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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-negative SUM159 cells have increased tumor-initiating capacity.A. ALDH+ cells from SUM159 cells shows lower mir-93 expression level in comparison to ALDH− cells as accessed by qRT-PCR. P<0.05; Error bars represent mean ± STDEV. B. A schematic of mir-93-Sensor-GFP lentiviral construct; C. SUM159 cells were transduced with the mir-93-sensor-GFP lentivirus and selected with hygromycin B, and cells were sorted based on the GFP expression. A serial dilution of mir-93-negative (sensor/GFP-positive) SUM159 cells and mir-93-positive (sensor/GFP-negative) SUM159 cells were injected into the 4th fatpads of NOD/SCID mouse. *p<0.05. D. mir-93-negative cells gave rise to tumors containing both mir-93-negative and mir-93-positive cell populations, but mir-93-positive cells only gave rise to tumors containing mir-93-positive cell populations.
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pgen-1002751-g001: mir-93-negative SUM159 cells have increased tumor-initiating capacity.A. ALDH+ cells from SUM159 cells shows lower mir-93 expression level in comparison to ALDH− cells as accessed by qRT-PCR. P<0.05; Error bars represent mean ± STDEV. B. A schematic of mir-93-Sensor-GFP lentiviral construct; C. SUM159 cells were transduced with the mir-93-sensor-GFP lentivirus and selected with hygromycin B, and cells were sorted based on the GFP expression. A serial dilution of mir-93-negative (sensor/GFP-positive) SUM159 cells and mir-93-positive (sensor/GFP-negative) SUM159 cells were injected into the 4th fatpads of NOD/SCID mouse. *p<0.05. D. mir-93-negative cells gave rise to tumors containing both mir-93-negative and mir-93-positive cell populations, but mir-93-positive cells only gave rise to tumors containing mir-93-positive cell populations.

Mentions: We have previously demonstrated that primary human breast cancers and established breast cancer cell lines contain subpopulations with stem cell properties that can be isolated by virtue of their expression of ALDH as assessed by the Aldefluor assay. These cells displayed increased tumor initiating capacity and metastatic potential compared to corresponding Aldefluor-negative cells [22]. mir-93 was shown as one of the most abundant miRNAs in ALDH− cells [21]. As assessed by qRT-PCR, mir-93 expression was significantly increased in the ALDH− compared to ALDH+ populations in SUM159 claudinlow and HCC1954 basal subtype of human breast cancer (Figure 1A and Figure S1A). As shown in Figure S3, mir-93 expression was lower in CSCs which were characterized by their expression of the CSC markers: ALDH+ or CD24−CD44+. To determine the relationship between mir-93 expression and tumor initiating capacity, we constructed a mir-93 sensor tagged with GFP (mir-93-sensor-GFP) containing a mir-93 target UTR coupled to GFP. In cells transfected with this vector, mir-93 expression results in degradation of GFP mRNA (sensor-negative), whereas mir-93-negative cells express GFP (sensor-positive) (Figure 1B). mir-93 expression was significantly higher in GFP-negative cells than GFP-positive cells (Figure S4) and the ALDH1A1 was much lower in GFP-negative cells than GFP-positive cells as accessed by western blot or immunohistochemical staining (Figure S5). Furthermore, GFP was significantly reduced by overexpression of mir-93 (Figure S6), demonstrating that the sensor reports mir-93 function. The relationship between mir-93 expression and tumor initiation was determined by introducing serial dilutions of sensor-positive (mir-93-negative) and sensor-negative (mir-93-positive) SUM159 cells into the mammary fatpads of NOD/SCID mice. As shown in Figure 1C, sensor-positive (mir-93-negative) cells had significantly higher tumor initiating capacity and CSC frequency than sensor-negative (mir-93-positive) cells. Moreover, mir-93-negative cells gave rise to tumors containing both mir-93-negative and mir-93-positive populations, whereas mir-93-positive cells gave rise only to small, slow growing tumors containing exclusively mir-93-positive populations (Figure 1D). Similar findings were seen using HCC1954 cells (data not shown). These studies demonstrated that in these breast cancer cell lines low mir-93 expression is associated with the CSC phenotype characterized by increased aldehyde dehydrogenase expression, tumor initiating capacity and the ability to generate heterogeneous tumors containing both stem cell and non-stem cell populations.


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-negative SUM159 cells have increased tumor-initiating capacity.A. ALDH+ cells from SUM159 cells shows lower mir-93 expression level in comparison to ALDH− cells as accessed by qRT-PCR. P<0.05; Error bars represent mean ± STDEV. B. A schematic of mir-93-Sensor-GFP lentiviral construct; C. SUM159 cells were transduced with the mir-93-sensor-GFP lentivirus and selected with hygromycin B, and cells were sorted based on the GFP expression. A serial dilution of mir-93-negative (sensor/GFP-positive) SUM159 cells and mir-93-positive (sensor/GFP-negative) SUM159 cells were injected into the 4th fatpads of NOD/SCID mouse. *p<0.05. D. mir-93-negative cells gave rise to tumors containing both mir-93-negative and mir-93-positive cell populations, but mir-93-positive cells only gave rise to tumors containing mir-93-positive cell populations.
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pgen-1002751-g001: mir-93-negative SUM159 cells have increased tumor-initiating capacity.A. ALDH+ cells from SUM159 cells shows lower mir-93 expression level in comparison to ALDH− cells as accessed by qRT-PCR. P<0.05; Error bars represent mean ± STDEV. B. A schematic of mir-93-Sensor-GFP lentiviral construct; C. SUM159 cells were transduced with the mir-93-sensor-GFP lentivirus and selected with hygromycin B, and cells were sorted based on the GFP expression. A serial dilution of mir-93-negative (sensor/GFP-positive) SUM159 cells and mir-93-positive (sensor/GFP-negative) SUM159 cells were injected into the 4th fatpads of NOD/SCID mouse. *p<0.05. D. mir-93-negative cells gave rise to tumors containing both mir-93-negative and mir-93-positive cell populations, but mir-93-positive cells only gave rise to tumors containing mir-93-positive cell populations.
Mentions: We have previously demonstrated that primary human breast cancers and established breast cancer cell lines contain subpopulations with stem cell properties that can be isolated by virtue of their expression of ALDH as assessed by the Aldefluor assay. These cells displayed increased tumor initiating capacity and metastatic potential compared to corresponding Aldefluor-negative cells [22]. mir-93 was shown as one of the most abundant miRNAs in ALDH− cells [21]. As assessed by qRT-PCR, mir-93 expression was significantly increased in the ALDH− compared to ALDH+ populations in SUM159 claudinlow and HCC1954 basal subtype of human breast cancer (Figure 1A and Figure S1A). As shown in Figure S3, mir-93 expression was lower in CSCs which were characterized by their expression of the CSC markers: ALDH+ or CD24−CD44+. To determine the relationship between mir-93 expression and tumor initiating capacity, we constructed a mir-93 sensor tagged with GFP (mir-93-sensor-GFP) containing a mir-93 target UTR coupled to GFP. In cells transfected with this vector, mir-93 expression results in degradation of GFP mRNA (sensor-negative), whereas mir-93-negative cells express GFP (sensor-positive) (Figure 1B). mir-93 expression was significantly higher in GFP-negative cells than GFP-positive cells (Figure S4) and the ALDH1A1 was much lower in GFP-negative cells than GFP-positive cells as accessed by western blot or immunohistochemical staining (Figure S5). Furthermore, GFP was significantly reduced by overexpression of mir-93 (Figure S6), demonstrating that the sensor reports mir-93 function. The relationship between mir-93 expression and tumor initiation was determined by introducing serial dilutions of sensor-positive (mir-93-negative) and sensor-negative (mir-93-positive) SUM159 cells into the mammary fatpads of NOD/SCID mice. As shown in Figure 1C, sensor-positive (mir-93-negative) cells had significantly higher tumor initiating capacity and CSC frequency than sensor-negative (mir-93-positive) cells. Moreover, mir-93-negative cells gave rise to tumors containing both mir-93-negative and mir-93-positive populations, whereas mir-93-positive cells gave rise only to small, slow growing tumors containing exclusively mir-93-positive populations (Figure 1D). Similar findings were seen using HCC1954 cells (data not shown). These studies demonstrated that in these breast cancer cell lines low mir-93 expression is associated with the CSC phenotype characterized by increased aldehyde dehydrogenase expression, tumor initiating capacity and the ability to generate heterogeneous tumors containing both stem cell and non-stem cell populations.

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