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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 is involved in the drug resistance and tumorigenicity of breast cancer cells.(a) Relative miR-27b expression levels in normal and luminal-type breast cancer tissues. Expression levels were normalized to those of RNU6B. Differences between groups were analysed using unpaired t-tests. (b) Schematic illustration of the miR-27b sensor construct used in the experiments shown in c–f. (c) Expression of Zs-DR 48 h after transfection of MCF7-luc Zs-DR-27bs cells with a negative control or miR-27b-specific LNA. Scale bar, 100 μm. (d) Flow cytometric analyses of Zs-DR expression 48 h after treatment of MCF7-luc Zs-DR-27bs cells with dimethylsulphoxide (DMSO) or docetaxel (DOC). (e) Isolation of tumourigenic MCF7-luc Zs-DR-27bs cells. After transplantation of MCF7-luc Zs-DR-27bs cells into 5-week-old NOD/SCID mice, the cells were isolated and cultivated in vitro. (f) Flow cytometric analyses of Zs-DR expression in tumourigenic MCF7-luc Zs-DR-27bs cells isolated from the mice described in e.
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f1: MiR-27b is involved in the drug resistance and tumorigenicity of breast cancer cells.(a) Relative miR-27b expression levels in normal and luminal-type breast cancer tissues. Expression levels were normalized to those of RNU6B. Differences between groups were analysed using unpaired t-tests. (b) Schematic illustration of the miR-27b sensor construct used in the experiments shown in c–f. (c) Expression of Zs-DR 48 h after transfection of MCF7-luc Zs-DR-27bs cells with a negative control or miR-27b-specific LNA. Scale bar, 100 μm. (d) Flow cytometric analyses of Zs-DR expression 48 h after treatment of MCF7-luc Zs-DR-27bs cells with dimethylsulphoxide (DMSO) or docetaxel (DOC). (e) Isolation of tumourigenic MCF7-luc Zs-DR-27bs cells. After transplantation of MCF7-luc Zs-DR-27bs cells into 5-week-old NOD/SCID mice, the cells were isolated and cultivated in vitro. (f) Flow cytometric analyses of Zs-DR expression in tumourigenic MCF7-luc Zs-DR-27bs cells isolated from the mice described in e.

Mentions: In agreement with a previous clinical study27 and our finding that miR-27b is downregulated in docetaxel-resistant luminal-type breast cancer cells25, a quantitative reverse transcription–PCR (qRT–PCR) analysis revealed that miR-27b expression was significantly lower in luminal-type breast cancer tissues (n=26) than normal tissues (n=9; Fig. 1a). In addition, according to the data in The Cancer Genome Atlas Research Network (http://cancergenome.nih.gov/), miR-27b was significantly downregulated in the luminal-type cancer patients who received the taxane-based adjuvant chemotherapy compared with normal breast tissues (Supplementary Fig. 1a, b). Because reduced miR-27b expression was observed in both non-recurrent and recurrent patients (Supplementary Table 1), we hypothesized that downregulation of miR-27b is associated with not only drug resistance, but also tumour initiation. To test this hypothesis, we investigated the expression of miR-27b in non-drug-resistant luminal-type breast cancer MCF7 cells after docetaxel treatment and tumour formation in immunodeficient mice. The expression level of miR-27b in the parental MCF7 cell line was comparable to that in two different normal breast tissue samples (Supplementary Fig. 1c, d). To monitor miR-27b expression, we prepared the MCF7 cells that expressed the sensor vector of miR-27b33343536. After MCF7 cells expressing firefly luciferase (MCF7-luc cells) were prepared by a lentiviral vector, these cells were then transfected with a destabilized fluorescent lentiviral reporter construct (ZsGreen1-DR), whose 3′-untranslated region (3′UTR) contained two miR-27b complementary sequences, to generate MCF7-luc Zs-DR-27bs cells (Fig. 1b and Supplementary Fig. 2a, b). In this system, Zs-DR-positive cells represented those in which miR-27b was downregulated (Fig. 1b). To confirm that the expression of Zs-DR was induced by miR-27b knockdown, endogenous miR-27b expression was inhibited by transfecting the MCF7-luc Zs-DR-27bs cells with a locked nucleic acid (LNA) probe. Treatment of the cells with the miR-27b-specific LNA (LNA-miR-27b; at least 5 nM) induced Zs-DR expression in MCF7-luc Zs-DR-27bs cells, whereas treatment with a control LNA (LNA-NC) did not (Fig. 1c). Next, this cell line was used to examine the effect of docetaxel on miR-27b expression. A flow cytometry analysis revealed that docetaxel treatment induced Zs-DR expression in MCF7-luc Zs-DR-27bs cells markedly (Fig. 1d). Elevated expression of Zs-DR was also observed after tumour formation in 5-week-old non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice that were injected in the hind legs with 105 MCF7-luc Zs-DR-27bs cells (Fig. 1e,f). Together with previous findings, these results suggest that downregulation of miR-27b is involved in the acquisition of docetaxel resistance and tumour seeding ability in 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)

MiR-27b is involved in the drug resistance and tumorigenicity of breast cancer cells.(a) Relative miR-27b expression levels in normal and luminal-type breast cancer tissues. Expression levels were normalized to those of RNU6B. Differences between groups were analysed using unpaired t-tests. (b) Schematic illustration of the miR-27b sensor construct used in the experiments shown in c–f. (c) Expression of Zs-DR 48 h after transfection of MCF7-luc Zs-DR-27bs cells with a negative control or miR-27b-specific LNA. Scale bar, 100 μm. (d) Flow cytometric analyses of Zs-DR expression 48 h after treatment of MCF7-luc Zs-DR-27bs cells with dimethylsulphoxide (DMSO) or docetaxel (DOC). (e) Isolation of tumourigenic MCF7-luc Zs-DR-27bs cells. After transplantation of MCF7-luc Zs-DR-27bs cells into 5-week-old NOD/SCID mice, the cells were isolated and cultivated in vitro. (f) Flow cytometric analyses of Zs-DR expression in tumourigenic MCF7-luc Zs-DR-27bs cells isolated from the mice described in e.
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Related In: Results  -  Collection

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f1: MiR-27b is involved in the drug resistance and tumorigenicity of breast cancer cells.(a) Relative miR-27b expression levels in normal and luminal-type breast cancer tissues. Expression levels were normalized to those of RNU6B. Differences between groups were analysed using unpaired t-tests. (b) Schematic illustration of the miR-27b sensor construct used in the experiments shown in c–f. (c) Expression of Zs-DR 48 h after transfection of MCF7-luc Zs-DR-27bs cells with a negative control or miR-27b-specific LNA. Scale bar, 100 μm. (d) Flow cytometric analyses of Zs-DR expression 48 h after treatment of MCF7-luc Zs-DR-27bs cells with dimethylsulphoxide (DMSO) or docetaxel (DOC). (e) Isolation of tumourigenic MCF7-luc Zs-DR-27bs cells. After transplantation of MCF7-luc Zs-DR-27bs cells into 5-week-old NOD/SCID mice, the cells were isolated and cultivated in vitro. (f) Flow cytometric analyses of Zs-DR expression in tumourigenic MCF7-luc Zs-DR-27bs cells isolated from the mice described in e.
Mentions: In agreement with a previous clinical study27 and our finding that miR-27b is downregulated in docetaxel-resistant luminal-type breast cancer cells25, a quantitative reverse transcription–PCR (qRT–PCR) analysis revealed that miR-27b expression was significantly lower in luminal-type breast cancer tissues (n=26) than normal tissues (n=9; Fig. 1a). In addition, according to the data in The Cancer Genome Atlas Research Network (http://cancergenome.nih.gov/), miR-27b was significantly downregulated in the luminal-type cancer patients who received the taxane-based adjuvant chemotherapy compared with normal breast tissues (Supplementary Fig. 1a, b). Because reduced miR-27b expression was observed in both non-recurrent and recurrent patients (Supplementary Table 1), we hypothesized that downregulation of miR-27b is associated with not only drug resistance, but also tumour initiation. To test this hypothesis, we investigated the expression of miR-27b in non-drug-resistant luminal-type breast cancer MCF7 cells after docetaxel treatment and tumour formation in immunodeficient mice. The expression level of miR-27b in the parental MCF7 cell line was comparable to that in two different normal breast tissue samples (Supplementary Fig. 1c, d). To monitor miR-27b expression, we prepared the MCF7 cells that expressed the sensor vector of miR-27b33343536. After MCF7 cells expressing firefly luciferase (MCF7-luc cells) were prepared by a lentiviral vector, these cells were then transfected with a destabilized fluorescent lentiviral reporter construct (ZsGreen1-DR), whose 3′-untranslated region (3′UTR) contained two miR-27b complementary sequences, to generate MCF7-luc Zs-DR-27bs cells (Fig. 1b and Supplementary Fig. 2a, b). In this system, Zs-DR-positive cells represented those in which miR-27b was downregulated (Fig. 1b). To confirm that the expression of Zs-DR was induced by miR-27b knockdown, endogenous miR-27b expression was inhibited by transfecting the MCF7-luc Zs-DR-27bs cells with a locked nucleic acid (LNA) probe. Treatment of the cells with the miR-27b-specific LNA (LNA-miR-27b; at least 5 nM) induced Zs-DR expression in MCF7-luc Zs-DR-27bs cells, whereas treatment with a control LNA (LNA-NC) did not (Fig. 1c). Next, this cell line was used to examine the effect of docetaxel on miR-27b expression. A flow cytometry analysis revealed that docetaxel treatment induced Zs-DR expression in MCF7-luc Zs-DR-27bs cells markedly (Fig. 1d). Elevated expression of Zs-DR was also observed after tumour formation in 5-week-old non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice that were injected in the hind legs with 105 MCF7-luc Zs-DR-27bs cells (Fig. 1e,f). Together with previous findings, these results suggest that downregulation of miR-27b is involved in the acquisition of docetaxel resistance and tumour seeding ability in 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