The proposed project uses a RNA interference-based loss-of-function genetic screen in cancer cells to identify and study proteins with a role in tumor cell invasion. A retroviral vector-based human RNAi library and a set of syntethics iRNAs will be used to suppress the expression of specific genes in cancer cells. The library targets around 8000 human genes selected for their association with cancer and other human diseases and include entire gene families suitable for being therapeutical targets. The genes in the set have been selected for their possible implication in actin dynamics and migration. I will check for abnormal invasion phenotypes on these cells in a modification of a classical cell-based in vitro invasion assay on 3-D collagen matrices in resp onse to hepatocyte growth factor/scatter factor (HGF/SF), a protein that induces the migration and invasion of various types of cancer cells. For this study I plan to use initially the PC-3 human prostate cancer cell line that invade well in response to HG F and is suitable for the use of RNAi in gene silencing. Later on results obtained will be confirmed in another model cell line, the BE colon carcinoma cell line, as well as in another prostate cancer line with a different metastatic potential. At the beginning the invasion system will be exploited to analyse in detail the contribution to tumour invasion of the Rho family of small GTPases followed later on with a high-throughput screen using a subset of RNAi constructs targeting all the human protein kinases and a selective large-scale screen with the whole library to identify new regulators of cancer cell invasion that would potentially serve as new therapeutical targets against cancer. This project will also complete the researcher and apos;s expertise in basic cancer research and extend it to the field of tumour metastasis, with scope to continue the researcher and apos;s career with the findings obtained from the project in a future independent position.
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