SUMOylated CPAP is required for IKK-mediated NF-κB activation and enhances HBx-induced NF-κB signaling in HCC.
OBJECTIVE
Constitutive activation of NF-κB is an important event involved in chronic inflammation in hepatocellular carcinoma (HCC). CPAP, which plays important roles in centrosomal functions, was previously identified as the transcriptional co-activator of NF-κB. However, the molecular mechanism is unclear. The goal of this study was to investigate the role of CPAP in activating the NF-κB pathway in HCC.


METHODS
SK-Hep1, HuH7, HepG2, HepG2X, Hep3B, and Hep3BX cells with CPAP overexpression or CPAP siRNA were used to evaluate activation of NF-κB under TNF-α stimulation by reporter assay, RT-PCR, Q-PCR, and Western blot analysis. In vivo SUMO modification of CPAP was demonstrated by an in situ PLA assay. Human HCC tissues were used to perform Q-PCR, Western blot, and IHC.


RESULTS
CPAP siRNA abolished the interaction between IKKβ and NF-κB, whereas overexpression of CPAP enhanced this interaction and finally led to augmented NF-κB activation by increasing the phosphorylation of NF-κB. CPAP could enter nuclei by associating with NF-κB. Furthermore, CPAP was SUMO-1 modified upon TNF-α stimulus, and this is essential for its NF-κB co-activator activity. SUMO-1-deficient CPAP mutant lost its NF-κB co-activator activity and failed to enter nuclei. Importantly, SUMOylated CPAP could synergistically increase the HBx-induced NF-κB activity.


CONCLUSIONS
CPAP is essential for the recruitment of the IKK complex to inactivated NF-κB upon TNF-α treatment. Expression of CPAP was positively correlated with a poor prognosis in HBV-HCC. CPAP has the potential to serve as a therapeutic target for inflammation and inflammation-related diseases.
Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan.
Shu-Ting
ST
Yang
Chia-Jui
CJ
Yen
Chien-Hsien
CH
Lai
Yih-Jyh
YJ
Lin
Kung-Chao
KC
Chang
Jenq-Chang
JC
Lee
Yao-Wen
YW
Liu
Pey-Yi
PY
Chang-Liao
Lu-Shin
LS
Hsu
Wen-Chang
WC
Chang
Wen-Chun
WC
Hung
Tang K
TK
Tang
Yi-Wen
YW
Liu
Liang-Yi
LY
Hung
D006528:Carcinoma, Hepatocellular / Q000209:etiology* / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase / Q000502:physiology*; D020672:I-kappa B Proteins / Q000378:metabolism; D008113:Liver Neoplasms / Q000209:etiology* / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000502:physiology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000502:physiology*; D010766:Phosphorylation; D025842:SUMO-1 Protein / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D058207:Sumoylation; D015534:Trans-Activators / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D054334:Viral Regulatory and Accessory Proteins
C414735:CENPJ protein, human; D020672:I-kappa B Proteins; D008869:Microtubule-Associated Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D025842:SUMO-1 Protein; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D000072000:NF-KappaB Inhibitor alpha; D051550:I-kappa B Kinase