Establishment and analysis of a novel mouse line carrying a conditional knockin allele of a cancer-specific FBXW7 mutation

Tsuneo Ikenoue,Tomoyuki Ohsugi,Xun Liu,Yumi Terakado,Kiyoshi Yamaguchi,Chi Zhu,Daisuke Matsubara,Takuma Shibata,Sachiko Kubo,Tomoki Fujii,Shigeru Kakuta,Yoichiro Iwakura,Yoichi Furukawa

doi:10.1038/s41598-018-19769-1

Abstract

F-box and WD40 domain protein 7 (FBXW7) is a component of the SKP1-CUL1-F-box protein (SCF) complex that mediates the ubiquitination of diverse oncogenic target proteins. The exploration of FBXW7 mutations in human primary cancer has revealed three mutation hotspots at conserved arginine residues (Arg465, Arg479, and Arg505) in the WD40 domain, which are critical for substrate recognition. To study the function of human FBXW7R465C, the most frequent mutation in human malignancies, we generated a novel conditional knockin mouse line of murine Fbxw7R468C corresponding to human FBXW7R465C. Systemic heterozygous knockin of the Fbxw7R468C mutation resulted in perinatal lethality due to defects in lung development, and occasionally caused an eyes-open at birth phenotype and cleft palate. Furthermore, mice carrying liver-specific heterozygous and homozygous Fbxw7R468C alleles cooperated with an oncogenic Kras mutation to exhibit bile duct hyperplasia within 8 months of birth and cholangiocarcinoma-like lesions within 8 weeks of birth, respectively. In addition, the substrates affected by the mutant Fbxw7 differed between the embryos, embryonic fibroblasts, and adult liver. This novel conditional knockin Fbxw7R468C line should be useful to gain a more profound understanding of carcinogenesis associated with mutation of FBXW7.

Highlights

F-box and WD40 domain protein 7 (FBXW7) is an F-box protein in the SKP1-CUL1-F-box protein (SCF) E3 ubiquitin ligase complex that controls the degradation of target substrates via the ubiquitin-proteasome pathway
FBXW7 is located on chromosome 4q32, where deletions are frequently observed in human cancers
Most of the mutations are missense mutations affecting arginine residues (Arg[465], Arg[479], and Arg505) within the WD40 domain, which is responsible for substrate recognition (Fig. 1A)[16], and R465C is the mutation most frequently detected in human primary tumours (Catalogue of Somatic Mutations in Cancer; http://www.sanger. ac.uk/cosmic)[16]

Summary

Results and Discussion

Generation of transgenic mice containing a conditional knockin allele of the Fbxw7R468C mutation. Real-time PCR analyses revealed no difference in the mRNA expression levels of some Fbxw[7] substrates between MEF cells from Fbxw7R468C/+ mice and those from wild-type mice (Figure S3) These data suggest that the R468C mutation affects the stability of specific target proteins in a cell type-specific manner. The expression of mTor, Srebp[1], and Mcl[1] was modestly increased in the livers of homozygous mice (Fig. 6D) These data suggest that the accumulation of these proteins may be involved in the abnormal development of the bile duct in Fbxw7Hep-R468C/R468C mice. No abnormal histological findings were detected in Alb-Cre;KrasLSL-G12D/+ (KrasHep-G12D/+) mice, carrying the liver-specific KrasG12D alone, until 8 months after birth, the heterozygous Fbxw7LSL-R468C mutation in combination with the oncogenic Kras mutation induced bile duct dilation and hyperplasia in some mice at the age of 8 months (Fig. 7A and B). This conditional knockin mouse line of the cancer-associated hotspot mutation of FBXW7 should be a useful tool for elucidating the mechanisms underlying human neoplasms that are associated with FBXW7 mutation

Materials and Methods

Author Contributions

Additional Information