論文の公開元へGenome-scale CRISPR/Cas9 screening for gemcitabine modulators in pancreatic cancer
概要
Pancreatic cancer ranks eleventh among cancers in terms of the diagnosis all over the world, however concerning the cancer-related death it grades seventh. This statistics implies high mortality rate and poor prognosis of the pancreatic cancer. In point of fact, five year overall survival rate of pancreatic cancer is less than 5%, more than 90% of the patients diagnosed with this cancer die and median overall survival for the patients is 8 to 12 months for locally advanced disease.
Currently chemotherapy is the first-line standard treatment for pancreatic cancer and gemcitabine plays a central role in this treatment. Gemcitabine is an analog of deoxycytidine which carries two flour atoms (2’,2’–difluoro-2’-deoxycytidine, dFdC). Upon entrance to the cells via human nucleoside transporters, gemcitabine becomes phosphorylated by deoxycytidine kinase, pyrimidine nucleoside monophosphate kinase and nucleoside diphosphate kinase to form gemcitabine mono-, di- and triphosphate, respectively. Consequently, gemcitabine triphosphate is incorporated into the DNA chains by DNA polymerases and causes a masked chain termination of DNA replication, where DNA replication is halted following incorporation of one base after gemcitabine. Also, gemcitabine diphosphate affects DNA synthesis by reducing dNTP pool of the cells via covalent binding to the active site of ribonucleotide reductase and thereby its inactivation.
Although gemcitabine has been used as the standard first-line treatment for pancreatic cancer its efficacy is still modest (5.6 months overall survival) which warrants exploration to discover candidates for combinatorial therapy along with gemcitabine. In the search for potential gemcitabine sensitizers, a wide range of studies including large-scale screenings have been performed. Large-scale screenings to discover gemcitabine cytotoxity regulators in pancreatic cancer cell lines have been performed utilizing RNA interference (RNAi) libraries. While RNAi screening provides a very powerful tool for detecting the genes involved in our process of interest, lack of the full ablation of the gene function may decrease sensitivity of RNAi approach for large-scale screenings. Since RNAi interferes with the function of a gene at the level of RNA a complete removal of the gene function is not achievable, as remnant of the gene product after RNAi application may be sufficient for performing the gene function. This flaw can be coped by the application of the techniques like CRISPR/Cas9 that provide gene knockout and in turn full cessation of the gene function. In fact, CRISPR/Cas9 system has shown to produce more positive hits, 2 to 5 folds, compared to shRNA system, in loss-of-function screening.
In this study, a CRISPR/Cas9 library (GeCKO-v2) consisting of 123,411 unique sgRNAs targeting 19,050 human genes, was utilized to identify modulators of gemcitabine action in Panc1 pancreatic carcinoma cell line. In short, Lentiviral library was prepared using GeCKO-v2 plasmid library, cultured Panc1 cells were transfected with prepared lentiviral library, infected cells were selected utilizing puromycin, selected cells were split to three parts, one part was used as the baseline control and two other parts were treated either with gemcitabine or its vehicle. Following the gemcitabine screening, the cells were collected and genomic DNA was extracted, next generation sequencing libraries were prepared and subjected to massively parallel sequencing. Raw sequencing data were analyzed and copy numbers of the sgRNAs were acquired.
Efficacy of the genome-scale CRISPR/Cas9 knockout experiment was assessed by GSEA employing Gene Ontology All gene sets from Molecular Signature Database (MSigDB). The result showed that the essential gene sets including Multi Organism Metabolic Process, Ribosomal Subunit and Translational Initiation were depleted in the knockout cells library after 22 days. Also, to obtain essential gene sets for Panc1 cells survival, GSEA employing Hallmark gene sets was performed and MYC-targets, DNA-repair, G2M-checkpoint and E2F-targets gene sets appeared as essential.
Inventories of the genes whose sgRNAs were depleted or enriched in the presence of gemcitabine were obtained employing RIGER algorithm, which ranks the gene based on the differential effects of sgRNAs targeting a gene. Genes involved in gemcitabine metabolism can be classified to two categories. Positive gemcitabine modulators, genes that act in accordance with gemcitabine action and negative gemcitabine modulators, genes that act apposed to gemcitabine action and reduce its effectiveness. As an in-silico validation of the gemcitabine screening, GSEA was performed utilizing the lists of gemcitabine positive and negative modulators. As expected, the positive modulators
were enriched in gemcitabine-treated cells, while negative modulators did not show any enrichment.
As a result of the screening, SH3D21 appeared as the top gemcitabine sensitizer and sensitizing activity of SH3D21 was validated in knockout and knockdown experiments. Panc1 cells carrying an sgRNA targeting SH3D21 showed more sensitivity to gemcitabine as compared to the control sgRNAs carrying cells. The EC50 of gemcitabine was reduced to 41.1 nM in SH3D21-knockout cells compared to that of the control cells (56.8 nM). Silencing SH3D21 gene with an siRNA targeting SH3D21 mRNA resulted in increased sensitivity of Panc1 cells to gemcitabine as well.
PANTHER analysis revealed that the proportion of the proteins with transporter activity was decreased among top 1000 depleted genes compared to those of the entire library. SLC28A3 gene, which codes for one of the known transporters of gemcitabine, was absent among top 1000 depleted genes and present among top 1000 enriched genes while three other known gemcitabine transporters did not show this pattern. The decrease in the proportion of the transporters in top 1000 depleted genes was not limited to the known gemcitabine transporters. On the other hand, the genes involved in endocytosis, like KIAA0196, were among the top enriched genes in gemcitabine-treated cells and one of the three viral proteins that were among top 1000 enriched genes was ERVFRD-1 that is involved in endosomal transport. These data suggested the possibility of involvement of other transporting molecules/mechanisms (e.g. endocytosis) in gemcitabine uptake by Panc1 cells. To verify this hypothesis, an endocytosis inhibitor, chlorpromazine, was used in cell viability assay in the presence of gemcitabine. The result showed that chlorpromazine decreased the efficacy of gemcitabine, confirming that endocytosis has a part in gemcitabine cellular uptake.
In brief, we found SH3D21 as a novel gemcitabine sensitizer that may act as a therapeutic target to improve gemcitabine efficacy. Also, MYC pathway appeared as an essential pathway for the survival of Panc1 cells in our study and might serve as a target for treatment of pancreatic cancer. Furthermore, we found that endocytosis is involved in gemcitabine cellular uptake, a finding that may help planning new strategies for gemcitabine delivery.
