Preliminary Study On The Value Of ATR In Ovarian Cancer Targeted Therapy And Its Synergistic Targets Screening

Background and objective:Ovarian cancer is still the most lethal gynecological malignancy,worldwide,accounting for 2.5%of all female cancers and 5%of female cancer-related deaths.Cancer statistics in 2021 showed that the estimated new cases and deaths were 21,410 and 13,770 in the United States,respectively.Due to the lack of effective screening methods,most ovarian cancer patients were late stage(FIGO 2014 Ⅲ-Ⅳ)at the time of diagnosis,and their 5-year survival rate were less than 30%.Classical first-line treatment for ovarian cancer includes debulking surgery,with the goal of residual disease-free(R0),and platinum-based combination chemotherapy.Unfortunately,the median progression-free survival(PFS)was only 18 months,and more than 80%of platinum-responsive patients will develop platinum-resistant cancer recurrence.Currently,maintenance therapy has emerged as an important strategy to prolong the PFS,including anti-angiogenic compounds such as the antibody bevacizumab or agents targeting DNA repair mechanisms like PARP(Poly-ADP Ribose Polymerase)inhibitors.However,as with platinum and taxane based chemotherapy,many patients will eventually become resistant to drug treatment.The prognosis of ovarian cancer patients remains extremely poor.There is consequently an urgent need to identify novel therapeutic strategy to advance ovarian cancer treatment.Genomic instability arises owning to defects in the DNA damage response(DDR)in the cell and/or increased replication stress,which makes it one of the key hallmarker of cancer.These changes promote the clonal evolution of cancer cells through the accumulation of driving factor aberrations,including changes in gene copy number,rearrangement and mutation;however,these defects also make cancer cells relatively specific vulnerabilities and become targets for malignant tumor treatment targets to improve the patient’s prognosis.BRCA mutant cancer cells are very sensitive to inhibitors of poly-ADP ribose polymerase(PARP).This discovery opens a new era in the research of biomarker-driven "synthetic lethal" treatment strategies for different cancers.PARP inhibitors olaparib and niraparib have been approved for clinical use.At present,the therapeutic field of anti-tumor drugs targeting DDR has rapidly expanded to include inhibitors of other DNA repair and replication key genes,such as ATR,ATM,CHK1 and CHK2,DNA-PK and WEE1.Research to optimize these treatment options is underway in a series of cancers,including the search for predictive biomarkers of targeted drug treatment response(other than BRCA mutations),evaluation of mechanisms of intrinsic and acquired resistance,and evaluation of reasonable treatment options,and Synergy of standard treatments(such as chemotherapy and radiotherapy),new molecular targeted drugs and immune checkpoint inhibitors,etc.In a series of breast cancer studies,high ATR expression and activation were significantly associated with higher tumor stage,mitotic index,pleomorphism,lymphovascular invasion,and poor survivorship.In addition,post-translational modifications of ATR contribute to ATR regulation and autophosphorylation and potentiate its action.The phosphorylation site of ATR is located at Ser428 and is crucial for proper ATR function.The high expression of p-ATR in esophageal cancer is associated with a poor prognosis.A review of the literature shows inhibition of ATR significantly enhances platinum drug response in endometrial,cervical,and ovarian cancer cell lines.In addition,studies have found that ATR inhibition preferentially targets tumor cells with homologous recombination defects,and the loss of TP53 function increases the lethality of adult mice with ATR deletion.It’s worth noting that 96%of TP53 gene mutations were found in ovarian cancer tissues through second-generation sequencing.These promising preclinical results and others have led to a number of clinical trials utilizing ATR-selective small-molecule inhibitors such as AZD6738,BAY1895344,and VE-822(VX-970,M6620),which are currently within phase Ⅰ/Ⅱ clinical trial stages in solid tumors and leukemia.However,the relationship between the expression of ATR in ovarian cancer and the clinical prognosis,its influence on the biological function of ovarian cancer cells,and the potential value as a target for ovarian cancer targeted therapy are still unclear.This study is divided into three parts,Part Ⅰ:the expression of ATR and p-ATR in ovarian cancer tissues and the value of predicting the malignant characteristics and prognosis of ovarian cancer;Part Ⅱ:the targeted inhibition of ATR on ovarian cancer cell lines Preliminary study on the biological function and its internal mechanism;Part Ⅲ:Screening the synergistic targets of ATR in the treatment of ovarian cancer.Part Ⅰ:The expression of ATR and p-ATR in ovarian cancer tissues and their relationship with the malignant characteristics and prognosis of ovarian cancer paientsObjective:Analyze the level of ATR mRNA in normal ovary and ovarian cancer tissues in the databases of GTEx and TCGA.Detect the expression of ATR and p-ATR in the primary tumor tissues of patients with ovarian cancer and their matched metastasis and recurrence tissues by immunohistochemistry.The aims of this study were to assess the expression,and clinical prognostic relationship of ATR and p-ATR in ovarian cancer.Methods:1.Apply the web analysis tool GEPIA to compare the levels of ATR mRNA in normal ovarian tissues and cancer tissues in GTEx and TCGA databases and their relationship with prognosis.2.The expression levels of ATR and p-ATR in ovarian cancer tissue microarray(TMA)was detected by Immunohistochemical staining.3.Use paired t-test to compare the expression of ATR,p-ATR in matched primary,metastatic and recurrent tissues of ovarian cancer.4.The Kaplan-Meier survival curve was used to analyze the correlation between the expression of ATR,p-ATR and the survival of ovarian cancer patients.Cox regression model was used to determine the value of ATR,p-ATR expression levels as independent predictors of overall survival.5.Analyze the clinical and follow-up data of 26 patients,and apply Fisher’s Exact Test to analyze the correlation between the expression of ATR,p-ATR and the clinicopathological characteristics of patients with ovarian cancer.6.Western blotting was used to determine the expression of ATR pathway related proteins in various ovarian cancer cell lines.7.GraphPad Prism v.8.0 software and SPSS 24.0 software were used for statistical analysis.Results:1.The levels of ATR mRNA in normal ovarian tissues were higher than that in ovarian cancer tissues,but its expression level was not related to progression-free survival and overall survival of patients with ovarian cancer.2.The results of immunohistochemistry showed that ATR was located in the cytoplasm and nucleus.For total ATR,the staining intensity pattern was scored as follows:0,no staining;1+,weak staining;2+,moderate staining;and 3+,intense staining.p-ATR mainly resided in the nucleus and was scored according to the percentage of cancer cells with positive nuclear staining.The staining patterns were categorized into six groups:0,no nuclear staining;1+,<10%of cells stained positive;2+,10%to 25%positive cells;3+,26%to 50%positive cells;4+,51%to 75%positive cells;5+,>75%positive cells.High expression of ATR is defined as score 3+,low expression is defined as score ≤2+;high expression of p-ATR is defined as score>3+,and low expression is defined as<3+.3.There were clear trends towards higher ATR(P=0.007)and p-ATR(P=0.01)expression in the recurrent tumors compared to their matched primary tumors.In contrast,there was no significant difference between metastatic tumors and their matched primary tumors,with p values of 0.326 for ATR and 0.972 for p-ATR.4.Kaplan-Meier survival analysis results showed that there was no statistical difference between the level of ATR expression and the disease-free survival and overall survival of patients with ovarian cancer.Compared with the p-ATR low expression group,the disease-free survival of patients in the high expression group(P=0.008)and overall survival(P=0.0002)were significantly shortened.5.There were no significant differences between p-ATR expression and tumor stage,grade,histologic subtype,or ascitic fluid content at surgery.6.In the Cox regression model,both in univariate and multivariate analysis,showed that high levels of p-ATR are a significant risk factor for shorter overall survival,with hazard ratios of 6.96 and 11.393,respectively.7.ATR pathway related proteins ATR,p-ATR,Chkl,p-Chk1 are all expressed in ovarian cancer cell lines A2780,OVCAR5,IGROV-1,SKOV3,OVCAR8 and CAOV-3,indicating that the ATR pathway is endogenous actived in ovarian cancer cell lines.Summary:1.The levels of ATR and p-ATR in recurrent ovarian cancer tissues were significantly higher than those of matched primary tumor tissues.2.The expression level of p-ATR could be served as an independent prognosis predictor for patients with ovarian cancer.Part Ⅱ:Study on the biological function changes and underlying mechanism after ATR inhibition in ovarian cancer cell lines.Objective:The expressions of ovarian cancer-related proto-oncogenes and DNA damage response-related genes were analyzed based on the TCGA database,and the genomes related to ATR expression were found through differential-expression analysis for further functional enrichment analysis.Interfering with the expression of ATR at the RNA level and inhibiting the function of ATR and p-ATR at the protein level,observing the effects on the proliferation,apoptosis,DNA damage,clonal formation and growth of ovarian cancer cell spheroids.The underlying mechanism of ATR participating in ovarian cancer cell biological behavior changes was preliminarily studied.Methods:1.cBioPortal web tool was used to analyze the mRNA levels of ovarian cancer-related proto-oncogenes and DNA damage response-related genes,and found the genomic groups that related to ATR in ovarian cancer tissues;2.The functional enrichment of ATR differential-expressed genes in ATR mRNA high level and no-alteration groups was analyzed by DAVID web tool;3.Assessment the effects of ATR-specific siRNA or highly selective inhibitors on the proliferation of ovarian cancer cells after interfering with the expression of ATR or inhibiting its function through MTT experiment;4.Western blot was performed to detect the effects of ATR siRNA or inhibitor VE-822 treatment on DNA damage pathways,cell cycle checkpoints,apoptosis and other related proteins in ovarian cancer cells;5.Detect the effect of ATR inhibitor VE-822 on the clonogenic ability of ovarian cancer cells through the clone formation experiment;6.Three-dimensional(3D)cell culture experiment was used to simulate the in vivo environment to detect the inhibitory effect of ATR inhibitor VE-822 on the growth of ovarian cancer cells;7.Using GraphPad Prism v.8.0 software and SPSS 24.0 software for statistical analysis,all experiments were repeated three times,and the data were expressed as mean±standard deviation.One-way ANOVA was used for comparison between multiple groups,and the LSD-t method was used for pairwise comparison between groups,with 0.05 as the test level.Results:1.At least one proto-oncogene was highly expressed in 91.1%of ovarian cancer tissues,TP53 gene mutation was found in 96%,and ATR mRNA level was increased in 21%;2.The top 150 genes whose expression were significantly elevated in the ATR mRNA high group were used for functional enrichment analysis.The results showed that they were related to cell cycle regulation,DNA damage repair,and response to ionizing radiation,among others.3.ATR-specific siRNA or highly selective inhibitor interferes with the expression of ATR or inhibits its function,and the proliferation ability of ovarian cancer cells is significant inhibited in a dose dependent manner.4.Western blot results showed that ATR siRNA reduces the expression level of ATR,while the ATR inhibitor VE-822 interferes with its function by inhibiting its phosphorylation.After ATR expression declines or phosphorylation is inhibited,the downstream p-Chk1,cell cycle G2/M arrest key proteins p-Cdc25c,and p-Cdc2 expression decrease.The expression of cleaved PARP and γH2AX proteins increase,which represent apoptosis and DNA damage,respectively.5.Compared with the control group,the clone formation ability of ovarian cancer cells treated with ATR inhibitor VE-822 was significantly inhibited and was concentration-dependent.6.In the 3D cell culture experiment,with the extension of time,the diameter of the ovarian cancer cell spheroids after ATR inhibitor VE-822 treatment were significantly reduced compared with the control group,and the difference was statistically significant(P<0.001).Summary:1.ATR plays a key role in the survival of ovarian cancer cells,targeting could reduce the ability of ovarian cancer cell proliferation,clone formation,and cell spheroid growth.2.Interfering with the expression of ATR or inhibiting its phosphorylation could increase the accumulation of DNA damage in ovarian cancer cells and relieve the mitotic cell cycle arrest,leading to cell apoptosis.Part Ⅲ:Screening the Synergistic Targets of ATR in the Treatment of Ovarian CancerObjective:Based on the concept of synthetic lethality,constructing a small molecule inhibitor library.Screening small molecule inhibitors that have a synergistic effect with the ATR inhibitor VE-822,explore its potential molecular mechanisms,and evaluate the value of the targets as biological markers of ovarian cancer.Methods:1.Preliminary screening:The ATR inhibitor VE-822 and the small molecule inhibitor in the library were used in SKOV3 and OVCAR8 cells individually and in combination at 4-fold increasing concentrations.Cell viability was detected by MTT,using GraphPad Prism v.8.0 to draw a dose-response curve and calculate IC50.Preliminary screening of target gene inhibitors that have a systemic effect with ATR inhibitors.2.Using a dose-response matrix assay,set the concentration gradient of the target small molecule inhibitor and VE-822,MTT detects cell viability,and SynergyFinder calculates the synergy score.3.Use ovarian cancer tissue microarray to detect the expression of target small molecule inhibitors in ovarian cancer tissues,analyze its relationship with the malignant characteristics and prognosis of ovarian cancer,and evaluate its value as a biomarker for ovarian cancer.4.Western blot and immunofluorescence experiments to detect changes in downstream pathway protein levels after combined application of target inhibitors and VE-822,and preliminary exploration of its synergistic mechanism.5.Clone formation,invasion experiments,3D cell culture to detect the synergy of target small molecule inhibitors and VE-822.6.Using GraphPad Prism v.8.0 software and SPSS 24.0 software for statistical analysis,all experiments were repeated three times,and the data were expressed as mean±standard deviation.One-way ANOVA was used for comparison between multiple groups,and the LSD-t method was used for pairwise comparison between groups,with 0.05 as the test level.Results:1.It was found that Weel,ATM,and PLK1 inhibitors,have synergistic effect with ATR inhibitor after the primary screening.2.Dose-response matrix experiment calculated the synergy score and found that the Weel inhibitor AZD1775 and the ATR inhibitor VE-822 have a higher synergistic score.3.The immunohistochemical analysis showed that the expression level of Weel in benign ovarian tumor tissues is lower than that in malignant tumor tissues,and its expression level is related to ovarian cancer tumor stage and patient prognosis.4.Western blot results showed that ATR and Weel pathway-related proteins are expressed in ovarian cancer cell lines.5.Immunofluorescence demonstrated that Weel is distributed in the cytoplasmic nucleus of ovarian cancer cells,and AZD1775 treatment significantly inhibits the proliferation and Weel expression of ovarian cancer cells.6.Immunofluorescence experiments showed that compared with the control group and the ATR inhibitor VE-822 and Weel inhibitor AZD1775 alone treatment groups,the combination group significantly inhibited the proliferation of ovarian cancer cells and increased the expression of yH2AX protein in ovarian cancer cells.7.Compared with a single medication,the expression of p-Chk1,p-Cdc2 and Survivin decreased significantly after the combined application of Weel inhibitor and ATR inhibitor,and the expression of cleaved PARP and γH2AX protein increased.8.Compared with the single application and the control group,the combined application significantly inhibited the clonal formation,invasion,and spheroid growth of ovarian cancer cells,and the difference was statistically significant(P<0.05).Summary:1.ATR inhibitor VE-822 and Weel inhibitor AZD1775 have a strong synergistic effect in inhibiting ovarian cancer cell proliferation,clone formation,and cell spheroid growth.2.The synergistic mechanism of VE-822 and AZD1775 is mainly through increasing the accumulation of DNA damage in ovarian cancer cells and reducing the activity of mitotic checkpoints,which leads to mitotic catastrophe and induces cell apoptosis.3.The expression level of Weel is related to the malignant characteristics of ovarian cancer and the prognosis of patients and may become a clinical biomarker.Conclusions:1.High levels of ATR and p-ATR expression are closely related to the recurrence of ovarian cancer patients.Higher expression of p-ATR is associated with shorter survival and could be served as an independent predictor of the worse prognosis of ovarian cancer patients.2.ATR in ovarian cancer cells may respond to replication stress and DNA damage through DNA damage repair and temporary mitotic arrest.Targeted inhibition of ATR can significantly reduce the proliferation of ovarian cancer cells and promote cell apoptosis.3.ATR inhibitor VE-822 and Wee1 inhibitor AZD1775 have significant synergistic effect in inhibiting ovarian cancer cell proliferation,clone formation,and cell spheroid growth.4.Weel could serve as a biomarker to predict the malignant characteristics and prognosis of ovarian cancer.