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


CONCLUSIONS
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.
Azorsa
David O
DO
Pharmaceutical Genomics Division, The Translational Genomics Research Institute, Scottsdale, Arizona 85259, USA. dazorsa@tgen.org
Gonzales
Irma M
IM
Basu
Gargi D
GD
Choudhary
Ashish
A
Arora
Shilpi
S
Bisanz
Kristen M
KM
Kiefer
Jeffrey A
JA
Henderson
Meredith C
MC
Trent
Jeffrey M
JM
Von Hoff
Daniel D
DD
Mousses
Spyro
S
D000368:Aged; D000470:Alkaloids / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002227:Carbazoles / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D020868:Gene Silencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D014162:Transfection; D000093542:Gemcitabine
D000470:Alkaloids; D000964:Antimetabolites, Antineoplastic; D002227:Carbazoles; D004791:Enzyme Inhibitors; C544549:PD 407824; D034741:RNA, Small Interfering; D003841:Deoxycytidine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C404329:SB 218078; D000093542:Gemcitabine