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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 initiates MET in SUM159 cells.A. pTRIPZ-SUM159-mir-93 cells were plated in 2-well chamber slides with (DOX) or without (CTRL) Doxycycline for 7 days. E-Cadherin and Vimentin were deleted by immunofluorescence staining. Expression of mir-93 in SUM159 cells causes them to assume a more epithelial appearance associated with a decrease in Vimentin and an increase in membrane localized E-Cadherin expression. The phase micrographs for CTRL and DOX are also shown. E-Cadherin, Green; Vimentin, Red; DAPI, Blue. A representative sample from 3 independent samples is shown. B. The effect of mir-93 expression on a panel of epithelial and mesenchymal markers at the mRNA level as accessed by qPCR. pTRIPZ-SUM159-mir-93 cells were plated with or without DOX, and ALDH+ and ALDH− cells were sorted at different times (12 hours, 1 day, 3 days, 8 days, 15 days) by Aldefluor assay. qRT-PCR was utilized to access the effects of mir-93 on mRNA expression of mesenchymal markers (Vimentin, N-Cadherin and Twist), epithelial markers (E-Cadherin and Claudin), and TGFβR2. *p<0.05; Error bars represent mean ± STDEV.
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pgen-1002751-g006: mir-93 initiates MET in SUM159 cells.A. pTRIPZ-SUM159-mir-93 cells were plated in 2-well chamber slides with (DOX) or without (CTRL) Doxycycline for 7 days. E-Cadherin and Vimentin were deleted by immunofluorescence staining. Expression of mir-93 in SUM159 cells causes them to assume a more epithelial appearance associated with a decrease in Vimentin and an increase in membrane localized E-Cadherin expression. The phase micrographs for CTRL and DOX are also shown. E-Cadherin, Green; Vimentin, Red; DAPI, Blue. A representative sample from 3 independent samples is shown. B. The effect of mir-93 expression on a panel of epithelial and mesenchymal markers at the mRNA level as accessed by qPCR. pTRIPZ-SUM159-mir-93 cells were plated with or without DOX, and ALDH+ and ALDH− cells were sorted at different times (12 hours, 1 day, 3 days, 8 days, 15 days) by Aldefluor assay. qRT-PCR was utilized to access the effects of mir-93 on mRNA expression of mesenchymal markers (Vimentin, N-Cadherin and Twist), epithelial markers (E-Cadherin and Claudin), and TGFβR2. *p<0.05; Error bars represent mean ± STDEV.

Mentions: SUM159 cells are derived from a “claudinlow” subtype of breast cancer which is characterized as having a high proportion of cells displaying “epithelial-mesenchymal transition (EMT)”. This state is characterized by loss of epithelial characteristics such as apical basal polarity and E-Cadherin expression and acquisition of mesenchymal characteristics, including loss of cell polarity and expression of Vimentin. We determined the effects of mir-93 expression on MET of SUM159 cells by assessing markers of these states at the protein and mRNA levels. SUM159 cells have a mesenchymal morphology and express Vimentin, but not the epithelial marker E-Cadherin, an effect not dependent on cell density (Figure 6A). Expression of mir-93 in these cells caused them to assume a more epithelial appearance associated with a decrease in Vimentin and an increase in E-Cadherin expression (Figure 6A). Similar effects were seen in the basal HCC1954 cell line (Figure S25) although these were less pronounced. To confirm and extend these results we determined the effect of mir-93 expression on mRNA expression of a wider panel of epithelial and mesenchymal markers. We also determined the time course of expression of epithelial and mesenchymal marker mRNAs expressed in ALDH+ stem cells and ALDH− non-stem cell populations. Expression of mir-93 in SUM159 cells resulted in a time dependent decrease in expression of mesenchymal markers, Vimentin, N-cadherin and Twist, and an increase in the epithelial markers E-Cadherin and Claudin (Figure 6B). Furthermore, although these effects were seen in ALDH− populations, they were even more pronounced in the ALDH+ stem cell compartment. Since TGFβ is a major inducer of the EMT [26], [27], we examined the effect of mir-93 expression on components of this pathway. Interestingly, expression of mir-93 significantly reduced expression of the mRNA for TGFβR2 in both ALDH+ and ALDH− SUM159 cells. This effect was seen as early as twelve hours, suggesting a potential role for down regulation of TGFβ signaling in inducing the MET.


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 initiates MET in SUM159 cells.A. pTRIPZ-SUM159-mir-93 cells were plated in 2-well chamber slides with (DOX) or without (CTRL) Doxycycline for 7 days. E-Cadherin and Vimentin were deleted by immunofluorescence staining. Expression of mir-93 in SUM159 cells causes them to assume a more epithelial appearance associated with a decrease in Vimentin and an increase in membrane localized E-Cadherin expression. The phase micrographs for CTRL and DOX are also shown. E-Cadherin, Green; Vimentin, Red; DAPI, Blue. A representative sample from 3 independent samples is shown. B. The effect of mir-93 expression on a panel of epithelial and mesenchymal markers at the mRNA level as accessed by qPCR. pTRIPZ-SUM159-mir-93 cells were plated with or without DOX, and ALDH+ and ALDH− cells were sorted at different times (12 hours, 1 day, 3 days, 8 days, 15 days) by Aldefluor assay. qRT-PCR was utilized to access the effects of mir-93 on mRNA expression of mesenchymal markers (Vimentin, N-Cadherin and Twist), epithelial markers (E-Cadherin and Claudin), and TGFβR2. *p<0.05; Error bars represent mean ± STDEV.
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Related In: Results  -  Collection

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

pgen-1002751-g006: mir-93 initiates MET in SUM159 cells.A. pTRIPZ-SUM159-mir-93 cells were plated in 2-well chamber slides with (DOX) or without (CTRL) Doxycycline for 7 days. E-Cadherin and Vimentin were deleted by immunofluorescence staining. Expression of mir-93 in SUM159 cells causes them to assume a more epithelial appearance associated with a decrease in Vimentin and an increase in membrane localized E-Cadherin expression. The phase micrographs for CTRL and DOX are also shown. E-Cadherin, Green; Vimentin, Red; DAPI, Blue. A representative sample from 3 independent samples is shown. B. The effect of mir-93 expression on a panel of epithelial and mesenchymal markers at the mRNA level as accessed by qPCR. pTRIPZ-SUM159-mir-93 cells were plated with or without DOX, and ALDH+ and ALDH− cells were sorted at different times (12 hours, 1 day, 3 days, 8 days, 15 days) by Aldefluor assay. qRT-PCR was utilized to access the effects of mir-93 on mRNA expression of mesenchymal markers (Vimentin, N-Cadherin and Twist), epithelial markers (E-Cadherin and Claudin), and TGFβR2. *p<0.05; Error bars represent mean ± STDEV.
Mentions: SUM159 cells are derived from a “claudinlow” subtype of breast cancer which is characterized as having a high proportion of cells displaying “epithelial-mesenchymal transition (EMT)”. This state is characterized by loss of epithelial characteristics such as apical basal polarity and E-Cadherin expression and acquisition of mesenchymal characteristics, including loss of cell polarity and expression of Vimentin. We determined the effects of mir-93 expression on MET of SUM159 cells by assessing markers of these states at the protein and mRNA levels. SUM159 cells have a mesenchymal morphology and express Vimentin, but not the epithelial marker E-Cadherin, an effect not dependent on cell density (Figure 6A). Expression of mir-93 in these cells caused them to assume a more epithelial appearance associated with a decrease in Vimentin and an increase in E-Cadherin expression (Figure 6A). Similar effects were seen in the basal HCC1954 cell line (Figure S25) although these were less pronounced. To confirm and extend these results we determined the effect of mir-93 expression on mRNA expression of a wider panel of epithelial and mesenchymal markers. We also determined the time course of expression of epithelial and mesenchymal marker mRNAs expressed in ALDH+ stem cells and ALDH− non-stem cell populations. Expression of mir-93 in SUM159 cells resulted in a time dependent decrease in expression of mesenchymal markers, Vimentin, N-cadherin and Twist, and an increase in the epithelial markers E-Cadherin and Claudin (Figure 6B). Furthermore, although these effects were seen in ALDH− populations, they were even more pronounced in the ALDH+ stem cell compartment. Since TGFβ is a major inducer of the EMT [26], [27], we examined the effect of mir-93 expression on components of this pathway. Interestingly, expression of mir-93 significantly reduced expression of the mRNA for TGFβR2 in both ALDH+ and ALDH− SUM159 cells. This effect was seen as early as twelve hours, suggesting a potential role for down regulation of TGFβ signaling in inducing the MET.

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