The Effect And Mechanism Of HJURP On The Progression And Enhanced Sensitivity Of Platinum And WEE1 Inhibitor In Ovarian Cancer

Background and objectiveOvarian cancer is the most lethal gynecologic malignancy,which usually presents at an advanced stage silently.High-grade serous cancer(HGSC),which is the most malignant subtype with a poor prognosis,accounts for 70%of epithelial ovarian cancer,severely threatening life and health of female compatriots.At present,complete surgical-debulking combined with platinum-based chemotherapy is still radical backbone for the treatment of HGSC patients.Although most of HGSC patients were platinum-sensitive in initial therapies,they were hard to escape finally from the misfortune of recurrence or chemoresistance along with disease progression.HJURP,a molecular chaperone of CENP-A,has been widely reported to participate in homologous recombination repair of DNA double-strand breaks,functioning crucially in chromosome stability and immortality of tumor cells.However,it’s unclear what the role HJURP plays in progression and chemoresistance of ovarian cancer still.WEE1 is a G2 cell cycle checkpoint,critical for genomic stabilization and DNA damage repair.As a specific small molecular inhibitor of WEE1,AZD1775 has shown potential antitumor effects in multiple preclinical research.Collectively,based on the correlation between HJURP and DNA damage repair and basic mechanism of WEE 1 inhibitor,we aim to explore the function and mechanism of HJURP in tumor progression and chemoresistance of ovarian cancer in the present study.Besides,the significance of HJURP regulation in AZD1775 single treatment or combination of AZD1775 and cisplatin would be further studied and discussed,enlightening new thoughts in targeted therapies of resistant or recurrent ovarian cancer.Contents and methodsTranscriptome sequence was carried out in serous ovarian cancer and fallopian tube fimbriae tissues,and HJURP was determined to be a closely-related gene in ovarian cancer progression according to sequencing data and literature review.Expression of HJURP in fallopian tube fimbriae and HGSC would be verified by qRT-PCR and Western blot.HJURP expression would be also detected by immunohistochemistry in tissue microarray.Then,the correlation between HJURP expression and clinicopathological parameters would be tested and the prognostic significance of HJURP expression would be analyzed by the retrospective cohort.The influence of HJURP on tumor cell proliferation and cell cycle would be detected by MTT assay,xenograft assay in nude mice and flow cytometry etc.Transwell assay would be performed to confirm its influence on metastatic capacity of tumor cells.The sensitivity of ovarian cancer towards platinum affected by HJURP regulation would be measured by MTT assay and colony formation with drug treatment of gradient concentration or apoptotic assay.Moreover,the complicated mechanism would be explored further via transcriptome sequence,bioinformatic analysis,correlation analysis and luciferase assay etc.The influence of HJURP silencing on therapeutic sensitivity towards WEE1 inhibitor and combinatory effect between cisplatin and WEE1 inhibitor would be measured by colony formation and apoptotic assay.ResultsWestern blot and qRT-PCR indicated that HJURP was up-regulated in ovarian cancer and patients with high HJURP level were apt to have unfavorable prognosis.HJURP knockdown could attenuate proliferation,metastasis and tumor formation ability in ovarian cancer cells.Cell cycle analysis revealed that HJURP silencing could mediate early G0/G1 stagnation.Western blot indicated that epithelial-mesenchymal transition(EMT)was affected by HJURP.Cisplatin treatment with gradient concentration illustrated that cell viability was impaired faster when HJURP was silenced in ovarian cancer.Colony formation,EdU assay and apoptotic assay indicated that combination of cisplatin and HJURP silencing could reach the highest level of proliferative inhibition or apoptotic induction in ovarian cancer cells.Immunofluorescence of yH2A.X revealed that HJURP participated in cisplatin-induced DNA damage repair.Transcriptome sequence was performed to figure out that MYC and WEE1 were regulated by HJURP,which was further verified by qRT-PCR and Western blot.Luciferase assay proved that MYC was an upstream transcription factor of WEE1,and rescued experiments indicated that HJURP could modulate platinum resistance partially through MYC/WEE1 axis.Treatment with gradient concentration of AZD1775 and colony formation assay unveiled that HJURP silencing could sensitize ovarian cancer cells towards AZD1775.The synergism of cisplatin and AZD1775 could be enhanced when HJURP was knocked down in colony formation assay.Conclusion1.HJURP was over-expressed in ovarian cancer,correlated to multiple clinicopathological parameters indicating malignant progression.Patients with high HJURP level usually had a worse prognosis.HJURP knockdown could inhibit proliferation and metastasis capacity of ovarian cancer cells.2.HJURP modulated cisplatin resistance of ovarian cancer though MYC/WEE1 axis indirectly.HJURP knockdown could attenuate DNA repair of cisplatin-induced damage and enhance the therapeutic effect of cisplatin.3.HJURP knockdown could increase antitumor effects of WEE1 inhibitor and facilitate the synergism of cisplatin and WEE1 inhibitor.Innovation of the present study1.The present study demonstrated the function of HJURP in malignant progression and prognostic value in ovarian cancer,which provided theoretic fundamentation for search of novel therapeutic target;2.The present study innovatively found that silencing HJURP could be a sensitizer for cisplatin and AZD1775 single agent treatment or combination of both.Besides,we found that HJURP could mediate chemoresistance through MYC/WEE1 axis partially;3.The present study innovatively discussed the function of HJURP in DNA damage repair induced by cisplatin in ovarian cancer,providing robust evidence for further targeted therapy.Limitation of the present study1.This study was a preclinical study and the backbone research was limited in vitro.Animal experiments were insufficient,and patient-derived tumor xenograft was not used in our study.The whole study was still far from actual clinical transformation;2.MYC/WEE1 axis was merely a partial indirect mechanism in the chemoresistance that HJURP mediated.There must be more complicated regulatory networks participating in the initiation of platinum resistance.The present study failed to find a direct target of HJURP.Further exploitation of molecular mechanism was insufficient.