Limits...
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

Identification of miR-27b targets.(a) Overview of the method used to identify potential miR-27b targets. (b) A qRT–PCR analysis of ENPP1 expression in MCF7-luc derivatives. Data are represented as the mean±s.d. of n=3 replicates. Expression levels were normalized to those of GAPDH. Statistical significance was determined by Student's t-test. (c) Flow cytometric analysis of the SP fraction of MCF7-luc anti-miR-27b-DR cells. (d) Immunoblot analysis of ENPP1 expression in MCF7-luc derivatives. β-Actin was used as a loading control. (e) Sequences of miR-27b and the miR-27b-binding site in the 3′UTR of ENPP1. The nucleotides shown in red were mutated in the ENPP1 3′UTR construct used in g. (f) Luciferase activity in MCF7 cells transfected with the pTK-GLuc reporter construct containing the wild-type 3′UTR of ENPP1 (50 ng), a miR-27b or nonspecific miRNA (miR-NC) expression vector and the pSV40-cLuc vector (50 ng). The ratio of Gaussia to Cypridina luciferase activity (GLuc/CLuc) was determined. Data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (g) Luciferase activity in MCF7 cells transfected with pTK-GLuc containing the wild-type (wt) or mutant 3′UTR of ENPP1 (50 ng), a miR-27b or nonspecific miRNA (miR-NC) expression vector, and pSV40-cLuc (50 ng). Data were normalized to luciferase activity in the corresponding cells transfected with miR-NC and are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (h) Flow cytometric analysis of ABCG2 expression in MCF7-GFP cells and MCF7-luc cells transfected with the ENPP1-MF expression vector. The red and blue lines indicate the results of control IgG-APC and ABCG2-APC, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4490376&req=5

f5: Identification of miR-27b targets.(a) Overview of the method used to identify potential miR-27b targets. (b) A qRT–PCR analysis of ENPP1 expression in MCF7-luc derivatives. Data are represented as the mean±s.d. of n=3 replicates. Expression levels were normalized to those of GAPDH. Statistical significance was determined by Student's t-test. (c) Flow cytometric analysis of the SP fraction of MCF7-luc anti-miR-27b-DR cells. (d) Immunoblot analysis of ENPP1 expression in MCF7-luc derivatives. β-Actin was used as a loading control. (e) Sequences of miR-27b and the miR-27b-binding site in the 3′UTR of ENPP1. The nucleotides shown in red were mutated in the ENPP1 3′UTR construct used in g. (f) Luciferase activity in MCF7 cells transfected with the pTK-GLuc reporter construct containing the wild-type 3′UTR of ENPP1 (50 ng), a miR-27b or nonspecific miRNA (miR-NC) expression vector and the pSV40-cLuc vector (50 ng). The ratio of Gaussia to Cypridina luciferase activity (GLuc/CLuc) was determined. Data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (g) Luciferase activity in MCF7 cells transfected with pTK-GLuc containing the wild-type (wt) or mutant 3′UTR of ENPP1 (50 ng), a miR-27b or nonspecific miRNA (miR-NC) expression vector, and pSV40-cLuc (50 ng). Data were normalized to luciferase activity in the corresponding cells transfected with miR-NC and are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (h) Flow cytometric analysis of ABCG2 expression in MCF7-GFP cells and MCF7-luc cells transfected with the ENPP1-MF expression vector. The red and blue lines indicate the results of control IgG-APC and ABCG2-APC, respectively.

Mentions: To identify the target of miR-27b that induces the generation of the SP fraction, a microarray analysis was combined with the use of miRNA target-predicting algorithms (miRWalk)40. Candidate miR-27b target genes that were upregulated (>2-fold, P<0.05) in MCF7-luc anti-miR-27b cells and downregulated in MCF7-luc miR-27b o.e. cells (<0.7-fold, P<0.05) compared with MCF7-luc anti-NC cells were identified (Fig. 5a and Supplementary Table 2). Among the candidates identified, we selected genes containing a miR-27b-binding site in their 3′UTR. Among these genes, ENPP1 showed the most drastic difference in expression between MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells (Supplementary Table 2). A qRT–PCR analysis coupled with cell sorting also revealed elevated expression of the ENPP1 and ABCG2 mRNAs in the SP fraction of MCF7 cells (Supplementary Fig. 8a–c). Consistent with these results, a qRT–PCR analysis revealed that ENPP1 expression in MCF7-luc anti-miR-27b cells was approximately sixfold higher than that in MCF7-luc anti-NC cells (Fig. 5b). Next, we established a docetaxel-resistant derivative of the MCF7-luc anti-miR-27b cell line (MCF7-luc anti-miR-27b-DR) by stepwise exposure to docetaxel. ENPP1 expression was even higher (approximately 15-fold) in MCF7-luc anti-miR-27b-DR cells, which had a large SP fraction (Fig. 5b,c). In addition, an immunoblot analysis revealed that ENPP1 was expressed at a higher level in MCF7-luc anti-miR-27b and anti-miR-27b-DR cells than MCF7-luc anti-NC cells (Fig. 5d).


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)

Identification of miR-27b targets.(a) Overview of the method used to identify potential miR-27b targets. (b) A qRT–PCR analysis of ENPP1 expression in MCF7-luc derivatives. Data are represented as the mean±s.d. of n=3 replicates. Expression levels were normalized to those of GAPDH. Statistical significance was determined by Student's t-test. (c) Flow cytometric analysis of the SP fraction of MCF7-luc anti-miR-27b-DR cells. (d) Immunoblot analysis of ENPP1 expression in MCF7-luc derivatives. β-Actin was used as a loading control. (e) Sequences of miR-27b and the miR-27b-binding site in the 3′UTR of ENPP1. The nucleotides shown in red were mutated in the ENPP1 3′UTR construct used in g. (f) Luciferase activity in MCF7 cells transfected with the pTK-GLuc reporter construct containing the wild-type 3′UTR of ENPP1 (50 ng), a miR-27b or nonspecific miRNA (miR-NC) expression vector and the pSV40-cLuc vector (50 ng). The ratio of Gaussia to Cypridina luciferase activity (GLuc/CLuc) was determined. Data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (g) Luciferase activity in MCF7 cells transfected with pTK-GLuc containing the wild-type (wt) or mutant 3′UTR of ENPP1 (50 ng), a miR-27b or nonspecific miRNA (miR-NC) expression vector, and pSV40-cLuc (50 ng). Data were normalized to luciferase activity in the corresponding cells transfected with miR-NC and are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (h) Flow cytometric analysis of ABCG2 expression in MCF7-GFP cells and MCF7-luc cells transfected with the ENPP1-MF expression vector. The red and blue lines indicate the results of control IgG-APC and ABCG2-APC, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490376&req=5

f5: Identification of miR-27b targets.(a) Overview of the method used to identify potential miR-27b targets. (b) A qRT–PCR analysis of ENPP1 expression in MCF7-luc derivatives. Data are represented as the mean±s.d. of n=3 replicates. Expression levels were normalized to those of GAPDH. Statistical significance was determined by Student's t-test. (c) Flow cytometric analysis of the SP fraction of MCF7-luc anti-miR-27b-DR cells. (d) Immunoblot analysis of ENPP1 expression in MCF7-luc derivatives. β-Actin was used as a loading control. (e) Sequences of miR-27b and the miR-27b-binding site in the 3′UTR of ENPP1. The nucleotides shown in red were mutated in the ENPP1 3′UTR construct used in g. (f) Luciferase activity in MCF7 cells transfected with the pTK-GLuc reporter construct containing the wild-type 3′UTR of ENPP1 (50 ng), a miR-27b or nonspecific miRNA (miR-NC) expression vector and the pSV40-cLuc vector (50 ng). The ratio of Gaussia to Cypridina luciferase activity (GLuc/CLuc) was determined. Data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (g) Luciferase activity in MCF7 cells transfected with pTK-GLuc containing the wild-type (wt) or mutant 3′UTR of ENPP1 (50 ng), a miR-27b or nonspecific miRNA (miR-NC) expression vector, and pSV40-cLuc (50 ng). Data were normalized to luciferase activity in the corresponding cells transfected with miR-NC and are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (h) Flow cytometric analysis of ABCG2 expression in MCF7-GFP cells and MCF7-luc cells transfected with the ENPP1-MF expression vector. The red and blue lines indicate the results of control IgG-APC and ABCG2-APC, respectively.
Mentions: To identify the target of miR-27b that induces the generation of the SP fraction, a microarray analysis was combined with the use of miRNA target-predicting algorithms (miRWalk)40. Candidate miR-27b target genes that were upregulated (>2-fold, P<0.05) in MCF7-luc anti-miR-27b cells and downregulated in MCF7-luc miR-27b o.e. cells (<0.7-fold, P<0.05) compared with MCF7-luc anti-NC cells were identified (Fig. 5a and Supplementary Table 2). Among the candidates identified, we selected genes containing a miR-27b-binding site in their 3′UTR. Among these genes, ENPP1 showed the most drastic difference in expression between MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells (Supplementary Table 2). A qRT–PCR analysis coupled with cell sorting also revealed elevated expression of the ENPP1 and ABCG2 mRNAs in the SP fraction of MCF7 cells (Supplementary Fig. 8a–c). Consistent with these results, a qRT–PCR analysis revealed that ENPP1 expression in MCF7-luc anti-miR-27b cells was approximately sixfold higher than that in MCF7-luc anti-NC cells (Fig. 5b). Next, we established a docetaxel-resistant derivative of the MCF7-luc anti-miR-27b cell line (MCF7-luc anti-miR-27b-DR) by stepwise exposure to docetaxel. ENPP1 expression was even higher (approximately 15-fold) in MCF7-luc anti-miR-27b-DR cells, which had a large SP fraction (Fig. 5b,c). In addition, an immunoblot analysis revealed that ENPP1 was expressed at a higher level in MCF7-luc anti-miR-27b and anti-miR-27b-DR cells than MCF7-luc anti-NC cells (Fig. 5d).

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