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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 targets stem cell regulatory genes.A. Schematic representation of the experimental design to identify the direct targets of mir-93 in SUM159 cells. B. Activity of the luciferase gene linked to the 3′UTR of AKT3, SOX4, or STAT3. The pMIR-REPORT firefly luciferase reporter plasmids with the wild-type 3′UTR sequences of AKT3, SOX4, or STAT3 were transiently transfected into pTRIPZ-mir-93-SUM159 cells and an internal control ACTB luciferase reporter was co-transfected for normalization. The cells were treated with or without DOX. Luciferase activities were measured after 48 hr. The relative luciferase activity was calculated as the ratio of (the results from the cells transfected by individual reporter)/(the results from the cells transfected by the internal control in the same cell group). The data are mean and standard deviation (SD) of separate transfections (n = 4). *p<0.05; Error bars represent mean ± STDEV.
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pgen-1002751-g004: mir-93 targets stem cell regulatory genes.A. Schematic representation of the experimental design to identify the direct targets of mir-93 in SUM159 cells. B. Activity of the luciferase gene linked to the 3′UTR of AKT3, SOX4, or STAT3. The pMIR-REPORT firefly luciferase reporter plasmids with the wild-type 3′UTR sequences of AKT3, SOX4, or STAT3 were transiently transfected into pTRIPZ-mir-93-SUM159 cells and an internal control ACTB luciferase reporter was co-transfected for normalization. The cells were treated with or without DOX. Luciferase activities were measured after 48 hr. The relative luciferase activity was calculated as the ratio of (the results from the cells transfected by individual reporter)/(the results from the cells transfected by the internal control in the same cell group). The data are mean and standard deviation (SD) of separate transfections (n = 4). *p<0.05; Error bars represent mean ± STDEV.

Mentions: In order to determine the cellular targets of mir-93 in BCSCs, ALDH+ and ALDH− populations of SUM159 cells were separated and cultured in suspension in the presence or absence of DOX for ten hours. Gene expression profiles in the four populations were determined utilizing Affymetrix oligonucleotide microarrays (Figure 4A). Of the 2,000 genes downregulated at least two-fold upon DOX treatment in the ALDH+ population (Table S1), 127 overlapped with the predicted target sequences of mir-93 including twenty-four genes known to be involved in stem cell regulation (Figure 4A and Table S2) including JAK1, SOX4, STAT3, AKT, E2H1 and HMGAZ. The downregulation of these genes in pTRIPZ-SUM159-mir-93, pTRIPZ-HCC1954-mir-93 cell lines and pTRIPZ-MC1-mir-93 were confirmed with customized PCR array plates (Figures S15, S16, S17). In contrast, only 352 genes were significantly downregulated by DOX in the ALDH− population (Table S3) with twelve of these genes (no stem cell genes) overlapping with the predicted mir-93 targets. These studies suggest that mir-93 regulates the CSC population by simultaneously targeting a number of stem cell regulatory genes. To confirm this, we utilized a luceriferase reporter assay to determine the effect of mir-93 on the expression of the stem cell regulatory genes AKT3, SOX4 and STAT3 selected from the expression profiling data. Expression of mir-93 reduced the level of these stem cell regulatory genes in SUM159 (Figure 4B) and HCC1954 cells (Figure S18) but not in luminal MCF7 and MDA-MB-453 cells (Figure S19). Furthermore, knockdown of STAT3 or SOX4 but not AKT3 decreases the proportion of ALDH+ SUM159 cells suggesting these genes play a role in the regulation of CSC self-renewal (Figure S20). The 127 genes in pTRIPZ-MCF7-mir-93 were also tested with customized PCR array plates, and interestingly, most of the stem cell genes were not knocked-down by mir-93 induction in the ALDH+ proportion of MCF7 (Figure S21).


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 targets stem cell regulatory genes.A. Schematic representation of the experimental design to identify the direct targets of mir-93 in SUM159 cells. B. Activity of the luciferase gene linked to the 3′UTR of AKT3, SOX4, or STAT3. The pMIR-REPORT firefly luciferase reporter plasmids with the wild-type 3′UTR sequences of AKT3, SOX4, or STAT3 were transiently transfected into pTRIPZ-mir-93-SUM159 cells and an internal control ACTB luciferase reporter was co-transfected for normalization. The cells were treated with or without DOX. Luciferase activities were measured after 48 hr. The relative luciferase activity was calculated as the ratio of (the results from the cells transfected by individual reporter)/(the results from the cells transfected by the internal control in the same cell group). The data are mean and standard deviation (SD) of separate transfections (n = 4). *p<0.05; Error bars represent mean ± STDEV.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002751-g004: mir-93 targets stem cell regulatory genes.A. Schematic representation of the experimental design to identify the direct targets of mir-93 in SUM159 cells. B. Activity of the luciferase gene linked to the 3′UTR of AKT3, SOX4, or STAT3. The pMIR-REPORT firefly luciferase reporter plasmids with the wild-type 3′UTR sequences of AKT3, SOX4, or STAT3 were transiently transfected into pTRIPZ-mir-93-SUM159 cells and an internal control ACTB luciferase reporter was co-transfected for normalization. The cells were treated with or without DOX. Luciferase activities were measured after 48 hr. The relative luciferase activity was calculated as the ratio of (the results from the cells transfected by individual reporter)/(the results from the cells transfected by the internal control in the same cell group). The data are mean and standard deviation (SD) of separate transfections (n = 4). *p<0.05; Error bars represent mean ± STDEV.
Mentions: In order to determine the cellular targets of mir-93 in BCSCs, ALDH+ and ALDH− populations of SUM159 cells were separated and cultured in suspension in the presence or absence of DOX for ten hours. Gene expression profiles in the four populations were determined utilizing Affymetrix oligonucleotide microarrays (Figure 4A). Of the 2,000 genes downregulated at least two-fold upon DOX treatment in the ALDH+ population (Table S1), 127 overlapped with the predicted target sequences of mir-93 including twenty-four genes known to be involved in stem cell regulation (Figure 4A and Table S2) including JAK1, SOX4, STAT3, AKT, E2H1 and HMGAZ. The downregulation of these genes in pTRIPZ-SUM159-mir-93, pTRIPZ-HCC1954-mir-93 cell lines and pTRIPZ-MC1-mir-93 were confirmed with customized PCR array plates (Figures S15, S16, S17). In contrast, only 352 genes were significantly downregulated by DOX in the ALDH− population (Table S3) with twelve of these genes (no stem cell genes) overlapping with the predicted mir-93 targets. These studies suggest that mir-93 regulates the CSC population by simultaneously targeting a number of stem cell regulatory genes. To confirm this, we utilized a luceriferase reporter assay to determine the effect of mir-93 on the expression of the stem cell regulatory genes AKT3, SOX4 and STAT3 selected from the expression profiling data. Expression of mir-93 reduced the level of these stem cell regulatory genes in SUM159 (Figure 4B) and HCC1954 cells (Figure S18) but not in luminal MCF7 and MDA-MB-453 cells (Figure S19). Furthermore, knockdown of STAT3 or SOX4 but not AKT3 decreases the proportion of ALDH+ SUM159 cells suggesting these genes play a role in the regulation of CSC self-renewal (Figure S20). The 127 genes in pTRIPZ-MCF7-mir-93 were also tested with customized PCR array plates, and interestingly, most of the stem cell genes were not knocked-down by mir-93 induction in the ALDH+ proportion of MCF7 (Figure S21).

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