Analysis of MicroRNA Expression in Breast Cancer Stem Cells

Institution: Stanford University
Investigator(s): Yohei Shimono, M.D., Ph.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12FB-0053 Award: $134,976
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2006)

Breast tumors contain a heterogeneous subpopulation of cancer cells. We have proposed that only a minority population of cancer cells, called breast cancer stem cells, have the extensive ability to self-renew and proliferate. Our laboratory has prospectively identified the solid cancer stem cells from human breast cancer. Cancer stem cells should be the target of cancer therapy because tumor can easily relapse or metastasize, if significant numbers of cancer stem cell remain after treatment. MicroRNAs (miRNAs) are short non-coding regulatory RNAs that are expressed in a tissue-specific and developmentally regulated manner whose expression is frequently aberrant in breast cancer. The ability of miRNA that simultaneously regulate many target genes makes them attractive candidates for regulating stem cell self-renewal and cell fate decisions. If so, then they may provide novel therapeutic targets for the treatment of breast cancer.

We hypothesize that miRNAs are key regulators of breast cancer stem cell self-renewal and differentiation. We further predict that we can identify key miRNAs that regulate the breast cancer stem cells by identifying species that are differentially expressed by the cancer stem cells, their non-tumorigenic progeny, or normal breast epithelial cells. Finally, we predict that anti-miRNA oligonucleotide that targets cancer stem cell specific miRNA will affect self-renewal and differentiation capacities of cancer stem cells and impair breast cancer progression. Our aims are;
#1. To explore the specifically expressed miRNAs in breast cancer stem cells.
#2. To identify the target protein controlled by the cancer stem cell specific miRNAs.
#3. To determine an effect of miRNA knockdown on breast cancer progression.

We will employ flow cytometry to isolate cancer stem cells, other cancer cells, and normal epithelial cells. The source of human breast tumor cells will be large primary tumor or pleural (lung) effusions of breast cancer patients, and xenograft human breast tumors grown in mice. Specific biomarkers will be used to collect cancer stem cells (CD44+CD24-/low Lineage-), other cancer cells that represent non-tumorigenic progeny (CD44+CD24+ Lineage-), or normal breast epithelial cells (ESA+Lineage-). Total RNA will be isolated and smaller RNA that contains miRNA will be enriched. Purified miRNA samples of cancer stem cell population and their progeny will be labeled and miRNA expression profiles will be analyzed by microarray. We will identify the candidate of miRNA target genes by using computational algorithm database, and analyze their expression by Western blotting. The effect of miRNA inhibitors, such as artificial oligonucleotides on the growth of cancer stem cells will be monitored using the mouse xenograft models.

This project will promote our understanding about breast cancer stem cells. It is proposed that tumor will relapse if the cancer stem cells remain after therapies. Our approach will contribute to clarify the difference between breast cancer stem cells and other breast cancer cells. Our project will be the first phase to test the concept of breast cancer stem cell therapy by using anti-miRNA approach.

Final Report (2009)

Human breast cancer contains a “cancer stem cell” population that has higher ability to form tumors when engrafted in mice as compared to the remaining non-tumorigenic breast cancer cells. Targeting breast cancer stem cells is important because current evidence indicates that breast cancer stem cells are more resistant to standard therapies and are responsible for recurrence and metastasis of breast cancer.

MicroRNAs (miRNAs) are a novel class of short RNA molecules and are able to regulate the expression of hundreds of target genes simultaneously. miRNAs control a variety of cell functions including cell proliferation, stem cell maintenance and differentiation. Abnormal expression of certain miRNAs in human cancers is associated with cancer progression and patients’ prognosis.

In this project, we investigate whether miRNAs are important regulators of breast “cancer stem cells”. To this end, we pursued three aims;
#1 systematic screening of expression of 460 miRNAs in human breast cancer stem cells and comparison with non-tumorigenic cancer cells,
#2 identification of the target genes regulated by miRNAs differentially expressed in human breast cancer stem cells,
#3 study of the effect of differentially expressed miRNAs on human breast cancer growth. All three aims were completed successfully.

We found 37 microRNAs that were differentially expressed between human breast cancer stem cells and non-tumorigenic cancer cells. Among them, we identified three clusters, miR-200c-141, miR-200b-200a-429 and miR-183-96-182, that were coordinately down-regulated both in human breast cancer stem cells and in normal human breast stem/progenitor cells. One of the down-regulated miRNAs, miR-200c, negatively controlled expression of BMI1, a known regulator of stem cell self-renewal. Most importantly, miR-200c strongly suppressed the ability of human breast cancer stem cells to drive tumor formation in vivo and the ability of normal mammary stem cells to form mammary ducts.

These data provide evidence that cancer stem cells share with normal stem cells molecular mechanisms regulating growth and differentiation. In addition, our results provide a molecular explanation, at least in part, for the increased tumorigenic ability displayed by cancer stem cells within human breast cancers.

Symposium Abstract (2007)
Breast cancer stem cells are a minority population of cancer cells that have higher tumorigenic capacity than other cancer cells. It is proposed that breast cancer stem cells maintain whole cancer mass and a tumor can easily regenerate if enough cancer stem cells remain after treatment. MicroRNAs (miRNAs) are genomically encoded short RNA species that can control expression of many target genes simultaneously. miRNAs are expressed in a tissue specific manner and recent accumulating evidence propose variety of roles including cell proliferation, apoptosis, differentiation, and stem cell maintenance. In addition, abnormal expression of miRNAs in human cancer is associated with cancer progression and patients’ prognosis.

In this project, we are exploring whether miRNAs are important regulators of breast cancer stem cell function. Single cell suspensions derived from human or mouse breast cancers were sorted by flow cytometry (FACS) to collect breast cancer stem cell population and other less-tumorigenic cancer cell population. Then we compared the expression of 460 miRNAs between cancer stem cells and non-tumorigenic cancer cells by using seven sets of human breast cancers, three sets of mouse breast cancers, and human normal breast tissue samples. miRNA expression profiling revealed that 30 out of 460 miRNAs were specifically expressed in breast cancer stem cell population compared to the non-tumorigenic cancer cells.

Currently, we are identifying the target genes of the breast cancer stem cell miRNAs by computational analysis to determine the underlying molecular and signaling pathways. We speculate that miRNAs and target genes identified in this project are involved in maintenance and/or differentiation of human breast cancer stem cells. This research may clarify the difference between breast cancer stem cells and other less-tumorigenic breast cancer cells. Inhibitors of miRNAs could be new class of therapeutic agents that target breast cancer stem cells to eliminate “the origin of breast cancer.”

The biology of cancer stem cells.
Periodical:Annual Review of Cell and Developmental Biology
Index Medicus: Annu Rev Cell Dev Biol
Authors: Lobo NA, Shimono Y, Qian D, Clarke MF
Yr: 2007 Vol: 23 Nbr: Abs: Pg:675-699

Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells.
Index Medicus: Cell
Authors: Shimono Y, Zabala M, et al., and Clarke MF
Yr: 2009 Vol: 138 Nbr: 3 Abs: Pg:592-603