Highly parallel identification of essential genes in cancer cells

Abstract

More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such information would be particularly valuable for identifying potential drug targets. Here, we report the development of an efficient, robust approach to perform genome-scale pooled shRNA screens for both positive and negative selection and its application to systematically identify cell essential genes in 12 cancer cell lines. By integrating these functional data with comprehensive genetic analyses of primary human tumors, we identified known and putative oncogenes such as EGFR, KRAS, MYC, BCR-ABL, MYB, CRKL, and CDK4 that are essential for cancer cell proliferation and also altered in human cancers. We further used this approach to identify genes involved in the response of cancer cells to tumoricidal agents and found 4 genes required for the response of CML cells to imatinib treatment: PTPN1, NF1, SMARCB1, and SMARCE1, and 5 regulators of the response to FAS activation, FAS, FADD, CASP8, ARID1A and CBX1. Broad application of this highly parallel genetic screening strategy will not only facilitate the rapid identification of genes that drive the malignant state and its response to therapeutics but will also enable the discovery of genes that participate in any biological process.oncogene ͉ pooled library ͉ RNAi ͉ screen ͉ shRNA A lthough human cancers harbor hundreds of genetic alterations, only a subset of these alterations is likely to impact tumor initiation or maintenance. Furthermore, genes that are not altered at the genomic level may play essential roles in tumor development. Thus, to identify genes with important roles in cancer, systematic functional assessment of genes for their contribution to specific cancer phenotypes is complementary to structural characterization of the cancer genome. Integrating both structural and functional approaches will provide insight into therapeutic targets for treating cancer.The recent development of RNAi libraries targeting the human and mouse genomes has enabled systematic genetic studies in mammalian cells by using arrayed and pooled screens (1-8). However, scaling up the application of this methodology to identify all essential genes across a diverse range of human cancers requires an integrated experimental and computational approach that is efficient, robust, and economical. Here, we describe the development and application of genome-scale high-throughput methods using our lentiviral RNAi library to systematically assess cancer gene function and to integrate structural and functional approaches in the study of cancer.

Genome-wide functional screen identifies a compendium of genes affecting sensitivity to tamoxifen

“…1B). In vitro functional studies as well as clinical data suggest that reduced PTEN activity causes resistance to tamoxifen (17). Five shRNAs targeting PTEN caused 4OHT resistance in the screen (Fig.…”

“…Sensitization effects were defined as shRNAs that returned DE scores of Z < −2. In addition to this approach we also used RNAi Gene Enrichment Ranking (RIGER) (17), as implemented in the Broad Institute's GENE-E software package. In brief, RIGER calculated the weighted sum of the two top-ranked shRNAs for each gene on the basis of the log fold change between three biological replicates for each condition, and provided a normalized enrichment score per gene.…”

Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration

“…The quantitative analysis of shRNA reads used both individual data review and a statistical method (RIGER) designed for the TRC shRNA library (21,22). We used the Second Best Rank feature of RIGER, which requires that two different shRNAs result in the same effect.…”

“…shRNAs were quantitated by highthroughput sequencing. Hits for gene targets were classified and analyzed using various methods, including RIGER (21). Additional details of the primary shRNA library screen, data analysis, and hit confirmation are described above and in the SI Methods.…”

The role of components of the chromatin modification machinery in carcinogenesis of clear cell carcinoma of the ovary (Review)

“…The C-terminus of the protein is capable of stimulating glucocorticoid receptor-dependent transcriptional activation. Additionally, ARID1 is an essential gene for FAS (TNF receptor superfamily, member 6)-mediated apoptosis (22). ARID1A is one of the most frequently deleted genes across all cancer types (23,24) and knockdown of this gene results in a failure of cell cycle arrest (23,25).…”