NEW YORK (GenomeWeb) – In an effort to help uncover the genetic mechanisms that underlie pancreatic cancer, researchers from Germany's Technical University Munich (TUM) have developed a method that combines traditional transgenesis and RNAi gene silencing to rapidly identify tumor-suppressor genes.
The approach, they wrote in a paper appearing in Molecular Cancer Research, is designed to overcomes many of the limitations of existing techniques and could potentially be adapted to use gene modulation technologies other than RNAi such as CRISPR/Cas9-based genome editing.
Pancreatic ductal adenocarcinoma (PDAC) is the most common forms of pancreatic cancer and one of the deadliest of all malignancies, with a five-year survival rate of about 5 percent. Despite ongoing research into the disease, only a few of its genetic drivers have been identified.
Mutations that activate the oncogene KRAS are found in 90 percent of PDAC cases and appear to initiate the development of the pancreatic intraepithelial neoplasms (PanINs) that often usher in the cancer. Yet in animal models, activation of KRAS alone rarely leads to the development of invasive PDAC, suggesting that additional genetic events are at play in the disease.
Recent studies have implicated the loss of the cell cycle regulator p16, as well as the tumor suppressor genes p53, in the development of PDAC, the TUM team wrote in Molecular Cancer Research. Still, there is a need to identify other genes involved in pancreatic tumorigenesis to "broaden our understanding of pancreatic cancer biology and eventually lead the way to more effective treatments."
Standard approaches for elucidating gene function involves the generation of transgenic and knockout mice carrying germline alterations of a gene candidate. And while valuable, these animal models are costly and time-consuming to produce and maintain. Further, since genes are manipulated during embryogenesis, they do not accurately represent the cancer-causing somatic mutations that occur in humans.
To address such issues, the TUM investigators developed a hybrid approach, starting by isolating from a mouse model an already well-established pancreatic ductal cell population in which oncogenic KRAS can be activated in vitro, then knocking down potential cancer-linked genes using lentiviral-delviered shRNAs. The cells are then implanted into the pancreata of mice, which are monitored for tumor growth.
To demonstrate their method, the scientists tested shRNAs against p16 and p53. When either gene was suppressed in KRAS -PDCs and the cells delivered into mice, animals experienced tumor growth, metastasis, and reduced survival, according to the paper. In contrast, KRAS activation alone did not result in tumor growth.
The study's authors stated that a key benefit of their approach is the rapid generation of a desired cell line carrying shRNAs against a gene of interested. Additionally, cell lines can be generated in parallel, allowing for the study of various genes and their effect on cancer simultaneously.
"An overall reduction of time-consuming and expensive generation of germline-altered animal models and subsequent breedings and genotyping, not to mention long-term backcrossing, will emerge as a consequence," they wrote.
They also noted that while shRNAs are used widely in screening assays, such studies often use cells that have already undergone genetic alteration or cells from established and immortalized lines.
Cells that have already been genetically manipulated may become "supersensitized" to minor oncogenic events, which could result in investigators overestimating the impact of a tumor suppressor gene, the TUM group wrote in their report. Meanwhile, long-term cultured cells, due to the number of genetic and epigenetic changes they harbor, "only partly reflect the cell of origin.
"In contrast, our model is designed to recapitulate truly somatic oncogene activation as we were able to avoid germline-activation of oncogenic KRAS," they continued. "In addition, one additional genetic hit was sufficient to induce tumor growth."
Overall, the approach represents "an expandable and powerful tool to screen for new tumor suppressor genes and will broaden our understanding of cancer biology," the researchers concluded. "Moreover, the principle of stepwise in vitro acquisition of genetic hits in primary pancreatic ductal cells may be transferrable to other techniques of gene modulation."