Functionalization of cancer-associated mutant alleles of human CDC4 (FBXW7)

Cancer is a leading cause of death worldwide. This somatic cell genetic disease is characterized by progressive accumulation of mutations in multiple genes. An important characteristic of cancer cells is an increased rate of gains and losses of chromosomes, termed Chromosomal Instability (CIN). One of the frequently mutated genes in a variety of cancers is FBXW7 (F-Box and WD repeat domain-containing 7), encoding the substrate-recognition component of a ubiquitin ligase complex. Fbxw7 targets a number of oncoproteins such as, Cyclin E, c-Myc, Notch1 and Aurora A for ubiquitin mediated degradation. Inactivation of FBXW7 has been linked to CIN in cancer cell lines. The majority of cancer-associated mutations in FBXW7 are monoallelic, missense substitutions whose phenotypic effects are difficult to predict. Interestingly, most of the mutations in FBXW7 cluster at three mutational hotspots, Arg465, Arg479 and Arg505. Located at β propeller-tip, these residues are critical for interaction with the Fbxw7 substrates. This study investigates the functional consequences of the substitutions at these residues. We individually tested the functional status of the R465C, R479Q and R505C variants of FBXW7 in three colorectal cancer cell lines in an HCT116 background. These cell lines had both, one or none of the alleles of FBXW7 inactivated by homologous recombination. Our data shows that the cell lines producing R465C, R479Q or R505C variants of FBXW7 failed to degrade Cyclin E, one of the major targets of FBXW7. These cell lines also exhibited a CIN phenotype, observed as an increase in the frequency of abnormal anaphases. These results show that mutations R465C, R479Q and R505C occurring in FBXW7 cause loss of function of the protein and act as dominant negative mutations.Science, Faculty ofGraduat