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Topotecan synergizes with CHEK1 (CHK1) inhibitor to induce apoptosis in ovarian cancer cells
© Kim et al.; licensee BioMed Central. 2015
Received: 19 September 2014
Accepted: 19 March 2015
Published: 28 March 2015
Topotecan (TPT) is a therapeutic option for women with platinum-resistant or -refractory ovarian cancer. However, the dose-limiting toxicity of TPT is myelosuppression. This led us to seek a combination treatment to augment TPT anti-cancer activity in a cancer-targeted manner. Ovarian serous cancers, a major subtype, show dysregulated DNA repair pathway and often display a high level of CHEK1 (CHK1), a cell cycle regulator and DNA damage sensor. CHEK1 inhibitors are a novel approach to treatment, and have been used as single agents or in combination chemotherapy in many cancers.
We evaluated the cellular effects of TPT in a panel of high grade serous (HGS) and non-HGS ovarian cancer cells. We then determined IC50s of TPT in the absence and presence of CHEK1 inhibitor, PF477736. Synergism between TPT and PF477736 was calculated based on cellular viability assays. Cytotoxic effect of the combined treatment was compared with apoptotic activities by Caspase3/7 activity assay and Western blotting of cleaved-PARP1 and γH2AX.
Non-HGS ovarian cancer cells were generally more sensitive to TPT treatment compared to HGS ovarian cancer cells. When combined with CHEK1 inhibitor, TPT potently and synergistically inhibited the proliferation of HGS ovarian cancer cells. This dramatic synergism in cellular toxicity was consistent with increases in markers of apoptosis.
Our findings suggest that the addition of CHEK1 inhibitor increases the response of ovarian cancer cells to TPT. Furthermore, reduced dosages of both drugs achieved maximal cytotoxic effects by combining TPT with CHEK1 inhibitor. This strategy would potentially minimize side effects of the drugs for extended clinical benefit.
Ovarian cancer consists of 4 major histologic subtypes including serous, clear cell, endometrioid, and mucinous . Each harbors defining patterns of molecular aberrations that could affect response to treatment and clinical outcome. Most ovarian cancers are diagnosed at advanced III or IV stage, and current standard of care includes surgical cytoreduction and platinum-based chemotherapy. Ovarian cancer has a high response rate, but most cancers eventually relapse and metastasize, becoming resistant and refractory to standard chemotherapy.
The most common subtype, high-grade serous (HGS) ovarian cancer, is characterized by defective DNA repair . Treatment approaches commonly include chemotherapies that induce DNA damage, since the ovarian cancer cell will die when it is incapable of recovering from injury to its DNA. Platinum chemotherapies, the hallmark of DNA damaging agents, have been combined with other cancer drugs such as taxanes, which alter microtubule function . Topoisomerase I and II inhibitors, such as camptothecin and etoposide, block DNA unwinding during replication, transcription, and chromatin remodeling, by preventing the rejoining process of the DNA strands and resulting in accumulation of DNA breaks. In particular, the topoisomerase I inhibitor topotecan (TPT) is active against many cancer types including platinum-sensitive and resistant ovarian cancers [4,5]. Unfortunately, side effects of TPT and other DNA-damaging agents include low blood counts, nausea, vomiting, diarrhea, and fatigue at doses that are required to induce tumor responses. These adverse events limit the dose that can be administered and the duration of treatment. Ideally, combination therapy with TPT should include agents that exploit defects in the cancer cells to augment tumor-specific cell death without increasing intolerable adverse effects to the patient.
Throughout the lifetime of a cell, DNA damage is introduced either exogenously or endogenously. Cellular sensing of DNA damage and its repair occur throughout cell cycle checkpoints. For example, p53 is a main checkpoint regulator in G1/S phase, while checkpoint kinase 1/2 (CHEK1/2, CHK1/2) is crucial in S and G2/M checkpoints. In HSG ovarian cancers, p53 is either null or mutated, increasing the cellular dependency on CHEK1/2 for DNA damage repair and survival. Supporting this notion, we recently showed that CHEK1 gene is overexpressed in nearly all cases of HGS ovarian cancer as compared to normal ovarian surface epithelium in The Cancer Genome Atlas (TCGA) dataset, suggesting that CHEK1 is required for cells to tolerate the defective DNA repair intrinsic to HGS cancers [2,6]. Upon DNA damage, CHEK1 is activated by ATR signaling and sequentially phosphorylated at residues S317 and S345 which subsequently induce autophosphorylation on S296 to trigger cell cycle arrest and DNA repair . Indeed, recent studies suggested that both pS345 CHEK1 and pS296 CHEK1 could be used as pharmacodynamic markers to monitor the CHEK1 inhibition upon treatment with CHEK1 inhibitor alone and combination treatments [8,9].
Inhibition of CHEK1/2 could therefore present a novel therapeutic strategy. Cancer cells with dysfunctional p53 may be particularly susceptible to such drugs. CHEK inhibition would cause premature entry into mitosis without adequate repair of DNA. This excessive DNA damage would lead to mitotic catastrophe and subsequent cancer cell death. For this reason, several CHEK1 inhibitors are under development as single agents as well as a combined therapy [10,11].
Since TPT is used as therapy for recurrent cisplatin resistant and refractory ovarian cancer, and increased expression levels of CHEK1 are observed in ovarian cancers, we hypothesized that inhibiting CHEK1 could be a means to enhance the anti-cancer activity of TPT preferentially in ovarian cancer cells, thus increasing their therapeutic index in cancer cells as compared to normal tissues.
Ovarian cancer cell lines, A2780, HeyA8, Igrov1, Skov3, Ovcar5, Ovcar3, Ovcar8, OV90, PEO1 and PEO4 were maintained in RPMI supplemented with 10% heat-inactivated FBS and 1X Pen/Strep.
Stock solutions of Topotecan HCl (Selleck, S1231) and PF477736 (Selleck, S2904) were prepared in DMSO and aliquots were stored at −80°C.
Cells were seeded in 96-well plates at a density of 1–2,000 cells/50 μl/well in triplicate. In general, the drug was added 24 hours after seeding and XTT assay was routinely performed in 3 days after drug treatment unless indicated. Cellular viability was assessed by incubating cultures with XTT freshly mixed with PMS (Sigma) and absorbances were read in a Tecan plate reader (Research Triangle Park, NC). Cellular proliferation was calculated relative to experimental negative controls and standard deviation was calculated from triplicates.
The cancer genome atlas data
Western blot analysis
Total protein was extracted from OC cell lines with 1% NP40 lysis buffer containing 150 mM NaCl, 50 mM TrisHCl, 10% glycerol, 1 X Halt proteinase inhibitor cocktail, 5 mM NaF, and 1 mM NaOrthovanadate. Protein concentrations were estimated using BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). The proteins were separated on the NuPage 4–12% gel (Invitrogen, Carlsbad, CA) and the band was visualized using either Lumina Classico or Crescendo Western HRP substrate system (Millipore) depending on the signal intensities. Antibodies Chk1 G-4 (Santa Cruz, sc-8408), phospho-Chk1 Ser345 (Cell Signaling, #2348), phospho-Chk1 Ser296 (Cell Signaling, #2349), cleaved PARP (Cell Signaling, #9541), phospho-H2AX (Cell Signaling, #5438), Topoisomerase I (Abcam, ab3825), and GAPDH (Millipore, MAB374) were used in this study, and the secondary antibodies ECL anti-rabbit IgG HRP and ECL anti-mouse IgG HRP (GE Healthcare) were used at 1:5000 dilutions.
Cells were seeded in 96-well white-walled plates at a density of 2,000 - 5,000 cells/50 μl/well and the drug in a 50 μl volume was added 24 hours after seeding. The caspase activity was measured after 16 hours additional incubation followed by adding 40 μl Caspase-Glo reagent (Promega) per well.
Cell cycle analysis
Cells were seeded at 8 X 105/60 mm plate and 8 hours prior to drug treatment. Fresh complete medium containing 0.2 μM TPT, 0.5 μM PF477736, or both drugs was added and cells were incubated for 16 hours. The procedure was performed according to manufacturer’s protocol (BD Pharmingen APC BrdU Flow kit). Following 30 minutes incubation after adding BrudU, cells were trypsinized and collected for subsequent fixation and permeabilization. Cells were analyzed by FACS Calibur (Becton Dickinson) after staining with APC-anti-BrdU and 7-AAD. Cell cycle was analyzed using FlowJo software.
HGS ovarian cancer cells are more resistant to TPT treatment than non-HGS cells
TOP1 is altered in a subset of ovarian serous tumors with an association with a poor overall survival
Co-treatment of TPT and PF477736 synergistically kills ovarian cancer cells
Combination index (CI) values of topotecan and PF477736 co-treatment in ovarian cancer cells
Total Dose (uM)
TPT + PF
0.25 + 0.5
0.125 + 0.25
0.063 + 0.125
0.032 + 0.063
Co-treatment with TPT and CHEK1 inhibitor increases apoptosis and DNA damage response
We also performed cell cycle analysis in both p53 null (Ovcar5) and p53 mutant (Ovcar8) cell lines to investigate whether inhibition of CHEK1 with PF477736 affects TPT-induced cell cycle arrest. In both cell lines, we observed that PF477736 abrogated TPT-induced cell cycle arrest (Figure 5C).
Taken together, co-treatment with TPT and PF477736 more potently induced DNA damage and apoptosis than either single treatment alone in ovarian cancer cells.
We recently found that CHEK1 gene was overexpressed in most of HGS ovarian cancer, and combined inhibition of pro-survival modulator, IKKε, cooperatively induced apoptosis in ovarian cancers . In this study, we have further extended this finding to evaluate CHEK1 inhibitor-based combination therapy. TPT is currently used for the salvage treatment of ovarian cancer, but myelosuppression is the dose-limiting toxicity. High doses of TPT are poorly tolerated, resulting in treatment delays or dose reductions. Therefore, we hypothesized that combining TPT with CHEK1 inhibitor may be an effective and feasible approach to maximize the therapeutic index between cancer cells and normal, allowing lower doses of both agents in order to minimize the dose-limiting toxicity. Here, we found that HGS ovarian cancer cell lines generally required higher concentrations of TPT, in the absence of CHEK1 inhibition, to achieve equivalent cytotoxicity compared to non-HGS ovarian cancer cell lines. When combined with CHEK1 inhibitor (PF477736), TPT synergistically produced maximal cytotoxic effects at reduced dosages.
Based on its mechanism of action, it has been hypothesized that CHEK1 inhibition would potentiate the effects of standard chemotherapy in a wide range of cell lines. In particular, CHEK1 inhibitors (e.g. V158411, AZD7762, MK-8776, GNE-900, CCT244747) have been combined with camptothecin and non-camptothecin inhibitors of topoisomerases [8,16-19]. While combined inhibitions generally showed synergistic effects, a study showed limited increase in cancer cell death with MK8776, reporting that CHEK1 depletion with siRNA or inhibition with MK-8776 only minimally sensitized Ovcar8 cells to TPT in colony formation assays . Combination of GNE-900 and camptothecin was minimally effective than either alone in vivo xenograft model . The reasons for this discrepancy may be due to different potency and penetrance of CHEK1 inhibitors as well as different measurements in biological assays.
Our current study provides insight into the nuances of how the combination of CHEK1 and topoisomerase inhibition may be most effectively used. Not all ovarian cancer cell lines were equally sensitive to either agent alone or in combination. Our data demonstrate that these agents may have the most therapeutic potential in HGS ovarian cancers, where the combination demonstrated the greatest degree of synergy. In addition, the order of drug administration was relevant, with the most cell death occurring when both drugs were given together, or when TPT was given first, followed by PF477736. Interestingly, the least effective sequence was administering PF477736 prior to TPT. Similarly, a study using CHEK1 inhibitor AZD7762 showed that gemcitabine administration either concurrent with or before AZD7762 resulted in maximal chemosensitization in pancreatic cancer model .
Several CHEK1 inhibitors (PF477736, MK-8776/SCH900776, and LY2606368) have completed phase 1 evaluation as single agents, but formal reports are not available. In addition, PF477736 (Pfizer) was evaluated in advanced solid tumors in combination with gemcitabine (NCT00437203) and was discontinued from development. CHEK1 inhibitor MK8776 (Merck) in combination with gemcitabine has also reported phase 1 evaluation in patients with solid tumors. Combining an anti-metabolite (gemcitabine) with MK8776 was well tolerated with few patients experiencing grade 3–4 adverse events, the most common being neutropenia (15%) .
TPT has been combined clinically with angiogenesis inhibitors bevacizumab or trabananib [21,22]. Such a strategy combines agents targeting two separate pathways (DNA replication and angiogenesis) important to cancer, without overlapping side effects so as not to limit dosage of either drug. The combination of TPT with CHEK1 inhibitor takes a different approach, by targeting a potential mechanism of resistance to TPT, the upregulated DNA repair by overexpressed CHEK1. This approach potentially widens the therapeutic index by preferentially targeting the ovarian cancer-specific dependence on upregulated or activated CHEK1, and allowing increased cancer cell killing at lower doses of each drug. In the future, it may also be possible to incorporate anti-angiogenic agents as well, due to non-overlapping toxicities.
Disrupting DNA repair mechanisms after DNA damaging chemotherapy is a strategy that we have used clinically . Carboplatin was given to induce DNA breaks, and the PARP inhibitor olaparib was administered in order to block repair of single-strand breaks, which progressed to double-strand breaks during DNA replication, causing cell death. This strategy was designed with the goal of achieving a higher therapeutic index in ovarian cancer cells known to harbor defective DNA repair pathways, particularly in cancers with BRCA mutations. Additionally, the increased potency was attained with reduced doses of each drug. In our current study, we achieved the same phenomenon in vitro, by co-treating with CHEK1 inhibitor and TPT. This combination synergistically inhibited cellular proliferation at sub-lethal concentrations for each drug given alone. The cytotoxic synergism was accordingly reflected in apoptotic activities and DNA damage response. Interestingly, we found that OV90 and PEO4 cells were resistant to TPT alone, and co-inhibition with TPT and PF477736 resulted in achieving equivalent cellular toxicity with reduced doses of both drugs. However, this co-inhibition still failed to attain complete cellular toxicity in OV90 and PEO4. Further studies are required to know why these two cell lines were so resistant to TPT and even to the co-treatment.
BRCA-mutated cancers tend to have the highest response rates to DNA damaging agents in general. Indeed, our results confirm that the BRCA-mutated cell line PEO1 was the most sensitive of HGS cancers to treatment with TPT or CHEK1 inhibitor alone. Most HGS ovarian cancers, however, do not have BRCA mutations, but do have elevated expression of CHEK1. Thus, combination therapy of CHEK1 inhibitor with DNA damaging agents such as TPT could introduce an analogous strategy for targeting non-BRCA mutated or BRCA-like HGS ovarian cancers.
Another potential marker for potency to CHEK1 inhibitor may be its autophosphorylation. Decreased levels of CHEK1 S296 autophosphorylation have been correlated with response to CHEK1 inhibition in colon, breast, and ovarian cancer cell lines [8,24]. As shown in this study, this marker is expressed in HGS ovarian cancer cell lines at steady states and CHEK1 gene is over-expressed in nearly all cases of TCGA HGS ovarian cancers . Therefore, this marker should be evaluated at the time of initial diagnosis and in the course of treatment, specifically in recurrent HGS ovarian cancers.
We are currently conducting a phase 2 clinical trial testing single agent CHEK1 inhibitor LY2606368 in women whose cancers are likely to have defects in DNA repair (NCT02203513). The trial will study 3 cohorts of cancers: BRCA-mutated, HGS ovarian cancer, and TNBC. We hypothesize that these types of cancers will be susceptible to CHEK1 inhibition due to their poor ability to repair double strand DNA breaks. The adverse events in this trial have not yet been analyzed. Among many CHEK1 inhibitors under clinical evaluation, an ideal CHEK1 inhibitor to use with TPT for combination treatment would exhibit minimal overlap in toxicity such as myelosuppression. Based on our data, however, it is possible that the anti-cancer effect of the combination will be achieved at lower doses of both agents, which would allow for safe administration of these two drugs, specifically targeted to patients with HGS ovarian cancer.
Our study shows that TPT synergistically induces cytotoxicity in combination with CHEK1 inhibitor especially in HGS ovarian cancer cells.
PEO1 and PEO4 cells were kindly provided by Dr. James Brenton (University of Cambridge). This work was funded by the Intramural Research Program, Center for Cancer Research, National Cancer Institute (C.M.A.).
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