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Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands
Published on: January 2, 2018
The cylindromatosis gene product, CYLD, interacts with MIB2 to regulate notch signalling
Neil Rajan1, Richard J R Elliott2, Alice Smith2
1The CRUK Gene Function Laboratory and Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, SW3 6JB, UK. Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, NE1 3BZ, UK.
Abstract:
CYLD, an ubiquitin hydrolase, has an expanding repertoire of regulatory roles in cell signalling and is dysregulated in a number of cancers. To dissect CYLD function we used a proteomics approach to identify CYLD interacting proteins and identified MIB2, an ubiquitin ligase enzyme involved in Notch signalling, as a protein which interacts with CYLD. Coexpression of CYLD and MIB2 resulted in stabilisation of MIB2 protein levels and was associated with reduced levels of JAG2, a ligand implicated in Notch signalling. Conversely, gene silencing of CYLD using siRNA, resulted in increased JAG2 expression and upregulation of Notch signalling. We investigated Notch pathway activity in skin tumours from patients with germline mutations in CYLD and found that JAG2 protein levels and Notch target genes were upregulated. In particular, RUNX1 was overexpressed in CYLD defective tumour cells. Finally, primary cell cultures of CYLD defective tumours demonstrated reduced viability when exposed to γ-secretase inhibitors that pharmacologically target Notch signalling. Taken together these data indicate an oncogenic dependency on Notch signalling and suggest potential novel therapeutic approaches for patients with CYLD defective tumours.
Insights
CYLD protein regulates Notch signaling, a pathway crucial in cancer. Loss of CYLD function in tumors leads to increased Notch signaling, suggesting new therapeutic targets for CYLD-defective cancers.
Area of Science:
- Cell Biology
- Cancer Biology
- Biochemistry
Background:
- CYLD is an ubiquitin hydrolase with diverse roles in cell signaling, implicated in various cancers.
- Dysregulation of CYLD is observed in numerous cancer types, highlighting its importance in tumorigenesis.
Purpose of the Study:
- To identify proteins interacting with CYLD using a proteomics approach.
- To elucidate the role of CYLD in regulating Notch signaling and its implications in cancer.
Main Methods:
- Proteomics to identify CYLD-interacting proteins.
- Coexpression and gene silencing (siRNA) studies to assess protein level changes and pathway activity.
- Analysis of patient tumor samples with CYLD mutations and cell culture experiments.
Main Results:
- Identified MIB2, an E3 ubiquitin ligase in Notch signaling, as a CYLD-interacting protein.
- CYLD expression stabilizes MIB2 and reduces JAG2 levels, while CYLD silencing increases JAG2 and Notch pathway activity.
- CYLD-deficient tumors show elevated JAG2, Notch target genes (RUNX1), and reduced viability upon Notch pathway inhibition.
Conclusions:
- CYLD negatively regulates Notch signaling, impacting cancer development.
- CYLD-defective tumors exhibit an oncogenic dependency on Notch signaling.
- Targeting Notch signaling with γ-secretase inhibitors may offer therapeutic strategies for CYLD-defective cancers.
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