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

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

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

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

The mammalian central nervous system (CNS) undergoes significant expansion postnatally,producing astrocytes,oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy,a condition commonly treated with valproate/valproic acid (VPA),a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs,however,may be essential for the differentiation of distinct subpopulations of neurons and glia. Here,we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7-P14,combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG,rostral migratory stream (RMS),and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed,however,that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA,OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity,we found that short term treatment with VPA in vivo reduced neurosphere numbers and size,a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor,Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively,these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered,especially in individuals whose brains are actively undergoing key postnatal time windows of development. View Publication

Background: The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). Method: Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4,KChIP2 and Kv4.3 were quantified from BCs. Results: BCs exhibited spontaneous beating (60.5??2.6bpm) and maximum-diastolic-potential (MDP) of 67.8??0.8mV (n=155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7??1.9 pA/pF at +40mV; n=14). Recovery of Ito from inactivation (at -80mV) showed slow kinetics (??1=200??110ms (12%) and ??2=2380??240ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. Conclusion: The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype. ?? 2013 Elsevier Ltd. View Publication

The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2),synthesised by PIKfyve,regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex,leading to even a mild reduction in PtdIns(3,5)P 2,results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity,using YM-201636,significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly,YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II,an effect that was potentiated by inhibition of lysosomal proteases,suggesting that alterations in autophagy could be a contributing factor to neuronal cell death. View Publication

Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study,we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4,hSox2,hKlf4,and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci,a hallmark of DM1,were detected in DM1 iPSCs,neural stem cells (NSCs),and terminally differentiated neurons and astrocytes. In conclusion,we have successfully established disease-specific human DM1 iPSC lines,NSCs,and neuronal lineages with pathognomonic intranuclear RNA foci,which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development. View Publication

We demonstrate substantial differences in 'adhesive signature' between human pluripotent stem cells (hPSCs),partially reprogrammed cells,somatic cells and hPSC-derived differentiated progeny. We exploited these differential adhesion strengths to rapidly (over approximately 10 min) and efficiently isolate fully reprogrammed induced hPSCs (hiPSCs) as intact colonies from heterogeneous reprogramming cultures and from differentiated progeny using microfluidics. hiPSCs were isolated label free,enriched to 95%-99% purity with textgreater80% survival,and had normal transcriptional profiles,differentiation potential and karyotypes. We also applied this strategy to isolate hPSCs (hiPSCs and human embryonic stem cells) during routine culture and show that it may be extended to isolate hPSC-derived lineage-specific stem cells or differentiated cells. View Publication

Raman spectra often contain undesirable,randomly positioned,intense,narrow-bandwidth,positive,unidirectional spectral features generated when cosmic rays strike charge-coupled device cameras. These must be removed prior to analysis,but doing so manually is not feasible for large data sets. We developed a quick,simple,effective,semi-automated procedure to remove cosmic ray spikes from spectral data sets that contain large numbers of relatively homogenous spectra. Although some inhomogeneous spectral data sets can be accommodated—it requires replacing excessively modified spectra with the originals and removing their spikes with a median filter instead—caution is advised when processing such data sets. In addition,the technique is suitable for interpolating missing spectra or replacing aberrant spectra with good spectral estimates. The method is applied to baseline-flattened spectra and relies on fitting a third-order (or higher) polynomial through all the spectra at every wavenumber. Pixel intensities in excess of a threshold of 3× the noise standard deviation above the fit are reduced to the threshold level. Because only two parameters (with readily specified default values) might require further adjustment,the method is easily implemented for semi-automated processing of large spectral sets. View Publication

BackgroundmicroRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years,the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.Methodology/ResultsUsing a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG,Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle,cell proliferation,p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p,hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).ConclusionOur findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers. View Publication