Profiling essential genes in human mammary cells by multiplex RNAi screening

Abstract

By virtue of their accumulated genetic alterations, tumor cells may acquire vulnerabilities that create opportunities for therapeutic intervention. We have devised a massively parallel strategy for screening short hairpin RNA (shRNA) collections for stable loss-of-function phenotypes. We assayed from 6000 to 20,000 shRNAs simultaneously to identify genes important for the proliferation and survival of five cell lines derived from human mammary tissue. Lethal shRNAs common to these cell lines targeted many known cell-cycle regulatory networks. Cell line-specific sensitivities to suppression of protein complexes and biological pathways also emerged, and these could be validated by RNA interference (RNAi) and pharmacologically. These studies establish a practical platform for genome-scale screening of complex phenotypes in mammalian cells and demonstrate that RNAi can be used to expose genotype-specific sensitivities.

Fig. 1

Experimental approach. (A) shRNA plasmids were packaged into retroviruses in triplicate and introduced into replicate target cell populations at a multiplicity of ~0.3 to achieve ~1 integrant per cell. Over a 2- week culture period, time points were collected on day 2 or day 4 after infection and then once each week for 2 weeks. (B) The shRNA guide strand and the barcode region were amplified from genomic DNA from screening pools. Polymerase chain reaction (PCR) products were gel-purified, labeled, and hybridized to multiplex arrays in competition with a common reference.

Fig. 2

Validation of genes essential to multiple cell lines. Cell viability assays (bars) were performed on cell lines (MCF-10A or MDA-MB- 435) expressing individual candidate shRNAs. Tables below the graphs show the level of target suppression, determined by quantitative real-time fluorescence PCR, with or without shRNA induction (indicated). An shRNA targeting luciferase (FF) and no shRNA serve as negative controls. (A) APC subunits ANAPC2 and ANAPC4 were suppressed by multiple hairpins in MCF-10A (1 to 5 for ANAPC4 and 1 to 4 for ANAPC2). Cell viability assays were carried out for 7 days after shRNA induction. (B) Nineteen additional inducible MCF- 10A cell lines were generated to validate shRNAs that were depleted in the screen. Viability assays were carried out for 11 days. (C) Validation of shRNAs that were depleted (left of the yellow line) or not depleted (right of the yellow line) in MDA-MB-435 cells. Viability assays were carried out for 11 days. (D) The Venn diagram illustrates the 166 depleted genes that were common to both screens and the 35 and 3 genes specifically depleted from MCF-10A and MDA-MB-435, respectively. shRNAs targeting P-TEFb components CDK9 and cyclin T2 were both depleted specifically in MCF-10A. The graph shows a dose-response curve for growth inhibition of MCF-10A and MDA-MB-435 cells by DRB over a 72-hour period.

Fig. 3

Cross-comparison of straight lethal screens in five different cell lines. (A) A heat map and dendrogram were generated by clustering based on shRNAs that showed depletion in at least one cell line. (B) shRNAs targeting selected complexes or pathways were chosen from (A) to highlight responses to EGFR, methyltransferase, and proteasome lesions. Pharmacological inhibition of these pathways by treatment with Tarceva (C), 5-aza-deoxycytidine (D), or MG-132 (E) was used to validate the predictions of the shRNA screen.