技术资料

Mechanisms that maintain cancer stem cells are crucial to tumour progression. The ID2 protein supports cancer hallmarks including the cancer stem cell state. HIFα transcription factors,most notably HIF2α (also known as EPAS1),are expressed in and required for maintenance of cancer stem cells (CSCs). However,the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. Here we report that DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Hypoxia downregulates this phosphorylation via inactivation of DYRK1A and DYRK1B. The activity of these kinases is stimulated in normoxia by the oxygen-sensing prolyl hydroxylase PHD1 (also known as EGLN2). ID2 binds to the VHL ubiquitin ligase complex,displaces VHL-associated Cullin 2,and impairs HIF2α ubiquitylation and degradation. Phosphorylation of Thr27 of ID2 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma,ID2 positively modulates HIF2α activity. Conversely,elevated expression of DYRK1 phosphorylates Thr27 of ID2,leading to HIF2α destabilization,loss of glioma stemness,inhibition of tumour growth,and a more favourable outcome for patients with glioblastoma. View Publication

Network activity homeostatically alters synaptic efficacy to constrain neuronal output. However,it is unclear how such compensatory adaptations coexist with synaptic information storage,especially in established networks. Here,we report that in mature hippocampal neurons in vitro,network activity preferentially regulated excitatory synapses within the proximal dendrites of CA3 neurons. These homeostatic synapses exhibited morphological,functional,and molecular signatures of the specialized contacts between mossy fibers of dentate granule cells and thorny excrescences (TEs) of CA3 pyramidal neurons. In vivo TEs were also selectively and bidirectionally altered by chronic activity changes. TE formation required presynaptic synaptoporin and was suppressed by the activity-inducible kinase,Plk2. These results implicate the mossy fiber-TE synapse as an independently tunable gain control locus that permits efficacious homeostatic adjustment of mossy fiber-CA3 synapses,while preserving synaptic weights that may encode information elsewhere within the mature hippocampal circuit. View Publication

Dendritic protein synthesis plays a critical role in several forms of synaptic plasticity,including BDNF (brain-derived neurotrophic factor)-mediated long-term synaptic potentiation (LTP). Dendritic transcripts are typically transported in a repressed state as components of large ribonucleoprotein complexes,and then translated upon stimulation at,or in the vicinity,of activated synapses. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) is a trans-acting factor involved in dendritic mRNA trafficking,but how the distribution of the protein in dendrites is regulated has not been characterized. Here we found that a fraction of hnRNP A2/B1 is present at the synapse under resting conditions in cultured hippocampal neurons. Accordingly,this ribonucleoprotein was detected in free mRNP,monosomal,and polyribosomal fractions obtained from synaptoneurosomes. Neuronal activity and BDNF treatment increased hnRNP A2/B1 protein levels in the cell body and dendritic compartments,and induced the delivery of this protein to synaptic sites. The activity-dependent accumulation of hnRNP A2/B1 at the synapse required,at least in part,the activation of TrkB receptors,presumably by BDNF. This neurotrophin also upregulated the hnRNP A2/B1 mRNA in the soma but was without effect on the abundance of neuritic hnRNP A2/B1 transcripts. These results show that the distribution of hnRNP A2/B1 is regulated by BDNF and by neuronal activity,an effect that may have a role in BDNF-induced synaptic plasticity events. View Publication

Defects in the control of Wnt signaling have emerged as a recurrent mechanism involved in cancer pathogenesis and acute myeloid leukaemia (AML),including the hematopoietic regeneration-associated WNT10B in AC133bright leukaemia cells,although the existence of a specific mechanism remains unproven. We have obtained evidences for a recurrent rearrangement,which involved the WNT10B locus (WNT10BR) within intron 1 (IVS1) and flanked at the 5' by non-human sequences whose origin remains to be elucidated; it also expressed a transcript variant (WNT10BIVS1) which was mainly detected in a cohort of patients with intermediate/unfavorable risk AML. We also identified in two separate cases,affected by AML and breast cancer respectively,a genomic transposable short form of human WNT10B (ht-WNT10B). The intronless ht-WNT10B resembles a long non-coding RNA (lncRNA),which suggests its involvement in a non-random microhomology-mediated recombination generating the rearranged WNT10BR. Furthermore,our studies supports an autocrine activation primed by the formation of WNT10B-FZD4/5 complexes in the breast cancer MCF7 cells that express the WNT10BIVS1. Chemical interference of WNT-ligands production by the porcupine inhibitor IWP-2 achieved a dose-dependent suppression of the WNT10B-FZD4/5 interactions. These results present the first evidence for a recurrent rearrangement promoted by a mobile ht-WNT10B oncogene,as a relevant mechanism for Wnt involvement in human cancer. View Publication

Neurotrophins and their receptors are frequently expressed in malignant gliomas,yet their functions are largely unknown. Previously,we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However,the role of Trk receptors has not been examined. In this study,we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here,we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC,not TrkA,and they also express neurotrophins NGF,BDNF,and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely,TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further,pharmacological inhibition of both ERK and Akt pathways blocked BDNF,and NT3 stimulated BTIC survival. Importantly,attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling,and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma. View Publication

Peripheral nerve injuries and neuropathies lead to profound functional deficits. Here,we have demonstrated that muscle-derived stem/progenitor cells (MDSPCs) isolated from adult human skeletal muscle (hMDSPCs) can adopt neuronal and glial phenotypes in vitro and ameliorate a critical-sized sciatic nerve injury and its associated defects in a murine model. Transplanted hMDSPCs surrounded the axonal growth cone,while hMDSPCs infiltrating the regenerating nerve differentiated into myelinating Schwann cells. Engraftment of hMDSPCs into the area of the damaged nerve promoted axonal regeneration,which led to functional recovery as measured by sustained gait improvement. Furthermore,no adverse effects were observed in these animals up to 18 months after transplantation. Following hMDSPC therapy,gastrocnemius muscles from mice exhibited substantially less muscle atrophy,an increase in muscle mass after denervation,and reorganization of motor endplates at the postsynaptic sites compared with those from PBS-treated mice. Evaluation of nerve defects in animals transplanted with vehicle-only or myoblast-like cells did not reveal histological or functional recovery. These data demonstrate the efficacy of hMDSPC-based therapy for peripheral nerve injury and suggest that hMDSPC transplantation has potential to be translated for use in human neuropathies. View Publication

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM,peritumor tissue (brain adjacent to tumor,BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased,regardless of the tumor cell presence,whereas GD3 correlated with neoplastic infiltration. In BAT,NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM. View Publication

Embryonal tumors with multilayered rosettes (ETMRs) are rare,deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified,in all cases,C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors,cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2,a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic,brain-specific DNMT3B isoform. View Publication

The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols,immaturity,and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional,and thereby stable,optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons,and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level. View Publication

BIX01294 (Bix) is known to be a euchromatic histone-lysine N-methyltransferase 2 inhibitor and treatment with Bix suppresses cancer cell survival and proliferation. In the present study,it was observed that sequential treatment with low-dose Bix notably increases glioblastoma cell migration and metastasis. It was demonstrated that U251 cells sequentially treated with low-dose Bix exhibited induced characteristic changes in critical epithelial-mesenchymal transition (EMT) markers,including E-cadherin,N-cadherin,β-catenin and zinc finger protein SNAI2. Notably,sequential treatment with Bix also increased the expression of cancer stem cell-associated markers,including sex determining region Y-box 2,octamer-binding transcription factor 4 and cluster of differentiation 133. Neurosphere formation was significantly enhanced in cells sequentially treated with Bix,compared with control cells (control: P=0.011; single treatment of Bix,P=0.045). The results of the present study suggest that accumulation of low-dose Bix enhanced the migration and metastatic potential of glioblastoma cells by regulating EMT-associated gene expression,which may be the cause of the altered properties of glioblastoma stem cells. View Publication

Triple-negative breast cancer (TNBC) has poor prognostic outcome compared with other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here,we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation,whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model,Bcl11a deletion substantially decreases tumour formation,even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level,Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus,BCL11A has an important role in TNBC and normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC-targeted therapies. View Publication

In culture conditions,human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks,which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons,the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study,we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. View Publication