Final Report Summary - TRANSPOSON GET NCRNA (Transposon based Forward Genetic Screen for the Identification of ncRNAs involved in Colorectal Cancer Metastasis) Colorectal cancer (CRC) is the second leading cause of cancer related deaths, mainly due to the lack of effective therapies to treat advanced metastatic CRC. The project “Transposon get ncRNA” aims to identify non-coding RNAs (ncRNA) involved in CRC metastasis through the development of a novel in vitro transposon based forward genetic screen. We have designed and successfully implemented a functional forward genetic screen comprised of a) in vitro transposition of CRC cells (the mutagenic tool) and of b) the modified anoikis resistance assay (the functional screening assay).Our results prove that both arms of the screen are effective: the transposon used is mutagenic and the in vitro selection assay is capable to select cells with a pro-metastatic advantage.In particular, we identified a novel molecular mechanism involving a protein-coding gene and a ncRNA important for cell survival during the modified anoikis resistance assay. We are now analyzing the clinical relevance of this finding in colorectal cancer specimens and we are performing in vivo mouse experiments to prove the significance of this mechanism during CRC metastasis. The described molecular mechanism, if supported by data from patient sample and in vivo studies, can be of future help for the design of novel therapeutic targets to eventually improve the outcome of patients with metastatic CRC.The support by CIG has been fundamental to develop this project and to advance Dr. Nicoloso’s career: it has provided her with the economic independence necessary to expand her research group and to support the experimental work. The results obtained are now being assembled to produce an original scientific publication and represent a solid platform on which additional grant projects can be developed.The data collected during the implementation of “Transposon get ncRNA” project supported by CIG has lead us to develop a similar screening but with high throughput analysis, and most of the group efforts are currently dedicated to this part. ADVANCEMENT BEYOND THE STATE OF THE ART IN THE FIELD:Transposons are currently used in forward mutagenic screens as carriers of protein coding gene traps (Ivics et al., 2009), which consist of splicing acceptors, splicing donors, poly adenylation sites and strong promoters. We assumed that this stratagem, although being powerful in inducing mutations (Starr et al., 2009), neglects the discovery of non-coding elements by targeting coding regions. Instead, our work represents a proof of concept that transposons devoid of protein coding gene traps can be used as a mutagenic tool targeting non-coding elements of the genome by simple insertional mutagenesis. Additionally, whereas all the transposon mutagenic screens have been conducted in vivo using tumorigenic mice models, we have shown that it can be used effectively also in vitro with cells that are then selected in a functional assay. Furthermore, the functional in vitro assay we have developed (forced Single Cell Suspension Assay, fSCS) is completely novel, and more stringent than the anoikis resistance assay that has been used so far to study one of the barriers to metastasis (Douma et al., 2004). By implementing the “Transposon get ncRNA” screen we have identified a novel regulatory mechanism involving the 3’UTR of a protein coding gene and a putative miRNA binding site, demonstrating that transposons can be harnessed for the discovery of novel genetic elements.IMPACTThe transposon mutagenic tool that we have implemented is straightforward and can be used to address different critical aspect of cancer biology. In fact, transposed cells can be subjected to various functional assays and by retrieving the insertion associated with the gain of function/surviving cells we can get insight into the genetic elements that regulate the process under investigation, for instance it can be used to evaluate chemoresistance. We are trying to exploit the forced Single Cell Suspension assay as an alternative assay that allows the selection and analyses of cancer cell populations with pro-metastatic features and in addition it can be easily used in high-throughput screens for drug compounds testing. The social value of the study lies on the fact that, by studying with alternative approaches colorectal cancer metastasis, we expect to identify novel mechanism that can be used in CRC patients management and eventually treatment.
