Significant progress has been made in deciphering the role of splicing factors in cancer including carcinogenesis and drug resistance. Splicing-based prognostic tools as well as therapeutic options hold great potential towards improvements in cancer therapy. However, gaining more in-depth molecular insight into the consequences of mutations in various components of the splicing machinery as well as of cellular effects of spliceosome inhibition is a prerequisite to establish the role of splicing in tumor progression and treatment options, respectively.
BackgroundRadiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening.MethodsWe developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells.ResultsOn 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated.ConclusionsWe developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid the identification of molecular targets for radiosensitization, thereby contributing to improving the efficacy of radiotherapy.
Background: Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in Western world males. PSA testing allows early detection of localized disease. Treatment options for patients with local disease include radiotherapy and surgery. However, local tumor control rates after radiotherapy are highly variable; and ∼40% of treated patients will experience disease recurrence and progression. Advanced prostate cancer has a very poor prognosis, emphasizing the need for more effective early local treatment, preventing advanced disease. Here, we set out to identify molecular targets of radiation susceptibility in PCa cells as a starting-point to increase the therapeutic index of radiotherapy. Methods: Targets were identified by high-throughput screening using a human whole genome siRNA library on the radioresistant PCa cell line PC-3 subjected to 4Gy irradiation. Combined effects of radiation and target gene silencing were quantified using a cell viability assay based on automated cell counting four days after irradiation. Candidate targets were validated using the colony formation assay (CFA) scored 8-10 days after irradiation. Results: We conducted two genome-wide siRNA screens with and without irradiation in PC-3 cells. We used a linear regression model to identify radiation susceptibility genes and selected 45 candidate targets with FDR < 10% for further research. In a conformation screen, multiple independent siRNAs silencing 17 genes induced ≥2-fold more cell death upon 4Gy irradiation compared to a negative control siRNA. Confirmed hits were validated independently using the CFA. The dose modifying factor at 80% clonogenic cell survival (DMF0.8) was calculated for each gene-specific siRNA compared to control siRNA from linear-quadratic survival curves. Sixteen candidate genes were validated with a DMF0.8 ≥ 1.3. This included the mitotic spindle assembly checkpoint component MAD2L2, which was previously reported as a radiosensitizing target in PCa cells (JCI 2011;121:2383-2390), thus validating our screens. Importantly, silencing of 9 genes sensitized PC-3 cells to irradiation more effectively than did silencing of the known radiation susceptibility gene PRKDC. Among these were genes involved in cell cycle control, ubiquitination, cell communication and G-protein coupled receptor signaling. Interestingly, silencing of two druggable genes, i.e. CPNE7 and GPR27, was particularly effective at low dose irradiation, resulting in a very high DMF0.8 of 4.6 and spectacular decreases in surviving fractions after 2Gy irradiation from 70% in controls to 37% and 23%, respectively. Conclusion: We have identified a set of novel candidate targets that could be used to potently enhance the efficacy of radiotherapy in prostate cancer. This could contribute significantly to improving overall success of prostate cancer intervention. Citation Format: Jasmina Hodzic, Ida van der Meulen, Renee de Menezes, Ilse Dingjan, Marielle JP Maas, Winald Gerritsen, Marcel Verheij, Ab A. Geldof, Baukelien van Triest, Victor W. van Beusechem. Identification of radiation susceptibility genes in prostate cancer by whole genome RNA interference screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2013-4434
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