Summary Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and non-tumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429 and miR-183-96-182 were down-regulated in human BCSCs, normal human and murine mammary stem/progenitor cells and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. MiR-200c inhibited the clonogenicity of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated down-regulation of three microRNA clusters and the similar functional regulation of clonogenicity by miR-200c provide a molecular link that connects breast cancer stem cells with normal stem cells.

Cancers originally develop from normal cells that gain the ability to proliferate aberrantly and eventually turn malignant. These cancerous cells then grow clonally into tumors and eventually have the potential to metastasize. A central question in cancer biology is, which cells can be transformed to form tumors? Recent studies elucidated the presence of cancer stem cells that have the exclusive ability to regenerate tumors. These cancer stem cells share many characteristics with normal stem cells, including self-renewal and differentiation. With the growing evidence that cancer stem cells exist in a wide array of tumors, it is becoming increasingly important to understand the molecular mechanisms that regulate self-renewal and differentiation because corruption of genes involved in these pathways likely participates in tumor growth. This new paradigm of oncogenesis has been validated in a growing list of tumors. Studies of normal and cancer stem cells from the same tissue have shed light on the ontogeny of tumors. That signaling pathways such as Bmi1 and Wnt have similar effects in normal and cancer stem cell self-renewal suggests that common molecular pathways regulate both populations. Understanding the biology of cancer stem cells will contribute to the identification of molecular targets important for future therapies.

Cancer is often viewed as a caricature of normal developmental processes, but the extent by which its cellular heterogeneity truly recapitulates multi-lineage differentiation processes of normal tissues remains unknown. Here, we implement “single-cell PCR gene-expression analysis” (SINCE-PCR) to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single-cell (n = 1), we show that transcriptional diversity of cancer tissues is largely explained by in vivo multi-lineage differentiation, not only by clonal genetic heterogeneity. Finally, we show that perturbations in gene-expression programs linked to multi-lineage differentiation strongly associate with patient survival. Guided by SINCE-PCR data, we develop two-gene classifier systems (KRT20 vs CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard-ratios superior to pathological grade and comparable to microarray-derived multi-gene expression signatures.

It is becoming recognized that screening of oncology drugs on a platform using two-dimensionally (2D)-cultured cell lines is unable to precisely select clinically active drugs; therefore three-dimensional (3D)-culture systems are emerging and show potential for better simulating the in vivo tumor microenvironment. The purpose of this study was to reveal the differential effects of chemotherapeutic drugs between 2D- and 3D-cultures and to explore their underlying mechanisms. We evaluated differences between 2D- and 3D-cultured breast cancer cell lines by assessing drug sensitivity, oxygen status and expression of Ki-67 and caspases. Three cell lines (BT-549, BT-474 and T-47D) developed dense multicellular spheroids (MCSs) in 3D-culture, and showed greater resistance to paclitaxel and doxorubicin compared to the 2D-cultured cells. An additional three cell lines (MCF-7, HCC-1954, and MDA-MB‑231) developed only loose MCSs in 3D, and showed drug sensitivities similar to those found in the 2D-culture. Treatment with paclitaxel resulted in greater increases in cleaved-PARP expression in the 2D-culture compared with the 3D-culture, but only in cell lines forming dense 3D-MCSs, suggesting that MCS formation protected the cells from paclitaxel-induced apoptosis. Hypoxia was observed only in the dense 3D-MCSs. BT-549 had fewer cells positive for Ki-67 in 3D- than in 2D-culture, suggesting that the greater G0-dormant subpopulation was responsible for its drug resistance in the 3D-culture. BT-474 had a lower level of caspase-3 in the 3D- than in the 2D-culture, suggesting that the 3D-environment was anti-apoptotic. Finally, we compared staining for Ki-67 and caspases in the 2D- and 3D-primary‑cultured cells originating from a patient-derived xenograft (PDX), fresh PDX tumor, and the patient's original tumor; 2D-cultured cells showed greater proportions of Ki-67-positive and caspase-3-positive cells, in agreement with the view that 3D-primary culture better represents characteristics of tumors in vivo. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important tumor characteristics in vivo, namely hypoxia, dormancy, anti-apoptotic features and their resulting drug resistance.

To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44 + cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.breast cancer | human-in-mouse cancer models | fused optical reporters | bioluminescence imaging C ancer stem cells (CSCs) were first identified in human leukemia (1, 2) and exhibited capacity to form tumors in immunodeficient mice. Because CSCs are characterized from various types of cancers, CD44 has been a useful marker for enriching CSCs not only for breast tumors but also a variety of other epithelial tumor models (3-17). We and others have previously reported that CSCs are more resistant to traditional cancer therapies (4,18,19). There is circumstantial evidence that CSCs may be involved in metastasis of solid tumors, including breast cancer. Breast CSCs (BCSCs) possess an "invasiveness" gene signature that correlates with poor overall survival and shortened metastasis-free survival in cancer patients (20). Importantly, BCSCs are enriched for cells that can undergo epithelial-mesenchymal cell transition (EMT), which likely plays a critical role in metastases in at least some tumors (21). The observation that microRNAs in normal breast stem cells and BCSCs can regulate both EMT and self-renewal further suggests that CSCs might somehow play a role in metastasis (22). Nonetheless, there remains uncertainty surrounding the contributions of CSCs to metastasis.Understanding the role of CSCs in metastasis requires a reliable, noninvasive measure of BCSC outgrowth and dissemination in representative and predictive models of human metastatic disease. Because of genetic differences in mouse tumors or genetic changes that occur with establishment of cell lines, the commonly used models to study metastases, including those involving human cancer cell lines, mouse tumor models, and/or metastatic tumor models via bloodstream injections, do not fully recapitulate human disease (9,(23)(24)(25). Here, by implanting patient tumors or BCSCs into mouse mammary fat pads and using noninvasive imaging strategies, we established represen...

Glial cell line derived neurotrophic factor (GDNF) signals through a multicomponent receptor complex consisting of RET receptor tyrosine kinase and a member of GDNF family receptor a (GFRa). Recently, it was shown that tyrosine 1062 in RET represents a binding site for SHC adaptor proteins and is crucial for both RAS/mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways. In the present study, we characterized how these two pathways diverge from tyrosine 1062, using human neuroblastoma and primitive neuroectodermal tumor cell lines expressing RET at high levels. In response to GDNF stimulation, SHC bound to GAB1 and GRB2 adaptor proteins as well as RET, and SHC and GAB1 were highly phosphorylated on tyrosine. The complex formation consisting of SHC, GAB1 and GRB2 was almost abolished by replacement of tyrosine 1062 in RET with phenylalanine. Tyrosine-phosphorylated GAB1 was also associated with p85 subunit of PI3-K, resulting in PI3-K and AKT activation, whereas SHC-GRB2-SOS complex was responsible for the RAS/ERK signaling pathway. These results suggested that the RAS and PI3-K pathways activated by GDNF bifurcate mainly through SHC bound to tyrosine 1062 in RET. Furthermore, using luciferase reporter-gene assays, we found that the RAS/ERK and PI3-K signaling pathways are important for activation of CREB and NF-kB in GDNF-treated cells, respectively. Oncogene (2000) 19, 4469 ± 4475.

MicroRNAs (miRNAs) are important regulators of stem and progenitor cell functions. We previously reported that miR-142 and miR-150 are upregulated in human breast cancer stem cells (BCSCs) as compared to the non-tumorigenic breast cancer cells. In this study, we report that miR-142 efficiently recruits the APC mRNA to an RNA-induced silencing complex, activates the canonical WNT signaling pathway in an APC-suppression dependent manner, and activates the expression of miR-150. Enforced expression of miR-142 or miR-150 in normal mouse mammary stem cells resulted in the regeneration of hyperproliferative mammary glands in vivo. Knockdown of endogenous miR-142 effectively suppressed organoid formation by BCSCs and slowed tumor growth initiated by human BCSCs in vivo. These results suggest that in some tumors, miR-142 regulates the properties of BCSCs at least in part by activating the WNT signaling pathway and miR-150 expression.DOI: http://dx.doi.org/10.7554/eLife.01977.001

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