Investigating The Therapeutic Effect Of Olaparib And Palbociclib In Ovarian Cancer And Its Underlying Mechanism

Among a series of malignant tumors affecting female reproduction,ovarian cancer is the most fatal and represents a prominent public health problem due to its frustrating long-term survival outcome and lack of substantial improvement for decades.According to the estimates of the World Health Organization,239000 new cases of ovarian cancer will be diagnosed each year,of which 152000 will die of the cancer.Ovarian cancer can be divided into various subtypes according to etiology and histological classification,and their clinical behavior and molecular genetic characteristics are different.Among the many subtypes,high-grade serous ovarian cancer(HGSOC)is the most common and deadliest.Its early symptoms are not obvious and spread widely in the peritoneal cavity,which greatly increases the difficulty of surgical resection.In addition,although HGSOC usually has a good initial response to platinum chemotherapy,it is almost inevitable to relapse and eventually develop drug-resistance.Genetically,in addition to p53 mutation,there are many recurrent driver mutations in HGSOC,which also play an important role in genomic instability and gene copy number changes.Therefore,there is an urgent need to identify molecular markers for patient stratification and targets to develop low-toxic and efficacious treatment strategies against this type of disease.Poly(ADP-ribose)polymerase(PARP)inhibitors are a group of pharmacological inhibitors targeting PARP and the first clinically approved drug designed on the basis of synthetic lethality.And they were first introduced as a cancer targeting strategy more than a dacade ago.PARP inhibitors has greatly improved the treatment of advanced ovarian cancer with extremely genetic complexity and defective DNA repair function through homologous recombination(HR)pathway.With the advancement in understanding of PARP biology,DNA repair mechanism and the action mechanism of PARP inhibitors,there is growing interest in combining PARP inhibitors with other anticancer therapies to develop more effective treatment regimens for ovarian cancer.It is well known that cyclin-dependent kinase 4 and 6(CDK4/6)inhibitors mainly block the phosphorylation of retinoblastoma protein(RB),thus preventing the cell cycle progression.For decades,three selective CDK4/6 inhibitors have been approved by the Federal Drug Administration(FDA)and the European Drug Administration(EMA)for the treatment of advanced and metastatic ER+/HER2-breast cancer.Among them,Palbociclib(PD0332991)and Ribociclib(LEE011)have been approved in combination with letrozole for first-line treatment.However,with the inevitable development of drug resistance,researchers are making efforts to explore potential biomarkers to predict the treatment response of CDK4/6 inhibitors so as to improve patient outcomes.Objective: Although PARP inhibitor Olaparib and CDK4/6 inhibitor Palbociclib are targeted drugs approved by FDA,and their therapeutic effects have been confirmed clinically,there are still patients who do not respond favorably to the two drugs or develop drug resistance.Therefore,there is an urgent need to develop new treatment strategies to improve existing clinical effects,especially when used in combination drug therapy.We also need to mine biomarkers to provide criteria for clinical treatment to distinguish treatment options for different types of patients.Methods: The effects of PARP inhibitor Olaparib and CDK4/6 inhibitor Palbociclib on ovarian cancer cell lines including HGSOC were examined in vitro.At the same time,the potential mechanism of drug synergism was explored,and the therapeutic effect of the combination of the two drugs was examined in vivo.The main process is as follows:(1)Co-drug reaction of cell lines: through the short-term drug sensitivity test of Olaparib and Palbociclib in 12 ovarian cancer cell lines,it was found that 5 cell lines had combined effect.The co-drug differences of 5 cell lines under long-term drug treatment were examined by clonogenic assay at 2D level.The synergistic effect of the two drugs in 5 cell lines was further proved by three-dimensional sphere assay at 3D level.Flow cytometry analysis was used to examine the expression of Annexin V-Propidium Iodide(PI)in 5 cell lines under the action of single drug and combined drug respectively,in order to characterize the effect of apoptosis induced by the combination of the two drugs.(2)study on the drug mechanism of CDK4/6 inhibitor Palbociclib: through RNA sequencing(RNA-Seq)analysis of A2780 cell line,it was found that Palbociclib could affect the E2 F and HR pathway,and draw the corresponding heat map.Quantitative reverse transcription-PCR analysis(q RT-PCR)was used to examine the expression of genes related to HR pathway in 5 cell lines in single-drug group and combined-drug group,which supported the results of RNA-Seq.(3)phenotypic verification of the combination of two drugs: The focal distribution of γH2AX and RAD51 in 5 cell lines was observed by immunofluorescence staining analysis to characterize the degree of double strand break damage and the level of HR repair.Comet assay was used to examine the DNA damage of 5 cell lines under single drug group and combined drug group,respectively.The length of tail distance represents the degree of DNA damage.The effects of the combination of the two drugs on the genomic stability of 5 cell lines were observed by metaphase chromosome spread assay.(4)Mining of combined therapy targets: through the further analysis of the results of RNA-Seq of A2780 cell line,the MYC pathway gene enrichment analysis(GSEA)map of Palbociclib single drug group and combined drug group was obtained.q RT-PCR assay was used to examine the expression of downstream genes of MYC pathway(LDHA and ODC1)in 5 cell lines in single drug group and combined drug group.The expression of MYC in 12 ovarian cancer cell lines was examined by western blot analysis and quantitatively analyzed.The RNA interference and short-term drug sensitivity test were used to study whether the 5 cell lines had the effect of drug combination in the case of MYC deletion.(5)Verification of the target of combined therapy: the MYC overexpression in IGROV1 and SKOV3 cell lines and the effect of combined drugs were examined by retrovirus transfection and short-term drug sensitivity test.Flow cytometry analysis was used to examine the apoptosis of IGROV1 and SKOV3 cell lines overexpressing MYC in single drug group and combined drug group,respectively.q RT-PCR assay was used to examine the expression of genes related to HR pathway in IGROV1 and SKOV3 cell lines overexpressing MYC in single drug group and combined drug group.The degree of DNA damage and the level of HR repair in IGROV1 and SKOV3 cell lines overexpressing MYC were examined by immunofluorescence staining analysis.q RT-PCR assay was used to examine the relationship between MYC and the expression of genes related to HR pathway in ovarian cancer cell lines.(6)in vivo verification of the combined strategy: the nude mouse model of tumor derived from A2780 cell line was established by subcutaneous tumorigenesis experiment.The experimental mice were treated with Olaparib,Palbociclib and their combination group by intraperitoneal injection and intragastric administration,respectively.During the treatment,the tumor volume was measured every two days.After the treatment,the tumor volume curve was drawn and the tumor was photographed.Immunohistochemical staining analysis was used to examine the expression of various indexes in the tumor after short-term treatment and quantitative analysis.The morphological characteristics of tumors in each group after long-term treatment were observed and quantitatively analyzed by histological staining analysis.Results: PARP inhibitor Olaparib and CDK4/6 inhibitor Palbociclib showed synergistic effect in 5 ovarian cancer cell lines,including HGSOC,and significantly induced more apoptosis.In A2780 cell line,compared with Palbociclib group,E2 F and HR pathway were significantly enriched in the control group.Palbociclib could relatively down-regulate the expression of genes related to HR pathway such as BRCA1,BRCA2 and RAD51 in 5 cell lines with combined drug response.Compared with PARP or CDK4/6 inhibitors alone,the combined treatment could increase the expression of γH2AX,decrease the expression of RAD51,aggravate the degree of DNA damage and reduce the genomic stability of cells.In A2780 cell line,compared with Palbociclib alone or the combination of two drugs,the two MYC pathways were significantly enriched in the control group.Palbociclib and the combination of drugs could substantially down-regulate the expression of LDHA and ODC1,the downstream genes of MYC,in 5 cell lines with combined drug response.It is worth noting that the expression of endogenous MYC in 5 ovarian cancer cell lines with co-drug reaction was significantly higher than that in other cell lines.There was a significant positive correlation between the expression of MYC and HR pathway-related genes in ovarian cancer cell lines.Palbociclib leads to HR deficiency in cell lines by down-regulating MYC-dependent genes related to HR pathway,which has a synthetic lethal effect with PARP inhibitors.In addition,in the mouse model of transplanted tumor of ovarian cancer cell line with high MYC expression,the combination of PARP inhibitor and CDK4/6 inhibitor could effectively inhibit the growth of tumor,and the changes of related biological indexes were consistent with those in vitro.Conclusions: The combination of PARP inhibitor Olaparib and CDK4/6 inhibitor Palbociclib provides a new treatment strategy for ovarian cancer,especially for patients with high-grade serous ovarian cancer.At the same time,the expression level of MYC can also be used as a biomarker to evaluate the utility of the combined strategy in ovarian cancer patients with high MYC expression or resistance to PARP inhibitor or CDK4/6 inhibitor monotherapy.