Mechanism Of Altered Polymerase Theta Expression Affects Chromosomal Instability In Salivary Adenoid Cystic Carcinoma

Background and ObjestivesSalivary adenoid cystic carcinoma（SACC）,accounting for about 10%of all salivary gland malignant tumors,frequently displays heterogeneity with aggressive behavior and treatment resistance.The gold-standard treatment for primary SACC is radical surgical resection and postoperative radiotherapy.However,patients treated with these strategies still show local recurrence and distant metastasis,and the long-term prognosis is still poor.Therefore,understanding of the mechanisms of SACC progression is critical to identify novel treatment strategies to control SACC progression and ultimately improve patient survival rates.Tumors are characterized by genomic instability（GIN）,which leads to a higher probability for genomic changes and the acquiring of deleterious mutations that may lead to tumor progression.As the characteristic of tumor,GIN includes chromosomal instability（CIN）and microsatellite instability（MSI）.In normal cells,GIN is repaired by DNA repair pathways.In human cells,the double-strain break（DSB）repair pathway is the major DNA repair pathway and includes the classical non-homologous end joining（NHEJ）and homologous recombination（HR）pathways as well as the error-prone repair pathway,the alternative non-homologous end-joining（alt-NHEJ）pathway.In mammals,most GIN are repaired by the c-NHEJ pathway,which is the most precise DSB repair pathway that maintains genomic stability throughout the cell cycle.When c-NHEJ and/or HR function are deficient,the alt-NHEJ pathway is activated.The alt-NHEJ pathway may cause GIN,including DNA sequence loss,insertion,chromosome translocation or chromosome rearrangement,and is regarded as a main driver of cancer progression.Moreover,mismatch repair（MMR）defect is the important reason of MSI.In previous study,we confirmed that POLQ,a key factor in alt-NHEJ pathway,was significantly correlated with poor patient survival,suggesting that POLQ might promote SACC progression through regulating genomic stability.Etoposide,which is a kind of topoisomerase II inhibitors,has been used in many cancer treatments by its ability to induce DSB.Olaparib,a small-molecule NAD+mimetic,mainly targeting another key protein in alt-NHEJ pathway,PARP1,is effective to advanced ovarian cancer and breast cancer.Furthermore,it is not clear about the regulatory mechanism of POLQ expression in SACC.Therefore,identifying the mechanism of POLQ regulating GIN in SACC and upstream direct regulator of POLQ is expected to improve SACC prognosis.Here,we used two SACC cell lines,SACC-83 and SACC-LM,to detect POLQ function and regulatory mechanism on GIN in SACC cells,tried to increase SACC sensitivity to DNA damage agent etoposide by inhibiting POLQ or POLQ related pathway,and find the mechanisms of up-regulation of POLQ in SACC cells.This study is composed of three parts.The first part will focus on the role of POLQ on GIN,CIN and MSI in SACC.The second part will analyze the mechanism of POLQ regulating CIN in SACC and the role of combination of anomalously expressed POLQ and etoposide on genomic stability,cell proliferation,migration,invasion and related pathways in vitro and in vivo.The last part will observe the effect of combination of etoposide and olaparib on genomic stability,cell proliferation,migration and invasion in vitro and in vivo,and search a new possible transcriptional regulator of POLQ by directly bounding to POLQ promoter.Methods1.We obtained the median expression of POLQ in tumor and normal samples from GEPIA（gene expression profiling interactive analysis）website;2.We constructed POLQ-anomalous-expressed SACC cell line models using transient transfection;3.Quantitative real-time polymerase chain reaction（q RT-PCR）and western blot（WB）were used to detect m RNA and protein expressions,respectively;4.DAPI was applied for nuclues staining to observe the shapes of nuclei;5.Giemsa staining was used to observe chromosomal stuctures in metaphase cells;6.CCK-8 cell proliferation assay was used to detect cell proliferation,and the migration and invasion capacity of SACC cells were examined by Transwell chamber assays;7.Quantitative real-time polymerase chain reaction-single strand conformation polymorphism（PCR-SSCP）was used to detect microsatellite instability;8.To initiate tumor xenograft,the human SACC cells lines,SACC-83 and SACC-LM,were implanted subcutaneously in the nude mice.Treatment started when the tumor size reached～50mm³.After 14 days’treatment,the mice were euthanized and the xenografts were measured and retrieved;9.Cytotoxicity assay was used to evaluate in vitro drug cytotoxicity,and the Bliss method was employed to calculate half maximal inhibitory concentration(IC₅₀);10.Immunofluorescence（IF）staining analysis was used to characterize the degree ofγH2AX;11.The biological prediction websites PROMO,Cistrome,JASPAR and GEPIA predicted transcriptional factors and related the potential binding sites on POLQ gene;12.Dual-luciferase reporter assay was used to confirm that POLQ gene was indeed regulated by the transcriptional factors predicted by the biological prediction websites;13.Immunohistochemistry（IHC）analysis was used to examine the distribution ofγH2AX,CASPASE-3 and KI-67 in xenografts;14.Mean±SEM deviation were the standard form for measurement data.The data between two groups were compared by unpaired two-tailed Student’s t-test.Data among multiple groups were compared by one-way analysis of variance（ANOVA）,followed by Tukey’s post hoc test.All experiments were performed at least three times in triplicate.The data were considered statistically significant when*p-values<0.05,and highly statistically significant when**p-values<0.01 or**p-values<0.001.Results1.The role of POLQ in SACC genomic stabilityCompared with homologous normal tissues,the POLQ expressions were relatively higher in glandular epithelial cancers;the expression level of POLQ was significantly higher in metastasis derivative SACC-LM than that in SACC-83;POLQ-anomalous-expressed SACC cell line models were successfully constructed;High expression and knockdown of POLQ strongly induced the formation of cell nucleus deformation;the frequency of chromosomal aberrations rose significantly in POLQ-anomalous-expressed SACC cell lines;POLQ positively regulated CENP-A,HJURP and TERT expressions in SACC cell lines;POLQ did not influence stability of five microsatellite markers.Moreover,high expression and knockdown of POLQ inhibited cell proliferation,migration and invasion in vitro and POLQ over-expression inhibited SACC xenograft growth and increased the degree ofγH2AX in vivo.2.Mechanism of POLQ regulating chromosomal stability and sensitivity to DNA damage in SACCIn POLQ-anomalous-expressed SACC cell lines,γH2AX levels were increased;With or without etoposide treatment,POLQ negatively regulated HR and c-NHEJ related proteins,RAD51 and KU70,and positively regulated alt-NHEJ related protein,PARP1.no change of MMR related protein MLH1 was found when POLQ was anomalously expressed in SACC-83.Moreover,Etoposide was highly toxic to SACC-83 cells in a dose-dependent manner,with an IC₅₀value of approximately 3μM;increasedγH2AX level in SACC-83 was closely coupled with the increased concentrations of etoposide;the sensitivities to etoposide in SACC cell lines were increased when POLQ were anomalously expressed;expression changes of POLQ strongly induced the formation of cell nucleus deformation in SACC cell lines with etoposide treatment;the frequency of chromosomal aberrations was increased significantly in SACC cell lines with etoposide treatment.Under etoposide treatment condition,expression changes of POLQ synergistically inhibited cell proliferation,migration and invasion in vitro and synergistic inhibitory effects on SACC cells were achieved by combination with POLQ suppression and etoposide in vivo.3.Effect of inhibiting POLQ-related pathway on DNA damage and the regulatory mechanism of POLQ expression in SACCOlaparib was highly toxic to SACC-83 cells in a dose-dependent manner,with an IC₅₀value of approximately 58μM.Under etoposide treatment condition,olaparib synergistically inhibited cell proliferation,migration and invasion in vitro and Synergistic inhibitory effects on SACC were achieved by combination with olaparib and etoposide in vivo,including improving the level ofγH2AX,inducing cell apoptosis as shown by the percentage of CASPASE-3 positive cells,and inhibiting cell proliferation as examined by KI-67 staining.Furthermore,transcriptional fatcors CEBPB,FOXM1,YY1 and ESR1 were predicted to bind to POLQ gene;as a result of q RT-PCR,CEBPB and FOXM1 were negtively regulated by PTEN,and YY1 and ESR1 were positively regulated by PTEN;dual-luciferase reporter assay showed that CEBPB and FOXM1 could bind to POLQ promoter region;CEBPB promoted POLQ in SACC cell lines.Conclusion1.POLQ were highly expressed in malignant SACC;POLQ-anomalous-expression promoted CIN not MSI in SACC;POLQ-anomalous-expression inhibited cell proliferation,migration and invasion in vitro,and tumor growth in vivo.2.POLQ influenced CIN by regulating DSB;POLQ negatively regulated HR and c-NHEJ activities and positively regulated alt-NHEJ activity in SACC;POLQ-anomalous-expression improved sensitivity of SACC to etoposide in vitro,and suppression of POLQ inhibited tumor growth in vivo.3.Suppression of POLQ-regulated pathway by olaparib improved sensitivity of SACC to DNA damage in vitro,and synergistically inhibited SACC chromosomal stability,and xenograft growth rates in vivo.Moreover,CEBPB was found as a possible factor regulated by PTEN and directly bounded to POLQ promoter.