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Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus,inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study,we investigated the effects of imetelstat (GRN163L),a potent telomerase inhibitor,on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro,telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally,imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat,but not control oligonucleotides,also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after textless4 weeks of treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in nude mice,concomitant with a reduction in the CSC levels. Differences between telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat,suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy. View Publication

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer worldwide. Signal transducers and activators of transcription 3 (STAT3) signaling is reported to promote tumor malignancy and recurrence in HNSCC. Cucurbitacins,triterpenoid derivatives,are strong STAT3 inhibitors with anticancer properties. Recent studies have shown aldehyde dehydrogenase 1 (ALDH1) to be a marker of cancer stem cells (CSC) in HNSCC. The aim of this study was to investigate the therapeutic effect of cucurbitacin I in HNSCC-derived CSCs. Using immunohistochemical analysis,we firstly showed that CD44,ALDH1,and phosphorylated STAT3 (p-STAT3) were higher in high-grade HNSCCs,and that triple positivity for CD44/ALDH1/p-STAT3 indicated a worse prognosis for HNSCC patients. Secondly,CD44(+)ALDH1(+) cells isolated from seven HNSCC patients showed greater tumorigenicity,radioresistance,and high expression of stemness (Bmi-1/Oct-4/Nanog) and epithelial-mesenchymal-transitional (Snail/Twist) genes as p-STAT3 level increased. Furthermore,we found that cucurbitacin I (JSI-124) can effectively inhibit the expression of p-STAT3 and capacities for tumorigenicity,sphere formation,and radioresistance in HNSCC-CD44(+)ALDH1(+). Notably,150 nmol/L cucurbitacin I effectively blocked STAT3 signaling and downstream survivin and Bcl-2 expression,and it induced apoptosis in HNSCC-CD44(+)ALDH1(+). Moreover,microarray data indicated that 100 nmol/L cucurbitacin I facilitated CD44(+)ALDH1(+) cells to differentiate into CD44?ALDH1? and enhanced the radiosensitivity of HNSCC-CD44(+)ALDH1(+). Xenotransplant experiments revealed that cucurbitacin I combined with radiotherapy significantly suppressed tumorigenesis and lung metastasis and further improved the survival rate in HNSCC-CD44(+)ALDH1(+)-transplanted immunocompromised mice. Taken together,our data show that cucurbitacin I,STAT3 inhibitor,reduces radioresistant,distant-metastatic,and CSC-like properties of HNSCC-CD44(+)ALDH1(+) cells. The potential of cucurbitacin I as a radiosensitizer should be verified in future anti-CSC therapy. View Publication

A relatively rare aldehyde dehydrogenase 1 (ALDH1)-positive stem cell-like" subpopulation of tumor cells has the unique ability to initiate and perpetuate tumor growth; moreover View Publication

BACKGROUND: Cancer stem cells (CSCs) are believed to be responsible for breast cancer formation and recurrence; therefore,therapeutic strategies targeting CSCs must be developed. One approach may be targeting signaling pathways,like Notch,that are involved in stem cell self-renewal and survival. MATERIALS AND METHODS: Breast cancer stem-like cells derived from cell lines and patient samples were examined for Notch expression and activation. The effect of Notch inhibition on sphere formation,proliferation,and colony formation was determined. RESULTS: Breast cancer stem-like cells consistently expressed elevated Notch activation compared with bulk tumor cells. Blockade of Notch signaling using pharmacologic and genomic approaches prevented sphere formation,proliferation,and/or colony formation in soft agar. Interestingly,a gamma-secretase inhibitor,MRK003,induced apoptosis in these cells. CONCLUSION: Our findings support a crucial role for Notch signaling in maintenance of breast cancer stem-like cells,and suggest Notch inhibition may have clinical benefits in targeting CSCs. View Publication

The longevity of organisms is maintained by stem cells. If an organism loses the ability to maintain a balance between quiescence and differentiation in the stem/progenitor cell compartment due to aging and/or stress,this may result in death or age-associated diseases,including cancer. Ewing sarcoma is the most lethal bone tumor in young patients and arises from primitive stem cells. Here,we demonstrated that endogenous Ewing sarcoma gene (Ews) is indispensable for stem cell quiescence,and that the ablation of Ews promotes the early onset of senescence in hematopoietic stem progenitor cells. The phenotypic and functional changes in Ews-deficient stem cells were accompanied by an increase in senescence-associated β-galactosidase staining and a marked induction of p16(INK4a) compared with wild-type counterparts. With its relevance to cancer and possibly aging,EWS is likely to play a significant role in maintaining the functional capacity of stem cells and may provide further insight into the complexity of Ewing sarcoma in the context of stem cells. View Publication

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24(low/?)). Since then,CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties,including aldehyde dehydrogenase (ALDH) activity,have also been used to isolate CSCs from malignant tissues. Recently,we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24,and CD133. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic,but ALDH+ cells are relatively more invasive. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,a fluorescent substrate of ALDH. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells. View Publication

Thyroid carcinoma is the most common endocrine malignancy and the first cause of death among endocrine cancers. We show that the tumorigenic capacity in thyroid cancer is confined in a small subpopulation of stem-like cells with high aldehyde dehydrogenase (ALDH(high)) activity and unlimited replication potential. ALDH(high) cells can be expanded indefinitely in vitro as tumor spheres,which retain the tumorigenic potential upon delivery in immunocompromised mice. Orthotopic injection of minute numbers of thyroid cancer stem cells recapitulates the behavior of the parental tumor,including the aggressive metastatic features of undifferentiated thyroid carcinomas,which are sustained by constitutive activation of cMet and Akt in thyroid cancer stem cells. The identification of tumorigenic and metastagenic thyroid cancer cells may provide unprecedented preclinical tools for development and preclinical validation of novel targeted therapies. View Publication

The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here,endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo,reduced mammosphere formation,and reduced the proportion of CD44(high)/CD24(low) breast tumor cells. Conversely,lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2,Nanog,and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog,KLF4,and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo. View Publication

BACKGROUND: Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy,a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. METHODS: Cancer stem cells were defined as CD44+/CD24? cells that could self-renew (ie,generate cells with the tumorigenic CD44+/CD24? phenotype),differentiate,invade,and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells,weakly tumorigenic parental MCF-7 cells,and MCF-7/MDR,an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry,with in vitro invasion assays,and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. RESULTS: Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg,CD44,TGFB1,and SNAI1). MCF-7/ADR cells were highly invasive,formed mammospheres,and were tumorigenic in mice. In contrast to parental MCF-7 cells,more than 30% of MCF-7/ADR cells had a CD44+/CD24? phenotype,could self-renew,and differentiate (ie,produce CD44+/CD24? and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1,CCNE1,and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field,difference = 6.69 cells per field,95% confidence interval = 4.82 to 8.55 cells per field,P textless .001). No enrichment in the CD44+/CD24? or CD133+ population was detected in MCF-7/MDR. CONCLUSION: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance. View Publication

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines,we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples,72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P textless 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor-initiating studies,where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly,tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations,but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer,ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy,significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%-90% reduction; P textless 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients,and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced,but not absolute,tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer. View Publication

The classification of breast cancer into multiple molecular subtypes has necessitated the need for biomarkers that can assess tumor progression and the effects of chemopreventive agents on specific breast cancer subtypes. The goal of this study was to identify biomarkers whose expression are altered along with estrogen receptor α (ERα) in the polyoma middle-T antigen (PyMT) transgenic model of breast cancer and to investigate the chemopreventive activity of phenethyl isothiocyanate (PEITC). The diet of PyMT female mice was fortified with PEITC (8 mmol/kg) and the mammary streak and/or gross tumors and metastases in lungs were subjected to immunohistochemical analyses for ERα,FOXA1,and GATA-3. FOXA1 is associated with luminal type A cancers,while GATA-3 is a marker of luminal progenitor cell differentiation. In both control and PEITC-treated groups,there was a progressive loss of ERα and FOXA1 but persistence of GATA-3 expression indicating that the tumors retain luminal phenotype. Overall,the PyMT induced tumors exhibited the entire gamut of phenotypes from ERα+/FOXA1+/GATA-3+ tumors in the early stage to ERα±/FOXA1-/GATA-3+ in the late stage. Thus,PyMT model serves as an excellent model for studying progression of luminal subtype tumors. PEITC treated animals had multiple small tumors,indicating delay in tumor progression. Although these tumors were histologically similar to those in controls,there was a lower expression of these biomarkers in normal luminal cells indicating delay in tumor initiation. In in vitro studies,PEITC depleted AldeFluor-positive putative stem/progenitor cells,which may partly be responsible for the delay in tumor initiation. View Publication

There is increasing evidence that breast cancers contain tumor-initiating cells with stem cell properties. The importance of estrogen in the development of the mammary gland and in breast cancer is well known,but the influence of estrogen on the stem cell population has not been assessed. We show that estrogen reduces the proportion of stem cells in the normal human mammary gland and in breast cancer cells. The embryonic stem cell genes NANOG,OCT4,and SOX2 are expressed in normal breast stem cells and at higher levels in breast tumor cells and their expression decreases upon differentiation. Overexpression of each stem cell gene reduces estrogen receptor (ER) expression,and increases the number of stem cells and their capacity for invasion,properties associated with tumorigenesis and poor prognosis. These results indicate that estrogen reduces the size of the human breast stem cell pool and may provide an explanation for the better prognosis of ER-positive tumors. View Publication