Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

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.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0806-3) contains supplementary material, which is available to authorized users.

The LIM Protein Ajuba Augments Tumor Metastasis in Colon Cancer

“…We demonstrate that tumor samples of CRC metastasis from the liver were highly proliferative, as shown by Ki-67 staining, and Ajuba was highly expressed in the actively proliferating cells. Nevertheless, others report Ajuba to be anti-proliferative in other cancers such as malignant mesothelioma and HCC [ 10 , 40 ]. The diverse functions of Ajuba are defined by its cellular localization.…”

AJUBA: A regulator of epidermal homeostasis and cancer

“…AJUBA overexpression was shown to inhibit hepatocellular carcinoma cell proliferation by diminishing β-catenin and YAP levels, preventing their translocation and expression of Cyclin D1 and CYR61, respectively. 112 Loss of AJUBA also enhanced migration and is correlated to prostate cancer metastasis. 113 In prostate cancer, overexpression of miR-193a-3p, an androgen receptor binding miRNA, induces migration of LNCaP and C4-2B cells by downregulating its target AJUBA.…”

The LIM protein Ajuba/SP1 complex forms a feed forward loop to induce SP1 target genes and promote pancreatic cancer cell proliferation

“…However, the exact role of Ajuba in tumor progression remains controversial. A number of literatures reported that Ajuba acts as an oncogene and promotes tumor cell growth and migration, while others showed that Ajuba functions as a tumor suppressor and inhibits tumor cell growth and migration [10, 16].…”