Functional characterization of FBXW7 mutations in colorectal cancer
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1
UKM Medical Molecular Biology Institute (UMBI), Malaysia
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2
Department of Surgery, Faculty of medicine, University of Malaya, Malaysia
Background
The somatic mutational landscape of colorectal cancer (CRC) has been widely profiled, yet is inadequately characterized functionally. One of the most frequent somatic mutations in CRC is in the F-box WD repeat domain-containing-7 (FBXW7) gene. FBXW7 encodes for F-box protein, the substrate-recognition subunit of the SKP1–Cullin1–F-box (SCF) E3 ubiquitin ligase. Mutations in the FBXW7 could inhibit the ubiquitin-mediated degradation of various oncoproteins, leading to the accumulation of its oncogenic substrates and, subsequently, cancer progression. Those mutations are also associated with chemotherapy resistance, although the mechanisms are poorly understood.
Methods
FBXW7 mutations were detected via whole genome sequencing (WGS) and validated using Sanger sequencing. FBXW7 expression in matched tumour-normal samples was analysed using real-time quantitative PCR (RT-qPCR). DDK-tagged expression plasmids of FBXW7 wild-type (wt), R479Q and G654fs mutations were introduced into 293T, HCT116, and SW1463 cell lines, and the transfected cells were assayed for FBXW7 gene expression, cell viability, response to 5-fluorouracil (5-FU) treatment, and protein expression using targeted antibody arrays.
Results
FBXW7 expression is down-regulated in CRC tissues compared to normal tissues. Introduction of R479Q and G654fs mutations into all the cell lines resulted in higher cell proliferation than in wt-FBXW7-transfected cells. On the other hand, restoring wt-FBXW7 in SW1463 cell line carrying the R479Q mutation inhibited cell proliferation and increased sensitivity to 5-FU treatment. Furthermore, ubiquitination levels of 38 proteins were decreased in both R479Q and G654fs transfected samples compared to the wild-type. The mutations were also observed to cause dysregulation of cancer-related proteins that are related to ubiquitin-mediated degradation.
Conclusion
Our results demonstrate that restoration of wt-FBXW7 in SW1463 influences cell viability and sensitizes SW1463 to 5-FU. The results also suggest FBXW7 inactivation caused by R479Q and G654fs mutations may contribute to the decreased levels of protein ubiquitination and dysregulation of cancer-related proteins, which could be the putative targets of FBXW7.
Keywords:
colorectal cancer,
protein expression,
Fbxw7,
5-fluorouracil,
Gene Expression
Conference:
International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18)
“Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019.
Presentation Type:
Oral Presentation
Topic:
Cancer
Citation:
Md Yusof
NF,
Ab Mutalib
N,
Mazlan
L and
Jamal
R
(2019). Functional characterization of FBXW7 mutations in colorectal cancer.
Front. Pharmacol.
Conference Abstract:
International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18)
“Seizing Opportunities and Addressing Challenges of Precision Medicine”.
doi: 10.3389/conf.fphar.2018.63.00037
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Received:
30 Sep 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Dr. Nurul-Syakima Ab Mutalib, UKM Medical Molecular Biology Institute (UMBI), Kuala Lumpur, Malaysia, syakima@ppukm.ukm.edu.my