Oncogene P28Gank Transcription Is Upregulated By IL-1β/Irak1Pathway Activation Via NFY-p300/CBP Complex In Heptocarcinoma

[Background and Purpose]The carcinogenesis of HCC is commonly associated with chronic liverinflammation caused by various etiologies. Cytokines such as TNFα, IL-1β, IL6in thechronic inflammatory microenvironment play an important role in carcinogenesis.Interleukin1beta (IL-1β) is a potent inflammatory cytokine that can activate NF-κBand other signaling pathways. NF-κB activation in pre-cancerous or inflammatorycells was recently shown to be an essential link between chronic inflammation andtumourigenesis of HCC. IL-1β exerts its biological function through binding to itstransmembrane receptor, interleukin1receptor, type1(IL-1RI). Upon binding ofIL-1β, IL-1RI recruits the adaptor protein MyD88. MyD88in turn recruits the IL-1receptor associated kinases, IRAK4and IRAK1. IRAK4phosphorylates IRAK1andreleases IRAK1into the cytosol, where it forms a complex with TRAF6and thenactivate NF-κB or Jun pathway.p28Gank (also known as gankyrin, PSMD10, or p28) was firstly identified as anoncogene in human liver cancers. Our previous studies have shown that the highexpression of p28Gank in HCC is a prognositic prediction factor for poor survival anddisease recurrence, which can promote EMT in liver cancer cells. Overexpression ofp28Gank can induce anchorage-independent cell growth and promote invasivenessand metastasis both in vivo and in vitro. p28Gank can bind to RB1and increase itsphosphorylation to accelerate the degradation of RB1. p28Gank can also bind toMDM2, enhance MDM2–p53interaction and promote p53ubiquitination anddegradation. p28Gank can activate AKT/PI3K/HIF1α pathway to acceleratehepatocellular carcinoma invasiveness and metastasis.As an oncogene, p28Gank mutation has not been found in any tumor so far,p28Gank-driven carcinogenesis is largely due to the abnormal high expression ofp28Gank in HCC. We assume that the aberrant transcriptional regulation of p28Gankmight contribute to the high expression of p28Gank in HCC. As a critical oncogene in HCC, the exact transcriptional regulation mechanisim of p28Gank is still known little.In the present study, we are trying to eluciate the exact transcription regulationmechanism of p28Gank, and to establish a possible linkage between IL-1β/IRAK1pathway and the high expression of p28Gank in HCC. It may contribute to explain themolecular mechanism of how the inflammatory response can promote carcinogenesisin the inflammation-related cancer, HCC.【Methods】1. Bioinformatics analysis of p28Gank promoter region and construction of p28GankpGL4luciferase reporter vector and its deletion-mutant plasmids;2. Drugs that affect p28Gank transcriptional activity are screened by pGL4dual-luciferase reporter gene system in HEK293. Moreover, these drugs arecomfirmed by quantitative PCR and Western Blot.3. p28Gank promoter binding proteins was found by gel shift assay (EMSA).Determination of protein binding sites and the type of the binding transcriptionfactors are made by competitive EMSA with DNA fragment or consensussequence of transcription factors. SuperShift EMSA is performed to confirm thethe types of transcription factors and transcriptional co-activators.4. Chromatin co-immunoprecipitation (ChIP) is used to confirm the in vivo binding oftranscription transcription factors and transcriptional co-activators to the p28Gankpromoter.5. Co-transfection of p28Gank luciferase reporter plasmid with p300, CBP, IRAK1, ofHDAC1, HDAC2expression plasmids in HEK293is performed to study theinduced transcription of p28Gank by p300, CBP and IRAK1or the transcriptionalinhibition of p28Gank by HDAC1and HDAC2.6. By RNA interference technique, p28GANK luciferase reporter vector isco-transfected with NFYA, NFYB, CBP, p300, IRAK1siRNA to comfirmreversely the involvement of NFYA, NFYB, CBP, p300, IRAK1in p28Ganktranscription.7. The distribution and change of NFY-p300/CBP complex in the nucleus orcytoplasm of HEK293after stimulation was studied by extraction of cytoplasmicand nuclear protein and the following Western blot detection.8. Immunoprecipitation and the following Western Blot are used to elucidate theacetylation of NFYA.9. Quantitative PCR, Western Blot and immunohistochemical staining are used todetect IL-1β, p28GANK and IRAK1expression in DEN induced SD rat HCCmodel.10. Quantitative PCR, Western Blot and immunohistochemical staining are used todetect p28Gank and IRAK1expression in normal liver, cirrhosis, peri-tumortissues and HCC samples. Survival analysis is perfomed by Kaplan-Meier method.【Results】1. Bioinformatics analysis: p28Gank core promoter is a bidirectional TATA-lesspromoter with the INR-DPE combinational structure. 2. We have constructed p28Gank promoter Luciferase reporter plasmid(pGL4-p28Gank-Luc) and its four mutants pGL4-p28Gank-295bp-Luc, pGL4p28Gank-ΔcSQ2-Luc, pGL4-p28Gank-ΔINR-Luc, pGL4-p28Gank-ΔDPE-Luc.The minimum range of p28Gank core promoter was determined. The functionof main cis-elements of p28Gank core promoter are confirmed.3. Dual-luciferase reporter gene system in HEK293was used to screen drugs whichcan affect the p28Gank transcriptional activity. We find that epidermal growthfactor (EGF) and interleukin-1β (IL-1β) can promote p28Gank transcription, andClass I&II histone deacetylase inhibitors TSA and NaBu can also improvep28Gank transcription, and proteasome inhibitor MG-132and calcium channelblocker amlodipine besylate (amlodipine, AML) and tetrandrine (TET) can inhibitp28Gank transcription.4. We prove that INR of p28GANK promoter can bind to proteins by gel shift assay(EMSA). The binding proteins is proved to be NF-Y transcription factors bycompetitive EMSA using consensus recognition sequence probes. NFY-p300/CBPcomplex binding to p28Gank promoter INR is comfirmed by SuperShift EMSA.ChIP experiments confirmed that NFY-p300/CBP complex can bind to p28Gankpromoter in vivo.5. Silencing NFYA, NFYB, irak1, p300and CBP by RNA interference in HEK293can decrease p28Gank transcriptional activity. Transfection of irak1, p300, CBPinto HEK293can increase p28Gank transcriptional activity, which confirms theregulatory function of NFY-p300/CBP complex and IRAK1to p28Ganktranscription.6. We illustrate the exact mechanism of p28Gank transcriptional activity stimulatedby IL-1β. IL-1β can activate IRAK1, and IRAK1can induce the high expressionof p300/CBP, the high expression of p300/CBP allows histone acetylation andNFYA acetylation, thereby activates p28Gank transcription.7. Based on the above results, we illustrate an exact molecular mechanism ofp28Gank transcriptional regulation.8. We confirm that IL-1β/IRAK1pathway is activated in the DEN induced SD ratHCC model, especially at the inflammation and carcigenesis phase. IL-1β/IRAK1pathway activation contributes to the highly transcriptional expression of p28Gankin this model.9. We confirm the high expression of IL-1β and IRAK1in tumor tissues of HCCpatients. IRAK1and p28Gank expression are significantly positive correlated atboth mRNA level or protein level in normal liver, cirrhosis and liver cancersamples. We analyze the p28Gank and IRAK1immunohistochemistry results on atissue array containing89cases of TNM I&II HCC samples, and find that jointp28Gank and IRAK1immunohistochemistry is a better predictive factor for HCCprognosis.[Conclusion]This study sheds light on the transcriptional regulation of oncogene p28Gank. Wefind that p28Gank core promoter a bidirectional TATA-less promoter with INR-DPE combinational structure, in which INR can bind to NFY-p300/CBP complex toregulate p28Gank transcription initiation. Transcriptional co-activator p300/CBP withhistone deacetylase activity can be recruited by NF-Y to promote transcription.HDAC1and HDAC2can inhibit p28Gank transcription by deacetylation of histonenearby nucleosomes. p300/CBP can also acetylate NFYA to promote p28Ganktranscription. The present study also illustrates the molecular mechanism of p28Ganktranscriptional upregulation by proinflammatory cytokine IL-1β. IL-1β can activatedownstream IRAK1, and IRAK1can increase the expression of p300/CBP, therebyactivate p28Gank transcription. We confirm that IL-1β/IRAK1pathway is activated inthe DEN induced SD rat HCC model, especially at the inflammation andcarcinogenesis phase. IL-1β/IRAK1pathway activation contributes to the highlytranscriptional expression of p28Gank in this animal model. We also find that IL-1β,IRAK1and p28Gank are upregulated in liver cancer samples of patients. Theexpression of IRAK1and p28Gank are closely correlated at both mRNA level andprotein level. Joint p28Gank and IRAK1immunohistochemistry is a better predictivefactor for HCC prognosis.To the best of our knowledge, this study is the first report about the molecularmechanism discovery of p28Gank transcriptional regulation. NFY-p300/CBP complexis the critical regulator to p28Gank of transcription initiation. For the first time, wereveal that IL-1β/IRAK1inflammatory signaling pathway contributes to thetranscriptional activation of p28Gank. It will deepen our understanding about thedevelopmental process from chronic hepatitis to liver cancer. This study provides anew interpretation on the involvement of IL-1β/IRAK1inflammatory signalingpathways and oncoprotein p28Gank in HCC carcinogenesis.