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.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.
