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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

Functional analysis of ENPP1 in MCF7-luc cells.(a) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. (b) Quantification of the SP fractions shown in a, 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. (c) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. (d) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. (e) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. 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. (f) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. (g) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.
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f6: Functional analysis of ENPP1 in MCF7-luc cells.(a) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. (b) Quantification of the SP fractions shown in a, 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. (c) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. (d) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. (e) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. 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. (f) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. (g) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.

Mentions: To confirm that ENPP1 induces generation of the SP fraction of MCF7 cells by regulating the expression and cell surface localization of ABCG2, a flow cytometric analysis of MCF7-ENPP1-MF cells was performed (Fig. 6a and Supplementary Fig. 8e). As expected, the SP fraction of the MCF7-ENPP1-MF cells was approximately fivefold larger than that of control cells transfected with a vector expressing GFP, and this increase was abolished after treatment with Ko143 (Fig. 6a,b). Because ENPP1 is not a transcription factor and localizes mainly to the cytoplasm and cell membrane41, we hypothesized that ENPP1 induces ABCG2 expression indirectly. Therefore, we performed a flow cytometric analysis using 293T cells overexpressing C-terminally HA-tagged ABCG2 (ABCG2-HA) and ENPP1-MF or firefly luciferase as a control. Overexpression of ENPP1-MF enhanced the cell surface localization of ABCG2 in 293T cells markedly (Fig. 6c). Next, we knocked down ENPP1 in MCF7-luc anti-miR-27b-DR cells using a lentivirus vector (Supplementary Fig. 8f) and examined its effects on the cell surface localization of ABCG2. A flow cytometry analysis revealed that knockdown of ENPP1 inhibited the cell surface localization of ABCG2 in these cells drastically (Fig. 6d). In addition, knockdown of ENPP1 reduced the docetaxel resistance of MCF7-luc anti-miR-27b-DR cells (Fig. 6e).


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)

Functional analysis of ENPP1 in MCF7-luc cells.(a) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. (b) Quantification of the SP fractions shown in a, 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. (c) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. (d) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. (e) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. 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. (f) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. (g) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: Functional analysis of ENPP1 in MCF7-luc cells.(a) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. (b) Quantification of the SP fractions shown in a, 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. (c) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. (d) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. (e) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. 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. (f) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. (g) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.
Mentions: To confirm that ENPP1 induces generation of the SP fraction of MCF7 cells by regulating the expression and cell surface localization of ABCG2, a flow cytometric analysis of MCF7-ENPP1-MF cells was performed (Fig. 6a and Supplementary Fig. 8e). As expected, the SP fraction of the MCF7-ENPP1-MF cells was approximately fivefold larger than that of control cells transfected with a vector expressing GFP, and this increase was abolished after treatment with Ko143 (Fig. 6a,b). Because ENPP1 is not a transcription factor and localizes mainly to the cytoplasm and cell membrane41, we hypothesized that ENPP1 induces ABCG2 expression indirectly. Therefore, we performed a flow cytometric analysis using 293T cells overexpressing C-terminally HA-tagged ABCG2 (ABCG2-HA) and ENPP1-MF or firefly luciferase as a control. Overexpression of ENPP1-MF enhanced the cell surface localization of ABCG2 in 293T cells markedly (Fig. 6c). Next, we knocked down ENPP1 in MCF7-luc anti-miR-27b-DR cells using a lentivirus vector (Supplementary Fig. 8f) and examined its effects on the cell surface localization of ABCG2. A flow cytometry analysis revealed that knockdown of ENPP1 inhibited the cell surface localization of ABCG2 in these cells drastically (Fig. 6d). In addition, knockdown of ENPP1 reduced the docetaxel resistance of MCF7-luc anti-miR-27b-DR cells (Fig. 6e).

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