Abstract
Cooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.
Highlights
Cancers frequently arise when normal cells accumulate multiple gene mutations that results in transformation[1]
Transduction of early passage mouse embryonic fibroblasts (MEFs)-LoxP-KC cells with a lentivirus delivering a single guide RNAs (sgRNAs) targeting Trp[53], but not a negative control sgRNA, induced anchorage independent growth in soft agar and formed tumors when the transduced cells were allografted into nude mice (Fig. 1C)
Mutation of Trp[53] in MEF-LoxP-KC cells was sufficient for transformation. These results suggest www.nature.com/scientificreports that MEF-LoxP-KC cells represent a platform for a robust genome-wide CRISPR/Cas[9] knockout screen to identify candidate genes whose mutations cooperate with oncogenic Kras to result in transformation
Summary
Cancers frequently arise when normal cells accumulate multiple gene mutations that results in transformation[1]. Unbiased genome-wide CRISPR/Cas[9] knockout screens have been applied to identify driver genes in different types of cancers, such as lymphoma[12], liver tumors[13], and breast cancer[14], but to our knowledge have not been performed to seek genes that cooperate with oncogenic Kras for transformation. We performed an unbiased genome-wide CRISPR/Cas[9] knockout screen in KrasG12D immortalized MEFs to search for genes that can cooperate with oncogenic Kras to drive growth in soft agar and in nude mice. From this screen, we identified several candidate genes whose mutation results in transformation. This suggests that in vitro transformation screens may fail to fully capture all of the required elements for in vivo tumorigenesis[15]
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