Investigating The Molecular Mechanism Underlying The Effective Treatment Of PTEN-deficient Endometrial Cancer By Combined Inhibition Of PI3K And PARP

Objective:Aberrant activation of the PI3K singaling pathway is frequently observed over 80%endometrial cancers.PTEN loss of function not only lead to PI3K signaling pathway activation,but also to dysfunction homologous recombination repair of DNA double-strand breaks.Combined inhibition of PARP and PI3K has been shown to effective in the treatment of BRCA1-related triple negative breast cancer?TNBC?,PTEN/P53 co-deletion hormone-insensitive advanced prostate cancer,PIK3CA-mutant and PIK3CA-WT ovarian cancer.Thus,the aim of this study was to investigate the molecular mechanism underlying the effective treatment of PTEN deficient endometrial cancer by co-inhibition of PI3K and PARP,and then find the suitable endometrial cancer patients for this combination therapy.Methods:?1?in vitro:four PTEN-deficient endometrial cancer cell lines?ishikawa,AN3CA,Nou-1,Hec108?and one PTEN-WT endometrial cancer cell line?Hec1A?were slected,cell proliferation assay and 3-Dimensional sphere culture was used to detect the inhibition efficiency after olaparib and BKM120 single agent or combination treatment.The effect of Olaparib and BKM120 single agent or in combination on DNA damage and repair process were observed by comet assay,immunofluorescence.Use chromosome spread assay to identify the chromosome integrity after olaparib and BKM120 alone or combination treatment.The PI3K/AKT/mTOR pathway and apoptosis associated protein expression were investigated by Western Blot technology.The effects of treatment on BRCA1/2mRNA level and protein level was detected by real-time RT-PCR and immunohistochemistrytechniques.?2?in vivo:the establishment of genetically engineered mouse model:primary injection of Ade-cre into 6-8 weeks old female mice(PTEN^loxp/loxpLKB1^loxp/loxp)uterine cavity.After 6 weeks injection of Ade-cre,tumor volume was detected by magnetic resonance imaging?MRI?.Mice was divided into four groups random,vehicle,olaparib,BKM120 single agent group and combination group.Animals were treated by oral gavage with BKM120?30mg/kg?every 6 days per week.Olaparib was resuspended for intraperitoneal administration at a dose of 50mg/kg every 6 days per week.After 3 weeks treatment,animal tumor volume was measured again by MRI.Mice were sacrificed and the tumor was used to detect the pathology after 4%paraformaldehyde fixing.Results:?1?combined inhibition of PI3K and PARP effectively bolcked the proliferation and the 3-Dimensional sphere formation of PTEN-deficient endometrial cancer cell lines.Compared with single agent,combination therapy results in a substantially DNA damage response and more reduced homologous recombination?HR?.Meanwhile,the combination of PI3K and PARP inhibitors synergized to suppress the PI3K signaling pathway when compared with single agent.?2?co-targeting PI3K and PARP shows synergized effects in treating PTEN and LKB1 co-deletion endometrial tumors.Based on the MRI method,we found this combination therapy effectively blocked tumor growth when compared to single treatment.In addition,none of the treatments caused weight loss in the tumor-bearing mice examined,showing there is no toxicity in this combination therapy.Consistently,reduced cellular proliferation and increased cell apoptosis showed tumor regression seen mice in treated with Olaparib and BKM120.Furthermore,combination treatment led to a substantial increase in DNA damage response and compromised HR repair.Conclusions:?1?PTEN-deficient endometrial cancer cells do not respond to PARP inhibitor Olaparib alone.?2?The effect of Olaparib and BKM120 combination treatment depends on PTEN status in endometrial cancer cells.?3?PI3K inhibition as a plausible strategy for expanding the utility of PARP inhibitors to endometrial cancers in a PTEN-deficient setting.?4?combined inhibition of PI3K and PARP effectively treat PTEN-deficient endometrial tumor and thus supply a plausible strategy for clinical trials.