Cancer-testis Oncogene MEIOB Sensitizes Triple-negative Breast Cancer To PARP1 Inhibitor By Inducing Homologous Recombination Deficiency

【Background】Breast cancer is one of the leading causes of female cancer mortality around the world.The incidence continues to rise by 3% per year in China,which is a serious threat to the health of women and causes huge burden for the society.Triple-negative breast cancer(TNBC)is the most aggressive subtype of breast cancer,and lack effective targeted therapies.TNBCs are characterized by a wide spectrum of genomic alterations such as loss of heterozygosity,some of which might be caused by HRD.One of the strongest risk factors of HRD is a deleterious mutation in the BRCA1 gene,which occurs in surpass 10% of patients with TNBC.Recently,PARP1 inhibitor,olaparib,has been approved by the FDA to treat ovarian cancer with mutations in BRCA genes.Therefore,inhibition of PARPs is a promising strategy for targeting breast cancers with defective HR repair.Analyses of tumor-derived genome sequences have shown that the loss of BRCA1 or BRCA2 yields a distinct pattern of HRD characterized by base-substitution mutations termed signature 3.However,a number of studies have suggested that some sporadic TNBC(BRCA wildtype)may bear substantial similarities to signature 3,including HRDs that might predispose to PARP1 inhibitor sensitivity.Thus,it is important to identify predictive biomarkers of PARP1 inhibitor sensitivity that is suitable for HRD patients without BRCA mutations.Based on our previous study,we have identified 876 new Cancer-testis genes(CT genes),including CT-nc RNA,in 19 different cancers and their expressions are limited to human germ cells and aberrantly upregulated in a variety of malignant tumors,such as breast,lung and melanoma.The tissue-restrictedcharacteristics endowed the CT genes as dominant potential targets for contraception and cancers without side-effects.Forexample,use of the CT antigen BRDT as a contraceptive targethas been proposed due to its high efficacy and the minimal sideeffects associated with this therapeutic strategy.In adult mammals,CTAs were intimately related to spermatogenesis due to their unusual restricted expression during different discrete events of spermatogenesis,such as cell cycle progression,meiosis and DNA repair process.Otherwise,ectopic expression of CT genes plays oncogenic roles in cancers,for instance,a cancer/testis lnc RNA THOR was identified to accelerate the onset of melanoma in zebrafish.In spermatogenesis,Jonathan et al discovered that MEIOB(meiosis specific with OB domains)forms a complex with SPATA22 and RPA,and act together during meiotic recombination.In stark contrast to the meiosis process,expressions of MEIOB and SPATA22 m RNA show mutually exclusive in several cancers including breast cancers,indicating their different roles in DNA repair process between spermatogenesis and tumorigenesis.We also found that MEIOBtranscriptional patterns show aberrant activation in various malignant cancers,especially enriched in TNBC based on TCGA database analysis.Thus,we devote to investigate whether MEIOB is involved in the DNA repair process and its oncogenic role in TNBCs.【Methods】Genotype-Tissue Expression(GTEx)and The Cancer Genome Atlas(TCGA)databases were used to quantify the expression of MEIOB.Cox regression analysis was applied to evaluate the association between MEIOB expression and prognosis in human TNBC.Cell proliferation and migration affected by MEIOB in TNBCs were also assessed in vitro.We measure the DNA damage degree by monitoring anti-γH2AX nucleus staining signals.We used NHEJ or HR GFP-reporter assays and cell cycle assessment to evaluate the way of DNA repair.We performed LC-tandem mass spectrometry to discover the proteins interact with MEIOB.Patient-derived xenograft(PDX)models were included to assess the sensibility of MEIOB-activated breast cancers to PARP1 inhibitor.IHC labeling and RNA-seq analysis were performed to determine the mechanisms mediating tumor growth inhibition in olaparib-treated PDX tumors.【Results】In this study,we firstly revealed their expression by RT-PCR in 16 normal tissues.In line with the result of GTEx database,MEIOB expression in the testis outdistances the other 15 normal tissues.We also found MEIOB was aberrantly activated in breast cancers based on the Cancer Genome Atlas data set analysis,and this activation rate was highest in TNBCs(activation ratio:11/97).In addition,Kaplan-Meier analysis revealed that high MEIOB expression is positively correlated with the reduced overall survival of breast cancer patients,and more associated with TNBC patient survival(HR=1.90 and 7.05,Figure).We also validated our hypothesis that MEIOB may affect breast cancer cell viability in multiple breast cancer cell lines.We knocked down MEIOB by si RNA in MDA-231 cell line,depletion of MEIOB in MDA-231 cells led to a significant decrease in both cell proliferation and migration.Then,we found that overexpression of MEIOB in both cells significantly increased cell proliferation and migration compared with control groups.Thus,MEIOB may act as an oncogene in TNBC tumorigenesis.These findings suggest a potential oncogenic role and practical values of MEIOB in breast tumorigenesis.Given the well-established role of MEIOB in meiotic recombination in spermatogenesis,we wanted to testthe effect of MEIOB on responding to DNA damage in tumorigenesis.We therefore treated SUM1315 cells with CDDP to induce DNA repair response,and then collected cells for IF analysis 12 h post CDDP-treatment to measure the DNA damage degree by monitoring anti-γH2AX nucleus staining signals.Results showed that,upon CDDP exposure,overexpression of MEIOB significantly decreased γ-H2 AX accumulation in cells.These studies suggested that MEIOB may promote DNA damage repair process.Further,our results showing that MEIOB overexpression resulted in cell cycle arrest at G1 phase strongly suggested that MEIOB may induce HR defect.To confirm this hypothesis,SUM1315 cells stably transfected with NHEJ or HR GFP-reporter constructs were transiently transfected with control or MEIOB plasmids.Twenty four hours later,the cells were re-transfected with the I-Sce I expression plasmid.After the additional 48 h incubation,cells were analyzed by Western blotting using anti-GFP antibody.GFP intensity was significantly upregulated in SUM1315-NHEJ-MEIOB cells compared with SUM1315-control cells.To find out the pathways affected by MEIOB in DNA damage repair process,we detected expression alternations of DNA repair-related molecular indicators in MEIOB overexpressed cells compared to the controls.Results showed that overexpression of MEIOB caused a decreased expression level of RAD51,a marker of HR repair process.To interrogate the mechanisms activating PARP1,we performed LC-tandem mass spectrometry and discovered proteins interact with MEIOB.MEIOB was purified with agarose beads from SUM1315 transfected with plasmid cloned MEIOB.As a result,YBX1,a protein physically interactwith PARP1 to inhibit its poly(ADP-ribose)degradation,was found to bind with MEIOB.This implies a HR-deficiency in MEIOB overexpressed cell lines.In addition,MEIOB can bind to YBX1 andstimulate PARP1.These findings suggest that MEIOB participates in DNA repair though the activation of PARP1 mediated repair process.Biallelic inactivation of BRCA1 or BRCA2 is associated with a pattern of genome-wide mutations known as signature 3.Patients bearing signature 3 including BRCA1/2 mutated tumors seem morelikelytorespondto PARP inhibiting therapy.Interestingly,we found MEIOB expression was positively associated with score of signature 3(p=0.02,corr=0.47).This phenomenon suggests us that MEIOB-activated patients bearingsubstantial mutation signature 3 is similar to BRCA-mutated TNBCs.Thus,we hypothesized that cancer cells overexpressing MEIOB would be more sensitive to PARP inhibitors than MEIOB-null cells.We found that MEIOB-null cells showed less sensitive to AG-14361 than MEIOB-activated cells.Similar results were also observed in cells treated with olaparib.In vivo,results showed that olaparib treatment on MEIOB-overexpressed tumors significantly decreased the tumor volume to as less as 30% of that in MEIOB-overexpressed groups.These results suggested that MEIOB-activated groups are more sensitive to olaparib compared with MEIOB-null ones.To determine the mechanisms mediating tumor growth inhibition in olaparib-treated PDX tumors,IHC labeling was performed using antibodies for cell proliferation marker(Ki67)and PARP1.After treatment with olaparib,a marked decrease in the number of Ki-67 positive proliferating cells was observed in the MEIOB-activated tumors compared with the MEIOB-null tumors.Originally,tumor tissues thatuntreated with olaparib shows high levels of Ki-67 positive proliferating cells in MEIOB-activated tumors compared with the MEIOB-null tumors.This result was consistent with the activated PARP1 by MEIOB in vitro.Yet,the overexpressed PARP1 were strongly inhibited by olaparib in MEIOB activated tumors.Not surprisingly,Gene Ontology(GO)analysisrevealed strong enrichment for genes involved in DNA repair processes after olaparib treatment.Representative gene expressions involved in the HR and NHEJ process were verified by RT-PCR.Expression of PARPBP,POLQ and RTEL1 participant in NHEJ and RAD51AP1,BRCA2,DMC1 and ESCO2 participant in HR were all inhibited by olaparib.Therefore,it seems that strong inhibition of tumor cell proliferation afterolaparibtreatment iscaused by the synthetic lethal effect.【Conclusion】Taken together,our study shows that MEIOB was strictly restricted to be expressed in testis and TNBCs.And overexpression or depletion of MEIOB can promote or suppress breast cancer cell vitality,respectively.Further,we discovered that MEIOB mediates the HR defect by activing the expression of PARP1 and that pharmacologic inhibition of PARP1 is able to reverse the effect of increased cell vitality led by MEIOB overexpression.In general,we demonstrated the oncogenic role of MEIOB in TNBC and it sensitizes the TNBC to PARP1 inhibitor.As we look to the future,this might be a worthy aspect to conduct thorough research to other CT genes especially those attended to HR in meiosis.