Journal Article

In vivo genome‐editing screen identifies tumor suppressor genes that cooperate with Trp53 loss during mammary tumorigenesis

Strasser, Andreas, Visvader, Jane E., Chen, Yunshun, Christie, Michael, Herold, Marco J., Tsai, Minhsuang, Capaldo, Bianca D., Lindeman, Geoffrey J., Smyth, Gordon K., Dawson, Caleb A., Whittle, James R., Surgenor, Elliot, Dekkers, Johanna F., Vaillant, François, Chen, Huei‐Rong, Heitink, Luuk, Milevskiy, Michael J. G.

2022

Overview

Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome‐wide CRISPR/Cas9 screen in Trp53+/– heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof‐of‐concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short‐guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53‐only mutants. This proof‐of‐principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis. TP53 mutations occur frequently in breast cancer. Here, we describe a framework to identify novel tumor suppressor genes that cooperate with loss of Trp53 in mammary tumorigenesis using an in vivo genome‐wide CRISPR/Cas9 screen. Hits were validated through genetic editing of mammary epithelial organoids in vitro and direct editing of ductal cells in vivo using engineered lentiviruses.

• Publisher: John Wiley & Sons, Inc,John Wiley and Sons Inc,Wiley
• Subject: Animals / axin1 / Breast cancer / Cell Transformation, Neoplastic - genetics / CRISPR / CRISPR-Cas Systems - genetics / CRISPR/Cas9 / Editing / Gene Editing / Gene expression / Genes, Tumor Suppressor / Genomes / Humans / intraductal / Keratin / Kinases / mammary organoids / Mice / Monoclonal antibodies / Mutagenesis / Mutants / Mutation / Organoids / Plasmids / Prkar1a / Protein kinase A / PTEN protein / Short Report / Trp53 / Tumor suppressor genes / Tumor Suppressor Protein p53 - genetics / Tumor Suppressor Protein p53 - metabolism / Tumorigenesis / Tumors