ABSTRACT
Ajuba has been found to be mutated or aberrantly regulated in several human cancers and plays important roles in cancer progression via different signaling pathways. However, little is known about the role of Ajuba in hepatocellular carcinoma (HCC). Here, we found an upregulation of Ajuba expression in HCC tissues compared with normal liver tissues, while a poor prognosis was observed in HCC patients with high Ajuba expression. Knockout of Ajuba in HCC cells inhibited cell growth in vitro and in vivo, suppressed cell migration, and enhanced the cell apoptosis under stress. Moreover, re-expression of Ajuba in Ajuba-deficient cells could restore the phenotype of Ajuba-deficient cells. In conclusion, these results indicate that Ajuba is upregulated in HCC and promotes cell growth and migration of HCC cells, suggesting that Ajuba could possibly be a new target for HCC diagnosis and treatment.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Movement/genetics , Gene Deletion , LIM Domain Proteins/deficiency , LIM Domain Proteins/genetics , Liver Neoplasms/genetics , Animals , Apoptosis , Cell Division/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Knockout Techniques , Humans , Liver Neoplasms/pathology , Mice , Mice, Nude , Neoplasm Transplantation , Up-RegulationABSTRACT
Aurora-A is a serine/threonine kinase, which is overexpressed in multiple human cancers and plays a key role in tumorigenesis and tumor development. In this study, we found that the receptor of activated C-kinase1 (RACK1), an important regulator of biological functions, interacted with Aurora-A and co-localized with Aurora-A at centrosomes. Moreover, RACK1 induces the auto-phosphorylation of Aurora-A in vitro and in vivo. Depletion of RACK1 impaired the activation of Aurora-A in late G2 phase, then inhibited the mitotic entry and leaded to multi-polarity, severe chromosome alignment defects, or centrosome amplification. Taken together, these results suggest that RACK1 is a new partner of Aurora-A and play a critical role in the regulation of the Aurora-A activity during mitosis, which may provide a basis for future anticancer studies targeting Aurora-A.
Subject(s)
Aurora Kinase A/metabolism , G2 Phase Cell Cycle Checkpoints/genetics , M Phase Cell Cycle Checkpoints/genetics , Neoplasm Proteins/metabolism , Receptors for Activated C Kinase/metabolism , Aurora Kinase A/chemistry , Aurora Kinase A/genetics , Catalytic Domain , Centrosome/metabolism , HeLa Cells , Humans , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Phosphorylation/genetics , Protein Binding , Protein Stability , Receptors for Activated C Kinase/chemistry , Receptors for Activated C Kinase/genetics , Signal Transduction/genetics , Spindle Apparatus/metabolism , TransfectionABSTRACT
Human hepatocellular carcinoma (HCC) is a malignant cancer. It is a challenge to develop anti-HCC drugs due to HCC's extreme aggressiveness and with the sensitivity of the liver to show severe adverse effects. More importantly, the precise mechanisms causing HCC pathogenicity are not known. Our previous study disclosed Nogo-B as a reticulon 4 (Rtn4) family member. In the present study, we first identified that Nogo-B played a critical role in HCC progression. We found, via in vitro and in vivo assays, that Nogo-B was expressed aberrantly in primary HCC tumor tissues and immortal HCC cells but was relatively scarce in the normal liver tissues or cells. Nogo-B knockout, via the CRISPR-Cas9 technique, resulted in significant suppression of HCC cell proliferation and tumor growth. Next-generation sequencing analysis showed that Nogo-B knockout have effects on interleukin-6 (IL-6) signaling pathway. Furthermore, we observed that IL-6 induced phosphorylation of STAT3 (pSTAT3) in wild-type HCC cells, but Nogo-B knockout could reduce IL-6-induced increase of pSTAT3, supporting that Nogo-B affects HCC tumor progression possibly via regulating the IL-6/STAT3 signaling pathway. In conclusion, Nogo-B is expressed aberrantly in HCCs and plays an oncogenic role. These findings support that Nogo-B may be a novel anti-HCC therapeutic target.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Nogo Proteins/genetics , Adult , Aged , Animals , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Disease Models, Animal , Disease Progression , Female , Gene Expression , Gene Knockout Techniques , Heterografts , Humans , Liver Neoplasms/metabolism , Male , Mice , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , Nogo Proteins/metabolism , Phosphorylation , STAT3 Transcription Factor/metabolism , Tumor BurdenABSTRACT
T-complex protein 10A homolog 2 (TCP10L) was previously demonstrated to be a potential tumor suppressor in human hepatocellular carcinoma (HCC). However, little is known about the molecular mechanism. MAX dimerization protein 1 (MAD1) is a key transcription suppressor that is involved in regulating cell cycle progression and Myc-mediated cell transformation. In this study, we identified MAD1 as a novel TCP10L-interacting protein. The interaction depends on the leucine zipper domain of both TCP10L and MAD1. TCP10L, but not the interaction-deficient TCP10L mutant, synergizes with MAD1 in transcriptional repression, cell cycle G1 arrest and cell growth suppression. Mechanistic exploration further revealed that TCP10L is able to stabilize intracellular MAD1 protein level. Consistently, the MAD1-interaction-deficient TCP10L mutant exerts no effect on stabilizing the MAD1 protein. Taken together, our results strongly indicate that TCP10L stabilizes MAD1 protein level through direct interaction, and they cooperatively regulate cell cycle progression. [BMB Reports 2016; 49(6): 325-330].
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cell Cycle Checkpoints/genetics , Proteins/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Cell Line, Tumor , HEK293 Cells , Humans , Immunoprecipitation , Leucine Zippers , Protein Binding , Protein Domains , Protein Stability , Proteins/chemistry , ProteolysisABSTRACT
Aurora-A, a centrosome-localized serine/threonine kinase, is over-expressed in multiple human cancers. We previously reported Zhang et al. (Biochem Biophys Res Commun 2007, 357:347-352) intramolecular inhibitory regulation of Aurora-A between its N-terminal (Nt) regulatory domain (amino acids 1-128, Nt) and C-terminal catalytic domain (aa 129-403, Cd). Here, we identified two essential sites located on the Nt of Aurora-A (Lys 99 and Lys 119) and demonstrate that mutation of either residue to Gly could cause the Nt and C-terminal lobes of the catalytic domain in Aurora-A to form a closed conformation, resulting in a loss of kinase activity. This inactive conformation was reversed by adding C26 peptide (274-299) or Ajuba, which is a required activator of Aurora-A. Over-expression of either mutant induced G2/M arrest. These results provide a basis for future anti-cancer studies targeting Aurora-A.
Subject(s)
Aurora Kinase A/antagonists & inhibitors , Aurora Kinase A/chemistry , Amino Acid Sequence , Amino Acids/metabolism , Aurora Kinase A/metabolism , Binding Sites , Cell Cycle Checkpoints , Enzyme Activation , HEK293 Cells , HeLa Cells , Humans , LIM Domain Proteins/metabolism , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Peptides/metabolism , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
Aurora-A is a centrosome-localized serine/threonine kinase that is overexpressed in multiple human cancers. We previously reported an intramolecular inhibitory regulation of Aurora-A between its N-terminal regulatory domain (Nt, amino acids [aa] 1-128) and the C-terminal catalytic domain (Cd, aa 129-403). Here, we demonstrate that although both Aurora-A mutants (AurA-K250G and AurA-D294G/Y295G) lacked interactions between the Nt and Cd, they also failed to interact with Ajuba, an essential activator of Aurora-A, leading to loss of kinase activity. Additionally, overexpression of either of the mutants resulted in centrosome amplification and mitotic spindle formation defects. Both mutants were also able to cause G2/M arrest and apoptosis. These results indicate that both K250 and D294/Y295 are critical for direct interaction between Aurora-A and Ajuba and the function of the Aurora-A complex in cell cycle progression.
