The role of the tumor suppressor gene, FBW7, and mechanisms of its inactivation in cancer

Abstract: The F3box protein FBW7 is a tumor suppressor and SCF ubiquitin ligase targeting several key oncoproteins for proteasomal degradation. In this thesis we addressed whether the FBW7 gene is inactivated by mutations in various human tumor types and explored alternative mechanism(s) for the inactivation of FBW7. We also explored how inactivation of FBW7 relates to substrate degradation (including cyclin E, Notch1 and c3 Myc) and its potential prognostic significance. Furthermore, we have investigated novel regulatory mechanism(s) for FBW7 expression and activity. Our results demonstrate that FBW7 is a general TSG, which is frequently inactivated by mutations (with an average mutation frequency of 6 ) in various malignancies. Heterozygous missense mutations altering specific arginines residues required for efficient substrate interaction are the most frequent mutations in FBW7. Our functional analysis indicates that heterozygous mutations might act in a dominant3negative manner. The highest mutation frequency was observed in cholangiocarcinomas and pediatric T3cell acute lymphocytic leukemias (T3ALL) (35 and 31 , respectively). We also found that FBW7 gene mutation is an infrequent event in several malignancies, including breast cancer and pediatric B3ALL. This finding prompted us to investigate whether alternative mechanism for regulation and inactivation of FBW7 occurs in cancer, including promoter hypermethylation and miRNA induced repression. Our results demonstrate that both the 5’3UTR and the 3’3UTR of FBW7 is epigenetically regulated. The promoter of FBW7? is frequently hypermethylated in primary breast tumors and its inactivation is associated with improved survival in certain patient subgroups. Similarly, mutational inactivation of FBW7 and/or NOTCH1 in T3ALL is also associated with increased overall survival. Analyses focusing on the 3’UTR of FBW7 revealed that FBW7 expression is regulated by miR327a, a putative oncogenic miRNA. miR327a was identified as a critical repressor of FBW7 expression during cell cycle progression with potential consequences for FBW73mediated turnover of cyclin E. These results indicate that miR327a serves an important cell cycle regulatory function by repressing FBW7 and at specific cell cycle stages, but releasing it from repression during the G1 to S3phase transition. In summary, our findings demonstrate that FBW7 is inactivated by several different 3 mechanisms, including mutation, deletion, promoter methylation and possibly miRNA3 induced repression of gene expression. Our pre3clinical analysis further suggests that inactivation of FBW7 in certain malignancies might be associated with improved survival, thus implicating FBW7 as a potential prognostic predictor in the some cancers.