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In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity
Published on: November 2, 2020
Phenotype-based screening of mechanistically annotated compounds in combination with gene expression and pathway
Mårten Fryknäs1, Linda Rickardson, Malin Wickström
1Department of Genetics and Pathology, Uppsala University, S-751 85 Uppsala, Sweden.
Abstract:
The squamous cell carcinoma HeLa cell line and an epithelial cell line hTERT-RPE with a nonmalignant phenotype were interrogated for HeLa cell selectivity in response to 1267 annotated compounds representing 56 pharmacological classes. Selective cytotoxic activity was observed for 14 of these compounds dominated by cyclic adenosine monophosphate (cAMP) selective phosphodiesterase (PDE) inhibitors, which tended to span a representation of the chemical descriptor space of the library. The PDE inhibitors induced delayed cell death with features compatible with classical apoptosis. The PDE inhibitors were largely inactive when tested against a cell line panel consisting of hematological and nonsquamous epithelial phenotypes. In a genome-wide DNA microarray analysis, PDE3A and PDE2A were found to be significantly increased in HeLa cells compared to the other cell lines. The pathway analysis software PathwayAssist was subsequently used to extract a list of proteins and small molecules retrieved from Medline abstracts associated with the hit compounds. The resulting list consisted of major parts of the cAMP-protein kinase A pathway linking to ERK, P38, and AKT. This molecular network may provide a basis for further exploitation of novel candidate targets for the treatment of squamous cell carcinoma.
Insights
Cyclic adenosine monophosphate (cAMP) selective phosphodiesterase (PDE) inhibitors show selective cytotoxicity against squamous cell carcinoma (SCC) HeLa cells, inducing apoptosis. These PDE inhibitors may offer novel therapeutic targets for SCC treatment.
Area of Science:
- Oncology
- Pharmacology
- Cell Biology
Background:
- Squamous cell carcinoma (SCC) is a prevalent cancer.
- Identifying selective therapeutic targets for SCC remains a challenge.
- The HeLa cell line is a well-established model for SCC research.
Purpose of the Study:
- To identify compounds with selective cytotoxic activity against SCC HeLa cells.
- To investigate the mechanism of action of identified compounds.
- To explore potential therapeutic targets for SCC.
Main Methods:
- Screening of 1267 annotated compounds against HeLa and hTERT-RPE cell lines.
- Assessing selective cytotoxic activity and apoptosis induction.
- Genome-wide DNA microarray analysis to identify differentially expressed genes.
- Pathway analysis using PathwayAssist to map molecular networks.
Main Results:
- 14 compounds, primarily cyclic adenosine monophosphate (cAMP) selective phosphodiesterase (PDE) inhibitors, exhibited selective cytotoxicity against HeLa cells.
- PDE inhibitors induced delayed cell death consistent with apoptosis.
- PDE3A and PDE2A were significantly upregulated in HeLa cells.
- Pathway analysis revealed a network involving the cAMP-protein kinase A pathway, ERK, P38, and AKT.
Conclusions:
- cAMP-selective PDE inhibitors demonstrate promising selective activity against SCC HeLa cells.
- The identified molecular network provides a basis for developing novel SCC therapeutic strategies.
- Targeting PDE enzymes and associated pathways may represent a new avenue for SCC treatment.
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