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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 promotes tumor growth by increasing CSCs in MCF7 cells.A. Mir-93 is expressed equally in CD24−CD44+ and bulk (non-CD24−CD44+) populations of MCF7 cells. B. 1×106 pTRIPZ-MCF7-mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. DOX alone, docetaxel alone or the combination increased the CD24−CD44+ population in vitro. C. 1000k pTRIPZ-MCF7-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. Treatment was initiated as indicated by the red arrow. DOX alone (1 mg/ml in drinking water) promoted MCF7 tumor growth in vivo; docetaxel (10 mg/kg i.p. once weekly) alone or the combination inhibits MCF7 tumor growth in vivo. D. Tumors from each group were collected. Analysis for CD24 and CD44 was performed on dissociated cells. DOX alone, docetaxel alone, or the combination increased the CD24−CD44+ populations in MCF7. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4th fatpads of secondary mice, which received no further treatment. Cells from DOX-, docetaxel-, or combination-treated tumors formed secondary tumors at all dilutions (50000, 5000, 500), whereas only higher numbers of cells (50000, 5000) obtained from control xenografts were able to generate tumors. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that tumor growth is significantly different between the control group and the group with the same colored curve.
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pgen-1002751-g003: mir-93 promotes tumor growth by increasing CSCs in MCF7 cells.A. Mir-93 is expressed equally in CD24−CD44+ and bulk (non-CD24−CD44+) populations of MCF7 cells. B. 1×106 pTRIPZ-MCF7-mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. DOX alone, docetaxel alone or the combination increased the CD24−CD44+ population in vitro. C. 1000k pTRIPZ-MCF7-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. Treatment was initiated as indicated by the red arrow. DOX alone (1 mg/ml in drinking water) promoted MCF7 tumor growth in vivo; docetaxel (10 mg/kg i.p. once weekly) alone or the combination inhibits MCF7 tumor growth in vivo. D. Tumors from each group were collected. Analysis for CD24 and CD44 was performed on dissociated cells. DOX alone, docetaxel alone, or the combination increased the CD24−CD44+ populations in MCF7. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4th fatpads of secondary mice, which received no further treatment. Cells from DOX-, docetaxel-, or combination-treated tumors formed secondary tumors at all dilutions (50000, 5000, 500), whereas only higher numbers of cells (50000, 5000) obtained from control xenografts were able to generate tumors. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that tumor growth is significantly different between the control group and the group with the same colored curve.

Mentions: Human breast cancer represents a heterogeneous set of diseases with distinct molecular profiles and clinical behaviors [24]. These subtypes may represent different cells of origin and/or differentiation state. It has been proposed that the most undifferentiated “claudinlow” tumors originate from and resemble normal mammary stem cells, whereas the triple-negative basal tumors arise from a more differentiated luminal progenitor cell and the most differentiated luminal tumors which express estrogen and progesterone receptors originate from and are composed of the most differentiated cells [24]. To determine the relationship between mir-93 expression and level of cellular differentiation, we compared the expression of mir-93 in claudinlow (SUM159), basal (HCC1954) and luminal (MCF7) cells. As shown in Figure S11, mir-93 levels correlate with postulated differentiation state of these cell lines. Furthermore, in the claudinlow SUM159 cells and basal HCC1954 cells, mir-93 expression is significantly lower in Aldefluor-positive as compared to Aldefluor-negative populations (Figure S11). In contrast, the CSC population in MCF7 cells characterized by the phenotype CD24−CD44+[25] expressed the same high level of mir-93 as did the other (non-stem) cells constituting the bulk population (Figure 3A, Figure S11). This suggests that mir-93 may play a different role in more differentiated luminal breast cancer than in the more undifferentiated claudinlow and basal subtype. Consistent with this, induction of mir-93 in MCF7 cells increased the CD24−CD44+ population (Figure 3B). Docetaxel also increased this population, as did the combination of mir-93 plus docetaxel (Figure 3B). In xenografts, induction of mir-93 accelerated the growth of MCF7 xenografts compared to control (Figure 3C), findings which were confirmed using two additional luminal cell lines MDA-MB-453 and T47D (Figures S12A and S13A). In contrast, docetaxel reduced tumor growth (Figure 3C). Analysis of treated MCF7 tumors confirmed that mir-93 induction increased the proportion of CD24−CD44+ cells and ALDH+ cells in tumors as did docetaxel or DOX plus docetaxel (Figure 3D). mir-93 expression level was significantly higher in DOX group compared to the control group at the end of treatment (Figure S14). mir-93 induction increased the proportion of ALDH+ cells from 1.01% to 9.5% in MDA-MB-453 tumors (Figure S12B) and from 1.26% to 3.84% in T47D tumors (Figure S13B). Furthermore, the calculated tumor initiating frequency was significantly increased after mir-93 induction (Figure 3E, Figures S12C and S13C). These results were confirmed and extended by demonstrating that mir-93 induction in primary tumors increased their tumor-initiating capacity when implanted into secondary recipients (Figure 3E, Figures S12C, S13C). Together, these experiments suggested that the effects of mir-93 on the CSC population differed in different molecular subtypes of breast cancer, an observation consistent with the hypothesis that miRNA effects might be differentiation state dependent.


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 promotes tumor growth by increasing CSCs in MCF7 cells.A. Mir-93 is expressed equally in CD24−CD44+ and bulk (non-CD24−CD44+) populations of MCF7 cells. B. 1×106 pTRIPZ-MCF7-mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. DOX alone, docetaxel alone or the combination increased the CD24−CD44+ population in vitro. C. 1000k pTRIPZ-MCF7-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. Treatment was initiated as indicated by the red arrow. DOX alone (1 mg/ml in drinking water) promoted MCF7 tumor growth in vivo; docetaxel (10 mg/kg i.p. once weekly) alone or the combination inhibits MCF7 tumor growth in vivo. D. Tumors from each group were collected. Analysis for CD24 and CD44 was performed on dissociated cells. DOX alone, docetaxel alone, or the combination increased the CD24−CD44+ populations in MCF7. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4th fatpads of secondary mice, which received no further treatment. Cells from DOX-, docetaxel-, or combination-treated tumors formed secondary tumors at all dilutions (50000, 5000, 500), whereas only higher numbers of cells (50000, 5000) obtained from control xenografts were able to generate tumors. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that tumor growth is significantly different between the control group and the group with the same colored curve.
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Related In: Results  -  Collection

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

pgen-1002751-g003: mir-93 promotes tumor growth by increasing CSCs in MCF7 cells.A. Mir-93 is expressed equally in CD24−CD44+ and bulk (non-CD24−CD44+) populations of MCF7 cells. B. 1×106 pTRIPZ-MCF7-mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. DOX alone, docetaxel alone or the combination increased the CD24−CD44+ population in vitro. C. 1000k pTRIPZ-MCF7-mir-93 cells were injected into the 4th fatpads of NOD/SCID mice. Treatment was initiated as indicated by the red arrow. DOX alone (1 mg/ml in drinking water) promoted MCF7 tumor growth in vivo; docetaxel (10 mg/kg i.p. once weekly) alone or the combination inhibits MCF7 tumor growth in vivo. D. Tumors from each group were collected. Analysis for CD24 and CD44 was performed on dissociated cells. DOX alone, docetaxel alone, or the combination increased the CD24−CD44+ populations in MCF7. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4th fatpads of secondary mice, which received no further treatment. Cells from DOX-, docetaxel-, or combination-treated tumors formed secondary tumors at all dilutions (50000, 5000, 500), whereas only higher numbers of cells (50000, 5000) obtained from control xenografts were able to generate tumors. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that tumor growth is significantly different between the control group and the group with the same colored curve.
Mentions: Human breast cancer represents a heterogeneous set of diseases with distinct molecular profiles and clinical behaviors [24]. These subtypes may represent different cells of origin and/or differentiation state. It has been proposed that the most undifferentiated “claudinlow” tumors originate from and resemble normal mammary stem cells, whereas the triple-negative basal tumors arise from a more differentiated luminal progenitor cell and the most differentiated luminal tumors which express estrogen and progesterone receptors originate from and are composed of the most differentiated cells [24]. To determine the relationship between mir-93 expression and level of cellular differentiation, we compared the expression of mir-93 in claudinlow (SUM159), basal (HCC1954) and luminal (MCF7) cells. As shown in Figure S11, mir-93 levels correlate with postulated differentiation state of these cell lines. Furthermore, in the claudinlow SUM159 cells and basal HCC1954 cells, mir-93 expression is significantly lower in Aldefluor-positive as compared to Aldefluor-negative populations (Figure S11). In contrast, the CSC population in MCF7 cells characterized by the phenotype CD24−CD44+[25] expressed the same high level of mir-93 as did the other (non-stem) cells constituting the bulk population (Figure 3A, Figure S11). This suggests that mir-93 may play a different role in more differentiated luminal breast cancer than in the more undifferentiated claudinlow and basal subtype. Consistent with this, induction of mir-93 in MCF7 cells increased the CD24−CD44+ population (Figure 3B). Docetaxel also increased this population, as did the combination of mir-93 plus docetaxel (Figure 3B). In xenografts, induction of mir-93 accelerated the growth of MCF7 xenografts compared to control (Figure 3C), findings which were confirmed using two additional luminal cell lines MDA-MB-453 and T47D (Figures S12A and S13A). In contrast, docetaxel reduced tumor growth (Figure 3C). Analysis of treated MCF7 tumors confirmed that mir-93 induction increased the proportion of CD24−CD44+ cells and ALDH+ cells in tumors as did docetaxel or DOX plus docetaxel (Figure 3D). mir-93 expression level was significantly higher in DOX group compared to the control group at the end of treatment (Figure S14). mir-93 induction increased the proportion of ALDH+ cells from 1.01% to 9.5% in MDA-MB-453 tumors (Figure S12B) and from 1.26% to 3.84% in T47D tumors (Figure S13B). Furthermore, the calculated tumor initiating frequency was significantly increased after mir-93 induction (Figure 3E, Figures S12C and S13C). These results were confirmed and extended by demonstrating that mir-93 induction in primary tumors increased their tumor-initiating capacity when implanted into secondary recipients (Figure 3E, Figures S12C, S13C). Together, these experiments suggested that the effects of mir-93 on the CSC population differed in different molecular subtypes of breast cancer, an observation consistent with the hypothesis that miRNA effects might be differentiation state dependent.

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