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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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mir93 promotes MET in normal breast epithelial cells.A. Single cells were isolated from normal breast tissues and stained with EpCAM-APC and CD49f-FITC for FACS sorting. After sorting, total RNA were isolated from different sorted groups (EpCAM+CD49f−, EpCAM+CD49f+, EpCAM−CD49f+, EpCAM−CD49f−) and mir-93 expression were measured by qRT-PCR. p<0.05; Error bars represent mean ± STDEV. B. Single cells were isolated from normal breast tissues and infected with mir-93-expressing lentiviruses in suspension. After one week, mammospheres were dissociated into single cells and plated in adherent culture in the absence (CTRL) or presence (DOX) of DOX for two weeks. Then, cells were dissociated and stained with EpCAM-APC and CD49f-FITC for FACS analysis. C. MCF10A cells were cultured in the absence (CTRL) or presence (DOX) of DOX for two weeks. Cells were then dissociated and stained with EpCAM-APC and CD49f-FITC for FACS analysis.
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pgen-1002751-g007: mir93 promotes MET in normal breast epithelial cells.A. Single cells were isolated from normal breast tissues and stained with EpCAM-APC and CD49f-FITC for FACS sorting. After sorting, total RNA were isolated from different sorted groups (EpCAM+CD49f−, EpCAM+CD49f+, EpCAM−CD49f+, EpCAM−CD49f−) and mir-93 expression were measured by qRT-PCR. p<0.05; Error bars represent mean ± STDEV. B. Single cells were isolated from normal breast tissues and infected with mir-93-expressing lentiviruses in suspension. After one week, mammospheres were dissociated into single cells and plated in adherent culture in the absence (CTRL) or presence (DOX) of DOX for two weeks. Then, cells were dissociated and stained with EpCAM-APC and CD49f-FITC for FACS analysis. C. MCF10A cells were cultured in the absence (CTRL) or presence (DOX) of DOX for two weeks. Cells were then dissociated and stained with EpCAM-APC and CD49f-FITC for FACS analysis.

Mentions: In addition to breast cancer cells, we also determined the effects of mir-93 expression on normal breast cell differentiation. We utilized flow cytometry to access expression of EpCAM and CD49f in breast epithelial cells obtained from reduction mammoplasties. It has previously been shown that mammary stem cells are contained within the EpCAM−CD49f+ population while double positive (EpCAM+CD49f+) cells are luminal progenitors, EpCAM+CD49f− more differentiated Luminal cells, while EpCAM−CD49f− constitute stromal cells [28]. We compared mir-93 expression levels in these four populations. Interestingly, we found that the highest level of mir-93 is expressed in the EpCAM+CD49f+ population (Figure 7A), which suggested mir-93 was required to maintain the cells as EpCAM+CD49f+. Furthermore, overexpression of mir-93 in freshly isolated normal breast cells or in immortalized non-transformed MCF-10A cells increased the proportion of cells expressing EpCAM (Figure 7B, 7C). These studies suggested that mir-93 played a role in maintaining normal breast cells in an epithelial (EpCAM+) state.


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)

mir93 promotes MET in normal breast epithelial cells.A. Single cells were isolated from normal breast tissues and stained with EpCAM-APC and CD49f-FITC for FACS sorting. After sorting, total RNA were isolated from different sorted groups (EpCAM+CD49f−, EpCAM+CD49f+, EpCAM−CD49f+, EpCAM−CD49f−) and mir-93 expression were measured by qRT-PCR. p<0.05; Error bars represent mean ± STDEV. B. Single cells were isolated from normal breast tissues and infected with mir-93-expressing lentiviruses in suspension. After one week, mammospheres were dissociated into single cells and plated in adherent culture in the absence (CTRL) or presence (DOX) of DOX for two weeks. Then, cells were dissociated and stained with EpCAM-APC and CD49f-FITC for FACS analysis. C. MCF10A cells were cultured in the absence (CTRL) or presence (DOX) of DOX for two weeks. Cells were then dissociated and stained with EpCAM-APC and CD49f-FITC for FACS analysis.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002751-g007: mir93 promotes MET in normal breast epithelial cells.A. Single cells were isolated from normal breast tissues and stained with EpCAM-APC and CD49f-FITC for FACS sorting. After sorting, total RNA were isolated from different sorted groups (EpCAM+CD49f−, EpCAM+CD49f+, EpCAM−CD49f+, EpCAM−CD49f−) and mir-93 expression were measured by qRT-PCR. p<0.05; Error bars represent mean ± STDEV. B. Single cells were isolated from normal breast tissues and infected with mir-93-expressing lentiviruses in suspension. After one week, mammospheres were dissociated into single cells and plated in adherent culture in the absence (CTRL) or presence (DOX) of DOX for two weeks. Then, cells were dissociated and stained with EpCAM-APC and CD49f-FITC for FACS analysis. C. MCF10A cells were cultured in the absence (CTRL) or presence (DOX) of DOX for two weeks. Cells were then dissociated and stained with EpCAM-APC and CD49f-FITC for FACS analysis.
Mentions: In addition to breast cancer cells, we also determined the effects of mir-93 expression on normal breast cell differentiation. We utilized flow cytometry to access expression of EpCAM and CD49f in breast epithelial cells obtained from reduction mammoplasties. It has previously been shown that mammary stem cells are contained within the EpCAM−CD49f+ population while double positive (EpCAM+CD49f+) cells are luminal progenitors, EpCAM+CD49f− more differentiated Luminal cells, while EpCAM−CD49f− constitute stromal cells [28]. We compared mir-93 expression levels in these four populations. Interestingly, we found that the highest level of mir-93 is expressed in the EpCAM+CD49f+ population (Figure 7A), which suggested mir-93 was required to maintain the cells as EpCAM+CD49f+. Furthermore, overexpression of mir-93 in freshly isolated normal breast cells or in immortalized non-transformed MCF-10A cells increased the proportion of cells expressing EpCAM (Figure 7B, 7C). These studies suggested that mir-93 played a role in maintaining normal breast cells in an epithelial (EpCAM+) state.

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