Use of Caenorhabditis elegans to evaluate inhibitors of Ras function in vivo

The human RAS genes constitute the most frequently mutated oncogenes in human cancers, and the critical role of aberrant Ras protein function in oncogenesis is well established. Consequently, considerable effort has been devoted to the development of anti-Ras inhibitors for cancer treatment. An important facet of molecularly targeted cancer drug discovery is the validation of a target-based mechanism of action, as well as the identification of potential off-target effects. This chapter describes the use of the nematode worm Caenorhabditis elegans for simple, inexpensive pharmacogenetic analysis of candidate molecularly targeted inhibitors of mutationally activated Ras, with a focus on the Ras>Raf>MEK>ERK mitogen-activated protein kinase pathway. This protein kinase cascade is well conserved from worms to humans and is well established as a critical player in the signaling events leading to vulval formation in C. elegans. Excess activity results in the development of a multivulva (Muv) phenotype, whose inhibition by test compounds can be characterized genetically as to the specific step of the pathway that is blocked. In addition, off-target activities can also be identified and characterized further using different strains of mutant worms. This chapter presents proof-of-principle analyses using the well-characterized MEK inhibitor U0126 to block the Muv phenotype caused by the constitutively activated Ras homolog C. elegans LET-60. It also provides a detailed description of protocols and reagents that will enable researchers to analyze on- and off-target effects of other candidate anti-Ras inhibitors using this system.