技术资料

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification,recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines,mouse xenograft models and matched human primary and metastatic tissues,we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER(+)),HER2(-) luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts,administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts,as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore,this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-$$B (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore,these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635-46. textcopyright2012 AACR. View Publication

Activation of the Smoothened (Smo) receptor mediates Hedgehog (Hh) signaling. Hh inhibitors are in clinical trials for cancer,and small-molecule Smo agonists may have therapeutic interests in regenerative medicine. Here,we have generated and validated a pharmacophoric model for Smo agonists and used this model for the virtual screening of a library of commercially available compounds. Among the 20 top-scoring ligands,we have identified and characterized a novel quinolinecarboxamide derivative,propyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido) benzoate,(GSA-10),as a Smo agonist. GSA-10 fits to the agonist pharmacophoric model with two hydrogen bond acceptor groups and four hydrophobic regions. Using pharmacological,biochemical,and molecular approaches,we provide compelling evidence that GSA-10 acts at Smo to promote the differentiation of multipotent mesenchymal progenitor cells into osteoblasts. However,this molecule does not display the hallmarks of reference Smo agonists. Remarkably,GSA-10 does not recognize the classic bodipy-cyclopamine binding site. Its effect on cell differentiation is inhibited by Smo antagonists,such as MRT-83,SANT-1,LDE225,and M25 in the nanomolar range,by GDC-0449 in the micromolar range,but not by cyclopamine and CUR61414. Thus,GSA-10 allows the pharmacological characterization of a novel Smo active site,which is notably not targeted to the primary cilium and strongly potentiated by forskolin and cholera toxin. GSA-10 belongs to a new class of Smo agonists and will be helpful for dissecting Hh mechanism of action,with important implications in physiology and in therapy. View Publication

Graft-versus-host disease (GVHD) is the main complication of allogeneic bone marrow transplantation. Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease the anticancer activity of the allogeneic graft. We previously identified Notch signaling in T cells as a new therapeutic target for preventing GVHD. Notch-deprived T cells showed markedly decreased production of inflammatory cytokines,but normal in vivo proliferation,increased accumulation of regulatory T cells,and preserved anticancer effects. Here,we report that γ-secretase inhibitors can block all Notch signals in alloreactive T cells,but lead to severe on-target intestinal toxicity. Using newly developed humanized antibodies and conditional genetic models,we demonstrate that Notch1/Notch2 receptors and the Notch ligands Delta-like1/4 mediate all the effects of Notch signaling in T cells during GVHD,with dominant roles for Notch1 and Delta-like4. Notch1 inhibition controlled GVHD,but led to treatment-limiting toxicity. In contrast,Delta-like1/4 inhibition blocked GVHD without limiting adverse effects while preserving substantial anticancer activity. Transient blockade in the peritransplant period provided durable protection. These findings open new perspectives for selective and safe targeting of individual Notch pathway components in GVHD and other T cell-mediated human disorders. View Publication

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer deaths among women in the United States,but its pathogenesis is poorly understood. Some epithelial cancers are known to occur in transitional zones between two types of epithelium,whereas others have been shown to originate in epithelial tissue stem cells. The stem cell niche of the ovarian surface epithelium (OSE),which is ruptured and regenerates during ovulation,has not yet been defined unequivocally. Here we identify the hilum region of the mouse ovary,the transitional (or junction) area between the OSE,mesothelium and tubal (oviductal) epithelium,as a previously unrecognized stem cell niche of the OSE. We find that cells of the hilum OSE are cycling slowly and express stem and/or progenitor cell markers ALDH1,LGR5,LEF1,CD133 and CK6B. These cells display long-term stem cell properties ex vivo and in vivo,as shown by our serial sphere generation and long-term lineage-tracing assays. Importantly,the hilum cells show increased transformation potential after inactivation of tumour suppressor genes Trp53 and Rb1,whose pathways are altered frequently in the most aggressive and common type of human EOC,high-grade serous adenocarcinoma. Our study supports experimentally the idea that susceptibility of transitional zones to malignant transformation may be explained by the presence of stem cell niches in those areas. Identification of a stem cell niche for the OSE may have important implications for understanding EOC pathogenesis. View Publication

The incidence of human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) has rapidly increased over the past 30 years,prompting the suggestion that an epidemic maybe on the horizon. Therefore,there is a clinical need to develop alternate therapeutic strategies to manage the growing number of HPV-positive HNSCC patients. High-risk HPV E6 inactivates p53 through two distinct mechanisms; association with E6AP to degrade p53 and association with p300 to block p300-mediated p53 acetylation and activation. In this study,we determined if targeting the E6-p300 interaction is an effective approach to reactivate p53 in HPV-positive HNSCC. Ectopic expression of the CH1 domain of p300 in HPV-positive HNSCC blocks the association between E6 and p300,increases total and acetylated p53 levels and enhances p53 transcriptional activity. Moreover,expression of p21,miR-34a and miR-200c are increased,demonstrating functional p53 reactivation. CH1 overexpression in HPV-positive HNSCC has a global anticancer effect resulting in a decrease in cell proliferation and clonogenic survival and an increase in apoptosis. The in vivo tumor-initiating ability of HPV-positive HNSCC is severely compromised with CH1 overexpression,in part through a reduction in the cancer-initiating cell population. A novel small-molecule CH1 inhibitor,CH1iB,reactivates p53 and potentiates the anticancer activity of cis-platinum in HPV-positive HNSCC cells. Our work shows that CH1-domain inhibitors represent a novel class of p53-reactivation therapeutics for managing HPV-positive HNSCC patients. View Publication

Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible,stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover,the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures,thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction,protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format,a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally,we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents. View Publication

A fundamental impediment to functional recovery from spinal cord injury (SCI) and traumatic brain injury is the lack of sufficient axonal regeneration in the adult central nervous system. There is thus a need to develop agents that can stimulate axon growth to re-establish severed connections. Given the critical role played by protein kinases in regulating axon growth and the potential for pharmacological intervention,small molecule protein kinase inhibitors present a promising therapeutic strategy. Here,we report a robust cell-based phenotypic assay,utilizing primary rat hippocampal neurons,for identifying small molecule kinase inhibitors that promote neurite growth. The assay is highly reliable and suitable for medium-throughput screening,as indicated by its Z'-factor of 0.73. A focused structurally diverse library of protein kinase inhibitors was screened,revealing several compound groups with the ability to strongly and consistently promote neurite growth. The best performing bioassay hit robustly and consistently promoted axon growth in a postnatal cortical slice culture assay. This study can serve as a jumping-off point for structure activity relationship (SAR) and other drug discovery approaches toward the development of drugs for treating SCI and related neurological pathologies. View Publication

Diabetes is the most common chronic disease in the world and causes complications with many diseases,such as heart disease and osteoporosis. Osteoporosis is a systemic bone disease characterized by imbalance in bone resorption and bone formation. Osteoclast is type of bone cell that functions in bone resorption and plays a critical role in bone remodeling. Rosiglitazone and pioglitazone,which belong to Thiazolidinediones(TZDs),are commonly used antidiabetic drugs. As PPARγ full agonists,they can activate PPARγ in a ligand-dependent way. Recent studies indicate that these PPARγ full agonists have some side effects,such as weight gain and bone loss,which may increase the risk of osteoporosis. In contrast,selective PPARγ Modulators (SPPARγMs) are novel PPARγ ligands that can activate PPARγ in different ways and lead to distinct downstream genes. Mice bone marrow cells were stimulated with recombinant mouse RANKL and M-CSF to generate osteoclasts. To determine the effect on osteoclasts formation,PPARγ ligands (Rosiglitazone,Fmoc-L-Leu,and Telmisartan) were added at the beginning of the culture. Rosiglitazone significantly increased the differentiation of multinucleated osteoclasts,while osteoclasts formation triggered by SPPARγMs was much less than that displayed by rosiglitazone. We found that the enhancement of PPARγ ligands may be associated with TRAF6 and downstream ERK signal pathway. We also demonstrated osteoclasts show characteristic M2 phenotype and can be further promoted by PPARγ ligands,especially rosiglitazone. In conclusion,reduced osteoclasts differentiation characteristic of SPPARγMs highlights SPPARγMs potential as therapeutic targets in diabetes,versus traditional antidiabetic drugs. View Publication

The bromodomain protein module,which binds to acetylated lysine,is emerging as an important epigenetic therapeutic target. We report the structure-guided optimization of 3,5-dimethylisoxazole derivatives to develop potent inhibitors of the BET (bromodomain and extra terminal domain) bromodomain family with good ligand efficiency. X-ray crystal structures of the most potent compounds reveal key interactions required for high affinity at BRD4(1). Cellular studies demonstrate that the phenol and acetate derivatives of the lead compounds showed strong antiproliferative effects on MV4;11 acute myeloid leukemia cells,as shown for other BET bromodomain inhibitors and genetic BRD4 knockdown,whereas the reported compounds showed no general cytotoxicity in other cancer cell lines tested. View Publication

BACKGROUND: Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.backslashnbackslashnMETHODS AND RESULTS: Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome,familial hypertrophic cardiomyopathy,and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome,hypertrophic cardiomyopathy,and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs,but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations.backslashnbackslashnCONCLUSIONS: We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects,long QT syndrome,hypertrophic cardiomyopathy,and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays. View Publication

Crosstalk between intracellular signaling pathways has been extensively studied to understand the pluripotency of human pluripotent stem cells (hPSCs),including human embryonic stem cells and human induced pluripotent stem cells (hiPSCs); however,the contribution of NAD(+) -dependent pathways remains largely unknown. Here,we show that NAD(+) depletion by FK866 (a potent inhibitor of NAD(+) biosynthesis) was fatal in hPSCs,particularly when deriving pluripotent cells from somatic cells and maintaining pluripotency. NAD and its precursors (nicotinamide [NAM] and nicotinic acid) fully replenished the NAD(+) depletion by FK866 in hPSCs. However,only NAM effectively enhanced the reprogramming efficiency and kinetics of hiPSC generation and was also significantly advantageous for the maintenance of undifferentiated hPSCs. Our molecular and functional studies reveal that NAM lowers the barriers to reprogramming by accelerating cell proliferation and protecting cells from apoptosis and senescence by alleviating oxidative stress,reactive oxygen species accumulation,and subsequent mitochondrial membrane potential collapse. We provide evidence that the positive effects of NAM (occurring at concentrations well above the physiological range) on pluripotency control are molecularly associated with the repression of p53,p21,and p16. Our findings establish that adequate intracellular NAD(+) content is crucial for pluripotency; the distinct effects of NAM on pluripotency may be dependent not only on its metabolic advantage as a NAD(+) precursor but also on the ability of NAM to enhance resistance to cellular stress. View Publication

Neurogenesis decreases during aging and following cranial radiotherapy,causing a progressive cognitive decline that is currently untreatable. However,functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover,we observed a marked increase in TGF-β1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. In co-cultures,irradiated brain endothelial cells induced the apoptosis of neural stem/progenitor cells via TGF-β/Smad3 signalling. Strikingly,the blockade of TGF-β signalling in vivo using a neutralizing antibody or the selective inhibitor SB-505124 significantly improved neurogenesis in aged and irradiated mice,prevented apoptosis and increased the proliferation of neural stem/progenitor cells. These findings suggest that anti-TGF-β-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging. View Publication