The Mechanism Study Of PARP Inhibitor-induced ALDH1A1 Expression Mediated Drug Resistance In BRCA2-/- Ovarian Cancer Cells

Introduction and ObjectiveOvarian epithelial cancer(EOC)is the most lethal malignancy of the female reproductive tract.Poly(ADP-ribose)polymerase(PARP)inhibitors are an exciting and promising new class of anticancer drugs that have been approved for ovarian cancer patients with BRCA1 or BRCA2 mutations.However,almost 100%patients receiving PARPi eventually develop tumor recurrence or progression.Therefore,understanding the mechanism underlying PARPi resistance,reversing the resistance and improving the benefit of PARP inhibitors in BRCA mutated EOCs is particularly important.Aldehyde dehydrogenase(ALDH)is often correlated with multi-chemotherapy resistance in various cancers.However,it is unknown whether ALDH activity affects the sensitivity of EOC cells to PARPi.In this study,we investigate whether ALDH1 A1,a member of ALDH family,participates in the PARP inhibitor resistance,the possible mechanism and the possibility of reversing this resistance.It might provide clues and help develop a new clinical target for recurrent PARPi-resistant ovarian cancer.Methods1.Generation of two BRCA2-/-PARP-inhibitor resistance EOC cell lines.2.Flow cytometry was used to detect ALDH activity diffrence between PARPi-resistance cell lines and their parent cell lines.3.Methylene blue assay was used to see if ALDH activity affects cells sensitivity to PARPi.4.RT-PCR detecting mRNA level gene expression of ALDH family gene and find out potential target gene ALDH1A1.Cells were transfected with plasmid or siRNA to overexpress or down-regulate ALDH1A1 then check cell sensitivity to PARPi.5.Western blot was conducted to check the relationship between BRD4,a possible upstream gene of ALDH1A1,and PARPi.Then use methylene blue assay and flow cytometry to check functional affect of BRD4 on ALDH1A1.6.Overexpress ALDH1A1 in three different DNA repair pathway reporter cell lines and flow cytometry was performed to check whether these pathway activity had changed.7.Methylene blue assay to check whether using selective ALDH1A1 inhibitor can reverse PARPi resistance.Results1.Successfully develop two BRCA2-/-PARPi-resistant EOC cell lines PEO1-R and Kura-R by intermittent,incremental,in vitro treating with PARPi Olaparib.2.Flow cytometry result shows that ALDH activity significantly increased in resistance cell line than parent cell line(P<0.01)and this is induced by Olaparib itself.3.Methylene blue assay shows higher ALDH activity was related to PARPi resistance(P<0.01).4.Real time PCR results show ALDH1A1 is the major member in the ALDH super family that induced by PARPi.Use Methylene blue cell viability assay we find overexpress ALDH1A1,BRCA2-/-EOC cells become resistant to PARPi Olaparib(P<0.01);similarly,knockdown ALDH1A1 expression in resistant cells can sensitize them to Olaparib,which indicates that ALDH1A1 is the main isozyme confers cells resistance to PARPi(P<0.01).5.Immunoblotting result shows that PARPi can positively regulate BET family protein BRD4.When using siRNA to down-regulate BRD4,PARPi can no longer induce ALDH1A1 expression.6.Immunofluorescence assay indicates ALDH1A1 can enhance DNA repair capability in BRCA2-/-EOC cells.Overexpress ALDH1A1 in three different DNA rapair pathway reporter cell line,showing HR,c-NHEJ and MMEJ activity,respectively and check activity change by flow cytometry,only enhancement of MMEJ activity is observed(P<0.01).7.Methylene blue assay results show after dual treatment of PARPi and ALDH1A1 selective inhibitor,no difference can be observed in BRCA Wildtype cells than single drug group(P>0.05).But significant synergism can be observed in BRCA2-/-cells(P<0.01)。8.By using intraperitoneal EOC xenografts model in nude mice,we can find the similar synergism in dual treatment group.Conclusions:1.PARP inhibitor can induce ALDH1A1 expression,and leads to higher ALDH enzyme acivity.2.The induction of PARPi to ALDH1A1 was mediated by positively regulating BRD4.3.ALDH1A1 can enhance microhomology-mediated end joining(MMEJ)pathway activity in BRCA2-/-ovarian cancer cells,which makes tumor cells repair drug-induced DNA damage more easily and eventually leads to drug resistance.4.Using selective ALDH1A1 inhibitor,resistant cells can again show sensitivity to PARPi.Resistance could be reversed.Taken together,our project indicates that ALDH1A1 mediates resistance to PARP inhibitor in BRCA2-/-EOC cells by enhancing MMEJ activity;By using selective ALDH1A1 inhibitor,resistance could be reversed,which gives a hint that ALDH1A1 could be a potential clinical target in treating PARPi resistance EOCs.