Abstract
BackgroundThe E3 ubiquitin ligase Fbxw7 functions as a general tumor suppressor by targeting several well-known oncoproteins for ubiquitination and proteasomal degradation. However, the clinical significance of Fbxw7 and the mechanisms involved in the anti-cancer effect of Fbxw7 in HCC are not clear.MethodThe Fbxw7 and YAP expression in 60 samples of surgical resected HCC and matched normal tumor-adjacent tissues were assessed using IHC or immunoblotting. Flow cytometry, caspase 3/7 activity assay, BrdU cell proliferation assay and MTT assay were used to detect proliferation and apoptosis of HCC cells. The regulatory effect of Fbxw7 on YAP in HCC cells was confirmed by qRT-PCR, immunoblotting and immunofluorescence. Co-immunoprecipitation was used to analyze interaction between YAP and Fbxw7. Nude mice subcutaneous injection, Ki-67 staining and TUNEL assay were used to evaluate tumor growth and apoptosis in vivo.ResultsIn this study, we found that Fbxw7 expression was impaired in HCC tissues and loss of Fbxw7 expression was correlated with poor clinicopathological features including large tumor size, venous infiltration, high pathological grading and advanced TNM stage. Additionally, we demonstrated that patients with positive Fbxw7 expression had a better 5-year survival and Fbxw7 was an independent factor for predicting the prognosis of HCC patients. We confirmed that Fbxw7 inhibited HCC by inducing both apoptosis and growth arrest. Elevated YAP expression was observed in the same cohort of HCC tissues. Pearson's correlation coefficient analysis indicated that Fbxw7 was inversely associated with YAP protein expression in HCC tissues. We also found that Fbxw7 regulated YAP protein abundance by targeting YAP for ubiquitination and proteasomal degradation in HCC. Furthermore, restoring YAP expression partially abrogated Fbxw7 induced HCC cell apoptosis and growth arrest in vitro and in vivo.ConclusionThese results indicate that Fbxw7 may serve as a prognostic marker and that YAP may be a potential target of Fbxw7 in HCC.
Highlights
Fbxw7 (F-box and WD repeat domain-containing 7) known as Fbw7, a well-known F-box protein in the SCF (SKP1-CUL1-F-box protein) E3 ligase complex, determines target specificity by recognizing and binding proteins, leading to their ubiquitination and proteasomal degradation [1]
We found that Fbxw7 regulated Yes-associated protein (YAP) protein abundance by targeting YAP for ubiquitination and proteasomal degradation in Hepatocellular carcinoma (HCC)
20 cases were subjected to immunoblotting for Fbxw7; we found that the Fbxw7 protein level in HCC tissues was significantly lower than that in the matched normal, tumor adjacent, tissues (P < 0.01, Figure 1A)
Summary
Fbxw (F-box and WD repeat domain-containing 7) known as Fbw, a well-known F-box protein in the SCF (SKP1-CUL1-F-box protein) E3 ligase complex, determines target specificity by recognizing and binding proteins, leading to their ubiquitination and proteasomal degradation [1]. Recent studies have identified several specific substrates of Fbxw including c-Myc [2], Cyclin E [3], c-Jun [4], SREBP1 (Sterol regulatory element binding protein-1) [5], mTOR (Mammalian target of rapamycin) [6], Notch-1 [7] and MCL-1 (Myeloid cell leukemia-1) [8] These target proteins typically contain the conserved CPD (Cdc phospho-degron) (L)X-pT/pS-P-(P)-X-pS/pT/E/D (X = any amino acid) motif and the amino residues in CPD must first be phosphorylated in order for Fbxw to efficiently recognize and mediate the ubiquitination of the target protein [1,9]. The clinical significance of Fbxw and the mechanisms involved in the anti-cancer effect of Fbxw in HCC are not clear
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