Dependence of Human Colorectal Cells Lacking the FBW7 Tumor Suppressor on the Spindle Assembly Checkpoint

Abstract

FBW7 (F-box and WD repeat domain containing 7), also known as FBXW7 or hCDC4, is a tumor suppressor gene mutated in a broad spectrum of cancer cell types. As a component of the SCF E3 ubiquitin ligase, FBW7 is responsible for specifically recognizing phosphorylated substrates, many important for tumor progression, and targeting them for ubiquitin-mediated degradation. Although the role of FBW7 as a tumor suppressor is well established, less well studied is how FBW7-mutated cancer cells might be targeted for selective killing. To explore this further, we undertook a genome-wide RNAi screen using WT and FBW7 knockout colorectal cell lines and identified the spindle assembly checkpoint (SAC) protein BUBR1, as a candidate synthetic lethal target. We show here that asynchronous FBW7 knockout cells have increased levels of mitotic APC/C substrates and are sensitive to knockdown of not just BUBR1 but BUB1 and MPS1, other known SAC components, suggesting a dependence of these cells on the mitotic checkpoint. Consistent with this dependence, knockdown of BUBR1 in cells lacking FBW7 results in significant cell aneuploidy and increases in p53 levels. The FBW7 substrate cyclin E was necessary for the genetic interaction with BUBR1. In contrast, the establishment of this dependence on the SAC requires the deregulation of multiple substrates of FBW7. Our work suggests that FBW7 knockout cells are vulnerable in their dependence on the mitotic checkpoint and that this may be a good potential target to exploit in FBW7-mutated cancer cells.

RNAi screen reveals synthetic lethality between cyclin G-associated kinase and FBXW7 by inducing aberrant mitoses

“…Mitotic duration was calculated for 100 cells for each siRNA. Although small, we observed a statistically significant difference in the median mitotic duration for the NT controls between the FBXW7-proficient and -deficient cells: 21 vs 27 min, respectively ( n =100, P <0.0001), perhaps suggesting a tendency for the FBXW7-null cells to have more problems aligning their chromosomes, as previously reported ( Rajagopalan et al , 2004 ; Bailey et al , 2015 ). For the cells treated with GAK RNAi, the median mitotic interval was increased to 30 min (95% CI 33.67–46.73) vs 33 min (43.03–57.59), respectively.…”

“…The severe cell cycle disruption with multipolar defects means that it is more likely to be a mitotic defect, although elucidating the exact mechanism of cell death has proven difficult. The GAK-FBXW7-deficient phenotype appears different to GAK inhibition in other cells ( Shimizu et al , 2009 ; Tanenbaum et al , 2010 ) and not related to loss of the spindle assembly checkpoint in HCT116 cells ( Bailey et al , 2015 ). Currently, there are no commercially available selective GAK inhibitors to allow the use of kinase inhibitors to simulate siRNA effects.…”

FBXW7 in Cancer: What Has Been Unraveled Thus Far?

“…Increasing lines of evidence have revealed that aberrant expression of F-box is associated with development, proliferation, angiogenesis, and metastasis of various malignancies [8]. Amongst the F-box family, F-box with 7 tandem WD40 repeats (FBXW7) which are responsible for recognizing and targeting various oncogenic substrates for ubiquitin-mediated degradation has been found to be deregulated in diverse cancers [13,14].…”

Overexpression of Fbxo6 inactivates spindle checkpoint by interacting with Mad2 and BubR1

“…FBPs superfamily includes 70 members [14], several of which played critical roles in cell cycle control, such as Fbxw1 (Beta-TrcP) [15], Fbxw5 [16], Fbxw7 [17], Fbxl1 (Skp2) [18], Fbxl2 [19], Fbxo4 [20], Fbxo5 [21] and Fbxo31 [22]. However, the function of FBPs during mitosis remains unclear.…”