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Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy, although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation. Treatment of lung tumor cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133, CD117, SSEA-3, TRA1-81, Oct-4, and nuclear beta-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres, maintain self-renewal capacity, and differentiate. Differentiated progenitors lost expression of CD133, gained CK 8/18 and acquired drug sensitivity. In the presence of drugs, differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice, which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF, bFGF, IL-6, IL-8, HGF, PDGF-BB, G-CSF, and SCGF-beta). CSCs also showed elevated levels of expression of human VEGFR2, FGFR2, CXCR1, 2 and 4 receptors. Moreover, human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors. These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy.

The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: \"rigidity dependent\" (those which show an increase in cell growth as extracellular rigidity is increased), and \"rigidity independent\" (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. These observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models.

The human genome generates many thousands of long noncoding RNAs (lncRNAs). A very small number of lncRNAs have been shown to be functional. Liu et al. carried out a large-scale CRISPR-based screen to assess the function of ∼17,000 lncRNAs in seven different human cell lines. A considerable number (∼500) of the tested lncRNAs influenced cell growth, suggesting biological function. In almost all cases, though, the function was highly cell type—specific, often limited to just one cell type. Science , this issue p. 10.1126/science.aah7111 A considerable fraction of long noncoding RNAs have highly cell type–specific biological functions. The human genome produces thousands of long noncoding RNAs (lncRNAs)—transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type–specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.

In 2004, a team led by Jonathan Tilly reported that mice contained oogonial stem cells (OSCs), suggesting that females may be able to generate new oocytes in adulthood—a concept that was, and still is, quite controversial even though those findings have since been replicated by others. In a new report, Tilly and colleagues now perfect the purification of mouse OSCs and, using this technique, they show that similar cells exist in women of reproductive age. They also show that the mouse and human OSCs are able to give rise to oocytes in vivo (in the case of the human cells after xenotransplantation into NOD-SCID mice), while also showing that the mouse OSCs can give rise to embryos after in vitro fertilization. Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro , these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1 n ) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo .

Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo . Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS , in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4 . This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.

Bone marrow stromal cells maintain the adult skeleton by forming osteoblasts throughout life that regenerate bone and repair fractures. We discovered that subsets of these stromal cells, osteoblasts, osteocytes, and hypertrophic chondrocytes secrete a C-type lectin domain protein, Clec11a, which promotes osteogenesis. Clec11a-deficient mice appeared developmentally normal and had normal hematopoiesis but reduced limb and vertebral bone. Clec11a-deficient mice exhibited accelerated bone loss during aging, reduced bone strength, and delayed fracture healing. Bone marrow stromal cells from Clec11a-deficient mice showed impaired osteogenic differentiation, but normal adipogenic and chondrogenic differentiation. Recombinant Clec11a promoted osteogenesis by stromal cells in culture and increased bone mass in osteoporotic mice in vivo. Recombinant human Clec11a promoted osteogenesis by human bone marrow stromal cells in culture and in vivo. Clec11a thus maintains the adult skeleton by promoting the differentiation of mesenchymal progenitors into mature osteoblasts. In light of this, we propose to call this factor Osteolectin.

Key Points Endoreplication occurs in multiple cell types in most animals and plants, typically in terminally differentiated cells. The final level of ploidy is highly cell type- and species-specific. Due to their increased genomic DNA content, endocycling cells can achieve large sizes or massive output of secreted protein products. Endocycle onset requires suppression of mitosis. This is achieved by different mechanisms in different species and cell types. These include transcriptional repression of mitotic cyclin expression, APC/C (anaphase promoting complex, also known as the cyclosome) -mediated degradation of mitotic cyclins and inhibition of cyclin-dependent kinase (CDK) activity. This inhibition often relies on CDK inhibitory proteins. Endoreplication onset and progression are also broadly affected by the activities of the E2F transcription factors and their co-repressors, the RB proteins, which affect the expression of genes involved in mitosis and DNA replication. Endocycle progression involves many of the same factors that control G1–S phase transitions and G1 length in mitotically proliferating cells. Key requirements are the suppression of mitotic CDK (M-CDK) activity, and the retention of oscillating S phase CDK (S-CDK) activity. Various upstream inputs control the speed of endocycling and the timing of endocycle exit. Together these two factors determine final ploidy and also affect cell size. Upstream inputs include nutrients and growth factors, target of rapamycin (TOR) signalling and (in plants) light exposure. Cross-species and cross-kingdom comparisons suggest that endocycles are an ancient cellular innovation that probably evolved many times. Endocycling cells successively replicate their genomes without segregating chromosomes during mitosis and thereby become polyploid. Lack of chromosome segregation typically results from downregulation of mitotic cyclin-dependent kinase activity. Endocycles probably evolved many times, and the various endocycle mechanisms found in nature highlight the versatility of the cell cycle control machinery. In endoreplication cell cycles, known as endocycles, cells successively replicate their genomes without segregating chromosomes during mitosis and thereby become polyploid. Such cycles, for which there are many variants, are widespread in protozoa, plants and animals. Endocycling cells can achieve ploidies of >200,000 C (chromatin-value); this increase in genomic DNA content allows a higher genomic output, which can facilitate the construction of very large cells or enhance macromolecular secretion. These cells execute normal S phases, using a G1–S regulatory apparatus similar to the one used by mitotic cells, but their capability to segregate chromosomes has been suppressed, typically by downregulation of mitotic cyclin-dependent kinase activity. Endocycles probably evolved many times, and the various endocycle mechanisms found in nature highlight the versatility of the cell cycle control machinery.

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.

Irisin, a recently identified myokine that is released from skeletal muscle following exercise, regulates body weight and influences various metabolic diseases such as obesity and diabetes. In this study, human recombinant nonglycosylated P-irisin (expressed in Escherichia coli prokaryote cell system) or glycosylated E-irisin (expressed in Pichia pastoris eukaryote cell system ) were compared to examine the role of recombinant irisin against pancreatic cancer (PC) cells lines, MIA PaCa-2 and Panc03.27. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-di phenyltetrazolium bromide] and cell colony formation assays revealed that irisin significantly inhibited the growth of MIA PaCa-2 and Panc03.27 in a dose-dependent manner. Irisin also induced G1 arrest in both cell lines. Scratch wound healing and transwell assays revealed that irisin also inhibited the migration of PC cells. Irisin reversed the activity of epithelial–mesenchymal transition (EMT) while increasing E-cadherin expression and reducing vimentin expression. Irisin activated the adenosine monophosphate-activated protein kinase (AMPK) pathway and suppressed the mammalian target of rapamycin (mTOR) signaling. Besides, our results suggest that irisin receptors exist on the surface of human MIA PaCa-2 and Panc03.27 cells. Our results clearly demonstrate that irisin suppressed PC cell growth via the activation of AMPK, thereby downregulating the mTOR pathway and inhibiting EMT of PC cells.