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SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs
Published on: June 28, 2019
A 'synthetic-sickness' screen for senescence re-engagement targets in mutant cancer backgrounds
Claire J Cairney1, Lauren S Godwin2, Alan E Bilsland1
1Institute of Cancer Sciences, Wolfson Wohl Cancer Research Centre, University of Glasgow, Glasgow, United Kingdom.
Abstract:
Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAβGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments.
Insights
Senescence induction shows promise for cancer therapy, but requires better agents. This study identified novel senescence inducers, revealing context-dependent effects and a potential KRAS-targeting mechanism via ECT2 in refractory cancers.
Area of Science:
- Cellular senescence
- Cancer biology
- Drug discovery
Background:
- Senescence is a key tumor suppressor mechanism, yet its therapeutic induction is limited by a lack of effective agents and understanding of underlying signaling.
- Targeting senescence offers a promising avenue for cancer therapy, particularly for immortalized and tumorigenic cells.
Purpose of the Study:
- To identify novel inducers of senescence phenotypes using a large-scale siRNA screen.
- To validate and characterize these inducers in different cancer cell contexts.
- To explore potential therapeutic strategies targeting specific cancer mutations.
Main Methods:
- Conducted a morphological siRNA screen in A375P melanoma cells to identify senescence inducers.
- Validated hits in HCT116 colorectal carcinoma cells and selected 16 robust candidates.
- Assessed senescence biomarkers (p21, 53BP1, SAβGal) and proliferation arrest in various HCT116 cell lines with cancer-relevant mutations.
- Investigated the role of oncogene ECT2 and KRAS mutations in senescence induction.
Main Results:
- Identified 16 robust inducers of senescence phenotypes.
- Demonstrated context-dependent induction of partial senescence, even without p21 or p53.
- Observed variable proliferation arrest based on genetic background; toxic effects in p21 null cells, no arrest in PI3K-mutant cells.
- Showed ECT2 induces partial senescence, with growth suppression and full senescence dependent on KRASG13D activation.
Conclusions:
- Partial senescence can be induced in a context-dependent manner, highlighting the complexity of senescence signaling.
- ECT2-mediated senescence induction offers a potential therapeutic strategy for KRAS-mutant cancers refractory to current treatments.
- Further research into senescence inducers and their signaling pathways is crucial for advancing cancer therapy.
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