Studies On The Anti-tumor Effect Of WEE1 Inhibition By MK1775 In Ovarian Cancer

Background:Ovarian cancer represents the most frequent cause of cancer-related deaths in women diagnosed with gynecologic malignancies.Due to the insidious onset,vague symptoms and rapid progresses,the majority of ovarian cancers are diagnosed at the advanced stage.In spite of the advancement occurring in both cytoreductive surgeries and chemotherapeutic techniques,the 5-year survival rate for ovarian cancer remains basically unchanged due to high metastatic and drug resistant properties.Thus,more effective therapy is urgently needed.WEE1,an important regulator for Cell cycle checkpoints,plays a critical role in maintaining the integrity of the cell genome.High expression of WEE1 has been reported in ovarian cancer,as well as melanomas,osteosarcoma,glioblastoma and breast cancer.Notably,WEE1 expression was significantly associated with poor overall survival of ovarian cancer patients with post-chemotherapy effusions,pointing to WEE1 as a potential novel therapeutic target for ovarian cancer.Objective:Determine both in vitro cytotoxicity and in vivo anti-tumor efficacy of MK1775 as single-agent in ovarian cancer.Detect the expression level of WEE1 in different immune subsets.And study the immune-modulating role of MK1775 in addition to direct cytotoxicity to cancer cells.Finnally provide the immunological rationale for the application of MK1775 in chemo-immunotherapy.Part Ⅰ In vitro anti-tumor activity of WEE1 inhibition by MK1775 as a mono-agent in ovarian cancer cellsMethods:1.The expression of WEE1 mRNA in ovarian cancer was determined by RT-PCR.(Since the expression of WEE1 in SKOV3 cell has been reported in previous studies,so it was not detected here.)2.MTT was conducted to determine the inhibitory effect of WEE1 inhibitor MK1775 on the cellular viabilities of ID8 and SKOV3 cells.3.Cell apoptosis assay was accomplished by cell staining with Annexin V and 7-AAD to characterize the role of MK1775 in tumor cell apoptosis under either WT p53 or mutant p53 status.4.Western blot was carried out to confirm the effect of MK1775 on the phosphorylation of CDK1.5.Cell cycle was conducted to determine the effect of MK1775 on cell cycle progression.Results:1.WEE1 expression in ID8 cell lines.WEE1 expression in ID8 cells was higher than melanoma B16 cell lines.2.WEE1 inhibit the viabilities of ID8 cells and SKOV3 cells in a dose dependent manner.The IC50 of MK1775 in ID8 cells was 750±196.33nM,the one in SKOV3 cells was 464.7±127.71nM.3.Treatment of 1μM MK1775 induce the cell apoptosis of ID8 cells(P24h=0.0229,P48h=0.0398)and SKOV3 cells(P24h=0.0011,P48h<0.0001).and SKOV3 cells are more sensitivity to the treatment of MK1775.4.Treatment of MK1775 down regulated the phosphorylation of CDC2.5.After MK1775 treatment,sub-G1 phase of ID8 cells was increase in both 0.5μM MK1775 group(p<0.0001)and 1μM MK1775 group(p<0.0001);G1 phase of ID8 cells was decreased in both 0.5 μM MK1775 group(p=0.0006)and 1μM MK1775 group(p<0.0001),S phase of ID8 cells was increased in both 0.5 μM MK1775 group(p=0.0012)and 1μM MK1775 group(p<0.0001);G2-M phase of ID8 cells were decreased in both 0.5 μM MK1775 group(p=0.0055)and 1μM MK1775 group(p=0.0007).The findings of cell cycle in SKOV3 cells after MK1775 treatment were similar to ID8 cells.And,the effect of MK1775 treatment on cell cycle of SKOV3 was obviously more significant than ID8 cells.Summaries:1.WEE1 inhibitor MK1775 showed in vitro cytotoxicity to ovarian cancer and the anti-tumor effect was dose dependent.2.Cell cycle analysis and western blot data indicated that MK1775 abrogated the G2-M checkpoint through inhibition of phosphorylation of CDK1 and induced apoptosis of ovarian cancer cells.3.The cytotoxic role of MK1775 as one single chemotherapy agent against ovarian cancer was established in either WT p53 or p53 defective cell lines.Part Ⅱ Expressions and significants of WEE1 in different subsets of immune cells and variety of tumor cells.Methods:1.Mouse T cells,B cells and CD11b+cells enrichment sets were used to isolate different subsets of immune cells from splenocytes of WT mice.Also,selections were carried out to get CD4+T cells,CD8+T cells and Treg cells.2.RT-PCR was conducted to detect the expression of WEE 1 in variety of tumor cells and different subsets of immune cells including T cells,B cells and CD11b+cells.3.RT-PCR was conducted to detect the expression of WEE1 in different subsets of T cells from WT mice.Results:1.Different subsets of immune cells were isolated and purified from splenocytes of mice.2.The expressions of WEE1 in immune subsets were significantly higher than in tumor cells(p<0.0001).3.No significant differences of WEE1 expressions were found in different subsets of T cells from WT mice.Summary:1.Expressions of WEE1 were detected in both tumor cells and immune cells indicating MK1775 may impact on the immune cells in addition to direct cytolytic effects on cancer cells.2.Higher expressions of WEE1 in immune cells than in tumor cells implied that immune cells may be more sensitive to MK1775 treatment.Part Ⅲ In vivo anti-tumor activities of MK1775 in ID8 ovarian cancer models and the immune-modulating role of MK1775 in tumor microenvironmentsMethods:1.C57BL/6 WT mice were used to establish the ID8 ovarian cancer models.2.Mice were divided into two groups when tumors were established.And MK1775 or DMSO was given daily.Tumor growth was monitored every seven days by measuring the abdominal circumferences and mice weight.Finally,the ascites from ID8-bearing mice were collected to measure the volume 30 days after MK1775 treatment.3.Flow cytometry was used to determine the proportions of different subsets of T cells in ovarian cancer tissues and in ascites from tumor bearing mice.4.Flow cytometry was used to determine the proportions of different subsets of T cells in splenocytes of tumor bearing mice.Results:1.The data from IVIS IMAGING system showed the ID8 ovarian cancer models were established successfully.2.The mice weight(p=0.0182)and abdominal circumferences(p=0.0291)of tumor bearing mice were significantly lower in MK1775 treated group than in control group.In line with the finding above,the volume of ascites was much lower in MK1775 treatment group(p=0.0172).3.The ratio of CD8+/CD4+T cells of ovarian cancer tissues had the trend to increase in MK1775 treatment group than control group(p=0.1136).The ratio of CD8+/CD4+T cells of ascites was significantly increased in MK1775 treatment group than control group(p=0.004).The absolute numbers of CD4+T cells in ovarian cancer tissues were significantly decreased in MK1775 treatment group(p=0.0497).The absolute numbers of CD4+T cells of ascites had the trend to decrease in MK1775 treatment group(p=0.1188).4.The absolute numbers of CD4+T cells(p=0.0172)and Treg cells in splenocytes were significantly decreased in MK1775 treated group(p=0.0209).Summary:1.In vivo antitumor effect of MK1775 was observed in ID8 ovarian cancer models.2.MK1775 played a subtle immune-modulating role in tumor microenvironment through increasing CD8+/CD4+T cells ratio and reducing the population of Treg cells.Part Ⅳ The study on the mechanism of MK1775 in immunemodulating activityMethods:1.pCDC2 staining was conducted to test the phosphorylation of CDC2 in CD4+T cells,CD8+T cells and Treg cells after MK1775 treatment.2.Cell proliferation assay was determined by eFluor?450 staining to analyze the effects of MK1775 on the proliferations of CD4+T cells and CD8+T cells.3.The in vitro induction and proliferation assay of Treg cells were carried out to determine the effects of MK1775 on the induction and proliferation of Treg cells in vitro.4.Flow Cytometry was used to detect the expression of CD69,CD44 and CD95 in Treg cells after MK1775 treatment.Cleaved-caspase 3 staining was used to analyze the apoptosis assay of Tregs.Results:1.The level of pCDC2 was significantly decreased in CD4+T cells(p<0.0001),CD8+T cells(p<0.0001),Treg cells(p<0.0001)after MK1775 treatment.2.MK1775 inhibited the proliferations of CD4+T cells and CD8+T cells.CD8+T cells were more tolerated to MK1775 treatment.At the high dose of 0.5μM MK1775,the proliferation of CD4+T cells was decreased by 54.7±2.982%compared with control group;the proliferation of CD8+T cells was only reduced by 7.933±0.569%.3.Negative correlations had been detected between the rates of Treg induction and the concentrations of M1775 treatment(r=-0.9116,p=0.0311).Also,negative relationships had been detected between the rates of Treg proliferations and the concentrations of M1775 treatment(r=-0.9826,p=0.0028).4.The expressions of CD69(p=0.0215),CD44(p=0.0006)and CD95(p=0.0001)were increased after MK1775 treatment.Elevated expressions of caspase 3 in iTreg with 0.1μM MK1775 treatment indicated MK1775 could suppress iTreg conversion(p=0.016).Summary:1.MK1775 inhibited the proliferations of CD4+T cells,CD8+T cells and Treg cells.CD8+T cells were more tolerated to MK1775 treatment.2.MK1775 suppressed the induction of Treg cells in a dose dependent manner.3.The elevated expressions of CD69,CD44 and CD95 in Treg cells indicated activation-induced cell death may play a partial role in the reduction of Treg cells caused by MK1775 treatment.Conclusions:1)Both in vitro and in vivo anti-tumor activities of MK1775 were confirmed in ovarian cancer.2)MK1775 impact on the proportions of different subsets of T cells through suppression of the proliferation of CD4+T cells,CD8+T cells and Treg cells,the inhibition of Treg induction as well as activation-induced Treg cell death.3)In addition to the direct cytotoxicity to ovarian cancer,MK1775 played subtle immune-modulating role in the tumor microenvironment.4)These data provided the immunological rationale for the application of MK1775 in chemo-immunotherapy.