Tu Kangsheng, Yang Wei, Li Chao, Zheng Xin, Lu Zhongtang, Guo Cheng, Yao Yingmin, Liu Qingguang
Department of Hepatobiliary Surgery, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, China.
Mol Cancer. 2014 May 17;13:110. doi: 10.1186/1476-4598-13-110.
The 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.
The 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.
In 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.
These results indicate that Fbxw7 may serve as a prognostic marker and that YAP may be a potential target of Fbxw7 in HCC.
E3泛素连接酶Fbxw7通过靶向几种著名的癌蛋白进行泛素化和蛋白酶体降解，发挥着一般肿瘤抑制因子的作用。然而，Fbxw7的临床意义以及Fbxw7在肝癌中抗癌作用的相关机制尚不清楚。
采用免疫组织化学（IHC）或免疫印迹法评估60例手术切除的肝癌组织及配对的癌旁正常组织中Fbxw7和YAP的表达。使用流式细胞术、半胱天冬酶3/7活性测定、BrdU细胞增殖测定和MTT测定来检测肝癌细胞的增殖和凋亡。通过实时定量聚合酶链反应（qRT-PCR）、免疫印迹和免疫荧光法证实Fbxw7对肝癌细胞中YAP的调控作用。采用免疫共沉淀法分析YAP与Fbxw7之间的相互作用。通过裸鼠皮下注射、Ki-67染色和TUNEL测定来评估体内肿瘤生长和凋亡情况。
在本研究中，我们发现肝癌组织中Fbxw7表达受损，Fbxw7表达缺失与不良临床病理特征相关，包括肿瘤体积大、静脉浸润、高病理分级和晚期TNM分期。此外，我们证明Fbxw7表达阳性的患者5年生存率更高，且Fbxw7是预测肝癌患者预后的独立因素。我们证实Fbxw7通过诱导凋亡和生长停滞来抑制肝癌。在同一组肝癌组织中观察到YAP表达升高。Pearson相关系数分析表明，肝癌组织中Fbxw7与YAP蛋白表达呈负相关。我们还发现Fbxw7通过靶向YAP进行泛素化和蛋白酶体降解来调节肝癌中YAP蛋白的丰度。此外，恢复YAP表达部分消除了Fbxw7在体外和体内诱导的肝癌细胞凋亡和生长停滞。
这些结果表明，Fbxw7可能作为一种预后标志物，而YAP可能是Fbxw7在肝癌中的潜在靶点。
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