Corilagin reduces resistance to PARP inhibitors by inhibiting the ERK signaling pathway in ovarian cancers

Background: Corilagin is a compound with hepatoprotective and antiviral activity extracted from Phyllanthus niruri L . Our previous work demonstrated that corilagin inhibits the growth of ovarian cancer cells by regulating the TGF-β/AKT/ERK signaling. Corilagin was also found to sensitize ovarian cancer cells to paclitaxel and carboplatin by inhibiting the Snail-glycolysis pathway. We have now studied whether corilagin could overcome resistance of ovarian cancer cells to poly ADP ribose polymerase inhibitors (PARPi). PARPi block DNA base excision repair and have been approved for treatment of ovarian cancers. Drug resistance has limited efficacy of PARPi. Methods: We have assessed the effect of corilagin alone and in combination with PARPi in two pairs of ovarian cancer cell lines - A2780CP/A2780CP_R and UWB1.289/UWB1.289_R - that are sensitive or resistant to PARPi. CulcuSyn software (BIOSOFT - Software for Science, Cambridge, U.K.) was used to calculate synergy between two drug combinations. Results: Corilagin was active against all four cell lines and enhanced BMN673 activity synergistically in both PARPi resistant cell lines. PARPi -BMN673 down-regulated the expression levels of PARP and up-regulated pH2AX, it decreased pERK activity in sensitive cell lines, but not in resistant cell lines. While corilagin affected DNA repair function to some extent, it inhibited pERK activity in both PARPi sensitive and resistant cells in a dose dependent manner. Corilagin, but not the BMN673, inhibited ZEB1 in resistant cells. Conclusions: Corilagin deserves further evaluation as a drug that could enhance the activity of PARPi in PARPi-resistant ovarian cancer cells. Corilagin a with and antiviral Our previous work demonstrated that corilagin inhibits the growth of ovarian cancer cells by regulating the TGF-β/AKT/ERK signaling pathways. 7 Corilagin was also found to sensitize ovarian cancer cells to paclitaxel and carboplatin by inhibiting the Snail-glycolysis pathway. 8 We have now studied whether corilagin can help to overcome resistance of ovarian cancer cells to PARPi. of ovarian carcinoma our downregulated suggesting that PARPi-resistance might through the ZEB1/EMT pathway in cancers.


Background
Poly ADP ribosylation is required for base excision repair following damage to DNA by alkylating agents. 1 This process is catalyzed primarily by a 113-kDa enzyme poly(ADP-ribose)polymerase-1 (PARP1), the major isoform of PARP. 2 PARP inhibitors (PARPi) block PARP activity in cells and have been used extensively in cancer therapy, particularly for cancers with defects in homologous recombination (HR) repair that mends DNA double strand breaks and limits genetic instability.
In ovarian cancer, up to 50% of all high grade serous ovarian cancers (HGSOC) have inactivating germline mutations, somatic mutations or epigenetic silencing of genes involved in HR DNA repair (e.g., BRCA1/2). 3 PARPi have been used to treat HR deficient, BRCA1/2-mutated, ovarian cancers. Mutations of other genes can predispose cancer cells to apoptosis induced by PARPi, including PALB2, ATM, BRIP1, CHEK2, and RAD51. 4 Three PARPi -olaparib, rucaparib and niraparibhave been approved by the U.S. Food and Drug Administration for maintenance therapy of remissions of recurrent ovarian cancers in women with germline BRCA mutations. 5 While long-lasting remissions have been observed, inevitably ovarian cancer cells become resistant to PARPi and there is an urgent need to find drugs that will prevent or reverse the emergence of PARPi resistance. 5 Corilagin is a compound with hepatoprotective and antiviral activity extracted from the herb Phyllanthus niruri L. 6 Our previous work demonstrated that corilagin inhibits the growth of ovarian cancer cells by regulating the TGF-β/AKT/ERK signaling pathways. 7 Corilagin was also found to sensitize ovarian cancer cells to paclitaxel and carboplatin by inhibiting the Snail-glycolysis pathway. 8 We have now studied whether corilagin can help to overcome resistance of ovarian cancer cells to PARPi.

Materials and Methods
Cancer cell line cultures. Cell proliferation assay. Sulforhodamine B (SRB) was used to detect the effect of drugs on growth of ovarian cancer cell lines, as previously described 7     the doses that produce 50%, 75% and 90% growth inhibition, respectively. Dm: Median-effect dose. m: The shape parameter for the dose-effect curve. r: The conformity parameter for goodness of fit to the median-effect principle of the massaction law.

BMN673 down-regulates the expression of PARP and up-regulates pH2AX.
Treatment with BMN673 decreased PARP protein expression and increased pH2AX, a marker of double strand DNA breaks, in these cell lines in a dose dependent manner, aside from A2780CP_R that did not express PARP and pH2AX ( Figure 5). Corilagin affected PARP to some extent, but increased pH2AX at high concentrations ( Figure 5).

12
GAPDH was used as a loading control. P/G means the ratio of PARP/GAPDH by bands scan, H/G means the ratio of pH2AX/GAPDH by bands scan.

Corilagin inhibits the pERK pathway in resistant cells, whereas BMN673 does not.
High concentrations of BMN673 inhibited pERK activity in sensitive A2780CP cells, but not in resistant A2780CP_R cells and UWB1.289/UWB1.289_R cells ( Figure   6).By contrast, corilagin inhibited pERK activity in both PARPi-sensitive and resistant cells in a dose dependent manner (Figure 6).

Discussion
While PARPi benefit both breast and ovarian cancer patients whose tumors have defects in DNA repair, the response is limited by the development of resistance. 12 Our study documents that corilagin, a novel natural product, enhances the sensitivity of the PARPi  9 This group also reported that BRD4i resensitizes cancer cells to overcome multiple mechanisms of acquired PARPi resistance. 10 There are also reports that Pglycoprotein and c-Met may be involved in PARPi resistance. 13,14 In our own study, the PARPi BMN673 down-regulated the expression of PARP and upregulated pH2AX, a biomarker for double strand DNA breaks, in both sensitive and resistant cancer cells. BMN673 down-regulated the expression levels of pERK only in PARPi-sensitive cells, but not in PARPi-resistant cells, suggesting a role for pERK in PARPi resistance. By contrast, corilagin inhibited pERK activity in both PARPsensitive and PARP-resistant cells ( Figure 6). Corilagin inhibited ZEB1 in A2780CP and A2780CP_R. BMN673 inhibited ZEB1 expression in the PARPi-sensitive cells, but not in the PARPi-resistant cell line (Figure 7). These observations may explain why corilagin has synergistic effects with PARPi when treating PARPi resistant cells.
Our previous study revealed corilagin inhibited multiple signaling pathways in ovarian cancer. Cyclin B1, Myt1, phospho-cdc2 and phospho-Weel were all down-regulated by treatment with corilagin. 7 Corilagin also inhibited TGF-β secretion and the TGF-βinduced stabilization of Snail; 7 Corilagin blocked the activation of both the canonical Smad and non-canonical ERK/AKT pathways. 7 Furthermore, corilagin sensitized ovarian cancer cells to paclitaxel and carboplatin treatment by primarily inhibiting the Snail-glycolysis pathways. 8 Several recent studies have focused on the role of EMT in chemo-resistance. EMT is a complex molecular program that regulates changes in cell morphology and function during embryogenesis and tissue development. EMT is associated with therapy resistance and tumor recurrence 11 . Several transcription factors, including the Snail/Slug family, Twist, δEF1/ZEB1, SIP1/ZEB2 and E12/E47, function as molecular switches for the EMT program. 11 Among these factors, the zinc finger E-box binding transcription factor ZEB1 is considered to play a crucial role in ovarian cancer 16 EMT. ZEB1 promotes EMT by repressing genes contributing to the epithelial phenotype while activating those associated with the mesenchymal phenotype. 15 Suppression of ZEB1 inhibits cell invasion and metastasis, restoration of paclitaxel sensitivity of chronic chemo-resistant ovarian carcinoma cells. 16 In our study, corilagin downregulated ZEB1, suggesting that PARPi-resistance might function through the ZEB1/EMT pathway in ovarian cancers.
In this paper, we document that corilagin interacts with PARPi to exert synergistic anti-

Declarations
Ethics approval and consent to participate：Not applicable.

Consent for publication：Not applicable.
Availability of data and materials：The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.     GAPDH was used as a loading control. P/G means the ratio of PARP/GAPDH by bands scan, H/G means the ratio of pH2AX/GAPDH by bands scan.