[Show abstract][Hide abstract] ABSTRACT: microRNAs are essential for self-renewal and differentiation of normal and malignant stem cells by regulating the expression of key stem cell regulatory genes. Here we report evidence implicating the microRNA100 (miR-100) in self-renewal of cancer stem-like cells (CSC). We found that miR-100 expression levels relate to the cellular differentiation state with lowest expression in cells displaying stem cell markers. Utilizing a tetracycline-inducible lentivirus to elevate expression of miR-100 in human cells, we found that increasing mir-100 levels decreased the production of breast CSCs. This effect was correlated with an inhibition of cancer cell proliferation in vitro and in mouse tumor xenografts due to attenuated expression of the CSC regulatory genes SMARCA5, SMARCD1 and BMPR2. Furthermore, miR-100 induction in breast CSC immediately upon their orthotopic implantation or intracardiac injection completely blocked tumor growth and metastasis formation. Clinically, we observed a significant association between miR-100 expression in breast cancer specimens and patient survival. Our results suggest that miR-100 is required to direct CSC self-renewal and differentiation.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have suggested similarities between cancer stem cells and the epithelial mesenchymal transition (EMT) state. In contrast, other studies suggest that these “states” are mutually exclusive. Our studies suggest that these divergent views may be explained by the existence of multiple stem cell states, which are regulated by microRNAs. Utilizing primary breast tissue and established cell lines, we demonstrate that both normal and malignant breast stem cells exist in distinct, inter-convertible states. The EMT-like state is characterized by expression of vimentin and N-cadherin, slug, snail and twist transcription factors. EMT-like CSCs have a mesenchymal morphology, are largely quiescent, invasive and characterized by expression of the CSC markers CD44+CD24- and are EpCAM-CD49f+. In contrast, the MET (mesenchymal epithelial transition) state of CSCs is characterized by an epithelial morphology and expression of E-cadherin and EpCAM. MET-like CSCs undergo self-renewal and express the CSC marker Aldehyde dehydrogenase (ALDH) and are EpCAM+CD49F+. A subpopulation of cells expressing both CD44+CD24- and ALDH may represent cells in transition between these states. This transition is regulated by signals in the microenvironment which in turn modulate microRNA networks. Expression induction of mir100 in MCF10A cells and several cancer cell lines resulted in a decrease of ALDH-positive CSC population with a concomitant increase in the CD24-CD44+ population accompanied by induction of EMT. We demonstrated mir100 effects are mediated by targeting BMPR2, SMARCA5 and SMARCD1, all of which may contribute to induction of EMT. Moreover, we show that mir100 overexpression induces cellular quiescence as shown by Ki67 and Brdu staining. Induction of mir100 expression immediately upon orthotopic implantation or after the tumors are established significantly reduced the subsequent tumor growth in NOD/SCID mice. In contrast of overexpression of mir100 stimulates cell invasion in vitro matrigel assay. The existence of multiple cancer stem cell states has important implications for understanding stem cell plasticity as well as tumor growth and metastasis. In addition, the existence of these states has implications for the development of CSC-targeting therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Breast cancer can be grouped by expression profiling into categories that differ in biological and clinical characteristics. Molecular characterization of these subtypes on the basis of stem cell markers has shown that basal and claudin-low tumor bear stem cell like characteristics with a higher population of CD44+/CD24- and CD49f+/EpCAM- cells, which also display characteristics of epithelial mesenchymal tumor transition (EMT). There are several genetic and epigenetic signaling pathways that regulate the stem cell like characteristics of these subtypes. We examined the role of microRNA221 (mir-221) in sub populations of normal breast cells isolated from reduction mammoplasties as well as in cancer cell lines from different molecular subtypes of breast cancer. We found that mir-221 drives cells towards more basal subtype and induces EMT in both normal mammary cells and breast cancer cell lines. On the basis of CD49f and ESA staining in the cells isolated from normal mammary tissue, we found high mir-221 expression in the CD49f+/EpCAM- population. Furthermore, we showed that ALDH+ cells from mammospheres expressed significantly higher mir-221 compared to ALDH- cells. In order to determine the effect of mir-221 on the stem cell population, we overexpressed mir-221 in cells from primary mammospheres and showed that overexpression of mir-221 increases the CD49f+/EpCAM- population with increased expression of mesenchymal biomarkers, an effect also seen in a non-transformed breast cell line MCF10A. Overexpression of mir-221 in luminal breast cancer cell lines MCF-7 and T47D cell lines increased the proportion of CD44+/CD24- cells. Furthermore, overexpression of mir-221 significantly stimulated the tumor growth of MCF7 xenographs in NOD/SCID mice. These studies suggest that mir-221 regulates breast CSCs by promoting EMT. This may play an important role in tumor behavior and treatment resistance.
[Show abstract][Hide abstract] 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 abstract][Hide abstract] ABSTRACT: There is increasing evidence that many human cancers, including breast cancer, are driven and maintained by a cellular subcomponent that displays stem cell properties. These “cancer stem cells” (CSCs) may also mediate tumor metastasis and contribute to treatment resistance and relapse. Our group was the first to describe a subpopulation in breast cancer that displayed stem cell properties and was characterized by expression of the cell surface markers ESA and CD44 in the absence of expression of the marker CD24. These cells have been termed “breast cancer stem cells” (BCSCs). More recently, we have demonstrated that breast cancer cells contain subpopulations with stem cell properties that can be isolated by virtue of their expression of Aldehyde dehydrogenase (ALDH) as assessed by the Aldefluor assay. Interestingly, these markers identify overlapping, but not identical cell populations. Recently it has been suggested that the cell surface markers EpCAM and CD49f can be used to define heterogeneous population of normal mouse and human mammary cells. It has been suggested that EpCAM-CD49f+ cells represent mammary stem cells. However, more recent study suggest that in addition to luminal progenitors, a portion of EpCAM+CD49f+ cells may also contain a population with stem cell characteristics. More and more studies have linked the EMT state to cancer stem cells and EMT cells are largely quiescent. As described previously by our lab, CD24-CD44+ and ALDH identify overlapping, but not identical cell populations. Our data showed that EpCAM+CD49f+ cells (MET-like) contain much more ALDH+ cells than other populations, and CD24-CD44+ cells are mainly contained in EpCAM-CD49f+ population (EMT-like). We demonstrated, among microRNAs differently expressed in Aldefluor-positive and Aldefluor-negative populations of breast cancer cell lines we identified micrRNA100 (mir100). We found that mir100 is significantly increased in Aldefluor-negative compared to Aldefluor-positive populations in both normal breast cells and breast cancer cells. Utilizing a tetracycline inducible lentivirus driving mir100 expression, we found that induction of mir100 expression decreased the ALDH-positive population but increased CD24-CD44+ population along with either increasing EpCAM- cells or CD49f+ cells in both normal and cancerous breast cells in vitro, which is a character of EMT. Furthermore, induction of mir100 expression immediately upon orthotopic implantation or after the tumor established significantly blocked subsequent tumor growth. Induction of mir100 expression immediately upon intra-cardiac injection completely blocked metastasis formation. Moreover, we showed that mir100 overexpression increased the quiescent cells but decreased proliferative cells by Ki67 and BrDu staining. We further identified BMPR2, SMARCA5 and SMARCD1 as mir100 targets in ALDH+ cells. These studies indicats the existence of alternative CSC state and mir100 plays a functional role in modulating breast cancer stem cell EMT state, suggesting that the acquisition of stem cell markers may reflect transition of CSC states rather than generation of CSCs from non-CSC populations. In addition, the existence of multiple stem cell states suggests the necessity of developing of therapeutic strategies capable of effectively targeting CSCs in all of these states.
[Show abstract][Hide abstract] ABSTRACT: There is increasing evidence that many human cancers, including breast cancer, are driven and maintained by a cellular subcomponent that displays stem cell properties. These “cancer stem cells” (CSCs) may also mediate tumor metastasis and contribute to treatment resistance and relapse. Our laboratory has identified cellular markers and developed in vitro and mouse models to isolate and characterize normal and malignant human mammary stem cells. We have previously demonstrated that breast cancer cell lines contain subpopulations with stem cell properties that can be isolated by virtue of their expression of Aldehyde dehydrogenase (ALDH) as assessed by the Aldefluor assay. We demonstrated, among microRNAs differently expressed in Aldefluor-positive and Aldefluor-negative populations of breast cancer cell lines we identified micrRNA100 (mir100). We found that mir100 is significantly increased in Aldefluor-negative compared to Aldefluor-positive populations. Utilizing a tetracycline inducible lentivirus driving mir100 expression, we found that induction of mir100 expression decreased the ALDH-positive population of SUM159 cells in vitro as well as in mouse xenografts where this reduction was associated with decreased tumor growth. Furthermore, induction of mir100 expression immediately upon orthotopic implantation or intracardiac injection completely blocked subsequent tumor growth and metastasis formation. These studies demonstrate that mir100 plays a functional role in the self-renewal and differentiation of breast cancer stem cells. Furthermore, the TET-inducible mir100 system allows for controlled regulation of the cancer stem cell population providing a valuable model to simulate the effects of CSC-directed therapies on breast cancer growth and metastasis.