Cancer proliferation gene discovery through functional genomics

Abstract

Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.

Fig. 1

Overview of the pool-based dropout screen with barcode microarrays. (A) Schematic of library construction and screening protocol. (B) Schematic of the HH barcode hybridization. (C) Comparison between HH amplicons (top) and full-hairpin PCR amplicons (bottom) on an HH probe microarray.

Fig. 2

Pool-based dropout screen for genes required for cancer cell viability. (A) Overview of shRNA pool behavior in the screen. For each cell line, shRNAs were ranked on the basis of their mean normalized log₂ Cy3/Cy5 ratios. The shaded rectangle indicates the log₂ ratio range within which an shRNA’s abundance was considered unchanged. (B) Clustering of the four cell lines with the antiproliferative shRNAs identified in the screen. The color scale represents mean normalized log₂ Cy3/Cy5 ratios of the probes. (C) Antiproliferative shRNAs and genes that scored in the screen for each cell line are shown. (D) Summary of the common shRNAs (blue) and genes (red) identified in the screen. Overlapping antiproliferative shRNAs/genes between pairwise combinations of cell lines are displayed (DLD-1 and HMEC have more overlapping genes than shRNAs because, in some cases, different sets of shRNAs targeting the same gene scored in each line).

Fig. 3

Genes commonly required for proliferation or survival of normal and cancer cells. Error bars represent SDs across triplicates. (A) Representative candidate shRNAs that reduce viability of all four cell lines. Multiple entries for the same gene indicate that multiple independent shRNAs have scored in the screen for that gene. For each shRNA, the normalized log₂ Cy3/Cy5 ratio (i.e., its relative abundance in the end sample versus initial sample) is given. (B) Examples of core cellular modules required in all four cell lines. Shown are the APC/C (top left), the COP9 signalosome (top right), the eIF3 translation initiation complex (bottom left), and E3 ubiquitin ligases (bottom right). Color scheme indicates genes required for viability of one (yellow), two (orange), three (red), or all four (black) cell lines. (C) Validation of selected shRNAs from the screen that reduce viability of all four cell lines. Candidate shRNAs were individually packaged into viruses and infected into cells in 96-well plates in independent triplicates. Cell viability was measured at day 9 after infection. All viability reductions were significant (P < 0.05), except where indicated by the number sign. FF, negative control shRNA targeting firefly luciferase.

Fig. 4

Genes selectively required for proliferation or survival of cancer cells. Error bars represent SDs across triplicates. (A) Identification of PPP1R12A (one shRNA) and PRPS2 (two shRNAs) as two genes that are selectively required by HCC1954 or DLD-1 cells, respectively, in the screen. (B and C) Validation of PPP1R12A (B) and PRPS2 (C) as selectively required for viability of HCC1954 or DLD-1 cells, respectively. Cells were either infected with individual retroviral shRNAs or transfected with individual siRNAs in triplicates. Cell viability was measured at 9 days after infection (shRNA) or 4 days after transfection (siRNA) (*, P < 0.05). Luc, negative control siRNA targeting luciferase. PLK1, positive control siRNA targeting polo-like kinase 1. (D) Normalized log₂ ratios of an MDM2 shRNA in the screen. (E) shRNA knockdown of MDM2 selectively impairs the viability of HMECs. Cell viability was measured 9 days after infection with retroviruses expressing five different MDM2 shRNAs (*, P < 0.05). (F) Differential sensitivity of the four cell lines to the MDM2 inhibitor nutlin-3. Cell viability is reflective of their p53 status (HMECs and HCT116 cells, p53 wild-type; HCC1954 and DLD-1 cells, p53 mutant). Cell viability was measured after 4 days of nutlin-3 treatment (*, P < 0.05). ctrl, control. (G) Normalized log₂ ratios of a BUB1 shRNA from the screen. (H) Enhanced sensitivity of HCC1954 cells to BUB1 knockdown. Both shRNA (left) and siRNA (right) knockdown of BUB1 reduce HCC1954 cell viability but have no effect on HMEC viability. Cell viability was measured 4 or 9 days after transfection or infection, respectively (*, P < 0.05).