| Background and objective: Ovarian cancer is currently the most common cause of death amongst gynecological malignancies,which seriously threatens women’s life and health.Its incidence rate is increasing year by year.The American Cancer Society estimates that approximately 22,530 new ovarian cancer cases and 13,980 deaths will occur in the United States in 2019 alone.The clinical features of ovarian cancer are hidden onset,late diaognosie,advanced malignancy,easy metastasis and poor prognosis.The current standard of advanced ovarian cancer treatment entails primary maximal debulking surgery followed by adjuvant combination platinum-paclitaxel based chemotherapy.This protocol,however,results in more than half of advanced ovarian cancer patients relapsing within the first five years,acquired resistance to standard chemotherapy,and cross-resistance to other functionally and structurally unrelated chemotherapeutic drugs.Despite diagnostic and therapeutic advances,the overall five-year survival rate for ovarian cancer patients has not significantly improved over the past three decades.Therefore,there is an urgent need to develop novel therapeutic strategies in order to improve the treatment of ovarian cancer.Cyclin-dependent kinases(CDKs)are are serine/threonine kinases that are involved in the regulation of cell cycle and transcriptional activity.CDK1,CDK2,CDK4 and CDK6 are involved in the regulation of cell cycle,while CDK8,CDK9,CDK12,CDK13,and CDK19 are involved in gene transcription.CDK7 and CDK20 are linked to both cell cycle and transcription processes.Recent studies have demonstrated that CDKs are frequently overexpressed in many cancer types and ultimately result in uncontrolled cell proliferation and drug resistance.Therefore,CDKs represent particularly attractive drug targets for human cancer treatment.The dual CDK4/6 inhibitor palbociclib(IBRANCE?)was approved by the Food and Drug Administration(FDA)as a first-line treatment of estrogen receptor(ER)positive and human epidermal growth factor receptor 2(HER2)negative advanced breast cancer.There are currently more than 100 phase I/II clinical trials investigating the effects of CDK inhibitors on various cancer types(http://clinicaltrials.gov/).Recently,CDK9 has emerged as an important therapeutic target in cancer,due to its crucial role in RNA transcription,elongation and other cellular processes.CDK9 and cyclin T constitute the positive transcription elongation factor b(P-TEFb)complex which promotes transcription elongation via phosphorylation of RNA polymerase II(RNAPII).CDK9 has been shown to play crucial roles in many types of human cancer,including leukemia,cervical cancer,prostate cancer,glioblastoma,breast cancer,melanoma,and lung cancer.However,the relationship between CDK9 expression and clinical prognosis,the relationship between CDK9 expression and drug resistance,and the therapeutic potential of targeting CDK9 in ovarian cancer,remains unclear.This study is divided into the following three parets: part I: CDK9 expression in ovarian cancer and its clinical significance;the second part: the biological function of CDK9 in ovarian cancer cell line and the preliminary study of its mechanism;part III: the function of CDK9 in ovarian cancer drug resistance and its mechanism.Part Ⅰ: CDK9 expression in ovarian cancer and its clinical significanceObjective: The relationship between the expression of CDK9 and the clinical pathology and prognosis of ovarian cancer patients was analyzed by detecting the expression level of CDK9 in primary ovarian cancer tissues and paied metastatic and recurrent tumor tissues,and combining with clinical data of patients.Methods: 1.Immunohistochemistry was used to detect the expression level of CDK9 in a unique ovarian cancer tissue microarray(TMA).The TMA was constructed with paired primary,metastatic,and recurrent tumor tissues from 26 ovarian cancer patients treated in Massachusetts General Hospital of Harvard Medical School in the United States.2.A paired t-test was used to compare the CDK9 scores among primary tumors,recurrent tumors,and tumors with metastasis.3.Kaplan-Meier survival analysis was used to explore the correlation between CDK9 expression level and disease survival for significance.4.Clinical data of 26 patients were collected,Pearson χ2 test was used to evaluate the relationship between CDK9 expression and clinical-pathological parameters of ovarian cancer patients.5.Western blot was used to detect the expression of CDK9 protein in different ovarian cancer cell lines.6.Statistical analysis was performed using Graph Pad Prism 7.0 software.The correlation between CDK9 expression level and disease survival were analyzed using Kaplan-Meier survival curves with log-rank tests for significance.A paired t-test was used to compare the CDK9 scores among primary tumors,recurrent tumors,and tumors with metastasis.The χ2 test was used to evaluate the relationship between CDK9 expression and clinical-pathological parameters of ovarian cancer patients.In all cases,results were presented as the mean ± standard deviation(SD)and P values < 0.05 were considered statistically significant.Results: 1.CDK9 was primarily localized in the nucleus.The nuclear staining patterns of CDK9 were graded into five groups: 1+,<10% of cells stained positives;2+,10–25% positive cells;3+,26–50% positive cells;4+,51–75% positive cells;5+,>75% positive cells.Patients were subdivided into two groups based on their CDK9 expression score in the primary ovarian cancer specimens,weak group(CDK9 staining score 1+-2+)and strong group(CDK9 staining score 3+-5+).2.The expression level of CDK9 in primary,metastatic and recurrent tissues were 2.58,3.23,and 3.19,respectively.P value of metastatic vs.primary = 0.003;P value of recurrent vs.primary = 0.001.3.Kaplan-Meier survival analysis showed that the patients with low CDK9 expression had significantly better prognosis in both disease-free survival and overall survival,with P values<0.001 compared to those with high expression of CDK9.Cox proportional hazards regression analysis revealed that elevated CDK9 expression in ovarian cancer was a significant risk factor for decreased disease-free survival [hazard ratio(HR),4.1;95% confidence interval(CI),1.56-10.73] and disease-free survival(HR,5.39;95% CI,1.6-18.19).4.No statistical differences were observed between CDK9 expression and the stage,grade,ascites,or histologic subtype of ovarian cancer patients(P values>0.05).5.CDK9 was expressed in ovarian cancer SKOV3,OVCAR8,A2780,IGROV-1,OVCAR5,and CAOV-3 cells.Summary: 1.Primary ovarian cancer tissues express lower leverls of CDK9 compared to patient-paired metastatic and recurrent tissues.2.Increased CDK9 correlates with poor prognosis in ovarian cancer patients.3.CDK9 is expressed in human ovarian cancer cell lines.Part Ⅱ: The biological function of CDK9 in ovarian cancer cell line and the preliminary study of its mechanismObjective: To explore the roles of CDK9 on cell proliferation,apoptosis,migration,clone formation,and 3D spheroid growth in ovrain cancer cells.The molecular mechanism of CDK9 participating in ovarian cancer cell biological behavior was preliminarily discussed.Methods: 1.5 days after CDK9 si RNA transfection or CDK9 inhibitor LDC000067 treatment,the cell viability of ovarian cancer cells was assessed by the MTT assay.2.Western bolt was applied to detect the expression level of CDK9,RNA transcription and apoptosis-related proteins after CDK9 si RNA transfection or CDK9 inhibitor LDC000067 treatment.3.The effect of CDK9 inhibitor LDC000067 on spheroid formation in ovarian cancer cells was detected by 3D cell culture assay.4.Clonogenic assay was conducted to evaluate the effect of CDK9 inhibitor LDC000067 on the clone formation in ovarian cancer cells.5.Wound healing assay was used to determin the effect of CDK9 inhibitor LDC000067 on cell migration in ovarian cancer cells.6.Statistical analysis was performed using Graph Pad Prism 7.0 software.One-way ANOVA test was used to analyze the multiple sets of data.LSD-t test was used for comparison between two groups.In all cases,results were presented as the mean ± standard deviation(SD)and P values < 0.05 were considered statistically significant.Results: 1.Over the 5-day observation period,there was a dose-dependent inhibition of cell viability in both ovarian cancer SKOV3 and OVCAR8 cells with increasing concentrations of CDK9 si RNA or CDK9 inhibitor LDC000067.2.Western blot showed that CDK9 si RNA significantly reduced CDK9 expression,whereas CDK9 inhibitor LDC000067 functionally inhibited CDK9 activity without apparent influence on CDK9 expression.Inhibition of CDK9 by si RNA or inhibitor both resulted in decreased levels of phosphorylated RNAPII and antiapoptotic protein Mcl-1,as well as increased level of the proapoptotic protein cleaved PARP and Bax.3.The spheroids formed from CDK9 inhibitor LDC000067 treated both ovarian cancer cells were significantly smaller than the untreated ovarian cancer cells with the statistical differences in a time-dependent manner(P values<0.05).4.The colony formation assay demonstrated that CDK9 inhibitor LDC000067 exposure caused a dose-dependent reduction in the number and size of colonies formed in both ovarian cancer cell lines compared to the untreated cells(P values<0.05).5.Wound healing assay showed that the addition of CDK9 inhibitor LDC000067 for 24,48,and 72 hours led to a time-dependent decrease in cell migration in both ovarian cancer cell lines compared to the untreated cells(P values<0.05).Summary: 1.Inhibition of CDK9 can suppress cell proliferation,spheroid growth,clonogenicity formation,and migration activity in ovarian cancer cells.2.Inhibition of CDK9 can suppress RNA transcription elongation and induce apoptosis.Part Ⅲ: The function of CDK9 in ovarian cancer drug resistance and its mechanismObjective: To explore the role of CDK9 in the development of drug resistance by establishing drug resistant ovarian cancer cell lines.To evaluate the impact of CDK9 on chemosensitivity of drug resistant ovarian cancer cells and its potential molecular mechanism.Methods: 1.Ovarian cancer SKOV3 and OVCAR8 cells were exposed to stepwise increased concentration of paclitaxel for a period of 6 months to establish paclitaxel resistant ovarian cancer cells SKOV3 TR and OVCAR8.2.Western bolt was applied to compare the expression level of CDK9,Pgp,s2 RNAPII,RNAPII,p-Stat3,and Stat3 between drug sensitive ovarian cancer cells and derivative drug resistant ovarian cancer cells.3.5 days after CDK9 si RNA transfection or CDK9 inhibitor LDC000067 treatment,the senstivity of chemotherapy durgs in drug resistant ovarian cancer cells was assessed by the MTT assay.4.The effect of the combination of palitaxel and CDK9 inhibitor LDC000067 on cell proliferation was assessed by calculating combination index(CI)values using Compusyn Software.5.Western bolt was applied to detect the expression level of CDK9,Stat3 signaling pathway,RNA transcription and apoptosis-related proteins after exposure to increasing concentration of CDK9 si RNA.6.Western bolt was applied to detect the expression level of CDK9,Stat3 signaling pathway,RNA transcription and apoptosis-related proteins after exposure to paclitaxel with or without CDK9 inhibitor LDC000067.7.The effect of CDK9 inhibitor LDC000067 on spheroid formation in drug resistant ovarian cancer cells was detected by 3D cell culture assay.8.Clonogenic assay was conducted to evaluate the effect of CDK9 inhibitor LDC000067 on the clone formation in drug resistant ovarian cancer cells.9.Wound healing assay was used to determin the effect of CDK9 inhibitor LDC000067 on cell migration in drug resistant ovarian cancer cells.10.Statistical analysis was performed using Graph Pad Prism 7.0 software. One-way ANOVA test was used to analyze the multiple sets of data.LSD-t test was used for comparison between two groups.In all cases,results were presented as the mean ± standard deviation(SD)and P values < 0.05 were considered statistically significant.Results: 1.Paclitaxel resistant ovarian cancer SKOV3 TR and OVCAR8 TR cells were resistant to paclitaxel whereas SKOV3 and OVCAR8 cells were sensitive to paclitaxel.2.Both the drug resistant and drug sensitive ovarian cancer cells presented a ubiquitous level of CDK9 expression,whereas SKOV3 TR and OVCAR8 TR expressed higher levels of CDK9 than parental sensitive cells SKOV3 and OVCAR8.Moreover,the expression levels of Pgp,s2 RNAPII,and p-Stat3 were also elevated in drug resistant ovarian cancer cells compared to the sensitive cells(P values<0.05).3.CDK9 si RNA or CDK9 inhibitor LDC000067 sensitize drug resistant ovarian cancer SKOV3 TR and OVCAR8 TR cells to paclitaxel.4.CDK9 inhibitor LDC000067 has a synergistic effect with paclitaxel on anti-proliferation and sensitize drug resistant ovarian cancer cells to paclitaxel(CI<0.7).CI>1.1 indicates antagonism;0.9–1.1 indicates additive;0.7–0.9 indicates moderate synergism;0.3–0.7 indicates synergism;<0.3 indicates strong synergism.5.Inhibition of CDK9 by si RNA resulted in decreased levels of phosphorylated RNAPII and Stat3,as well as induced apoptosis in drug resistant ovarian cancer cells.The antiapoptotic protein Mcl-1 decreased,and the proapoptotic protein Bax increased in a dose-dependent manner.6.Western blot showed that paclitaxel and CDK9 inhibitor LDC000067 combination have an increased suppression effect compared with either agent alone,which resulted in decreased levels of phosphorylated RNAPII and Stat3,as well as induced apoptosis in drug resistant ovarian cancer cells.The antiapoptotic protein Mcl-1 decreased,and the proapoptotic protein Bax and cleaved PARP increased in a dose-dependent manner.7.The spheroids formed from CDK9 inhibitor LDC000067 treated both drug resistant ovarian cancer cells were significantly smaller than the untreated ovarian cancer cells with the statistical differences in a time-dependent manner(P values<0.05).8.The colony formation assay demonstrated that CDK9 inhibitor LDC000067 exposure caused a dose-dependent reduction in the number and size of colonies formed in both drug resistant ovarian cancer cell lines compared to the untreated cells(P values<0.05).9.Wound healing assay showed that the addition of CDK9 inhibitor LDC000067 for 24,48,and 72 hours led to a time-dependent decrease in cell migration in both drug resistant ovarian cancer cell lines compared to the untreated cells(P values<0.05).Summary: 1.Drug resistant ovarian cancer cell lines exhibit higher expression of CDK9 than their parental sensitive cell lines.2.Inhibition of CDK9 sensitize drug resistant ovarian cancer cells to paclitaxel.3.Inhibition of CDK9 can suppress RNA transcription elongation,Stat3 signaling pathway,and induce apoptosis in drug resistant ovarian cancer cells.4.Paclitaxel and CDK9 inhibitor LDC000067 combination have a synergistic antiproliferative effect in drug resistant ovarian cancer cells.5.Inhibition of CDK9 can suppress spheroid growth,clonogenicity formation,and migration activity in drug resistant ovarian cancer cells.Conclusion: 1.Increased expression of CDK9 is closely related to the recurrence and metastasis of ovarian cancer,and negatively correlates with the prognosis of patients.2.CDK9 can activate RNA transcription elongation,suppress apoptosis,thus promoting the proliferation of ovarian cancer cells.3.CDK9 may be involved in the development of drug resistance in ovarian cancer. Inhibition of CDK9 can effectively reverse the sensitivity of drug resistant ovarian cancer cells to paclitaxel.4.CDK9 can be a novel prognostic biomarker and a promising therapeutic target for ovarian cancer patients. |
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