Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer

Abstract

Background: Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement.

Methods: In order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine.

Results: Results from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine.

Conclusion: These findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine.

Figure 1

HT-RNAi kinase screening for identification of sensitizers to gemcitabine. HT-RNAi screens were performed on MIA PaCa-2 cells transfected with a siRNA library targeting 572 kinases followed by treatment with either vehicle or 5 nM or 10 nM gemcitabine. Cell viability was assessed and normalized to control wells. (A) Scatterplot of the log₂ values of cell viability for gemcitabine plus siRNA treated cells versus vehicle plus siRNA treated cells showed CHK1 as a significant hit. (B) Plot of log₂ ratios of gemcitabine/vehicle for each siRNA treated with either 5 nM or 10 nM gemcitabine. (C) Empirical Probability Distribution of log₂ ratios of gemcitabine/vehicle (5 nM and 10 nM). Hit areas are highlighted in red. (D) Venn diagram of gene hits from both the 5 nM (highlighted in pink) and 10 nM (highlighted in yellow) gemcitabine synthetic lethal RNAi screen.

Figure 2

Validation of gene silencing by CHK1 siRNA. MIA PaCa-2 cells were transfected with either CHK1 or control siRNA and allowed to grow for 48–72 hrs. (A) Total RNA from the siRNA treated MIA PaCa-2 cells was isolated at 48 hrs and analyzed by qRT-PCR for CHK1 expression. CHK1 expression for each siRNA treatment was compared to untreated cells. GAPDH was used as an internal control for all the samples and fold change was calculated by normalizing all the data to GAPDH expression. (B) Lysates from CHK1 siRNA treated MIA PaCa-2 cells were prepared at 72 hrs post transfection and analyzed by western blot for expression of CHK1 protein using an anti-CHK1 antibody. (C) CHK1 siRNA treated cells showed decreased growth of MIA PaCa-2 cells at 72 hours after siRNA transfection when compared to no siRNA treatment or non-silencing siRNA treatment. Cell images were taken at 20× magnification.

Figure 3

Validation of CHK1 as a sensitizing target to gemcitabine in pancreatic cancer cells. MIA PaCa-2 and BxPC3 pancreatic cancer cells were transfected with either CHK1, CHK2 or non-silencing siRNA. After 24 hours, cells were treated with varying concentrations of gemcitabine and incubated for an additional 72 hours. Cell number was assessed and data was normalized to siRNA plus vehicle control and plotted. Silencing of CHK1 showed potentiation of gemcitabine response in (A) MIA PaCa-2 and (C) BxPC3 cells as seen by the shift in the dose response curves. Silencing of CHK2 did not affect the response to gemcitabine in either (B) MIA PaCa-2 cells or (D) BxPC3 cells. Data is representative of three independent experiments.

Figure 4

Kinetic analysis of CHK1 siRNA induced sensitization of gemcitabine response. MIA PaCa-2 cells were transfected with either CHK1 siRNA or non-silencing siRNA and at 24 hours post transfection, cells were treated with either vehicle or 10 nM gemcitabine. Growth was assessed by impedance measurements at 1-hour intervals and cell index was plotted as a function of time. (A) Treatment of cells with non-silencing siRNA and either vehicle or gemcitabine showed a slight decrease in cell growth by gemcitabine. (B) Pretreatment with CHK1 siRNA caused a pronounced decrease in cell growth in the gemcitabine treated cells compared to the vehicle treated cells. Data is representative of three independent experiments.

Figure 5

CHK1 inhibitors potentiate gemcitabine response. Treatment of MIA PaCa-2 cells with the CHK1 inhibitors (A) SB 218078 or (B) PD 407824 in combination with varying concentrations of gemcitabine resulted in a shift of the dose response curves suggesting potentiation of the gemcitabine response. Cell number was assessed and data was normalized to vehicle control and plotted. Data is representative of three independent experiments.

Figure 6

Schematic of the role of CHK1 and ATR in sensitization to gemcitabine. Genes identified as synergistic to gemcitabine in the RNAi kinase screens are shown in red. Gemcitabine induced DNA damage results in the phosphorylation and activation of serine/threonine-protein kinase CHK1 by ATR. The activated CHK1 then phosphorylates Cdc25A, leading to cell cycle arrest in G2/M. This rapid response via CHK – Cdc25A pathways additionally is followed by the p53-mediated maintenance of G1/S arrest. Tumor suppressor p53 plays a key role in the G2/M checkpoint arrest as well. In the maintenance stage, ATR phosphorylates Ser15 of p53 directly and Ser20 through activation of CHK1. Phosphorylated p53 activates its target genes, including cyclin-dependent kinase inhibitor 1A (p21), which binds to cyclin-dependent kinase 2 (Cdk2) and cyclin-dependent kinase 4 (Cdk4). Map was constructed with MapEditor (GeneGO).