Protein translation control in cancer - Mechanistic dissection, in vivo quantification and therapeutic implications
Nuclear Medicine, Radiotherapy, Radiobiology
Final Report Abstract
Genome-wide screening techniques give deep insights into the mechanisms of cancer growth and the communication of cells. This project screened for factors relevant to the activation of the cell signaling molecule NFkB since this transcription factor is highly relevant for the growth of lymphoma, leukemia and other types of cancer. For this, a genetic reporter system was used in a large library of cells that carried specific knock-out lesions of essentially all genes of the genome in order to identify variants that would not be able to activate this reporter. The reporter was validated and characterized regarding dose-response function and kinetics. Control genes were successfully knocked out and inhibited the activation of the reporter, and this conferred resistance against the drug ganciclovir. However in the main experiments the results from genome sequencing indicated that cells were non-specifically selected, indicating that the drug used for killing of cells with functional reporter was also effective in neighboring cells due to cell-cell interactions (bystander effect). This is useful for the development of screening systems in better suited cells, e.g. cells growing in suspension, or the development of better reporter assays with genes not prone to the bystander effect. Work ongoing after the project will address these issues in order to glean insights into NFkB signaling in normal and cancer cells. The second part of the project aimed to develop a radiolabeling strategy for the protein translation inhibitor puromycin with the isotope F-18 (positron emitter with a half-life of 108 min). Since cancer cells have abnormally increased protein translation and this might be exploited for therapeutic modulation, non-invasive longitudinal imaging of protein translation is highly relevant for development of such treatments. Radioactive puromycin would be incorporated into newly synthesized cells and thus allow to non-invasively measure protein translation in cancer patients in order to characterize the tumor and identify the best treatment. Due to the high sensitivity of positron emission tomography in comparison to other imaging modalities, only low amounts of the toxic puromycin would be sufficient for this measurement, avoiding toxicity problems with the molecule. For this goal, a precursor molecule with a Boc protection group and Tosyl activation of the hydroxyl group to be labeled was synthesized and incubated with the pre-treated (purified and dried) radioactive isotope. Unfortunately, under all experimental conditions (e.g. different temperatures, solvents and reaction times) the intended reaction only occurred to a low amount insufficient for future application. These insights will be used for development of better-suited, automated multi-step radiolabeling approaches of this radiotracer that eventually will enable imaging studies of puromycin biodistribution and protein translation.
