b was downregulated in CD24low cell fraction compared with CD24high cell fraction (Supplementary Fig. 7a). We also found that CD44low/CD24high cell fraction was decreased in MCF7-luc anti-miR-27b cells (Supplementary Fig. 7b); therefore we next investigated the role of miR-27b for the generation of CD44high/CD24low cells using MCF7-luc-ZT1 cells (Fig. 1e,f). Because, a flow cytometry analysis revealed that approximately 90% of MCF7-luc-ZT1 cells showed CD24low antigen phenotype (Fig. 4a, middle panel) and mommsphere culture conditions, which are used for the enrichment of CSCs in breast cancer cells32, induced the generation of CD44high/CD24low cell fraction in MCF7-luc-ZT1 cells (Fig. 4a, right panel). Compared with CD44low/CD24high fraction, elevated expression of Zs-DR was observed in CD44high/CD24low fraction (Fig. 4a, right panel). Furthermore, in ZR75-1-luc anti-miR-27b cells, docetaxel treatment significantly induced the increase of CD44high/CD24low cells (Fig. 4b, upper panel). Because the lentivirus vector expressing the miR-27b antisense sequence also expressed green fluorescent protein (GFP), we confirmed the efficiency of knockdown of miR-27b by monitoring GFP expression (Fig. 4b, lower panel). Exposure of ZR75-1-luc anti-miR-27b cells to 5 nM docetaxel induced a clear increase in the number of GFPhigh cells (Fig. 4b, right pannel). These results suggest that miR-27b inhibits the generation of the SP fraction from the main population of cells, and that the SP fraction derived from cells with low miR-27b expression has CSC properties such as chemoresistance and high tumour seeding ability. These findings also indicate that loss or downregulation of miR-27b is essential but not sufficient for the generation of the CD44high/CD24low fraction of luminal-type breast cancer cells.

Bottom Line: Here, we identified microRNA-27b (miR-27b) as a key regulator for the generation of a side-population in breast cancer cells that showed CSC properties, and also found that the anti-type II diabetes (T2D) drug metformin reduced this side-population via miR-27b-mediated repression of ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1), which is involved in T2D development.ENPP1 induced the generation of the side-population via upregulation of the ABCG2 transporter.ENPP1 was also identified as a substrate of the 26S proteasome, the activity of which is downregulated in CSCs.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Tokyo 104-0045, Japan.

ABSTRACT
Cancer stem cells (CSCs) have been identified in various types of cancer; however, the mechanisms by which cells acquire CSC properties such as drug resistance and tumour seeding ability are not fully understood. Here, we identified microRNA-27b (miR-27b) as a key regulator for the generation of a side-population in breast cancer cells that showed CSC properties, and also found that the anti-type II diabetes (T2D) drug metformin reduced this side-population via miR-27b-mediated repression of ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1), which is involved in T2D development. ENPP1 induced the generation of the side-population via upregulation of the ABCG2 transporter. ENPP1 was also identified as a substrate of the 26S proteasome, the activity of which is downregulated in CSCs. Overall, these results demonstrate that a T2D-associated gene plays an important role in tumour development and that its expression is strictly controlled at the mRNA and protein levels.

No MeSH data available.


Related in: MedlinePlus