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A study of the mechanism of action of novel inhibitors of tumour cell invasion McHardy, Lianne M.

Abstract

Metastasis is the leading cause of death in cancer patients. Tumour invasion and migration are critical aspects of metastatic progression. A forward chemical genetics project was initiated in an effort to identify novel compounds that inhibit tumour invasion. After screening a natural extract library, two novel inhibitors were identified: motuporamine C (MotC) and strongylophorine-26 (STP-26). Structure-activity studies identified dihdyromotuporamine C (dhMotC) as a potent and easily synthesized analogue. In this work, the mechanism of activity of dhMotC and STP-26 was investigated. It was found that both dhMotC and STP-26 affect cellular shape. dhMotC induced thick central actin stress fibres and large focal adhesions and caused cells to contract. STP-26 also induced adhesion formation but it reduced stress fibres and caused increased cell spreading. Both inhibitors activated Rho GTPase, a result which was shown to mediate, in large part, the anti-invasion activity of these molecules. Motuporamines also induce the formation of membrane-rich inclusions at the peri-nuclear region in cells. Motuporamines cause an increase in lysosomal pH and inhibit lysosomal function, such that EGFR/EGF complexes internalized in the presence of dhMotC do not get degradad. This inhibition of EGF degradation is not dependent on Rho activity. However, the anti-invasive activity of motuporamine analogues correlates well with their ability to induce the formation of membrane-rich inclusions. Thus, alteration in membrane trafficking or degradation of cellular membranes may be mechanistically related to the anti-invasion effect of motuporamines. A systematic genome-wide yeast haploinsufficiency screen was employed in an effort to identify possible targets of dhMotC. Yeast screening resulted in a list of 21 mutant strains which showed increased drug sensitivity. Sphingolipid biosynthesis was identified as a target in yeast cells. By testing other genes from the list, ARF1 was identified as a target that partially mediates the anti-invasive activity in human cells. The results of this body of work show that Rho and ARF1 are important molecular players in the mechanism of tumour cell invasion. This knowledge will contribute to the development of future anti-metastasis therapies and to the development of small molecules for use as biological probes to investigate the molecular basis of metastasis.

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