P28～(GANK) Regulates NF-κB/RelA Signalling Pathway Uniquely

Hepatocelluar carcinoma (HCC) is one of the most prevalent malignant cancers in our country. The development and progression of HCC is a complicated process involving multiple genes and multiple steps in human bodies. Up to now, though some suppressors in HCC were identified such as p53 and p16INK4a, and confirmed their specific inactivation, few specific activated oncogenes in HCC was found and reported. In 2000, a novel gene named gankyrin with repeated sequences coding'ankyrin'motif was cloned in human HCC by Japanese scientists Fujita and his colleagues using subtractive hybridization method. We found this gankyrin gene was identical to the p28 (Nas6p) gene reported by Hori in 1998 by searching the GenBank data, thus named it as p28^GANK in our work.p28^GANK(also known as PSMD10, p28, Nas6p, and Gankyrin), a non-ATPase subunit of the 19S/PA700 complex from the 26S proteasome, contains five ankyrin repeats mediating proteins interaction. 20S proteasome is a protease degrading some incorrectly folding proteins or cell cycle regulators. Little is known about the mechanism how oncoprotein p28^GANK relates with proteins degradation by 26S proteasome. p28^GANK regulates the CDK4/CyclinD1/p16INK4a/Rb1/E2F-1 pathway which connects with tumor development and progression, and p28^GANK-transfected cells are tumorigenic in nude mice. Recently, Nagao et al has identified MAGE-A4 binds to gankyrin and suppresses its oncogenic activity through yeast two-hybrid screen. p28^GANK also directly binds to MDM2, which increases p53-MDM2 association, inducing the ubiquitylation and degradation of p53. Our previous results demonstrate that the significant downregulation of p28^GANK in HCC cells by adenovirus-delivered siRNA to target p28^GANK (Adsip28^GANK) inhibits cancer cells growth, and results in apoptosis eventually in vitro and in vivo.The nuclear factor-κB (NF-κB) families have key roles in inflammation, immune response, oncogenesis and protection against apoptosis. NF-κB is the key transcription factor that modulates the apoptotic response and is essential for p53-dependent apoptosis.Activation of the NF-κB pathway in the cytoplasm can be divided into classical pathway-activating the inhibitors of NF-κB (IκBs) kinsases (IKKs) and promoting the degradation of the IκBs and non-classcical pathway-the post-translational processing of the NFKB2 gene product p100 to p52. The inhibitor of NF-κB (IκB) family of proteins is believed to regulate NF-κB activity by cytoplasmic sequestration. However, much less is known about that cytoplasmic retention of NF-κB by other cellular proteins except IκB proteins renders the pathway unresponsive to activation.Here we find that p28^GANK, an ankyrin repeat oncoprotein commonly overexpressed in hepatocellular carcinoma (HCC), regulate NF-κB pathway through a novel molecular mechanism. We demonstrated that p28^GANK is a nuclear-cytoplasmic shuttling protein, bound to NF-κB heterodimer (RelA/p50), suppressed NF-κB transcriptional activity and consequently rendered the pathway unresponsive to activation by tumor necrosis factor-α(TNF-α), lipopolysaccharide (LPS) and Interleukin-1β(IL-1β). p28^GANK sequestered it in the cytoplasm by accelerating its export from the nucleus through a chromosomal region maintenance-1 (CRM-1)-dependent pathway. Notably, adenovirus-mediated short interfering RNAs that reduced the expression of p28^GANK (Adsip28^GANK) in human HCC cell lines HepG2 promotes sustained transcriptional activation of NF-κB and nuclear-retranslocation, while no phosphorylation of IκBαand no degradation of IκBα, IκBβand IκBεwas observed duing this process. Furthermore, Adsip28^GANK does not lead to activation of IκB kinases and the inducible proteolytic processing of p100. Regulation of NF-κB/RelA activity by p28^GANK is not involved in IKKβand IκB proteins, and is unresponsive to pro-inflammatory stimuli and independent of p53-Ribosomal S6 Kinase 1 (RSK1) signalling. These results highlight a rather unusual pathway of NF-κB regulated by p28^GANK that is different from traditional manners, indicating that p28^GANK, as a novel NF-κB activity regulator unlike IκB proteins, plays a unique role in cytoplasmic sequestration of NF-κB unresponsive to activation.