A Preliminary Study On X-linked Inhibitor Of Apoptosis Protein (XIAP)’s Proliferation-related Biological Effects On Maliganant Tumors

【Background】As we all know, XIAP is involved in the regulation of apoptosis by inhibiting caspaseactivity[1]. XIAP expression increases in a variety of malignant tumors, including prostatecancer[2], acute and chronic leukemia[3,4], and other malignancies[5-7]. Although involvedin apoptosis regulation[8,9], it also has other non-apoptosis biological functions, such asparticipating in signal transduction, being involved in copper metabolism, being involvedin ubiquitination[11]. In the inhibitor of apoptosis protein （IAP） family members, XIAP isthe most potential cancer therapeutic target. Chemical inhibitors or small interfering RNA（siRNA） inhibition of XIAP expression can inhibit the proliferation of tumor cells[12,13],but these studies did not clarify whether XIAP is a direct or indirect inhibitor of tumor cellproliferation. XIAP protein contains three BIRs （baculovirus IAP repeats） located in the N-terminaldomain and the C-terminal RING （Really Interesting New Gene） domain[14]. XIAP cancombine caspase3/7/9to regulate their function in apoptosis[14]. XIAP RING domain’s E3ubiquitin ligase activity connects labeled target proteins to ubiquitin molecules, andpromote protein ubiquitination and degradation. By adjusting the type of the target proteindegradation pathway, domain of XIAP RING structure plays different biological functions,including cell death （14）, inflammation （15）, and migration （16）. Our recent studiesconfirmed the the XIAP knockout or low expression can inhibit HCT116cell migrationand invasion, and restore the expression of XIAP can reverse this change （17）. This is thefirst report that XIAP has the ability to regulate migration and invasion of malignant cells.Our further studies confirmed XIAP’s binding with RhoGDIα, inhibition the stable ofRhoGDIα protein through the SUMOylation at138histidine, thereby affecting the activityof small Rho GTP, tumor cell beta-actin polymerization, cytoskeletal rearrangements, andcell migration （17,18）. Therefore, the discovery and identification of XIAP non-cellapoptosis biological functions have important biological significance.【Objective】The purpose of the study is to investigate the role of XIAP in regulating themolecular mechanisms of tumor cell proliferation based on our previous studies.【Method】（1） By liposome transfection, HA-XIAP, HA-XIAP ΔRING, HA-XIAP ΔBIR,HA-XIAP H467A, and pcDNA3.0empty plasmids were transfected into HCT116XIAP^-/-cells, and antibiotic selection was used to obtain stably transfected cell lines;（2） By softagar colony formation assay, we investigated colony formation ability ofstably-transfected cell lines;（3） By ATP activity detection assay, we investigated cellproliferation rate in different stably-transfected cell lines;（4） PI staining flow cytometryassay was used to determine cell cycle distribution;（5） Western blot assay was used todetermine the expression of cell cycle regulatory proteins;（6） RT-PCR assay was carried out to determine the transcription of NFAT gene;（7） By luciferase activity assay, weinvestigated NFAT dependent transcription activity;（8） Stable transfection withdomin-negative mutant DN-Akt plasmid was used to inhibit the activity of AKT andobserve its effect in cell colony formation;（9） The translocation of transcription factorswere determined after the separation of nuclear and cytoplasmic proteins;（10） The role ofc-Jun in XIAP-mediated cyclin D1transcription was determined by liposome transfectionwith a c-Jun dominant-negative mutant, TAM67;（11） Co-immunoprecipitation was usedto determine the binding of c-Jun and PP2A proteins in cells;（12） Liposomal transfectionof cyclin E-specific shRNA plasmid was performed to reduce the expression of cyclin E;（13） Soft agar colony formation assay was carried out to determine the effect of cyclin Eexpression inhibition on cell colony formation;（14） To detect the role of E2F1in cyclin Etranscription regulated by XIAP, cells were transfected cyclin E promoter reporter plasmidwith E2F1mutant, and luciferase activity was detected;（15） Co-immunoprecipitationexperiment was used to detect the binding capacity between XIAP BIR structure domainand E2F1protein.【Results】1. XIAP’ different domains and E3ligase activity regulated tumor cell proliferation（1） Soft agar colony formation experimental results show that the quantity of coloniesformed in XIAP^-/-（vector） cells was significantly lower than that of wt （vector） cells.Transfection of HA-XIAP into XIAP^-/-cells increased the quantity of colonies. However,the transfection of HA-XIAP H467A into XIAP^-/-had no observed effect on colonyformation. XIAP^-/-（HA-△RING） colony formation increased significantly.（2） The resultsof cell proliferation assayed by ATPase activity determination were consistent with theresults of the soft agar clone formation assay. The results showed that XIAP regulatedtumor cell clone forming ability by affecting cell proliferation rate.2. XIAP BIR domain regulated NFAT1transcription factor protein stability and itsbiological function （1） Western blot showed that the phosphorylation level of Akt at308and473washigher in XIAP^-/-（vector） and XIAP^-/-cells （HA-△BIR） than other cells.（2） Thesuppression of Akt phosphorylation by transfection with the Akt kinase dominant negativemutant plasmid （Dominant negative mutant Akt, DN-Akt） lead the clone formation abilityof XIAP^-/-（HA-△BIR） cells back to normal.（3） Western Blot results showed that theprotein expression level of NFAT1in XIAP^-/-（vector） and XIAP^-/-cells （the HA-ΔBIR）was lower than WT cells, the luciferase reporter gene assay showed that NFAT-dependenttranscriptional activity was less than XIAP^-/-（HA-XIAP） cells.（4） In XIAP^-/-（vector） andXIAP^-/-cells （HA-ΔBIR）, NFAT1protein degradation rate was faster than that of the WTcells.（5） The XIAP lack of BIR domain had the increased MDM2phosphorylation. Theregulatory function of XIAP BIR domain on MDM2phosphorylation was dependent onincreased phosphorylation of Akt phosphorylation. The above results showed that there isinteraction between the apoptosis mediated by XIAP signal pathway and cell survivalsignaling pathways mediated by Akt.3. XIAP RING domain deletion affected cyclin E gene transcription and its biologicalfunction（1） Flow cytometry assay showed that the XIAP RING structure domain deletionaffected G1/S phase transition in tumor cells, leadting to significantly reduced number ofcells in G0/G1phase.（2） Western blot results showed that cyclin E expression wasincreased in cells deficient of XIAP RING structure domain.（3） RT-PCR and promoterreporter gene analysis showed that the expression of cyclin E was at the transcriptionallevel.（4） The transfection of cyclin E-specific shRNA into XIAP^-/-（HA-△RING） cellsinhibited the abnormal cell proliferation ability.（5） Promoter reporter gene analysisshowed that the deletion of XIAP RING structure domain affected the expression of cyclinE through increased transcription activativity of E2F1transcription factor.（6） InCo-immunoprecipitation, XIAP was found to bind to E2F1through its BIR domain.（7）Sub-celluar protein localization analysis showed that the RING domain of XIAP affects itsown cell localization. The above results showed that XIAP can enhance its transcription activity on its downstream genes through the binding of BIR domain to E2F1. Theabnormal expression of XIAP without BIR structure may affect the cell proliferation oftumor cells.4. XIAP regulated cyclin D1gene transcription and its biological function through itsE3ligase activity（1） The ratio of cells in G0/G1phase in XIAP^-/-（vector） cells was significantlyincreased compared with that in wt （vector） cells reaching84.58%; transfection ofHA-XIAP into XIAP^-/-cells reduce this ratio to69.31%, with no significant differencecompared with that of WT （vector） cells （71.51%）; the G0/G1phase ratio in XIAP^-/-cellstransfected with HA-XIAP H467A was78.94%, similar to that of of XIAP^-/-（vector） cells.（2） Western blot results showed that XIAP E3ligase activity can affect the expression ofcyclin D1expression, with no effect on other cell cycle regulatory proteins.（3） RT-PCRand gene promoter report confirmed the effect of XIAP E3ligase enzyme activity oncyclin D1transcription. Transcription factor translocation analysis showed that both c-Junphosphorylation and AP-1transcription factor may be involved in XIAP E3ligase activityregulation on cyclin D1transcription.（4） Luciferase reporter gene analysis showed thatXIAP E3ligase activity can affect the activity of AP-1transcription.（5） The dominantnegative mutant of c-Jun TAM67was able to change the regulatory effect of XIAP E3ligase activity on cyclin D1expression.（6） Co-immunoprecipitation confirmed thecombination between PP2A and c-Jun in the cell.（7） Inhibition of PP2A activity enabledincreased c-Jun phosphorylation and cyclin D1expression. The above results indicatedthat XIAP E3ligase activity affected G1/S phase transition and proliferation of tumor cellsthrough a PP2A/c-Jun/cyclin D1pathway.【Conclusion】In addition to its regulation on apoptosis, XIAP protein could regulate cancer cellproliferation, and the RING domain, BIR domains, and E3ligase activity played differentroles in regulating tumor cell growth. XIAP E3ligase activity affected tumor cell proliferation through its regulation on cyclin D1expression. The E3ligase activity ofXIAP can inhibit the phosphorylation of PP2A, thereby affecting the regulation of PP2Aon c-Jun phosphorylation, and the further regulation on the transcription of cyclin D1. Thedeletion of RING domain in XIAP can cause the high expression of cyclin E in tumor cells,in which the BIR domain can be combined with E2F1to promote its transcriptionalactivity, thereby affecting the transcription of cyclin E and tumor cell proliferation. XIAPBIR domain deletion induced enhanced Akt phosphorylation activity in the tumor cells,thereby affecting the MDM2phosphorylation, NFAT1protein stability, and tumor cellproliferation. These results suggest that XIAP’s E3ligase activity is a potential cancertherapy target, and that XIAP-targeted tumor therapy need to consider the differentbiological functions of different structural domains, and that there are complicatedrelationship between XIAP and AKT-mediated celluar proliferation-regulatory pathways.