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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

ENPP1 is a substrate of the 26S proteasome.(a) ENPP1 expression in MCF7-luc cells stably expressing ENPP1-MF or anti-NC as a control, detected by qRT–PCR. Expression levels were normalized to those of GAPDH and data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (b) A schematic illustration of the approach used in the experiments shown in c–e. (c) Immunoblot analyses of ENPP1 expression in MCF7-luc anti-NC and MCF7-luc ENPP1-MF cells treated with or without MG132 for 24 h. (d) Immunoblot analyses of ENPP1 in MCF7-luc ENPP1-MF cells grown under adherent (Ad) or mammosphere (Ma) culture conditions for the indicated times. (e) ENPP1 expression in the indicated MCF7-luc derivatives grown under adherent (Ad) or mammosphere (Ma) culture conditions. (f) Growth of the indicated MCF7-luc cell derivatives. An MTT assay was performed to determine the numbers of cells at each time point. Data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (g) Immunofluorescent detection of ENPP1-MF and GFP in MCF7-luc ENPP1-MF cells in paraffin-embedded sections of primary tumour xenografts. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 50 μm. (h) Bioluminescent images of tumours in NOD/SCID mice injected with MCF7-luc shENPP1 cells that were treated with or without docetaxel (DOC). Alternatively, the mice were injected with MCF7-luc Zs-DR-27bs cells as a technical control. Representative images are shown for each cohort.
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f7: ENPP1 is a substrate of the 26S proteasome.(a) ENPP1 expression in MCF7-luc cells stably expressing ENPP1-MF or anti-NC as a control, detected by qRT–PCR. Expression levels were normalized to those of GAPDH and data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (b) A schematic illustration of the approach used in the experiments shown in c–e. (c) Immunoblot analyses of ENPP1 expression in MCF7-luc anti-NC and MCF7-luc ENPP1-MF cells treated with or without MG132 for 24 h. (d) Immunoblot analyses of ENPP1 in MCF7-luc ENPP1-MF cells grown under adherent (Ad) or mammosphere (Ma) culture conditions for the indicated times. (e) ENPP1 expression in the indicated MCF7-luc derivatives grown under adherent (Ad) or mammosphere (Ma) culture conditions. (f) Growth of the indicated MCF7-luc cell derivatives. An MTT assay was performed to determine the numbers of cells at each time point. Data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (g) Immunofluorescent detection of ENPP1-MF and GFP in MCF7-luc ENPP1-MF cells in paraffin-embedded sections of primary tumour xenografts. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 50 μm. (h) Bioluminescent images of tumours in NOD/SCID mice injected with MCF7-luc shENPP1 cells that were treated with or without docetaxel (DOC). Alternatively, the mice were injected with MCF7-luc Zs-DR-27bs cells as a technical control. Representative images are shown for each cohort.

Mentions: To determine the molecular mechanism by which ENPP1 induces drug resistance in only a small population of breast cancer cells, we investigated the fate of this protein in MCF7-luc cells stably expressing ENPP1-MF (MCF7-luc ENPP-MF), which was generated using a lentivirus vector. Because the lentivirus vector expressing ENPP1 also expressed GFP, a polyclonal cell line was established by flow cytometric sorting of GFP-positive cells. The expression of ENPP1-MF in stable transfectants was confirmed by immunoblotting at day 7 after infection (Supplementary Fig. 10a). However, after 2 weeks of culture, ENPP1-MF expression in these cells was reduced markedly. At the same time point, ENPP1 mRNA expression was tenfold higher in the MCF7-luc ENPP1-MF cells than the control cells (MCF7-luc anti-NC; Fig. 7a), suggesting that the stability of ENPP1 is affected by post-translational modifications.


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)

ENPP1 is a substrate of the 26S proteasome.(a) ENPP1 expression in MCF7-luc cells stably expressing ENPP1-MF or anti-NC as a control, detected by qRT–PCR. Expression levels were normalized to those of GAPDH and data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (b) A schematic illustration of the approach used in the experiments shown in c–e. (c) Immunoblot analyses of ENPP1 expression in MCF7-luc anti-NC and MCF7-luc ENPP1-MF cells treated with or without MG132 for 24 h. (d) Immunoblot analyses of ENPP1 in MCF7-luc ENPP1-MF cells grown under adherent (Ad) or mammosphere (Ma) culture conditions for the indicated times. (e) ENPP1 expression in the indicated MCF7-luc derivatives grown under adherent (Ad) or mammosphere (Ma) culture conditions. (f) Growth of the indicated MCF7-luc cell derivatives. An MTT assay was performed to determine the numbers of cells at each time point. Data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (g) Immunofluorescent detection of ENPP1-MF and GFP in MCF7-luc ENPP1-MF cells in paraffin-embedded sections of primary tumour xenografts. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 50 μm. (h) Bioluminescent images of tumours in NOD/SCID mice injected with MCF7-luc shENPP1 cells that were treated with or without docetaxel (DOC). Alternatively, the mice were injected with MCF7-luc Zs-DR-27bs cells as a technical control. Representative images are shown for each cohort.
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Related In: Results  -  Collection

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f7: ENPP1 is a substrate of the 26S proteasome.(a) ENPP1 expression in MCF7-luc cells stably expressing ENPP1-MF or anti-NC as a control, detected by qRT–PCR. Expression levels were normalized to those of GAPDH and data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (b) A schematic illustration of the approach used in the experiments shown in c–e. (c) Immunoblot analyses of ENPP1 expression in MCF7-luc anti-NC and MCF7-luc ENPP1-MF cells treated with or without MG132 for 24 h. (d) Immunoblot analyses of ENPP1 in MCF7-luc ENPP1-MF cells grown under adherent (Ad) or mammosphere (Ma) culture conditions for the indicated times. (e) ENPP1 expression in the indicated MCF7-luc derivatives grown under adherent (Ad) or mammosphere (Ma) culture conditions. (f) Growth of the indicated MCF7-luc cell derivatives. An MTT assay was performed to determine the numbers of cells at each time point. Data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student's t-test. (g) Immunofluorescent detection of ENPP1-MF and GFP in MCF7-luc ENPP1-MF cells in paraffin-embedded sections of primary tumour xenografts. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 50 μm. (h) Bioluminescent images of tumours in NOD/SCID mice injected with MCF7-luc shENPP1 cells that were treated with or without docetaxel (DOC). Alternatively, the mice were injected with MCF7-luc Zs-DR-27bs cells as a technical control. Representative images are shown for each cohort.
Mentions: To determine the molecular mechanism by which ENPP1 induces drug resistance in only a small population of breast cancer cells, we investigated the fate of this protein in MCF7-luc cells stably expressing ENPP1-MF (MCF7-luc ENPP-MF), which was generated using a lentivirus vector. Because the lentivirus vector expressing ENPP1 also expressed GFP, a polyclonal cell line was established by flow cytometric sorting of GFP-positive cells. The expression of ENPP1-MF in stable transfectants was confirmed by immunoblotting at day 7 after infection (Supplementary Fig. 10a). However, after 2 weeks of culture, ENPP1-MF expression in these cells was reduced markedly. At the same time point, ENPP1 mRNA expression was tenfold higher in the MCF7-luc ENPP1-MF cells than the control cells (MCF7-luc anti-NC; Fig. 7a), suggesting that the stability of ENPP1 is affected by post-translational modifications.

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