Mechanisms of Neoplastic Transformation in Human Cells

Duke University, Durham NC

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Abstract

Cancer is derived from the accumulation of mutations in genes that derail the normal growth regulatory mechanisms of cells. One of the most commonly mutated set of genes are those belonging to ras family. These genes encode G-proteins that transfer growth-promoting signal from extracellular queues to the intracellular machinery. Mutations in these genes found in cancers leave the protein in a constitutively active state that results in the constant stimulation of three main classes of proteins, or effectors: Raf1, PI3-kinase and a group of proteins termed RalGEFs. Studies with murine cells categorically demonstrate that the most potent effector of ras in oncogenic transformation is Raf1. We recently developed the first system to test the effect of genetic changes in normal primary human cells on the processes of transformation in vitro and tumour growth in vivo. Using this novel system we tested whether mutations that leave ras capable of activating only one of the three effectors perturb the ability of oncogenic (12V) H-ras to transform human cells. We find that unlike the situation in murine cells, the mutation 37G, which leaves the RalGEFs pathway intact, was the only mutation that did not totally abolish the ability of H-ras12V to support the tumourigenic phenotype of anchorage-independent growth of human cells. RalGEFs had previously been envisioned to play only a small role in transformation. These results suggest that transformation as classically defined in NIH 3T3 murine cells is not representative of the transformation process in human cells. Dissecting the transformation process in human cells will be critical to the understanding of the process of oncogenesis. We propose to test whether the RalGEF pathway is essential while the Raf1 pathway is dispensable for transformation in vitro and tumourigenesis in vivo by: 1) Determine the role of RalGEFs in oncogenesis. 2) Determine what role the Raf1 pathway plays in oncogenesis. 3) Determine the role of effector proteins in tumourous growth of human cells in vivo The accomplishment of the above aims will define a role for the RalGEF and Raf1 pathways in human cancer, which ultimately may help define proteins to targets for the treatment of human cancers.