Fig 1.
The schematic diagram of the siRNA screen is illustrated on the left and the microarray gene expression study on the right. The data from the two studies were analyzed as described in the methods section. The top hits from the siRNA screen and the differentially expressed genes were compared at the genetic level to identify genes that may be targeted to enhance cisplatin activity. The two lists were separately processed through Pathway Studio and the resulting pathways were compared subsequently to determine the most critical pathways to be targeted in combination with cisplatin.
Table 1.
Pathways essential for A549 cell survival and cisplatin sensitivity.
Fig 2.
Top 100 hits from the genome-wide siRNA screen based on gene score.
The survival index is a measure of cell viability relative to negative controls; % viability with CDDP represents the amount of cell death induced in the presence of both gene knockdown and cisplatin; sensitization is the difference between the survival index and % viability with CDDP; gene score takes into account both the survival index and level of cisplatin activity enhancement (see Methods).
Fig 3.
Most lethal targets identified in the siRNA screen.
The 100 genes with the lowest gene score are listed; ranked by lowest to highest survival index. While a few of these genes may still sensitize A549 cells to cisplatin treatment, gene knockdown in the absence of cisplatin would cause at least 70% loss in cell viability.
Fig 4.
Differentially expressed genes in A549 cells following cisplatin treatment at its IC10.
The heat map represents the gene expression profile for the differentially expressed genes identified between untreated (blue) and cisplatin-treated (orange) cells (A). Each column displays data from one microarray chip. Genes that were further validated by qPCR are highlighted with a black vertical line beside the gene. The fold-changes in mRNA expression are plotted in the box plot (B; blue = over-expression, red = under-expression after cisplatin treatment) and the averaged values are tabulated from six individual experiments at each time-point (C). All changes observed were statistically significant (p<0.05) except for FNTA.
Table 2.
Over-expressed genes from Affymetrix Exon 1.0 Array matched with siRNA Screen Data.
Fig 5.
Validation studies assessing the effects of ALDH3A1, RRM2B, CABYR, FHL2, and NINJ1 silencing on cisplatin activity in A549 and H460 cell lines.
Knockdown of each candidate target was confirmed via qPCR (A; *p<0.05 relative to scramble control). The representative image for each tested condition is displayed in B. The plating efficiency of each condition is plotted in separate graphs for each cell line (C; data shown as mean ± SEM; n = 3; *p<0.05).
Table 3.
Potential cisplatin activity enhancers selected from the siRNA screen and microarray gene expression studies and their known roles in cancer biology.
Table 4.
siRNA Sequences Used for Target Validation.
Table 5.
Pathway enrichment analysis of cisplatin-potentiating gene targets from the siRNA screen.
Table 6.
Pathway enrichment analysis of differentially expressed genes following low-dose cisplatin treatment.
Fig 6.
Pathways enriched in both the siRNA screen top hits and the microarray differentially expressed genes.
Of the siRNA screen hits and the microarray gene list, the double stranded homologous repair (A) and the INO80 chromatin remodelling (B) pathways were enriched in both datasets. The schematic diagram of the two pathways is displayed on the left while genes from the siRNA screen (purple) and the microarray study (green) that map to these pathways are illustrated based on cellular localization (right).
Fig 7.
Fitness measurements for cisplatin treated yeast mutant strains.
WT fitness (white bar) was used to predict the effects of cisplatin on mutant strains assuming a simple multiplication of the mutant fitness defect and the effect of cisplatin on WT. Significantly lower fitness values were observed for strains deleted for RAD1, RAD18, RAD52, CTF8, MRE11 or HTZ1 (Holm-Bonferroni corrected p-value <0.05) but not for other mutants tested. Raw p-values are indicated above each comparison. Human gene orthologues of each yeast mutant are indicated below. Errors bars are standard error of the mean.