Abstract
BackgroundAberrant activation of β-catenin and Yes-associated protein (YAP) signaling pathways has been associated with hepatocellular carcinoma (HCC) progression. The LIM domain protein Ajuba regulates β-catenin and YAP signaling and is implicated in tumorigenesis. However, roles and mechanism of Ajuba expression in HCC cells remain unclear. The E3 ligase Hakai has been shown to interact with other Ajuba family members and whether Hakai interacts and regulates Ajuba is unknown.MethodsHCC cell lines stably depleted of Ajuba or Hakai were established using lentiviruses expressing shRNAs against Ajuba or Hakai. The effects of Ajuba on HCC cells were determined by a number of cell-based analyses including anchorage-independent growth, three dimension cultures and trans-well invasion assay. In vivo tumor growth was determined in a xenograft model and Ajuba expression in tumor sections was examined by immunohistochemistry. Co-immunoprecipitation, confocal microscopy and immunoblot assay were used to examine the expression and interaction between Ajuba and Hakai.ResultsDepletion of Ajuba in HCC cells significantly enhanced anchorage-independent growth, invasion, the formation of spheroids and tumor growth in a xenograft model, suggesting a tumor suppressor function for Ajuba in HCC. Mechanistically, Ajuba depletion triggered E-cadherin loss and β-catenin translocation with increased Cyclin D1 levels. In addition, depletion of Ajuba upregulated the levels of YAP and its target gene CYR61. Furthermore, siRNA-mediated knockdown of either β-catenin or YAP attenuated the pro-tumor effects by Ajuba depletion on HCC cells. Notably, Ajuba stability in HCC cells was regulated by Hakai, an E3 ligase for E-cadherin. Hakai interacted with Ajuba via its HYB domain and induced Ajuba neddylation, which was antagonized by the neddylation inhibitor, MLN4924, but not MG132. We further show that overexpression of Hakai in HCC cells markedly increased anchorage-independent growth, spheroid-formation ability and tumor growth in xenografts whereas Hakai depletion resulted in these opposite effects, indicating an oncogenic role for Hakai in HCC. Hakai also induced β-catenin translocation with increased levels of Cyclin D1.ConclusionsOur data suggest a role for Ajuba and Hakai in HCC, and uncover the mechanism underlying the regulation of Ajuba stability.
Highlights
Aberrant activation of β-catenin and Yes-associated protein (YAP) signaling pathways has been associated with hepatocellular carcinoma (HCC) progression
We further show that overexpression of Hakai in HCC cells markedly increased anchorageindependent growth, spheroid-formation ability and tumor growth in xenografts whereas Hakai depletion resulted in these opposite effects, indicating an oncogenic role for Hakai in HCC
We further show that Ajuba protein turnover in HCC cells is mediated by E3 ubiquitin ligase Hakai via neddylation, while Hakai promoted HCC cell growth both in vitro and in vivo
Summary
Aberrant activation of β-catenin and Yes-associated protein (YAP) signaling pathways has been associated with hepatocellular carcinoma (HCC) progression. Somatic mutations in the β-catenin and Axin genes are involved in the aberrant activation of the Wnt/β-catenin signaling in HCC [1, 5], cytoplasmic accumulation and/or nuclear translocation of β-catenin were found to be independent of β-catenin mutations in the subclass of more aggressive tumors [4]. In addition to β-catenin signaling, the emerging role of the Hippo tumor suppressor cascade in liver tumorgenesis has been well established [6, 7]. YAP and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators, are the main downstream effectors of the mamalian Hippo signaling pathway. A growing number of studies document the oncogenic roles of YAP as well as TAZ in liver tumorgenesis and progression [8,9,10,11,12,13]
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