The Sensitizing Effect Of Mir-509-3 On PARP Inhibitors In High-grade Serous Ovarian Cancer Based On PDX Models

BackgroundOvarian cancer is a type of malignant tumor that seriously threatens the life and health of women.In the United States,more than 14,000 thousands women are died from ovarian cancer whose fatality rate ranks the fifth,following lung cancer,breast cancer,colorectal cancer and bladder cancer.In China,about 25,000 women die from ovarian cancer each year.This could be explained by the fact that three-quarters of ovarian cancer patients were already suffering from advanced metastasis at the time of diagnosis.What's worse is that even accepted initial treatment based on cytoreductive surgery and platinum-based adjuvant chemotherapy,70%of patients eventually relapsed and developed acquired chemotherapy resistance.High-grade serous ovarian cancer(HGSOC)accounts for 70%of ovarian cancers,and is characterized by loss of p53 function,DNA damage response defects and genomic instability.Approximately 35%of HGSOC patients carry the BRCA1/2 mutation,both of which play an essential role in the homologous recombination(HR)repair pathway and another 6%-10%patients may carry the other HR pathway gene mutations including ATM,CHEK1,CHEK2 and ATR,etc.Polyphosphate diphosphate polymerase inhibitor(PARP inhibitor,PARPi)is a small-molecule target drug which can cause"synthetic lethality" effect when used in the patients with HR-related gene deficiency,especially BRCA 1/2 mutant tumors.With reference to the conclusions of several international multi-center phase ? clinical trials,a variety of PARPis have been approved by drug administration for maintenance treatment of ovarian cancer,which has greatly improved the patients'treatment prospects.However,primary insensitivity or acquired resistance to PARPi has become an urgent problem in the application of PARPi.MiRNA(microRNA)is a large family of non-coding RNAs,which is regarded as an important transcriptional regulator,and its effects on tumor development have been confirmed by a great many studies.Transferring miRNA mimics or target antagonists into human tissues via transport media is expected to achieve a new method for the treatment of malignant tumors or other diseases,whose initial outcome has been obtained in preclinical research.Previous studies have confirmed that some miRNAs might play an synergistic role in the sensitization of platinum or PARPi.However most of current studies were achieved through cellular experiments which inevitably contained many uncertainties in actually accomplishing clinical transformation.In this study,we integrately analysed the miRNA expression profiles and clinical data in TCGA ovarian cancer cohort and proved several miRNAs related to HGSOC prognosis and platinum response in which miR-509-3 was selected as the study object.The in vitro and in vivo cel lular experiments revealed miR-509-3's effect on suppressing cancer and its mechanism of action in HGSOC.Furthermore,patient-derived xenograft(PDX)models of HGSOC were established to verify the PARPi sensitization effect of miR-509-3 and moreover a PARPi response prediction system was proposed for further validation.The detailed research content was as follows:1.Effect of miR-509-3 on HGSOC prognosis and proliferation,invasion and migration abilities.2.Role of miR-509-3 in HGSOC's responses to platinum and PARP inhibitor.3.MiR-509-3 sensitized HGSOC to PARPi by directly targeting HMGA2 and RAD51.4.Sensitization of miR-509-3 on PARPi in PDX models.PART 1 Effect of miR-509-3 on HGSOC prognosis and proliferation,invasion and migration abilitiesObjective:The vast majority of ovarian cancer patients would be resistant to first-line platinum chemotherapy which deadly impacts the survival and recurrence rates.Bioinformatics analysis of miRNA expression profiles and clinical data of TCGA ovarian cancer patients proved miR-509-3 as the most closely related to platinum sensitivity of HGSOC,whose impact on the prognosis of HGSOC patients was also confirmed in this part.What's more,we preliminarily discussed miR-509-3's effects on the basic biological behavior of HGSOC including proliferation,invasion and migration ability.Methods:The HGSOC patients in TCGA cohort were divided into platinum-sensitive and platinum-resistant groups.The miRNA sequencing data of the two groups were analyzed for differential expression using the DEGseq R package,in which the most significant up-and down-regulated miRNAs were plotted in heat map.RT-qPCR was used to detect the miRNA-509-3 expression in 126 HGSOC cases from Qilu Hospital,which was compared between platinum-sensitive and platinum-resistant groups by t test.The Kaplan-Meier survival curve and Log-rank test were used to compare the difference in OS or PFS between miR-509-3 high-expression or low-expression group in TCGA and Qilu Hospital independent cohorts.Transient infection was used to upregulate miR-509-3 and control NC in A2780,HEY and UWB1.289 ovarian cancer cell lines,And then we performed transwell assay,MTT cell proliferation assay,cell cycle detection and plate clongenic assay to verify miR-509-3's effects on HGSOC proliferation,invasion and migration ability in vitro.After construction of miR-509-3 overexpressing cell lines by lentivirus transfection,nude mice were injected intraperitoneally to form tumors to prove the effect of miR-509-3 on HGSOC metastasis in vivo.Western Blot detected changes in epithelial-mesenchymal transition(EMT)and cycle-related proteins in miR-509-3 overexpressing cells.Results:The comparison of miRNA expression profiles in two groups of TCGA database illustrated that mir-509-3 was up-regulated in platinum-sensitive patients.Constently,in the cases of TCGA and Qilu Hospital cohorts,miR-509-3 expression in platinum-sensitive HGSOC patients was significantly higher than that in platinum-resistant patients and the OS and PFS of miR-509-3 high-expressing group was prolonged than that of low-expressing group to a great extent.MiR-509-3 was the independent factor of OS and PFS in HGSOC patients.After overexpressing miR-509-3,the proportion of cells in G0/G1 phase increased while that of S phase cells decreased.The relative cell viability and colony formation number were also lower than those in NC group,and the cycle-related proteins CDK4,CDK6,CCND1 and p21 expression were down-regulated.In vitro,miR-509-3 significantly decreased cells that passed through the Transwell compartment than NC group;in vivo experiments revealed that miR-509-3 over-expressing cells formed much fewer tumor metastases in the nude mice's abdominal cavities.Western Blot showed that the mesenchymal cell marker N-cad declined,while the epithelial cell marker E-cad increased.Meanwhile,miR-509-3 down-regulated the protein expression levels of ZEB1,?-Catenin,Vimentin,Snail and Slug.Conclusions:MiR-509-3 is responsible for the platinum sensitivity and prognosis of HGSOC.MiR-509-3 can prolong overall survival and reduce the risk of relapse in HGSOC.MiR-509-3 inhibits the growth and proliferation of HGSOC through G1 phase arrest and regulates the EMT pathway to impair its invasion and migration,which makes it a tumor suppressor in HGSOC.PART 2 Role of miR-509-3 in HGSOC's responses to platinum and PARP inhibitorObjective:Platinum resistance is an important cause of poor prognosis in HGSOC patients.MiR-509-3 has been previously confirmed to be associated with platinum sensitivity of HGSOC.This part aims to further explore its effect on platinum sensitivity in HGSOC cells through in vitro experiments.Considering platinum sensitivity's clinical significance in predicting PARPi response and their similar function mechanism,we are much more interested in the effect of miR-509-3 on the PARPi sensitivity of HGSOC which may offer novel potential therapeutic targets.Methods:MTT method was used to draw the growth curve of miR-509-3 overexpressing or control cell lines when exposed to cisplatin or Olaparib gradiently to verify the effects of miR-509-3 on the sensitivity to cisplatin or Olaparib.The constructed stably overexpressing miR-509-3 HEY and UWB1.289 cells or control cells were used to xenograft tumors subcutaneously in nude mice.After tumor formation,they accepted intraperitoneal injection of Olaparib(50mg/kg)for 14 days and then were sacrificed.The size and weight of the tumors were measured and compared among different test groups.Results:Olaparib' s IC50 of HEY was significantly lower than that of UWB1.289(10uM-30uM vs 50uM-70uM).When cells were treated with different concentrations of cisplatin or Olaparib,the relative cell viability ofHEY and UWB1.289 overexpressing miR-509-3 was significantly decreased than the corresponding NC cell,and the growth curve exhibited a larger slope and a faster downward trend.In vivo,the tumor burden of DMSO/miR-509-3 group was significantly lower than that of untreated DMSO/NC group(HEY 0.76±0.05g vs 0.39±0.14g,P<0.01;UWB1.289,1.02±0.17g vs 0.48±0.26g,P<0.01).Comparison between Olaparib/NC group and DMSO/NC group confirmed that Olaparib injection significantly inhibited the tumorigenic activity of HEY and UWB1.289(HEY 0.76±0.05g vs 0.33±0.14g,P<0.01;UWB1.289,1.02±0.17g vs 0.41 0.14g,P<0.01).Last but not least,the average tumor burden of Olaparib/miR-509-3 group(HEY,0.12±0.06g;UWB1.289,0.15±0.008g)was unsurprisingly the lowest(P<0.05).Conclusions:Cell line BRCA status could not accurately predict its sensitivity to PARPi.MiR-509-3 has a sensitizing effect on platinum and PARPi in both BRCA wild-type and BRCA-deficient cell lines which indicates its potential application value in synergistic therapy to reverse PARPi resistance and improve sensitivity of HGSOC patients.PART 3 MiR-509-3 sensitized HGSOC to PARPi by directly targeting HMGA2 and RAD51Objective:The above in vivo or in vitro cell phenotypic experiments have clearly confirmed that miR-509-3 plays a definitive role in slowing HGSOC growth and proliferation,inhibiting invasion and migration,and increasing sensitivity to platinum and PARPi,whose downstream mechanism is unclear.It is well known that miRNA mainly functions by binding to the 3' UTR of the target genes to degrade or inhibit the mRNA translation This section intends to predict and verify the downstream target genes of miR-509-3 to explore the exact mechanism of its biological function.Methods:Potential candidate targets of miR-509-3 were predicted from online databases TargetScan 7.1 and miRDB.After transient infecting miR-509-3 mimics to A2780,HEY,and UWB1.289 cell lines,RNA and protein were extracted to detect the target gene mRNA or protein expression levels respectively by RT-qPCR or Western Blot.Meanwhile,in subcutaneous-forming tumor tissues of nude mice,immunohistochemical method was used to detect protein level alteration of target genes in condition of miR-509-3 overexpressing in vivo.Referring the 3'UTR binding sites of the target genes predicted by TargetScan,3'UTR wild-type or mutant pmirGLO vectors were constructed respectively in the dual-luciferase system experiment.What's more,forty clinical samples of HGSOC were collected to probe the correlations of target genes' mRNAs levels and miR-509-3 expression.Cell function experiments after transiently transfecting small interfering RNA(siRNA)were performed to verify whether the target genes had a PARPi-sensitizing effect on HGSOC and then we designed protein co-immunoprecipitat ion(CoIP)to look for target genes' PARPi sensitization downstream molecule.Phenotype rescue or mechanism rescue experiments were performed by transiently transducing the target gene's cDNA/pENTER and miR-509-3 mimics/nc to further complete the pathway of miR-509-3's tumor-suppressor role.We exposed miR-509-3 overexpressing or control cells to different doses of radiation to induce DNA damage and then Western Blot was used to compare the expressions of related proteins in the HR repair pathway.Immunofluorescence confocal imaging was applied to count the number of RAD51 nuclear fluorescent foci.Results:TargetScan 7.1 and miRDB both predicted that HMGA2 and RAD51 were the target genes of miR-509-3 with potential binding sites on the 3'UTR of the two mRNAs respectively.The dual-luciferase system assay confirmed that miR-509-3 could directly bind to two sites of HMGA2 3' UTR and two sites of RAD51 3'UTR.After miR-509-3 overexpressing,HMGA2 and RAD51 were down-regulated in cellular RNA or protein levels as well as in tumor tissues.In the correlation analysis of 40 clinical tissue samples,the expressions of HMGA2 and RAD51 were both negatively correlated with the level of miR-509-3(HMGA2:Pearson r=-0.3844,P=0.01 RAD51:Pearson r=-0.3476,P=0.03).Since the role of RAD51 in Olaparib response has been extensively studied,only Olaparib sensitization of HMGA2 was discussed in this part.After inferring the HMGA2 expression,the sensitivity of HEY and UWB1.289 to Olaparib was increased,accompanying by the up-regulation of total ATM protein and activated ATM protein.And the results of Co-IP showed that HMGA2 and ATM could bind directly to achieve regulation.When we transiently transferred HMGA2 cDNA plasmid to miR-509-3 overexpressing cells,miR-509-3's sensitizing Olaparib and inhibiting invasion abilities were all weakened and ATM expression was also up-regulated.Similarly,transient transfection of RAD51 cDNA plasmid also showed phenotypic rescue phenomenon.When the cells were exposed to a gradient dose of radiation,the expression level of HR-related proteins increased with radiation dose.And the HR-related proteins in miR-509-3 overexpressing cells were significantly suppressed,which were lower than that in control cells.Additionally,the number of RAD51 nuclear immunofluorescence foci was also attenuated by miR-509-3.Conclusions:HMGA2 and RAD51 are downstream targets of miRNA-509-3.In HGSOC,miR-509-3 can interfere with the HR repair ability of cells by directly down-regulating RAD51 and impairing HMGA2-ATM axes,thereby increasing the sensitivity to PARPi,which implies synergistic therapeutic potential with PARPi.PART 4 Sensitization of miR-509-3 on PARPi in PDX modelsObjective:At present,most of the PARPi related mechanism researches are completed with the help of commercial stably-passaged cell lines.Due to the common heterogeneity of HGSOC,traditional cell line experiments cannot accurately reflect the actual situation of clinical patients.Patient-derived tumor xenograft model(PDX)is a method in which surgically resected tumor tissues from cancer patients are transplanted immediately to immune-deficient animals in order to maintain the original histological and molecular biological characteristics to the greatest extent.The response of the models to anticancer treatments is closely related to the actual efficacy in patients which confers it great potential in the preclinical efficacy prediction of conventional and novel anticancer therapies.Therefore,our study intends to establish PDX models of HGSOC to confirm the synergistic therapeutic effect of mir-509-3 and Olaparib at the level of patient-derived tumor tissues.Methods:The transplanted live tumor masses were obtained from fresh tumor tissues(labeled as PO generation)which was diagnosed as HGSOC through intraoperative rapid pathology and then ground into tissue homogenate to be ectopic implanted into NCG mice subcutaneously.When tumors formed,we cryopreserved and passaged the tumor tissues to construct stably-passaging PDX models in which 9 PDXs' abdominal implantation models were adopted.The abdominal tumor-bearing mice were treated by groups with the synthetic miR-509-3 overexpressing adeno-associated virus(AAV)and Olaparib intraperitoneal injection and then the tumor burdens of mice from different treatment groups were measured and compared among the groups.What's more,whole exon sequencing(WES)was performed to analyze the mutations of ovarian cancer susceptibility genes and DDR-related genes in patients PO of PDX cases.Meanwhile,HE and immunohistochemical staining were used to detect the histomorphological characteristics and the expressions of p53,RAD51 and HMGA2 in different-generation tumor tissues.Results:Different-generation PDX tumors shared similar histomorphologic features.The mutation rate of p53 in 9 PDX cases detected by WES was 88.89%(8/9)while immunohistochemical tests revealed that all the patients were p53 strongly positive.And 44.44%(4/9)cases carried HR-related genes mutation,3 of which were BRCA1/2 mutation.Of the 4 cases with HR-related gene mutations,3 cases with BRCA1/2 mutations responded well to Olaparib,while the other case with a non-HR core gene,the LIG1 mutation,was not sensitive to Olaparib treatment.In 5 HR wild-type cases,the effective rate of Olaparib was only 40%(2/5).The response rate of Olaparib was 80%(4/5)in patients with primary low RAD51 expression(less than 50%),which was much higher than 25%(1/4)in patients with high RAD51 expression.Interestingly,all Olaparib sensitive cases maintained low expression of RAD51 in the P3 generation tissues.When taking miR-509-3 treatment into comparison,we found that two cases(PDX1 and PDX9)showed increased sensitivity to Olaparib after treatment with AAV-miR-509-3 intraperitoneally injection and one case's(PDX8)primary Olaparib resistance was reversed.And in these 3 cases,the RAD51 expression of P3 tumor was significantly lower than that of P2.Conclusions:HR core gene test and RAD51 functional detect can more accurately achieve Olaparib sensitivity prediction,which may optimize the clinical drug strategies in HGSOC patients.Moreover,the combined application of miR-509-3 can enhance the synthetic lethality of PARPi in some certain HGSOC patients,which indicates a novel potential target for the synergistic therapy of PARPi.Conclusions1.MiR-509-3 is associated with platinum sensitivity and good prognosis of HGSOC.2.MiR-509-3 plays the sensitization role of PARPi by interfering with HR repair in HGSOC.3.In some PDX cases of HGSOC,the combination of miR-509-3 and PARPi can enhance the synthetic lethality effect which suggests the potential for synergistic treatment4.HR core gene analysis and RAD51 functional detect exhibited higher accuracy in PARPi response prediction of HGSOC,which may be applied to guide the therapy strategies of clinical patients.