技术资料

Rapamycin analogs,temsirolimus and everolimus,are approved for the treatment of advance renal cell carcinoma (RCC). Currently approved agents inhibit mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). However,the mTOR kinase exists in two distinct multiprotein complexes,mTORC1 and mTORC2,and both complexes may be critical regulators of cell metabolism,growth and proliferation. Furthermore,it has been proposed that drug resistance develops due to compensatory activation of mTORC2 signaling during treatment with temsirolimus or everolimus. We evaluated Ku0063794,which is a small molecule that inhibits both mTOR complexes. Ku0063794 was compared to temsirolimus in preclinical models for renal cell carcinoma. Ku0063794 was effective in inhibiting the phosphorylation of signaling proteins downstream of both mTORC1 and mTORC2,including p70 S6K,4E-BP1 and Akt. Ku0063794 was more effective than temsirolimus in decreasing the viability and growth of RCC cell lines,Caki-1 and 786-O,in vitro by inducing cell cycle arrest and autophagy,but not apoptosis. However,in a xenograft model there was no difference in the inhibition of tumor growth by Ku0063794 or temsirolimus. A potential explanation is that temsirolimus has additional effects on the tumor microenvironment. Consistent with this possibility,temsirolimus,but not Ku0063794,decreased tumor angiogenesis in vivo,and decreased the viability of HUVEC (Human Umbilical Vein Endothelial Cells) cells in vitro at pharmacologically relevant concentrations. Furthermore,expression levels of VEGF and PDGF were lower in Caki-1 and 786-O cells treated with temsirolimus than cells treated with Ku0063794. View Publication

BACKGROUND: The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them,fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However,downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study,we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2,an inhibitor of protein kinase C (PKC),GF109203X (GFX),increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β),suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator,phorbol 12-myristate 13-acetate whereas Gö6976,a selective inhibitor of PKCα,β,and γ isoforms could not counteract the effect of PMA. Intriguingly,functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ,PKCε,and ζ,the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ,and the phosphorylation of AKT was reduced by PKCε in hPS cells. CONCLUSIONS/SIGNIFICANCE: Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT,mitogen-activated protein kinase/ERK-1/2 kinase (MEK),PKC/ERK-1/2 kinase,and PKC/GSK-3β. Addition of GFX with a MEK inhibitor,U0126,in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells. View Publication

AIM To investigate the expression status of periostin in colorectal cancer (CRC) to lay a foundation for managing this disease. METHODS Periostin expression status was detected by immunohistochemistry staining in 720 CRC specimens. The relationship between the periostin protein and clinicopathological factors and prognosis was subsequently determined. RESULT The periostin protein was expressed significantly higher in CD133+ tumor cells compared to CD133- tumor cells. Positively expressed periostin was observed in 218 (30.28%) of the 720 cases. Spearman correlation analysis showed that periostin expression has a linear correlation to the tumor size,histological type,lymph node metastasis,TNM stage,and postoperative liver metastasis (p=0.02,0.035,0.001,0.001,and 0.001,respectively). Multivariate analysis showed that histological type,lymph node metastasis,TNM stage,and periostin were found to be related to liver metastasis (p=0.01,0.035,0.01,and 0.001,respectively). In the Cox regression test,the histological grade,Lymph node metastasis,TNM stage,and periostin were detected as independent prognostic factors (p=0.01,0.001,0.001,and 0.001,respectively). CONCLUSION Periostin was found to be related to the liver metastasis of CRC and may be a potential target for CRC. View Publication

Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors,but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue,we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct,which inhibits signaling through all Notch receptors,and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease,but not a complete abrogation,of these cells in dnMAML-expressing cells. Interestingly,when assessed in secondary assays in vitro or in vivo,there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool,which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population. View Publication

Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context,we explored the growth and differentiation potential of mesenchymal progenitors (MPs) derived in vitro from human embryonic stem cells (hESCs). Similar to MPs isolated from bone marrow,hESC derived MPs (hESC-MPs) efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1,hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5,MEF2C,HAND2 and MYOCD. Nevertheless,NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes,raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that,although hESC-MP derived cells expressed a suite of cardiac related genes,they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes,but treated cells retain a mesenchymal like phenotype. View Publication

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

Platelet-derived growth factor receptors (PDGFR) α and β have been suggested as potential targets for treatment of rhabdomyosarcoma,the most common soft tissue sarcoma in children. This study identifies biologic activities linked to PDGF signaling in rhabdomyosarcoma models and human sample collections. Analysis of gene expression profiles of 101 primary human rhabdomyosarcomas revealed elevated PDGF-C and -D expression in all subtypes,with PDGF-D as the solely overexpressed PDGFRβ ligand. By immunohistochemistry,PDGF-CC,PDGF-DD,and PDGFRα were found in tumor cells,whereas PDGFRβ was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFRα signaling associated with genes involved in the reactivation of developmental programs,PDGF-DD/PDGFRβ signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFRβ in vascular stroma and the alveolar subtype and with presence of metastases. Functional validation of our findings was carried out in molecularly distinct model systems,where therapeutic targeting reduced tumor burden in a PDGFR-dependent manner with effects on cell proliferation,vessel density,and macrophage infiltration. The PDGFR-selective inhibitor CP-673,451 regulated cell proliferation through mechanisms involving reduced phosphorylation of GSK-3α and GSK-3β. Additional tissue culture studies showed a PDGFR-dependent regulation of rhabdosphere formation/cancer cell stemness,differentiation,senescence,and apoptosis. In summary,the study shows a clinically relevant distinction in PDGF signaling in human rhabdomyosarcoma and also suggests continued exploration of the influence of stromal PDGFRs on sarcoma progression. View Publication

In this study,we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor $$-positive (ER$$(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ER$$,HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly,expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover,aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5,indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT,stemness and ultimately tumor progression. Importantly,pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ER$$ expression,downregulation of HER-2/Neu,inhibition of EMT and impaired self-renewal ability,resulting in the suppression of distant metastases. Taken together,our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ER$$(+) breast cancer cells. Moreover,this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ER$$(+) breast cancer patients resistant to conventional endocrine therapy. View Publication

Induced pluripotent stem cells (iPSCs) hold great clinical potential for regenerative medicine. Much work has been done to investigate the mechanisms of their generation,focusing on the cell nucleus. However,the roles of specific organelles and in particular mitochondria in the potential mechanisms of nuclear reprogramming remain unclear. In this study,we sought to determine the role of mitochondrial metabolism transition in nuclear reprogramming. We found that the mitochondrial cristae had remodeled in iPSCs. The efficiency of iPSC generation was significantly reduced by down-regulation of mitochondrial inner membrane protein (IMMT),which regulates the morphology of mitochondrial cristae. Moreover,cells with the oxidative phosphorylation (OXPHOS) advantage had higher reprogramming efficiency than normal cells and the glycolysis intermediate lactic acid enhanced the efficiency of iPSCs generation. Our results show that the remodeling of mitochondrial cristae couples with the generation of iPSCs,suggesting mitochondrial metabolism transition plays an important role in nuclear reprogramming. View Publication

Most forms of chemotherapy employ mechanisms involving induction of oxidative stress,a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However,recent studies have shown that relative redox levels in primary tumors can be heterogeneous,suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies,we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First,the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed ROS-low"). Second� View Publication

Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background,yet the landscapes of in vivo tissues remain largely uncharted. Here,we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages,lineages,and cellular environments. They also reveal global features of the epigenome,related to nuclear architecture,that also vary across cellular phenotypes. Specifically,developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture,and discuss their implications for lineage fidelity,cellular senescence,and reprogramming. ?? 2013 Elsevier Inc. View Publication

A major goal of current hematopoiesis research is to develop in vitro methods suitable for the measurement and characterization of stem cells with long-term in vivo repopulating potential. Previous studies from several centers have suggested the presence in normal human or murine marrow of a population of very primitive cells that are biologically,physically,and pharmacologically different from cells detectable by short-term colony assays and that can give rise to the latter in long-term cultures (LTCs) containing a competent stromal cell layer. In this report,we show that such cultures can be used to provide a quantitative assay for human LTC-initiating cells" based on an assessment of the number of clonogenic cells present after 5-8 weeks. Production of derivative clonogenic cells is shown to be absolutely dependent on the presence of a stromal cell feeder. When this requirement is met� View Publication