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      MicroRNA93 Regulates Proliferation and Differentiation of Normal and Malignant Breast Stem Cells

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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.

          Author Summary

          Recent evidence suggests that many cancers, including those of the breast, are maintained by a population of cancer cells that display stem cell properties. These “cancer stem cells” may also contribute to tumor metastasis, treatment resistance, and relapse. Recently, miRNAs (small non-coding RNAs) have been reported to be capable of functioning as oncogenes or tumor suppressors. miRNA93 (mir-93) is frequently overexpressed in human cancer but, paradoxically, has been found to function as a tumor suppressor in some contexts. 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 by regulating their proliferation and differentiation states. In less differentiated tumors, enforced expression of mir-93 completely blocks tumor development in mammary fat pads and development of metastases following intracardiac injection in mouse xenografts by reducing breast cancer stem cells. However, the effect of mir-93 on the cancer stem cell population is dependent on the cellular differentiation state, with mir-93 expression increasing the cancer stem cell population in more differentiated breast tumors. These studies demonstrate that miRNAs can regulate breast stem cell proliferation and differentiation, an observation with important biological and clinical implications.

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          Most cited references 19

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          Identification of pancreatic cancer stem cells.

          Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. Although data have been provided to support this theory in human blood, brain, and breast cancers, the identity of pancreatic cancer stem cells has not been determined. Using a xenograft model in which primary human pancreatic adenocarcinomas were grown in immunocompromised mice, we identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD44, CD24, and epithelial-specific antigen (ESA). Pancreatic cancer cells with the CD44(+)CD24(+)ESA(+) phenotype (0.2-0.8% of pancreatic cancer cells) had a 100-fold increased tumorigenic potential compared with nontumorigenic cancer cells, with 50% of animals injected with as few as 100 CD44(+)CD24(+)ESA(+) cells forming tumors that were histologically indistinguishable from the human tumors from which they originated. The enhanced ability of CD44(+)CD24(+)ESA(+) pancreatic cancer cells to form tumors was confirmed in an orthotopic pancreatic tail injection model. The CD44(+)CD24(+)ESA(+) pancreatic cancer cells showed the stem cell properties of self-renewal, the ability to produce differentiated progeny, and increased expression of the developmental signaling molecule sonic hedgehog. Identification of pancreatic cancer stem cells and further elucidation of the signaling pathways that regulate their growth and survival may provide novel therapeutic approaches to treat pancreatic cancer, which is notoriously resistant to standard chemotherapy and radiation.
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            Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature.

            Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function, as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally, we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines, establish techniques that can facilitate the characterization of regulatory pathways of CSCs, and identify potential stem cell markers and therapeutic targets.
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              Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy

              Introduction The phenotypic and functional differences between cells that initiate human breast tumors (cancer stem cells) and those that comprise the tumor bulk are difficult to study using only primary tumor tissue. We embarked on this study hypothesizing that breast cancer cell lines would contain analogous hierarchical differentiation programs to those found in primary breast tumors. Methods Eight human breast cell lines (human mammary epithelial cells, and MCF10A, MCF7, SUM149, SUM159, SUM1315 and MDA.MB.231 cells) were analyzed using flow cytometry for CD44, CD24, and epithelial-specific antigen (ESA) expression. Limiting dilution orthotopic injections were used to evaluate tumor initiation, while serial colony-forming unit, reconstitution and tumorsphere assays were performed to assess self-renewal and differentiation. Pulse-chase bromodeoxyuridine (5-bromo-2-deoxyuridine [BrdU]) labeling was used to examine cell cycle and label-retention of cancer stem cells. Cells were treated with paclitaxol and 5-fluorouracil to test selective resistance to chemotherapy, and gene expression profile after chemotherapy were examined. Results The percentage of CD44+/CD24- cells within cell lines does not correlate with tumorigenicity, but as few as 100 cells can form tumors when sorted for CD44+/CD24-/low/ESA+. Furthermore, CD44+/CD24-/ESA+ cells can self-renew, reconstitute the parental cell line, retain BrdU label, and preferentially survive chemotherapy. Conclusion These data validate the use of cancer cell lines as models for the development and testing of novel therapeutics aimed at eradicating cancer stem cells.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                June 2012
                June 2012
                7 June 2012
                : 8
                : 6
                Affiliations
                [1 ]Comprehensive Cancer Center, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America
                [2 ]Centre de Recherche en Cancérologie de Marseille, Laboratoire d'Oncologie Moléculaire, UMR891 Inserm/Institut Paoli-Calmettes, Université de la Méditerranée, Marseille, France
                [3 ]Program in Genetics and Bioinformatics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America
                Baylor College of Medicine, United States of America
                Author notes

                Conceived and designed the experiments: SL CG DB GJH MSW. Performed the experiments: SL SHP CG II RM-T SB SPM LS JK SJO AH KJZ. Analyzed the data: SL CG II HK SGC EC-J MSW. Contributed reagents/materials/analysis tools: SL DB GJH MSW. Wrote the paper: SL MSW.

                Article
                PGENETICS-D-12-00979
                10.1371/journal.pgen.1002751
                3369932
                22685420
                Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 15
                Categories
                Research Article
                Biology

                Genetics

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