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MicroRNA93 regulates proliferation and differentiation of normal and malignant breast stem cells.

Liu S, Patel SH, Ginestier C, Ibarra I, Martin-Trevino R, Bai S, McDermott SP, Shang L, Ke J, Ou SJ, Heath A, Zhang KJ, Korkaya H, Clouthier SG, Charafe-Jauffret E, Birnbaum D, Hannon GJ, Wicha MS - PLoS Genet. (2012)

Bottom Line: In "claudin(low)" SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFβ signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion.The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated "luminal" phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties.These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Cancer Center, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA. sulingl@med.umich.edu

ABSTRACT
MicroRNAs (miRNAs) play important roles in normal cellular differentiation and oncogenesis. microRNA93 (mir-93), a member of the mir106b-25 cluster, located in intron 13 of the MCM7 gene, although frequently overexpressed in human malignancies may also function as a tumor suppressor gene. Using a series of breast cancer cell lines representing different stages of differentiation and mouse xenograft models, we demonstrate that mir-93 modulates the fate of breast cancer stem cells (BCSCs) by regulating their proliferation and differentiation states. In "claudin(low)" SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFβ signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion. Enforced expression of mir-93 completely blocks tumor development in mammary fat pads and development of metastases following intracardiac injection in mouse xenografts. The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated "luminal" phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties. These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications.

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mir-93 inhibits tumor growth and metastasis by decreasing CSCs in SUM159 cells.A. SUM159 cells were transduced with the pTRIPZ-mir-93 lentivirus and selected with Puromycin for 7 days. Tetracycline (DOX) induces mir-93 expression in suspension-cultured SUM159 cells by 10 hours; B. 1×106 SUM159 cells or pTRIPZ-SUM159 -mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, with (DOX) or without (CTRL) DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. Cells were utilized for Aldefluor assay and stained for Annexin V-APC and DAPI for apoptosis assay. C. 100 k pTRIPZ-SUM159-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. The treatment started as indicated by the red arrow. DOX alone (1 mg/ml in drinking water), or docetaxel (10 mg/kg i.p. once weekly) alone, or the combination inhibits SUM159 tumor growth in vivo (note: The Y-axis is on a logarithmic scale). D. Tumors from each group were collected. ALDH was accessed by the Aldefluor assay on viable dissociated cells and by ALDH1 immunohistochemistry on fixed sections. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4th fatpads of secondary mice, which received no further treatment. F. 10k pTRIPZ-SUM159-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. G. 200k pTRIPZ-SUM159-mir-93-Luc cells in 100 ul of PBS were injected into the left ventricle of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. Metastasis formation was monitored using bioluminescence imaging. Quantification of the normalized photon flux, measured at weekly intervals following inoculation. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that the tumor growth or metastasis is significantly different between the control group and the group with the same colored curve.
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pgen-1002751-g002: mir-93 inhibits tumor growth and metastasis by decreasing CSCs in SUM159 cells.A. SUM159 cells were transduced with the pTRIPZ-mir-93 lentivirus and selected with Puromycin for 7 days. Tetracycline (DOX) induces mir-93 expression in suspension-cultured SUM159 cells by 10 hours; B. 1×106 SUM159 cells or pTRIPZ-SUM159 -mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, with (DOX) or without (CTRL) DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. Cells were utilized for Aldefluor assay and stained for Annexin V-APC and DAPI for apoptosis assay. C. 100 k pTRIPZ-SUM159-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. The treatment started as indicated by the red arrow. DOX alone (1 mg/ml in drinking water), or docetaxel (10 mg/kg i.p. once weekly) alone, or the combination inhibits SUM159 tumor growth in vivo (note: The Y-axis is on a logarithmic scale). D. Tumors from each group were collected. ALDH was accessed by the Aldefluor assay on viable dissociated cells and by ALDH1 immunohistochemistry on fixed sections. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4th fatpads of secondary mice, which received no further treatment. F. 10k pTRIPZ-SUM159-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. G. 200k pTRIPZ-SUM159-mir-93-Luc cells in 100 ul of PBS were injected into the left ventricle of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. Metastasis formation was monitored using bioluminescence imaging. Quantification of the normalized photon flux, measured at weekly intervals following inoculation. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that the tumor growth or metastasis is significantly different between the control group and the group with the same colored curve.

Mentions: We utilized a tetracycline (TET) inducible mir-93 construct tagged with RFP (pTRIPZ-mir-93-RFP) to determine the functional role of mir-93 in CSCs. mir-93 levels were significantly increased by ten hours following tetracycline induction in these cells (Figure 2A). Induction of mir-93 was associated with a significant decrease in the CSC population as assessed by the Aldefluor assay (Figure 2B), which were also seen in two basal breast cancer cell lines HCC1954 and SUM149 (Figure S1B and Figure S7). Furthermore, this decrease did not result from induction of apoptosis in these cells as assessed by Annexin V staining (Figure 2B). Our group and others have previously shown that CSCs were relatively resistant to cytotoxic chemotherapy. Consistent with this, addition of the cytotoxic agent docetaxel resulted in a relative increase in the percentage of Aldefluor-positive cells (Figure 2B), an increase associated with induction of apoptosis in the bulk cell population (42.8% versus 1.1% control) (Figure 2B). The relative increase in the Aldefluor-positive population seen with docetaxel treatment was abrogated by simultaneous mir-93 expression (Figure 2B). These experiments suggested that unlike cytotoxic agents which primarily target the bulk cell population, mir-93 overexpression was able to reduce the CSC population. Moreover, this did not appear to result from increased CSC apoptosis suggesting a potential role for mir-93 in promoting differentiation of CSCs. Furthermore, since the TET-inducible mir-93 system allows for the controlled regulation of CSC populations, it provides a valuable tool for assessing the role of CSCs in tumor growth in mouse xenograft models. Furthermore, the ability to regulate the CSC population during different phases of tumor growth allows for the assessment of the role of these cells in tumor initiation and maintenance.


MicroRNA93 regulates proliferation and differentiation of normal and malignant breast stem cells.

Liu S, Patel SH, Ginestier C, Ibarra I, Martin-Trevino R, Bai S, McDermott SP, Shang L, Ke J, Ou SJ, Heath A, Zhang KJ, Korkaya H, Clouthier SG, Charafe-Jauffret E, Birnbaum D, Hannon GJ, Wicha MS - PLoS Genet. (2012)

mir-93 inhibits tumor growth and metastasis by decreasing CSCs in SUM159 cells.A. SUM159 cells were transduced with the pTRIPZ-mir-93 lentivirus and selected with Puromycin for 7 days. Tetracycline (DOX) induces mir-93 expression in suspension-cultured SUM159 cells by 10 hours; B. 1×106 SUM159 cells or pTRIPZ-SUM159 -mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, with (DOX) or without (CTRL) DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. Cells were utilized for Aldefluor assay and stained for Annexin V-APC and DAPI for apoptosis assay. C. 100 k pTRIPZ-SUM159-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. The treatment started as indicated by the red arrow. DOX alone (1 mg/ml in drinking water), or docetaxel (10 mg/kg i.p. once weekly) alone, or the combination inhibits SUM159 tumor growth in vivo (note: The Y-axis is on a logarithmic scale). D. Tumors from each group were collected. ALDH was accessed by the Aldefluor assay on viable dissociated cells and by ALDH1 immunohistochemistry on fixed sections. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4th fatpads of secondary mice, which received no further treatment. F. 10k pTRIPZ-SUM159-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. G. 200k pTRIPZ-SUM159-mir-93-Luc cells in 100 ul of PBS were injected into the left ventricle of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. Metastasis formation was monitored using bioluminescence imaging. Quantification of the normalized photon flux, measured at weekly intervals following inoculation. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that the tumor growth or metastasis is significantly different between the control group and the group with the same colored curve.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3369932&req=5

pgen-1002751-g002: mir-93 inhibits tumor growth and metastasis by decreasing CSCs in SUM159 cells.A. SUM159 cells were transduced with the pTRIPZ-mir-93 lentivirus and selected with Puromycin for 7 days. Tetracycline (DOX) induces mir-93 expression in suspension-cultured SUM159 cells by 10 hours; B. 1×106 SUM159 cells or pTRIPZ-SUM159 -mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, with (DOX) or without (CTRL) DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. Cells were utilized for Aldefluor assay and stained for Annexin V-APC and DAPI for apoptosis assay. C. 100 k pTRIPZ-SUM159-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. The treatment started as indicated by the red arrow. DOX alone (1 mg/ml in drinking water), or docetaxel (10 mg/kg i.p. once weekly) alone, or the combination inhibits SUM159 tumor growth in vivo (note: The Y-axis is on a logarithmic scale). D. Tumors from each group were collected. ALDH was accessed by the Aldefluor assay on viable dissociated cells and by ALDH1 immunohistochemistry on fixed sections. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4th fatpads of secondary mice, which received no further treatment. F. 10k pTRIPZ-SUM159-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. G. 200k pTRIPZ-SUM159-mir-93-Luc cells in 100 ul of PBS were injected into the left ventricle of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. Metastasis formation was monitored using bioluminescence imaging. Quantification of the normalized photon flux, measured at weekly intervals following inoculation. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that the tumor growth or metastasis is significantly different between the control group and the group with the same colored curve.
Mentions: We utilized a tetracycline (TET) inducible mir-93 construct tagged with RFP (pTRIPZ-mir-93-RFP) to determine the functional role of mir-93 in CSCs. mir-93 levels were significantly increased by ten hours following tetracycline induction in these cells (Figure 2A). Induction of mir-93 was associated with a significant decrease in the CSC population as assessed by the Aldefluor assay (Figure 2B), which were also seen in two basal breast cancer cell lines HCC1954 and SUM149 (Figure S1B and Figure S7). Furthermore, this decrease did not result from induction of apoptosis in these cells as assessed by Annexin V staining (Figure 2B). Our group and others have previously shown that CSCs were relatively resistant to cytotoxic chemotherapy. Consistent with this, addition of the cytotoxic agent docetaxel resulted in a relative increase in the percentage of Aldefluor-positive cells (Figure 2B), an increase associated with induction of apoptosis in the bulk cell population (42.8% versus 1.1% control) (Figure 2B). The relative increase in the Aldefluor-positive population seen with docetaxel treatment was abrogated by simultaneous mir-93 expression (Figure 2B). These experiments suggested that unlike cytotoxic agents which primarily target the bulk cell population, mir-93 overexpression was able to reduce the CSC population. Moreover, this did not appear to result from increased CSC apoptosis suggesting a potential role for mir-93 in promoting differentiation of CSCs. Furthermore, since the TET-inducible mir-93 system allows for the controlled regulation of CSC populations, it provides a valuable tool for assessing the role of CSCs in tumor growth in mouse xenograft models. Furthermore, the ability to regulate the CSC population during different phases of tumor growth allows for the assessment of the role of these cells in tumor initiation and maintenance.

Bottom Line: In "claudin(low)" SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFβ signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion.The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated "luminal" phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties.These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Cancer Center, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA. sulingl@med.umich.edu

ABSTRACT
MicroRNAs (miRNAs) play important roles in normal cellular differentiation and oncogenesis. microRNA93 (mir-93), a member of the mir106b-25 cluster, located in intron 13 of the MCM7 gene, although frequently overexpressed in human malignancies may also function as a tumor suppressor gene. Using a series of breast cancer cell lines representing different stages of differentiation and mouse xenograft models, we demonstrate that mir-93 modulates the fate of breast cancer stem cells (BCSCs) by regulating their proliferation and differentiation states. In "claudin(low)" SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFβ signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion. Enforced expression of mir-93 completely blocks tumor development in mammary fat pads and development of metastases following intracardiac injection in mouse xenografts. The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated "luminal" phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties. These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications.

Show MeSH
Related in: MedlinePlus