The Role And Transcriptional Regulation Of DNA Polymerase Iota In Bladder Cancer

Bladder cancer (BC) or urothelial cell carcinoma is one of the most frequent cancers inworldwide. BC represents the second urological malignancy following prostate cancer indeveloped countries; moreover, BC is the most common urological tumor in China.Significant researches revealed that mature urothelial carcinoma, which is highly recurrenceand heterogeneity, contain numerous genetic and epigenetic abnormalities. The propertiesresult to the difficulty of prevention, cure and prognosis in bladder cancer. Recentlydiscovered DNA polymerase iota (Pol ι) is one of the Y family DNA polymerases, whichbelongs to translesion synthesis (TLS) to maintain the genomic stability. Pol ι has thelowest fidelity among eukaryotic polymerases, because it lacks3’ to5’ exonuclease activity.Error-prone Pol ι expression and function may be highly regulated in normal mammaliancells, whereas the abnormal expression will induce genetic mutation in DNA replication tocause carcinogenesis. Previous research discovered that overexpression of Pol ι leads toUV-induced hypermutagenesis in breast cancer cells. Pol ι was also found to besignificantly overexpressed in a variety of primary tumor tissues, including glioma, prostate,uterus, stomach and rectal cancers. These reports suggest that the hypermutagenesis by Polι may have an important significance in bladder cancer development. Understanding thetranscriptional regulation of Pol ι and its function will provide an insight into the target ofprevention and cure in bladder cancer. However, the regulation of Pol ι expression and thecharacter of Pol ι in tumorigenesis remain to be obscure.To investigate the regulation mechanism of Pol ι expression and its possible clinicalsignificance in bladder cancer, we carried out this study and the main results weresummarized as below: 1. c-Jun is a critical transcriptional factor for human POLI gene.(1) Bioinformatics analysis and identification of human POLI promoter region.The5′flanking region of human POLI gene was analyzed by bioinformatics and the5’-deletion mutants were subcloned into pGL3-basic. The human POLI minimal promoterlocated at the-275/+63region by assessing the activity of luciferase in HEK293cells. Thebioinformatics analysis demonstrated that the POLI promoter contains a putativenon-canonical AP-1binding site.(2) c-Jun protein increased the transcriptional activity of human POLI promoter.Cotransfection with c-Jun expression plasmid increased the transcriptional activity ofhuman POLI promoter; meanwhile mutation in the putative c-Jun binding site reducedtranscriptional activity in HEK293cells.(3) c-Jun protein binds to human POLI promoter in vitro and in vivo.Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitationanalysis (ChIP) revealed that c-Jun protein interacted with the-275/+63promoter region ofhuman POLI gene in vitro and in vivo.2. JNK/c-Jun signaling regulates Pol ι expression.(1) The transcriptional activity of human POLI promoter was enhanced in DNAdamage agent ultraviolet ray (UV) induced HEK293cells. Pol ι protein also increased andwas correlated with the activity of c-Jun and JNK in the UV-induced HEK293cells.(2) JNK special inhibitor (SP600125) and c-Jun RNA interference both inhibited theexpression of Pol ι in HEK293cells treated with or without UV by Western Blot assay. Theresults confirmed that JNK/c-Jun regulated the basal and UV-induced Pol ι expression.(3) Western Blot assay showed that Pol ι expression was higher in T24bladder cancercells than RT4cells involved into the activity of c-Jun, and was suppressed by SP600125inT24and RT4cells. These data suggest that JNK/c-Jun signaling regulates Pol ι expressionin general mechanism.3. Nuclear protein ATR and XPC promote p-JNK to regulate Pol ι expression inresponse to UV damage.(1) Inhibition of DNA damage response protein ATR and XPC by RNA interferenceboth reduced the activity of JNK/c-Jun and decreased the expression of Pol ι protein in UV-induced HEK293cells. Furthermore, Western Blot and Real-time PCR assay showedthat the inhibition of ATR or XPC was fail to affect MKK4or MKK7activating JNK/c-Junpathway, whereas enhanced the expression of MKP-1inhibiting JNK/c-Jun activity. Theresults suggest that ATR and XPC induce the activity of JNK/c-Jun to enhance Pol ιexpression in response to DNA damage in HEK293cells, and prompt that there is acrosstalk between intranuclear DNA damage response and JNK/c-Jun pathway.(2) Inhibition of ATR and XPC by RNA interference both reduced the activity ofJNK/c-Jun and decreased the expression of Pol ι protein in UV-induced T24bladder cancercells. The results support that ATR and XPC promote Pol ι expression through JNK/c-Junpathway in response to DNA damage in bladder cancer cells.4. Elevated expression of Pol ι is associated with activity of c-Jun and the pathologyclassification in bladder cancer clinical samples.The paraffin-embedded bladder urothelial carcinoma tissues were obtained bytransurethral bladder tumor resection and radical cystectomy. The overexpression of Pol ιwas found to be concordant with the activity of c-Jun in bladder cancer samples byimmunohistochemistry. Statistical analysis indicated that Pol ι expression and c-Jun activitywere associated with the pathological grade of bladder cancer tissues.5. The hypermutagenesis of Pol ι is regulated by JNK/c-Jun in bladder cancer cells.The pSupFG1plasmids irradiated by UV were transfected into T24bladder cancercells. There was a UV dose-dependent increase in the frequency of SupF gene mutants inT24cells and a significant decrease of mutation frequency in T24cells pretreated withSP600125. Mutation spectrum obtaining from T24cells was consistent with the enzymaticproperties of Pol ι published in vitro kinetic analyses. The results suggested thatoverexpressed Pol ι controlled by JNK/c-Jun plays an important role of hypermutagenesisin bladder cancer cells.In summary, the core promoter region of human POLI gene was mapped in-275/+63.Transcription factor c-Jun binds to POLI promoter to up-regulate the promoter activity andPol ι expression. Furthermore, JNK/c-Jun pathway regulates Pol ι expression, and DNAdamage response protein ATR and XPC influence JNK/c-Jun pathway and regulate DNA damage induced Pol ι expression, through reducing the inhibitory signal MKP-1rather thanactivating MKK4and MKK7. In addition, elevated expression of Pol ι was associated withactivity of c-Jun and the malignancy in human bladder cancer. Moreover, overexpression ofPol ι activated by JNK/c-Jun promoted UV-induced hypermutagenesis in T24cells. Theresults support that dysregulation of Pol ι by JNK/c-Jun is involved in carcinogenesis andprompt that Pol ι may function as a mutator in bladder cancer. This study provides a cluefor further claritying the regulation mechanism and the clinical significance of Pol ιexpression in bladder cancer, and founds a basis for seeking a novel anticancer strategy.