In Vitro Insertional Mutagenesis Screen Identifies Novel Genes Driving Breast Cancer Metastasis

• Published in: Molecular cancer research 2022-10, Vol.20 (10), p.1502-1515
• Main Authors: Miskey, Csaba, Botezatu, Lacramioara, Temiz, Nuri A, Gogol-Doring, Andreas, Bartha, Aron, Gyorffy, Balazs, Largaespada, David A, Ivics, Zoltan, Sebe, Attila
• Format: Article
• Language: English
• ISSN: 1541-7786; 1557-3125
• DOI: 10.1158/1541-7786.MCR-21-0772

Metastasis, a complex, multistep process, is responsible for the overwhelming majority of cancer-related deaths. Despite its devastating consequences, it is not possible to effectively treat cancer that has spread to vital organs, the mechanisms leading to metastasis are still poorly understood and the catalogue of metastasis promoting genes is still incomprehensive. To identify new driver genes of metastasis development, we performed an in vitro Sleeping Beauty transposon-based forward genetic screen in non-metastatic SKBR3 human breast cancer cells. Boyden chamber- based matrix invasion assays were used to harvest cells that acquired a de novo invasive phenotype. Using targeted RNA-sequencing data from eighteen pools of invasive cells, we carried out a gene-centric candidate gene prediction and identified established and novel metastasis driver genes. Analysis of these genes revealed their association with metastasis related processes and we further established their clinical relevance in metastatic breast cancer. Two novel candidate genes, GIT2 and MUSK, were functionally validated as metastasis driver genes in a series of in vitro and in vivo experimental metastasis models. We propose that our robust and scalable approach will be a useful addition to the toolkit of methodological resources used to identify genes driving cancer metastasis. Implications: Novel metastasis drivers were identified in a human breast cancer cell line by performing an in vitro, Sleeping Beauty transposon-based forward genetic screen and an RNA fusion-based candidate gene prediction.