The α-enolase (ENO1) plays pivotal roles in several types of cancer, but its clinical significance, functional role, and possible mechanism in colorectal cancer (CRC) have remained unclear. Expression level of ENO1 in CRC tissues was examined by qRT-PCR, Western blot, and immunohistochemistry. The effects of ENO1 on cell growth were investigated by MTT, colony formation, flow cytometry assays, and in vivo tumorigenic capacity analysis. The impacts of ENO1 on cell migration and invasion were also explored by scratch-healing, Transwell or Matrigel chamber assays, and in vivo metastatic capacity analysis. Our results showed that the expression level of ENO1 was significantly elevated in CRC tissues. High expression level of ENO1 was associated with disease progression in CRC patients. Overexpression of ENO1 in HCT116 cell line promoted cell proliferation, migration, and invasion in vitro as well as tumorigenesis and metastasis in vivo. In other hand, ablation of ENO1 in HCT116 cells led to totally reverse effects. Mechanistically, we revealed ENO1 could regulate AMPK/mTOR signaling pathway. AMPK pathway activation or mTOR pathway suppression blocked these ENO1 induced alterations. Together, our results demonstrated that ENO1 is a potent promoter of CRC genesis and metastasis at least in part though regulating AMPK/mTOR pathway. These findings also suggested that ENO1 may be a promising therapeutic target in CRC patients.

FBXW7 (F-box and WD40 domain protein 7) is a tumor suppressor frequently inactivated in human cancers. The precise molecular mechanisms by which FBXW7 exerts antitumor activity remain under intensive investigation and are thought to relate in part to FBXW7-mediated destruction of key cancer-relevant proteins. Enolase 1 (ENO1) possesses oncogenic activity and is often overexpressed in various human cancers, besides its critical role in glycolysis. However, the detailed regulatory mechanisms of ENO1 expression remain unclear. Here we show that the elevated expression of ENO1 was identified in FBXW7-depletion HCT116 cells through two-dimensional protein electrophoresis and mass spectrometry assays (2DE-MS). Subsequent western blotting and immunohistochemical assays confirmed that ENO1 expression reversely correlates with FBXW7 expression in several cells and colon cancer tissues. Furthermore, we show that FBXW7 physically binds to ENO1 and targets ENO1 for ubiquitin-mediated degradation. Functionally, we found that FBXW7 suppresses the ENO1-induced gene expression, lactate production, cell proliferation and migration. These findings suggest that ENO1 is a novel substrate of FBXW7, and its activity can be negatively regulated by FBXW7 at the posttranslational level. Our work provides a novel molecular insight into FBXW7-directed tumor suppression through regulation of ENO1.

Ubiquitin specific protease 35 (USP35) is a member of deubiquitylases (DUBs). It remains largely unknown about the biological role and the regulation mechanism of USP35. Here, we first identified miR let-7a as a positive regulator of USP35 expression and showed that USP35 expression positively correlates with miR let-7a expression in different cancer cell lines and tissues. Then, we showed that USP35 expression was decreased dramatically in the tumor tissues compared with the adjacent non-cancerous tissues. USP35 overexpression inhibited cell proliferation in vitro and inhibited xenograft tumor growth in vivo. Furthermore, we revealed that USP35 acts as a functional DUB and stabilizes TNFAIP3 interacting protein 2 (ABIN-2) by promoting its deubiquitination. Functionally, both ABIN-2 and USP35 could inhibit TNFα-induced NF-κB activation and overexpression of ABIN-2 alleviated USP35-loss induced activation of NF-κB. Collectively, our data indicated that miR let-7a-regulated USP35 can inhibit NF-κB activation by deubiquitination and stabilization of ABIN-2 protein and eventually inhibit cell proliferation. Overall, our study provides a novel rationale of targeting miR let-7a-USP35-ABIN-2 pathway for the therapy of cancer patients.

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