NUT midline carcinoma (NMC) is an aggressive type of squamous cell carcinoma that is defined by the presence of BRD-NUT fusion oncogenes, which encode chimeric proteins that block differentiation and ...maintain tumor growth. BRD-NUT oncoproteins contain two bromodomains whose binding to acetylated histones is required for the blockade of differentiation in NMC, but the mechanisms by which BRD-NUT act remain uncertain. Here, we provide evidence that MYC is a key downstream target of BRD4-NUT. Expression profiling of NMCs shows that the set of genes whose expression is maintained by BRD4-NUT is highly enriched for MYC upregulated genes, and MYC and BRD4-NUT protein expression is strongly correlated in primary NMCs. More directly, we find that BRD4-NUT associates with the MYC promoter and is required to maintain MYC expression in NMC cell lines. Moreover, both siRNA knockdown of MYC and a dominant-negative form of MYC, omomyc, induce differentiation of NMC cells. Conversely, differentiation of NMC cells induced by knockdown of BRD4-NUT is abrogated by enforced expression of MYC. Together, these findings suggest that MYC is a downstream target of BRD4-NUT that is required for maintenance of NMC cells in an undifferentiated, proliferative state. Our findings support a model in which dysregulation of MYC by BRD-NUT fusion proteins has a central role in the pathogenesis of NMC.
Regarding to the ASTER‐validation cohort, ASPECTS and age were the most robust predictors and they formed the initial model. But the predictive performance was significantly improved by adding ...baseline NIHSS score, history of vascular risk factors and onset‐to‐reperfusion time to build an updated final model. This could help identifying subgroup of patients with defavorable outcome and building a prediction tool to improve care management.
Background and purpose
Intravenous thrombolysis plus mechanical thrombectomy (IVT + MT) is the best current management of acute stroke due to large‐vessel occlusion and results in optimal reperfusion for most patients. Nevertheless, some of these patients do not subsequently achieve functional independence. The aim was to identify baseline factors associated with 3‐month independence after optimal reperfusion and to validate a prediction model.
Methods
All consecutive patients with intracranial anterior large‐vessel occlusion, with indication for IVT + MT and achieving optimal reperfusion (defined as modified Treatment in Cerebral Ischaemia score 2b–3), from the THRACE trial and the ETIS registry, were included in order to identify a prediction model. The primary outcome was 3‐month independence modified Rankin Scale (mRS) score ≤ 2. Multivariate inferences invoked forward logistic regression, multiple imputation and bootstrap resampling. Predictive performance was assessed by c‐statistic. Model validation was conducted on patients from the ASTER trial.
Results
Amongst 139 patients (mean age 65.5 years; 54.3% female), predictors of 3‐month mRS ≤ 2 (n = 82) were younger age odds ratio 0.62 per 10‐year increase; 95% confidence interval (CI) 0.53–0.72 and higher Alberta Stroke Program Early Computed Tomography Score (ASPECTS) (odds ratio 1.65 per 1‐point increase; 95% CI 1.47–1.86) with c‐statistic 0.77. Model validation (n = 104/181 patients with 3‐month mRS ≤ 2) demonstrated a moderate discrimination (c‐statistic 0.74; 95% CI 0.66–0.81) combining age and ASPECTS. The validation model was improved by the adjunction of three candidate variables that were found to be predictors. Addition of baseline National Institutes of Health Stroke Scale (NIHSS) score, history of vascular risk factor and onset‐to‐reperfusion time significantly improved discrimination (c‐statistic 0.85; 95% CI 0.83–0.87).
Conclusions
After optimal reperfusion, younger age, higher ASPECTS, lower NIHSS score, shorter onset‐to‐reperfusion time and absence of vascular risk factor were predictive of independence and could help to guide patient management.
An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about ...2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3-NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4-NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3-NUT and BRD4-NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4-NUT and BRD3-NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD-NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3-NUT and BRD4-NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD-NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.
Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of ...activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.
Growing evidence suggests that microRNAs (miRNAs) facilitate the cross-talk between transcriptional modules and signal transduction pathways. MYC and NOTCH1 contribute to the pathogenesis of lymphoid ...malignancies. NOTCH induces MYC, connecting two signaling programs that enhance oncogenicity. Here we show that this relationship is bidirectional and that MYC, via a miRNA intermediary, modulates NOTCH. MicroRNA-30a (miR-30a), a member of a family of miRNAs that are transcriptionally suppressed by MYC, directly binds to and inhibits NOTCH1 and NOTCH2 expression. Using a murine model and genetically modified human cell lines, we confirmed that miR-30a influences NOTCH expression in a MYC-dependent fashion. In turn, through genetic modulation, we demonstrated that intracellular NOTCH1 and NOTCH2, by inducing MYC, suppressed miR-30a. Conversely, pharmacological inhibition of NOTCH decreased MYC expression and ultimately de-repressed miR-30a. Examination of genetic models of gain and loss of miR-30a in diffuse large B-cell lymphoma (DLBCL) and T-acute lymphoblastic leukemia (T-ALL) cells suggested a tumor-suppressive role for this miRNA. Finally, the activity of the miR-30a-NOTCH-MYC loop was validated in primary DLBCL and T-ALL samples. These data define the presence of a miRNA-mediated regulatory circuitry that may modulate the oncogenic signals originating from NOTCH and MYC.
The Notch pathway is an evolutionary conserved signaling cascade that has an essential role in melanoblast and melanocyte stem cell homeostasis. Notch signaling is emerging as a key player in ...melanoma, the most deadly form of skin cancer. In melanoma, Notch1 is inappropriately reactivated and contributes to melanoma tumorigenicity. Here, we propose a novel mechanism by which Notch1 promotes the disease. We found that Notch1 directly regulates the transcription of neuregulin1 (NRG1) by binding to its promoter region. NRG1 is the ligand for ERBB3 and 4, members of the epidermal growth factor family of receptors that are involved in the genesis and progression of a number of cancers. Notch1 and NRG1 expression are associated in melanoma and inhibition of NRG1 signaling leads to melanoma cell growth inhibition and tumor growth delay. Mechanistically, these effects are associated with the inhibition of the PI3Kinase/Akt signaling pathway and with the accumulation of p27(Kip1). On the other end, addition of recombinant NRG1 can partially restore melanoma cell growth that is inhibited by Notch1 ablation. Taken together, our findings underline a new, previously undescribed autocrine signaling loop between Notch1 and NRG1 that controls melanoma growth and provide experimental evidence that the targeting of Notch and ERBB signaling may represent a novel potential therapeutic approach in melanoma.
Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the ...inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. Here, we show that F3/contactin acts as a functional ligand of Notch. This
trans-extracellular interaction triggers γ-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination.
Melanoma is the most lethal form of skin cancer, and the incidence and mortality rates are rapidly rising. Epidemiologically, high numbers of nevi (moles) are associated with higher risk of melanoma ...1. The majority of melanomas exhibit activating mutations in the serine/threonine kinase BRAF 2–4. BRAF mutations may be critical for the initiation of melanoma 5; however, the direct role of BRAF in nevi and melanoma has not been tested in an animal model. To directly test the role of activated BRAF in nevus and melanoma development, we have generated transgenic zebrafish expressing the most common BRAF mutant form (V600E) under the control of the melanocyte mitfa promoter. Expression of mutant, but not wild-type, BRAF led to dramatic patches of ectopic melanocytes, which we have termed fish (f)-nevi. Remarkably, in p53-deficient fish, activated BRAF induced formation of melanocyte lesions that rapidly developed into invasive melanomas, which resembled human melanomas and could be serially transplanted. These data provide direct evidence that BRAF activation is sufficient for f-nevus formation, that BRAF activation is among the primary events in melanoma development, and that the p53 and BRAF pathways interact genetically to produce melanoma.
Notch receptors are involved in cell-fate determination in organisms as
diverse as flies, frogs and humans. In Drosophila melanogaster
, loss-of-function mutations of Notch produce a 'neurogenic' ...phenotype
in which cells destined to become epidermis switch fate and differentiate
to neural cells. Upon ligand activation, the intracellular domain of Notch
(ICN) translocates to the nucleus, and interacts directly with
the DNA-binding protein Suppressor of hairless (Su(H)) in flies, or recombination
signal binding protein Jκ (RBP-Jκ) in mammals,
to activate gene transcription. But the precise mechanisms
of Notch-induced gene expression are not completely understood. The gene
mastermind has been identified in multiple genetic screens for modifiers
of Notch mutations in Drosophila. Here
we clone MAML1, a human homologue of the Drosophila gene
Mastermind, and show that it encodes a protein of 130 kD localizing to
nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian
NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jκ, and
amplifies NOTCH-induced transcription of HES1. These studies provide
a molecular mechanism to explain the genetic links between mastermind
and Notch in Drosophila and indicate that MAML1 functions as
a transcriptional co-activator for NOTCH signalling.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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