Side-population (SP) cells within cancers and cell lines are rare cell populations known to enrich cancer stem-like cells. In this study, we characterized SP cells from the human breast cancer cell line MCF7 as a model for cancer stem-like cells. Compared with non-SP cells, MCF7 SP cells had higher colony-formation ability in vitro and greater tumorigenicity in vivo , suggesting that MCF7 SP cells enrich cancer stem-like cells. cDNA microarray analysis of the SP cells indicated higher expression of ATP-binding cassette transporters and genes involved in quiescence, which were confirmed by quantitative RT-PCR and flow cytometry cell cycle analysis. To identify signal pathways important for cancer stem-like cells, we analyzed cDNA microarray data and identified nine pathways that were altered in the SP cells. To analyze the protein signaling networks, we used reverse-phase signaling pathway protein microarray technology and identified three signaling proteins that are significantly different between MCF7 SP and non-SP cells. Notably, signaling of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR), signal transduction and activator of transcription (STAT3), and phosphatase and tensin homolog (PTEN) was confirmed to be critical for MCF7 SP cell survival and proliferation by pathway specific inhibitors, selected gene knockdown, and in vivo tumorigenicity assay. The STAT3 pathway was found to be positively regulated by mTOR signaling, whereas PTEN served as a negative regulator of both STAT3 and mTOR signaling. This study suggests the existence of prosurvival signaling pathways critical for cancer stem-like cell maintenance, which could be selectively targeted for inhibiting cancer stem-like cells for improved treatment.

Nanog and Oct4 are essential transcription factors that regulate self-renewal and pluripotency of ES cells. However, the mechanisms by which Nanog and Oct4 modulate ES cell fate remain unknown. Through characterization of endogenous Nanog and Oct4 protein complexes in mouse ES cells, we found that these transcription factors interact with each other and associate with proteins from multiple repression complexes, including the NuRD, Sin3A and Pml complexes. In addition, Nanog, Oct4 and repressor proteins co-occupy Nanog-target genes in mouse ES cells, suggesting that Nanog and Oct4 together may communicate with distinct repression complexes to control gene transcription. To our surprise, of the various core components in the NuRD complex with which Nanog and Oct4 interact, Mta1 was preferred, whereas Mbd3 and Rbbp7 were either absent or present at sub-stoichiometric levels. We named this unique Hdac1/2- and Mta1/2-containing complex NODE (for Nanog and Oct4 associated deacetylase). Interestingly, NODE contained histone deacetylase (HDAC) activity that seemed to be comparable to NuRD, and retained its association with Nanog and Oct4 in Mbd3(-/-) ES cells. In contrast to Mbd3 loss-of-function, knockdown of NODE subunits led to increased expression of developmentally regulated genes and ES-cell differentiation. Our data collectively suggest that Nanog and Oct4 associate with unique repressor complexes on their target genes to control ES cell fate.

Pluripotence in embryonic stem (ES) cells and the early mouse embryo is controlled by several key transcription factors such as Oct4, Sox2, Foxd3, Nanog, and signaling molecules such as STAT3, FGF4, FGF4 receptor, and LIF (4,11,18,22,32,35,51). The regulation of expression of these key factors is thus crucial for the maintenance of pluripotence and embryonic development. However, little is known about the upstream factors that regulate the expression of these genes. Oct4, a member of the POU homeodomain family of transcription factors, is the best studied of these factors. Recent studies have shown that Oct4 plays a critical role in embryonic development and cellular differentiation (38,43). Oct4 is expressed in totipotent and pluripotent stem cells of the pregastrulation embryo, primordial germ cells, and oocytes (37,44,47). Oct4 is also highly expressed in ES and embryonic carcinoma cell lines, such as P19 and F9 cells, and is rapidly down-regulated by differentiation induced with retinoic acid (RA) (6, 31). In ES cells, a less than twofold increase in the level of Oct4 mRNA causes the cells to differentiate into primitive endoderm and mesoderm, whereas reduction to less than 50% of normal levels triggers differentiation into trophectoderm (36). Moreover, targeted disruption of the Oct4 gene in mice results in embryonic death at the blastocyst stage and compacted morula cells that do not differentiate along the pluripotent inner cell mass lineage but instead differentiate into trophectodem (32).

The proper maintenance of telomeres is essential for genome stability. Mammalian telomere maintenance is governed by a number of telomere binding proteins, including the newly identified CTC1-STN1-TEN1 (CST) complex. However, the in vivo functions of mammalian CST remain unclear. To address this question, we conditionally deleted CTC1 from mice. We report here that CTC1 null mice experience rapid onset of global cellular proliferative defects and die prematurely from complete bone marrow failure due to the activation of an ATR-dependent G2/M checkpoint. Acute deletion of CTC1 does not result in telomere deprotection, suggesting that mammalian CST is not involved in capping telomeres. Rather, CTC1 facilitates telomere replication by promoting efficient restart of stalled replication forks. CTC1 deletion results in increased loss of leading C-strand telomeres, catastrophic telomere loss and accumulation of excessive ss telomere DNA. Our data demonstrate an essential role for CTC1 in promoting efficient replication and length maintenance of telomeres.

consults and holds stock in Ideaya, and cofounded and holds stock in Cedilla Therapeutics. G.G. receives research funding from IBM and Pharmacyclics and is an inventor on multiple patent applications related to bioinformatic tools, including applications related to MuTect, ABSOLUTE, MSMuTect, MSMutSig and MSIClass. Y.E.M. is an inventor on patent applications related to the bioinformatic tools, MSMuTect, MSMutSig and MSIClass. The Broad Institute filed a US patent application related to the target described in this manuscript.

SummarySLX4 interacts with several endonucleases to resolve structural barriers in DNA metabolism. SLX4 also interacts with telomeric protein TRF2 in human cells. The molecular mechanism of these interactions at telomeres remains unknown. Here, we report the crystal structure of the TRF2-binding motif of SLX4 (SLX4TBM) in complex with the TRFH domain of TRF2 (TRF2TRFH) and map the interactions of SLX4 with endonucleases SLX1, XPF, and MUS81. TRF2 recognizes a unique HxLxP motif on SLX4 via the peptide-binding site in its TRFH domain. Telomeric localization of SLX4 and associated nucleases depend on the SLX4-endonuclease and SLX4-TRF2 interactions and the protein levels of SLX4 and TRF2. SLX4 assembles an endonuclease toolkit that negatively regulates telomere length via SLX1-catalyzed nucleolytic resolution of telomere DNA structures. We propose that the SLX4-TRF2 complex serves as a double-layer scaffold bridging multiple endonucleases with telomeres for recombination-based telomere maintenance.

Embryonic stem (ES) cells are capable of unlimited symmetrical self-renewal and have the potential to differentiate into any cell type in the body (15,16,18,26). The pluripotency of ES cells is maintained by several key regulatory factors, which establish precise patterns of gene expression and are characteristic of the undifferentiated phenotype of ES cells. The transcription factor Oct4 belongs to the POU homeodomain family and plays an essential role in the maintenance of ES cell pluripotency (4,34,37,40). Oct4 is highly expressed in ES cells and embryo carcinoma (EC) cells and is down-regulated upon differentiation of either cell type (35,42). Precise levels of Oct4 are required to sustain stem cell self-renewal, and either up-or down-regulation induces divergent developmental programs. A less than twofold increase in expression causes differentiation into primitive mesoendoderm. In contrast, repression of Oct4 induces loss of pluripotency and differentiation into trophectoderm (35).Oct4 also plays a pivotal role during early development of embryos. Oct4 is expressed throughout the morula and inner cell mass of the blastocyst and is restricted to primordial germ cells after gastrulation. Oct4 is only expressed in the germ cell lineage in adults (4, 41, 44). Oct4-deficient embryos die at the blastocyst stage due to the loss of pluripotency of inner cell mass cells. In the absence of a true inner cell mass, trophoblast proliferation is not maintained and they differentiate into trophectoderm (33). Oct4 regulates the expression of several other genes, including Rex1, Utf1, and Sox2, of which the last was also found to be essential for ES cell maintenance (2, 36).Recently a new transcription factor, Nanog, belonging to the ES cell regulatory network was discovered (8, 31). Nanog is a homeodomain protein that directs propagation of undifferentiated ES cells. The expression profile of Nanog is very similar to that of Oct4 in ES cells and during early embryonic development. Nanog mRNA is present in pluripotent mouse and human ES cell lines and absent from differentiated cells (6, 12). Nanog expression is restricted to the inner cell mass in preimplantation embryos and restricted to embryonic ectoderm of postimplantation embryos (21,31). Endogenous Nanog and elevated Nanog from transgene expression act in parallel with cytokine stimulation of Stat3 to drive ES cell self-renewal, bypassing Stat3 and maintaining Oct4 levels (8, 31). Nanogdeficient inner cell mass failed to generate epiblast and only produced parietal endoderm-like cells (31). Nanog-deficient ES cells lost pluripotency and differentiated into an extraembryonic endoderm lineage (31). The level of Nanog expression is tightly correlated with the undifferentiated state of ES cells (22). These findings establish a central role for Nanog in the maintenance of ES cell pluripotency and the epiblast stage of embryonic development.Differentiation of ES cells is accompanied by down-regulation of the whole series of transcription factors and signaling molecules th...

Accumulating evidence indicates that breast cancer is caused by cancer stem cells and cure of breast cancer requires eradication of breast cancer stem cells. Previous studies with leukemia stem cells have shown that NF-κB pathway is important for leukemia stem cell survival. In this study, by using MCF7 sphere cells as model of breast cancer stem-like cells, we evaluated the effect of NF-κB pathway specific inhibitors on human breast cancer MCF7 sphere cells. Three inhibitors including parthenolide (PTL), pyrrolidinedithiocarbamate (PDTC) and its analog diethyldithiocarbamate (DETC) were found to preferentially inhibit MCF7 sphere cell proliferation. These compounds also showed preferential inhibition in term of proliferation and colony formation on MCF7 side population (SP) cells, a small fraction of MCF7 cells known to enrich in breast cancer stem-like cells. The preferential inhibition effect of these compounds was due to inhibition of the NF-κB activity in both MCF7 sphere and MCF7 cells, with higher inhibition effect on MCF7 sphere cells than on MCF7 cells. PDTC was further evaluated in vivo and showed significant tumor growth inhibition alone but had better tumor growth inhibition in combination with paclitaxel in the mouse xenograft model than either PDTC or paclitaxel alone. This study suggests that breast cancer stem-like cells could be selectively inhibited by targeting signaling pathways important for breast cancer stem-like cells.