Research ArticleExperimental Studies

PARP Inhibitor Sensitizes BRCA-mutant Pancreatic Cancer to Oxaliplatin by Suppressing the CDK1/BRCA1 Axis

CHORONG KIM, DANBEE KIM, DA SOL LEE, SEONMIN LEE, CHANGHOON YOO and KYU-PYO KIM
Anticancer Research December 2023, 43 (12) 5523-5534; DOI: https://doi.org/10.21873/anticanres.16754

Abstract

Background/Aim: Currently, olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, has been approved as maintenance therapy for patients with germline BRCA mutations and metastatic pancreatic cancer. However, platinum-based chemotherapy, which induces synthetic lethality with PARP inhibitor treatment, is still controversial. Hence, we aimed to examine a platinum-based drug in combination with a PARP inhibitor and generate data regarding the use of a PARP inhibitor in the overall treatment of pancreatic cancer. Materials and Methods: Using the Capan-1 cell line (BRCA2-mutant pancreatic cancer cell line), we evaluated the combinatorial effects of olaparib, a PARP inhibitor, and oxaliplatin by cell viability, combination index, western blotting, immunocytochemistry, flow cytometry, apoptosis assays and in vivo experiments. Results: Capan-1 cells showed high sensitivity to olaparib due to the alteration in PARP activity, which led to cell death through the accumulation of oxaliplatin-induced DNA damage. Beyond DNA damage, oxaliplatin also suppressed the CDK1/BRCA1 signaling axis, which induced defects in homologous recombination repair. Additionally, inhibition of CDK1, a biomarker for oxaliplatin efficacy, induced cell death regardless of the BRCA mutation profile. Conclusion: Oxaliplatin may be used in combination with olaparib in PDAC patients with DNA damage repair mutations. Our findings highlight CDK1 as a potential therapeutic target for pancreatic cancer.

Key Words:

Research on the development of pancreatic ductal adenocarcinoma (PDAC) has largely focused on genetic aberrations. Genomic analyses serve to identify pathways with therapeutic potential for PDAC. The most common genetic mutations in PDAC are observed in KRAS (90%), CDKN2A (90%), TP53 (70%), and SMAD4 (55%). DNA damage repair (DDR) related gene mutations, such as those in BRCA1/2, PALB2, and ATM have been identified in familial PDAC (17%) (1-4). Multiple studies have highlighted the DDR pathways’ therapeutic potential for DDR-mutated PDAC. Among the various DDR pathway related genes, BRCA is a tumor suppressor involved in the homologous DNA repair (HR) mechanism in the double-stranded DDR (dsDDR) pathway. BRCA plays an important role in maintaining genome integrity, and the loss of BRCA function causes genome instability, thereby promotes cancer (5). In particular, cancers with BRCA mutations undergo inefficient DNA repair and thus, are sensitive to platinum-based anti-cancer drugs (6). Synthetic lethality occurs when two genetic mutations that are not lethal individually combine and become lethal. Treatment using a synthetic lethality approach can selectively target genetically modified cancer cells. Defects in BRCA1/2 genes required for HR repair become synthetically lethal when poly(ADP-ribose) polymerase (PARP) is inhibited. PARP, the synthetic lethality partner of BRCA1/2 mutations, is an enzyme that catalyzes the ADP ribosylation of various cellular proteins and is a DNA nick sensor involved in DNA repair, maintenance of genomic integrity, cell apoptosis, and cell survival (7, 8). Olaparib, a PARP inhibitor, is an FDA approved drug for the treatment of breast and ovarian cancers with BRCA mutations (9, 10). The effects of PARP inhibitors in BRCA-mutant ovarian and breast cancers, as well as prostate, pancreatic, and non-small cell lung cancer have been shown in many clinical trials conducted since 2009 (11). Recent clinical studies have been conducted using a combination of a cytotoxic agent and a PARP inhibitor in various cancers. For patients who had breast or ovarian cancer with BRCA mutation, the phase III VELIA/GOG-3005 trial (ClinicalTrials.gov identifier: NCT02470585) examined the efficacy of a treatment with veliparib and front-line chemotherapy (carboplatin and paclitaxel) and maintenance (12). In the phase III Pancreas Cancer Olaparib Ongoing (POLO) trial (ClinicalTrials.gov identifier: NCT02184195), treatment with olaparib as a maintenance was initiated after 16 weeks of continuous first-line platinum-based chemotherapy induction in patients with germline BRCA1/2 mutant PDAC. Although treatment with olaparib prolonged progression-free survival for patients with germline BRCA mutations, no statistically significant increase in overall survival was observed (13). However, it is still controversial which chemotherapy is appropriate as therapeutic partner for PARP inhibition because of the unknown mechanism of action. In this study, we identified not only oxaliplatin as synergistic partner of olaparib via synthetic lethality in BRCA2-mutant PDAC cells, but also the potential of CDK1 as therapeutic target in pan-PDAC.

Materials and Methods

Cell lines and culture. The human PDAC cell line Capan-1 was obtained from the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, Republic of Korea). The human PDAC cell line MIAPaCa-2 was obtained from Asan Preclinical Evaluation center for cancer therapeutiX (APEX, Seoul, Republic of Korea). The cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (WELGENE, Gyeongsan, Republic of Korea) and 1% penicillin/streptomycin (Gibco). All cells were maintained in an incubator at 37°C with 5% CO2.

Cell viability assay and IC50 assay. 1×103 cells were seeded in triplicate in 50 μl per well in 96-well cell culture plates and incubated for 24 h in a 37°C incubator with 5% CO2. Olaparib (Selleckchem, Houston, TX, USA), cisplatin, and oxaliplatin (Sigma-Aldrich, Saint Louis, MO, USA) were diluted to 2x of the final concentration, and 50 μl of each drug solution was added to the cells. Cisplatin, oxaliplatin, and olaparib were used at concentrations of 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μM for 24, 48, 72, 96, and 120 h. The cell viability assay was performed as previously described and half-maximal inhibitory concentration (IC50) was calculated (14).

Western blotting. The western blotting assay was performed as previously described (14). The antibodies against PARP (#9532), phospho-histone H2A.X ser139 (#80312), phospho-BRCA1 ser1524 (#9009), cdc2 (POH1) (#9116), XRCC1 (#2735), 53BP1 (#4937), and phoshpo-cdc2 Tyr15 (#9111) were from Cell Signaling (Danvers, MA, USA). The antibody against β-actin (A5441) was from Sigma-Aldrich. The antibodies against BRCA1 (sc-6954) and XRCC4 (sc-271087) were from Santa Cruz Biotechnology (Dallas, TX, USA). Rad51 (ab133534) and RPA70 (ab79398) were from Abcam (Cambridge, UK) and PAR (4335-MC-100) was from Trevigen (Minneapolis, MN, USA). The numerical intensity of the bands was calculated using the program ImageJ v1.52a.

Analysis of synergistic effects. The Capan-1 and MIAPaCa-2 cells (1×103 cells in 50 μl per well) were seeded in 96-well plates and incubated for 24 h at 37°C. After 24 h, the cells were exposed to olaparib for 6 h before treatment with oxaliplatin or cisplatin. Combinatorial responses were analyzed using a constant drug combination ratio (2×) at the IC50 concentration of a single drug. After 48 h, cell viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA). For cell viability data analysis, the test values were normalized to the negative control values (vehicle control). The potential synergism between olaparib and oxaliplatin or cisplatin was assessed by calculating the combination index (CI) value based on the Chou–Talalay method (CalcuSyn software, Biosoft, Cambridge, UK). CI values below 0.9 indicate synergy.

Immunofluorescent staining. The Capan-1 cells (2×104 cells in 200 μl per well) were seeded on glass coverslips (Nunc, Naperville, IL, USA) in eight-well plates and incubated for 24 h at 37°C. After 24 h, the cells were exposed to olaparib for 6 h before treatment with oxaliplatin. Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After fixation, the cells were permeabilized with 0.1% Triton X-100/phosphate-buffered saline (PBS) for 10 min. Then, the cells were washed three times with PBS and blocked with 1% skim milk in 0.1% Tween-20/PBS containing 22.52 mg/mL glycine for 30 min at room temperature. The cells were incubated with primary antibodies overnight at 4°C. The cells were then washed three times with 0.1% Tween-20/PBS and incubated with the secondary antibody (1:1,000 dilution; Alexa 488-conjugated antibody, Invitrogen, Camarillo, CA, USA) at 37°C for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Invitrogen). The slides were rinsed with distilled water (D.W.) and mounted using Vectamount solution (Vector Laboratories, Newark, CA, USA). The specimens were viewed using a laser scanning Confocal Microscope LSM880 (Carl Zeiss, Jena, Germany). The antibodies used were as follows; phospho-histone H2A.X ser139 antibody (1:200 dilution; Cell Signaling), 53BP1 (1:100 dilution; Cell Signaling), and Rad51 (1:1,000 dilution; Abcam).

Flow cytometry analysis. Apoptosis was examined using a commercial Annexin V-FITC kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Capan-1 cells were treated with olaparib or oxaliplatin for 48 h. The cells were harvested at a density of 1×106 cells/mL and rinsed with PBS, followed by centrifugation at 300 × g for 10 min at 4°C. The following methods have been previously described in detail (15). Finally, the apoptotic cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA) and the FlowJo V10 software (FlowJo). The FACS Canto II was operated by the Flow Cytometry Core Facility, Research Development Support Center, Asan Medical Center.

Xenografts. Female 4-week-old BALB/c nude mice were purchased from Central Lab. Animal Inc. (Seoul, Republic of Korea). All mice were provided a sufficient acclimation period for stabilizing to their new environment. Capan-1 cells (5×106) were subcutaneously injected into the right flank of all mice and, body weights and tumor volumes were measured twice a week. Tumor volume was calculated according to the formula (length × width × width)/2. After completing the last treatment, all mice were euthanized and the tumors were harvested for further analysis.

For experiments of combined treatment with oxaliplatin and olaparib, when the tumors reached approximately 90 to 100 mm3, the animals were randomly grouped into vehicle control, oxaliplatin alone, olaparib alone, or olaparib-oxaliplatin combination groups comprised of 6 mice each. Oxaliplatin (5 mg/kg) was diluted in D.W. and administered intraperitoneally twice a week for four weeks. Olaparib (75 mg/kg) was diluted with 3% DMSO/30% PEG-300/ddH2O buffer and administered orally five days a week for four weeks. The experiments were approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Science (protocol code: IACUC-2021-02-298 and date of approval: October 26, 2021).

For experiments involving treatment with Ro-3306, CDK1 inhibitor, when the tumors reached approximately 100 mm3, the animals were randomized into control and Ro-3306 treatment groups of four mice each, followed by oral treatment of Ro-3306 (4 mg/kg) for 28 days three times a week. Ro-3306 was diluted with 10% DMSO/40% PEG-300/50% saline. The experiment was approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Science (protocol code: IACUC-2022-12-103 and date of approval: April 15, 2022).

Statistical analysis. Data were analyzed by two-tailed Student’s t-tests using the GraphPad Prism software v5.01 (GraphPad, San Diego, CA, USA). Results with p<0.05 were considered statistically significant.

Results

BRCA2 mutant cells are resistant to oxaliplatin but susceptible to olaparib. BRCA1/2 mutations in PDAC cell lines were assessed using the Cancer Cell Line Encyclopedia (CCLE) database. Among various PDAC cell lines, only Capan-1 cells had a frameshift mutation (6174delT) causing truncation of the BRCA2 protein C-terminus (Table I). Several reports have demonstrated that cells with BRCA1/2 mutations are sensitive to platinum-based agents. To compare the drug sensitivity based on the BRCA mutation profile, Capan-1 (BRCA2 mt) and MIAPaCa-2 (BRCA2 wt) cells were exposed to cisplatin, oxaliplatin, and olaparib. Cell viability was decreased in dose- and time-dependent manners (Figure 1). The IC50 values of cisplatin were not different between Capan-1 and MIAPaCa-2 cells (Figure 1A). Interestingly, Capan-1 cells were more resistant to oxaliplatin than MIAPaCa-2 cells. The IC50 value of oxaliplatin at 72 h was 5.128 μM in Capan-1 cells, which was about two-fold higher than that in MIAPaCa-2 cells (2.65 μM) (Figure 1B). The IC50 of olaparib was 88.75 μM in MIAPaCa-2 cells, which was four times higher than that in Capan-1 cells (20.73 μM) (Figure 1C). These data suggested that Capan-1 cells with BRCA2 mutations were more resistant to oxaliplatin but were more sensitive to olaparib than MIAPaCa-2 cells with wild-type BRCA2.

View this table:
Table I.

Status of BRCA2 mutations in pancreatic cancer cell lines.

Figure 1.
Figure 1.

BRCA2-mutant pancreatic cancer cell lines are highly sensitivity to olaparib. (A) IC50 curve of cisplatin, (B) IC50 curve of oxaliplatin, (C) IC50 curve of olaparib in the BRCA2-mutant pancreatic cancer cell line (Capan-1) and BRCA2 wild-type pancreatic cancer cell line (MIAPaCa-2). Cells were treated with cisplatin, oxaliplatin, and olaparib at the indicated concentrations (1-200 μM) for 72 h, and cell viability was measured using the CellTiter-Glo assay. Each value indicates the mean±S.D (n=3).

Olaparib sensitizes Capan-1 cells to oxaliplatin. The BRCA2-mutant PDAC cells were resistant to oxaliplatin and sensitive to olaparib compared to BRCA2-wt PDAC cells. To observe the PARP1 response to oxaliplatin, Capan-1 and MIAPaCa-2 cells were exposed to olaparib or oxaliplatin. First, we observed that the PARP expression level in Capan-1 cells under physiological conditions was 50% less than that observed in MIAPaCa-2 cells (**p<0.01) (Figure 2A). Next, the levels of poly(ADP-ribose) (PAR), the active form of PARP1, were observed using western blot. Oxaliplatin significantly increased the level of PAR in Capan-1 cells compared with MIAPaCa-2 cells. The PAR increase in response to oxaliplatin was completely suppressed by the combination of olaparib and oxaliplatin (Figure 2B). Additionally, the combination treatment of olaparib and oxaliplatin decreased cell viability (Figure 2C). The synergistic effects of the combination of olaparib and oxaliplatin were assessed according to the CI in Capan-1 and MIAPaCa-2 cells. Only Capan-1 cells showed synergistic effects of various concentration combinations. In particular, the combination of olaparib and oxaliplatin (CI=0.37) showed the greatest synergistic effects at 30 μM olaparib and 5 μM oxaliplatin (Figure 2D). Our findings demonstrate that defects in the DNA repair system due to BRCA1/2 mutation were compensated by the alteration in the PARP1 response to oxaliplatin in the cells with BRCA2 mutations. The alteration in the PARP1 activity makes Capan-1 cells sensitive to olaparib and the combination of olaparib and oxaliplatin induced synergistic cell death.

Figure 2.
Figure 2.

Poly(ADP-ribose) polymerase 1 (PARP1) expression under physiological conditions and activity after treatment with olaparib or oxaliplatin. (A) Western blot showing expression of PARP1 in Capan-1 and MIAPaCa-2 cells. (B) Western blot showing the activity of PARP1 by detecting PAR after treatment with olaparib and/or oxaliplatin. (C) Quantification of viability of Capan-1 and MIAPaCa-2 cells treated with the indicated doses for 48 h (n=3, *p<0.05, **p<0.01, ***p<0.001). (D) Combination index (CI) of olaparib and oxaliplatin in Capan-1 and MIAPaCa-2 cells (left). Fa-CI plot of oxaliplatin and olaparib in Capan-1 and MIAPaCa-2 cells (right).

The combination of olaparib and oxaliplatin induces BRCAness via suppression of the CDK1-BRCA1 axis. Previous studies have reported that oxaliplatin and cisplatin regulate CDK1 during the cell cycle (16). We examined whether oxaliplatin regulates CDK1 in comparison with cisplatin in Capan-1 and MIAPaCa-2 cells. Oxaliplatin decreased the levels of p-CDK1, the active form of CDK1, by 1.8-fold compared to cisplatin (*p<0.05) (Figure 3A). Additionally, the activity of BRCA1, a HR repair protein regulated by CDK1, was also assessed. Oxaliplatin also decreased the levels of p-BRCA1, the active form of BRCA1, by 4-fold in comparison with cisplatin (**p<0.01) (Figure 3A, lanes 3 and 4). Combined treatment with olaparib and oxaliplatin reduced the expression levels of p-CDK1 and p-BRCA1 by more than 2-fold compared to oxaliplatin treatment alone (**p<0.01 and ***p<0.001, respectively) (Figure 3A, lanes 4 and 6). Unlike oxaliplatin, cisplatin alone or combination in combination with olaparib did not affect p-BRCA1 levels (Figure 3A, lanes 3 and 5). These data suggest that BRCAness through strong inhibition of the CDK1-BRCA1 axis in BRCA2-mutant cell lines was only observed after combined olaparib and oxaliplatin treatment. Western blotting was performed to confirm the effects of olaparib and oxaliplatin combination treatment on proteins involved in various DDR mechanisms. RPA70 expression levels increased 1.7-fold after oxaliplatin treatment, suggesting replication stress induced by double strand breaks (DSBs) (*p<0.05). Compared to oxaliplatin treatment alone, combined treatment with oxaliplatin and olaparib decreased the expression levels of RPA70 5-fold (**p<0.01) (Figure 3B). In BRCA2-mutant cells, the RAD51 expression levels were not affected by anti-cancer drugs. Inhibition of PARP by olaparib reduced the expression levels of XRCC1, a base excision repair effector, and XRCC4, an non-homologous DNA end joining mediator, by more than 2-fold (*p<0.05) (Figure 3B). Taken together, the combination of olaparib and oxaliplatin leads to the significant accumulation of DNA damage and disruption of the DDR pathway in BRCA2-mutant cells.

Figure 3.
Figure 3.

Combination of olaparib and oxaliplatin suppresses CDK1/BRCA1 activity in BRCA2-mutant pancreatic cancer cells. (A) Validation using western blotting of the effects of indicated drug treatments on CDK1 expression and phosphorylation, BRCA1 expression and phosphorylation. (B) DNA damage repair involved pathways after treatment with olaparib and/or oxaliplatin were confirmed using western blot (n=3, *p<0.05, **p<0.01, ***p<0.001).

Combination of olaparib and oxaliplatin-induced apoptosis via aggravation of DNA damage and suppression of the DNA repair system. We assessed the level of DNA damage in Capan-1 and MIAPaCa-2 cells after exposure to olaparib, oxaliplatin, and combined treatment of olaparib and oxaliplatin. Capan-1 cells treated with the combination of olaparib and oxaliplatin showed a 2.4-fold increase in phospho-γH2AX, a surrogate marker for DSBs, compared to cells treated with oxaliplatin alone (**p<0.01) However, MIAPaCa-2 cells showed no significant differences in the levels of phospho-γH2AX when treated with oxaliplatin alone or a combination of olaparib and oxaliplatin (Figure 4A). Consistent with the western blot results, cells treated with oxaliplatin alone showed a 40% higher accumulation of phospho-γH2AX than vehicle-treated control cells. This accumulation of phospho-γH2AX increased by 25% after olaparib and oxaliplatin combination treatment (*p<0.05) (Figure 4B, top). Treatment with oxaliplatin alone increased the accumulation of 53BP1, a DSB repair effector, by 20% in the vehicle control group and olaparib alone group. Olaparib and oxaliplatin combination treatment increased the accumulation of 53BP1 in the nucleus by 40% (**p<0.01) (Figure 4B, middle). Formation of RAD51 foci, which indicates BRCA-dependent DSB repair and a loss of PARP1 function, was also observed; however, in BRCA2-mutant PDAC cells, RAD51 foci were not formed, regardless of drug treatment (p>0.05) (Figure 4B, bottom). These findings suggest that inhibition of PARP1 by olaparib significantly aggravates oxaliplatin-induced DNA damage and enhances sensitivity to oxaliplatin in PDAC cells with BRCA2 mutations. Next, we hypothesized that accumulation of DNA damage and defects in DDR proteins might lead to apoptosis. The apoptotic effect was greatest in cells treated with a combination of olaparib and oxaliplatin (**p<0.01) (Figure 4C).

Figure 4.
Figure 4.
Figure 4.

Combination of olaparib and oxaliplatin induces DNA damage foci formation and apoptosis in BRCA2-mutant pancreatic cancer cells. The Capan-1 cells were treated with olaparib and/or oxaliplatin for 48 h. (A) The expression levels of p-γH2AX. (B) Immunocytochemistry for p-γH2AX, 53BP1, and Rad51 foci in Capan-1 cells. (C) Effect of olaparib and oxaliplatin combination on apoptosis in Capan-1 cells (n=3, *p<0.05, **p<0.01).

Combination of olaparib and oxaliplatin inhibits tumor growth in a BRCA2-mutant pancreatic cancer xenograft model. According to the results observed, we confirmed that combination of olaparib and oxaliplatin induced synthetic lethality, which led to improved therapeutic efficacy in a PDAC xenograft model using Capan-1 cells. The combination treatment group of olaparib and oxaliplatin showed significant inhibition of tumor growth compared to the vehicle control group, oxaliplatin alone, or olaparib alone groups (6 mice/group, *p<0.05, **p<0.01, **p<0.001) (Figure 5A, B and D). There was no weight loss in either the drug-treated or control groups, indicating that there was no toxicity (Figure 5C). The oxaliplatin alone (59.2%) and olaparib alone (49.2%) treatment groups showed tumor growth inhibition compared to the vehicle control group. Together, these findings indicated that treatment with the combination of olaparib and oxaliplatin improved the anti-cancer effect compared with single treatment in vivo.

Figure 5.
Figure 5.

Olaparib and oxaliplatin combination therapy improves the anti-cancer effect on a Capan-1 xenograft model. The Capan-1 xenograft mouse model was treated with vehicle (3% DMSO/30% PEG-300/ddH2O), oxaliplatin (5 mg/kg), olaparib (75 mg/kg), or a combination of these two agents by oral gavage or intraperitoneal injection for 30 days. (A) The tumor growth curve. (B) Tumor weights. (C) Body weights. (D) Tumor mass (6 mice/group, *p<0.05, **p<0.01, ***p<0.001).

Ro-3306 suppresses the growth of pan-PDAC cells regardless of BRCA2 mutation. To validate whether CDK1 could be therapeutic target, we first examined the anti-tumor effect in PDAC cell lines with or without BRCA2 mutations. Capan-1 and MIAPaCa-2 cells were treated with Ro-3306, a CDK1 inhibitor, for 48 or 72 h. Cell viability was reduced in dose- and time-dependent manners with no significant difference between Capan-1 and MIAPaCa-2 cells (Figure 6A). Next, we evaluated the anti-tumor effect of Ro-3306 in a BRCA2-mutant PDAC xenograft model. Tumor growth in the Ro-3306-treated group was inhibited by approximately 22% compared to the control group (Figure 6B). Our findings demonstrated that CDK1 inhibition has therapeutic efficacy in vivo.

Figure 6.
Figure 6.

Ro-3306, a CDK1 inhibitor, shows anti-tumor effect in vitro and in vivo in pancreatic ductal adenocarcinoma with BRCA2 mutation. (A) IC50 of Ro-3306. Capan-1 and MIAPaCa-2 cells were treated with Ro-3306 for 48 and 72 h. Following that, cell viability was analyzed using the CellTiter-Glo assay (n=3). (B) Tumor growth curve and tumor growth inhibition (%). (C) Body weights of the mice (4 mice/group, *p<0.05).

Discussion

Here, we demonstrate the synergistic anti-tumor effects of combination treatment with olaparib and oxaliplatin in a BRCA2-mutant PDAC cell line. Our study showed 1) inhibition of PARP1 activity by olaparib and oxaliplatin-induced DNA damage inhibited the CDK1/BRCA1 axis, which caused the accumulation of DNA damage, and finally, apoptosis increased by defects in the DNA repair system. These results suggested oxaliplatin could be a synthetic lethality partner with olaparib in DDR-mutant PDAC. 2) CDK1 inhibition by Ro-3306 showed tumor suppression in PDAC with or without BRCA2 mutation. Our data suggests that CDK1 may be a potential anti-cancer therapy in pan-PDAC.

Previous studies have reported that combination treatment with a PARP inhibitor and platinum-based chemotherapy induced synthetic lethality, which improved the anti-tumor effect in patients with DDR mutations and sensitivity to platinum-based chemotherapy (7, 17, 18). However, our data demonstrated that Capan-1 cells (BRCA2 mt) showed higher resistance to oxaliplatin than MIAPaCa-2 cells (BRCA2 wt). A recent publication has reported that some of Capan-1 cells with BRCA2 mutation showed resistance to platinum-based chemotherapy. To investigate how the clones acquired resistance, 14 clones from twelve million Capan-1 cells showing resistance to platinum-based chemotherapy drug, cisplatin, were isolated, and in 7 clones, BRCA2 protein expression was recovered and became function via a second mutation (19). To identify whether the Capan-1 cells used in our experiments have a mutation, we performed sequencing, and identified a mutation that led to the truncation of BRCA2 (data not shown). We observed that the levels of PAR, as response to oxaliplatin, increased strongly, demonstrating that the PARP-related DDR, induced by oxaliplatin, performed perfectly and cells with defects in the HR repair system were well protected.

The phase III POLO study in patients with metastatic PDAC and germline BRCA (gBRCA) mutation, who had received platinum-based chemotherapy, provided a clinical basis for treating with a PARP inhibitor as maintenance therapy (13), but not a clear basis for the results of platinum-based drugs as combination treatment partner of PARP inhibitor. In this study, we compared oxaliplatin and cisplatin, and found mechanisms of oxaliplatin-specific BRCAness induction. Oxaliplatin suppressed the activity of BRCA1, a CDK1 downstream signaling protein, through CDK1 inhibition. Inhibition of BRCA1 sequentially suppressed other DNA repair involved enzymes, and eventually resulted in the loss of HR activity. Then, when PARP was inhibited simultaneously, DNA single-strand breaks that could not be repaired were converted to DNA DSBs, followed by inhibition of HR activity and accumulation of DNA damage, which induced apoptosis. Cisplatin is also known to regulate CDK1 (16), showed neither effective inhibition of the CDK1-BRCA1axis, nor effective induction of synthetic lethality with the PARP inhibitor (data not shown).

The regulation of CDK1 activity was regarded as an important mediator in the induction of BRCAness by oxaliplatin. Although drugs targeting other CDK subtypes, rather than CDK1, are used in chemotherapy (20-22), there are no previous studies in PDAC, and data on whether CDK1 is suitable for anti-cancer targeting is still insufficient. Therefore, we performed an experiment using CDK1 inhibitors to identify whether CDK1 was suitable as target for PDAC and showed the potential of CDK1 to be a therapeutic target for pan-PDACs regardless of the BRCA2 mutation. However, despite these promising results, extensive research is needed to understand why CDK1 inhibition has not been used clinically and what treatment conditions would be suitable clinically.

In conclusion, we showed synergy of oxaliplatin and olaparib in Capan-1 xenograft, which is a BRCA2-deficient human PDAC cell line-based model. In addition, CDK1 inhibition with Ro-3306 suppressed PDAC cell lines regardless of DDR mutations.

Acknowledgements

The Authors thank Dr. Baek-Yeol Ryoo and Jae Ho Jeong, Department of Oncology, Asan Medical Center for their helpful comments. The Authors also thank the core facilities in Asan Institute for Life Sciences, Asan Medical Center. This paper was modified and developed from the master’s degree thesis of the co-first author, DANBEE KIM, available at: https://oak.ulsan.ac.kr/bitstream/2021.oak/9917/2/200000594378.pdf

Footnotes

  • Authors’ Contributions

    CK: Conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing–original draft, writing–review and editing. DK: Conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing–original draft, writing–review and editing. DSL: Conceptualization, data curation, visualization, writing–review and editing. SL: Conceptualization, data curation, visualization, writing–original draft, writing–review and editing. CY: Conceptualization, resources, writing–original draft, writing–review and editing. KPK: Conceptualization, funding acquisition, project administration, resources, supervision, writing–original draft, writing–review and editing.

  • Conflicts of Interest

    The Authors declare no potential conflicts of interest in relation to this study.

  • Funding

    This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number: 2021R1A6A1A03040260) and a Korea Medical Device Development Fund grant funded by the Korean government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (grant number: KMDF_PR_20200901_0128,1711138236).

  • Received July 18, 2023.
  • Revision received October 5, 2023.
  • Accepted October 6, 2023.

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PARP Inhibitor Sensitizes BRCA-mutant Pancreatic Cancer to Oxaliplatin by Suppressing the CDK1/BRCA1 Axis
CHORONG KIM, DANBEE KIM, DA SOL LEE, SEONMIN LEE, CHANGHOON YOO, KYU-PYO KIM
Anticancer Research Dec 2023, 43 (12) 5523-5534; DOI: 10.21873/anticanres.16754
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