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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

MiR-27b regulates the resistance of breast cancer cells to docetaxel.(a) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. (b,c) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC50 value. Data are represented as the mean±s.d. of n=3 replicates. (d) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. (e) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. (f) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n=3 replicates. Statistical significance was determined by Student's t-test.
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f2: MiR-27b regulates the resistance of breast cancer cells to docetaxel.(a) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. (b,c) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC50 value. Data are represented as the mean±s.d. of n=3 replicates. (d) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. (e) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. (f) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n=3 replicates. Statistical significance was determined by Student's t-test.

Mentions: To investigate its role in the regulation of CSC properties further, the effects of knockdown of miR-27b on the drug resistance and tumorigenicity of MCF7 cells were examined. A miR-27b knockdown MCF7-luc cell line (MCF7-luc anti-miR-27b) was generated using a lentiviral vector expressing an antisense miR-27b sequence (Fig. 2a). To confirm the suppression of miR-27b, MCF7-luc anti-miR-27b cells were transfected with a sensor vector (pTK-GLuc-27bs) harbouring a secreted gaussia luciferase reporter and two miR-27b complementary sequences in its 3′UTR (Supplementary Fig. 2a, b). A reporter assay showed that the luciferase activity in MCF7-luc anti-miR-27b cells expressing pTK-GLuc-27bs was fivefold higher than that in control cells (MCF7-luc anti-NC) expressing this reporter (Supplementary Fig. 2c). A similar assay was also performed using a luciferase reporter construct harbouring the 3′UTR of the gene encoding peroxisome proliferator-activated receptor gamma (PPARG), which has been reported as a direct target of miR-27b37. After transfection with the PPARG reporter construct, luciferase activity was significantly higher in MCF7-luc anti-miR-27b cells than control cells (Supplementary Fig. 3a, b). Overall, these results demonstrate that the lentivirus vector system inhibited the function of miR-27b efficiently.


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)

MiR-27b regulates the resistance of breast cancer cells to docetaxel.(a) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. (b,c) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC50 value. Data are represented as the mean±s.d. of n=3 replicates. (d) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. (e) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. (f) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n=3 replicates. Statistical significance was determined by Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490376&req=5

f2: MiR-27b regulates the resistance of breast cancer cells to docetaxel.(a) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. (b,c) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC50 value. Data are represented as the mean±s.d. of n=3 replicates. (d) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. (e) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. (f) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n=3 replicates. Statistical significance was determined by Student's t-test.
Mentions: To investigate its role in the regulation of CSC properties further, the effects of knockdown of miR-27b on the drug resistance and tumorigenicity of MCF7 cells were examined. A miR-27b knockdown MCF7-luc cell line (MCF7-luc anti-miR-27b) was generated using a lentiviral vector expressing an antisense miR-27b sequence (Fig. 2a). To confirm the suppression of miR-27b, MCF7-luc anti-miR-27b cells were transfected with a sensor vector (pTK-GLuc-27bs) harbouring a secreted gaussia luciferase reporter and two miR-27b complementary sequences in its 3′UTR (Supplementary Fig. 2a, b). A reporter assay showed that the luciferase activity in MCF7-luc anti-miR-27b cells expressing pTK-GLuc-27bs was fivefold higher than that in control cells (MCF7-luc anti-NC) expressing this reporter (Supplementary Fig. 2c). A similar assay was also performed using a luciferase reporter construct harbouring the 3′UTR of the gene encoding peroxisome proliferator-activated receptor gamma (PPARG), which has been reported as a direct target of miR-27b37. After transfection with the PPARG reporter construct, luciferase activity was significantly higher in MCF7-luc anti-miR-27b cells than control cells (Supplementary Fig. 3a, b). Overall, these results demonstrate that the lentivirus vector system inhibited the function of miR-27b efficiently.

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