The Varied Roles of Notch in Cancer Aster, Jon C; Pear, Warren S; Blacklow, Stephen C
Annual review of pathology,
01/2017, Letnik:
12, Številka:
1
Journal Article
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Notch receptors influence cellular behavior by participating in a seemingly simple signaling pathway, but outcomes produced by Notch signaling are remarkably varied depending on signal dose and cell ...context. Here, after briefly reviewing new insights into physiologic mechanisms of Notch signaling in healthy tissues and defects in Notch signaling that contribute to congenital disorders and viral infection, we discuss the varied roles of Notch in cancer, focusing on cell autonomous activities that may be either oncogenic or tumor suppressive.
Notch receptors participate in a conserved pathway in which ligands expressed on neighboring cells trigger a series of proteolytic cleavages that allow the intracellular portion of the receptor to ...travel to the nucleus and form a short-lived transcription complex that turns on target gene expression. The directness and seeming simplicity of this signaling mechanism belies the complexity of the outcomes of Notch signaling in normal cells, which are highly context and dosage dependent. This complexity is reflected in the diverse roles of Notch in cancers of various types, in which Notch may be oncogenic or tumor suppressive and may have a wide spectrum of effects on tumor cells and stromal elements. This review provides an overview of the roles of Notch in cancer and discusses challenges to clinical translation of Notch targeting agents as well as approaches that may overcome these hurdles.
Ligands stimulate Notch receptors by inducing regulated intramembrane proteolysis (RIP) to produce a transcriptional effector. Notch activation requires unmasking of a metalloprotease cleavage site ...remote from the site of ligand binding, raising the question of how proteolytic sensitivity is achieved. Here, we show that application of physiologically relevant forces to the Notch1 regulatory switch results in sensitivity to metalloprotease cleavage, and bound ligands induce Notch signal transduction in cells only in the presence of applied mechanical force. Synthetic receptor-ligand systems that remove the native ligand-receptor interaction also activate Notch by inducing proteolysis of the regulatory switch. Together, these studies show that mechanical force exerted by signal-sending cells is required for ligand-induced Notch activation and establish that force-induced proteolysis can act as a mechanism of cellular mechanotransduction.
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•Development of single-molecule assays to monitor Notch proteolysis under force•Exposure of the Notch1 masked proteolytic site occurs in a physiological force regime•Dll4-induced activation of Notch1 requires the application of mechanical force•Non-native tethering can substitute for receptor-ligand complexes in signaling
Gordon et al. develop and apply single-molecule and high-throughput magnetic tweezers assays to show that mechanical force is needed to unmask the metalloprotease site in the Notch “activation switch” and induce Notch signaling in cells. Synthetic signaling systems also show a ligand endocytosis requirement even without native ligand-receptor interactions.
Translocation events are frequent in cancer and may create chimeric fusions or 'regulatory rearrangements' that drive oncogene overexpression. Here we identify super-enhancer translocations that ...drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in alternate ACC lineages.
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to ...formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.
The main oncogenic driver in T-lymphoblastic leukemia is NOTCH1, which activates genes by forming chromatin-associated Notch transcription complexes. Gamma-secretase-inhibitor treatment prevents ...NOTCH1 nuclear localization, but most genes with NOTCH1-binding sites are insensitive to gamma-secretase inhibitors. Here, we demonstrate that fewer than 10% of NOTCH1-binding sites show dynamic changes in NOTCH1 occupancy when T-lymphoblastic leukemia cells are toggled between the Notch-on and -off states with gamma-secretase inhibiters. Dynamic NOTCH1 sites are functional, being highly associated with Notch target genes, are located mainly in distal enhancers, and frequently overlap with RUNX1 binding. In line with the latter association, we show that expression of IL7R , a gene with key roles in normal T-cell development and in T-lymphoblastic leukemia, is coordinately regulated by Runx factors and dynamic NOTCH1 binding to distal enhancers. Like IL7R , most Notch target genes and associated dynamic NOTCH1-binding sites cooccupy chromatin domains defined by constitutive binding of CCCTC binding factor, which appears to restrict the regulatory potential of dynamic NOTCH1 sites. More remarkably, the majority of dynamic NOTCH1 sites lie in superenhancers, distal elements with exceptionally broad and high levels of H3K27ac. Changes in Notch occupancy produces dynamic alterations in H3K27ac levels across the entire breadth of superenhancers and in the promoters of Notch target genes. These findings link regulation of superenhancer function to NOTCH1, a master regulatory factor and potent oncoprotein in the context of immature T cells, and delineate a generally applicable roadmap for identifying functional Notch sites in cellular genomes.
Understanding the interactions between tumor and the host immune system is critical to finding prognostic biomarkers, reducing drug resistance, and developing new therapies. Novel computational ...methods are needed to estimate tumor-infiltrating immune cells and understand tumor-immune interactions in cancers.
We analyze tumor-infiltrating immune cells in over 10,000 RNA-seq samples across 23 cancer types from The Cancer Genome Atlas (TCGA). Our computationally inferred immune infiltrates associate much more strongly with patient clinical features, viral infection status, and cancer genetic alterations than other computational approaches. Analysis of cancer/testis antigen expression and CD8 T-cell abundance suggests that MAGEA3 is a potential immune target in melanoma, but not in non-small cell lung cancer, and implicates SPAG5 as an alternative cancer vaccine target in multiple cancers. We find that melanomas expressing high levels of CTLA4 separate into two distinct groups with respect to CD8 T-cell infiltration, which might influence clinical responses to anti-CTLA4 agents. We observe similar dichotomy of TIM3 expression with respect to CD8 T cells in kidney cancer and validate it experimentally. The abundance of immune infiltration, together with our downstream analyses and findings, are accessible through TIMER, a public resource at http://cistrome.org/TIMER .
We develop a computational approach to study tumor-infiltrating immune cells and their interactions with cancer cells. Our resource of immune-infiltrate levels, clinical associations, as well as predicted therapeutic markers may inform effective cancer vaccine and checkpoint blockade therapies.
MYD88(L265P) and CXCR4(WHIM) mutations are highly prevalent in Waldenström's macroglobulinemia. MYD88(L265P) triggers tumor-cell growth through Bruton's tyrosine kinase, a target of ibrutinib. ...CXCR4(WHIM) mutations confer in vitro resistance to ibrutinib.
We performed a prospective study of ibrutinib in 63 symptomatic patients with Waldenström's macroglobulinemia who had received at least one previous treatment, and we investigated the effect of MYD88 and CXCR4 mutations on outcomes. Ibrutinib at a daily dose of 420 mg was administered orally until disease progression or the development of unacceptable toxic effects.
After the patients received ibrutinib, median serum IgM levels decreased from 3520 mg per deciliter to 880 mg per deciliter, median hemoglobin levels increased from 10.5 g per deciliter to 13.8 g per deciliter, and bone marrow involvement decreased from 60% to 25% (P<0.01 for all comparisons). The median time to at least a minor response was 4 weeks. The overall response rate was 90.5%, and the major response rate was 73.0%; these rates were highest among patients with MYD88(L265P)CXCR4(WT) (with WT indicating wild-type) (100% overall response rate and 91.2% major response rate), followed by patients with MYD88(L265P)CXCR4(WHIM) (85.7% and 61.9%, respectively) and patients with MYD88(WT)CXCR4(WT) (71.4% and 28.6%). The estimated 2-year progression-free and overall survival rates among all patients were 69.1% and 95.2%, respectively. Treatment-related toxic effects of grade 2 or higher included neutropenia (in 22% of the patients) and thrombocytopenia (in 14%), which were more common in heavily pretreated patients; postprocedural bleeding (in 3%); epistaxis associated with the use of fish-oil supplements (in 3%); and atrial fibrillation associated with a history of arrhythmia (5%).
Ibrutinib was highly active, associated with durable responses, and safe in pretreated patients with Waldenström's macroglobulinemia. MYD88 and CXCR4 mutation status affected responses to this drug. (Funded by Pharmacyclics and others; ClinicalTrials.gov number, NCT01614821.).
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