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

Csaba Miskey, Lacramioara Botezatu, Nuri A. Temiz, Andreas Gogol-Döring, Áron Bartha, Balázs Györffy, David A. Largaespada, Zoltán Ivics, Attila Sebe

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


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 catalog 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 nonmetastatic 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 18 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, G protein–coupled receptor kinase interacting ArfGAP 2 (GIT2) and muscle-associated receptor tyrosine kinase (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 methodologic resources used to identify genes driving cancer metastasis.

Original languageEnglish (US)
Pages (from-to)1502-1515
Number of pages14
JournalMolecular Cancer Research
Issue number10
StatePublished - Oct 2022

Bibliographical note

Funding Information:
This work was supported by a grant from the LOEWE Center for Cell and Gene Therapy Frankfurt funded by the Hessische Ministerium fu€r Wissenschaft und Kunst to C. Miskey (High-throughputintegration site analysis of viral-, andtransposon-based vectors by computation-assisted hemi specific PCR and Illumina deep sequencing).

Funding Information:
D.A. Largaespada reports grants from American Cancer Society during the conduct of the study; other support from NeoClone Biotechnologies, Inc., Recombinetics, and Inc., Styx Therapeutics, Inc.; and other support from Luminary Therapeutics, Inc. outside the submitted work. No disclosures were reported by the other authors.

Publisher Copyright:
© 2022 American Association for Cancer Research Inc.. All rights reserved.


  • Breast Neoplasms/genetics
  • DNA Transposable Elements/genetics
  • Female
  • Humans
  • Mutagenesis
  • Mutagenesis, Insertional
  • Protein-Tyrosine Kinases/genetics
  • RNA
  • Receptors, G-Protein-Coupled/genetics

PubMed: MeSH publication types

  • Research Support, Non-U.S. Gov't
  • Journal Article


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