Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells

Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy worldwide. Development of chemoresistance and peritoneal dissemination of EOC cells are the major reasons for low survival rate. Targeting signal transduction pathways which promote therapy resistance and metastatic dissemination is the key to successful treatment. Members of the ErbB family of receptors are over-expressed in EOC and play key roles in chemoresistance and invasiveness. Despite this, single-targeted ErbB inhibitors have demonstrated limited activity in chemoresistant EOC. In this report, we show that dacomitinib, a pan-ErbB receptor inhibitor, diminished growth, clonogenic potential, anoikis resistance and induced apoptotic cell death in therapy-resistant EOC cells. Dacominitib inhibited PLK1-FOXM1 signalling pathway and its down-stream targets Aurora kinase B and survivin. Moreover, dacomitinib attenuated migration and invasion of the EOC cells and reduced expression of epithelial-to-mesenchymal transition (EMT) markers ZEB1, ZEB2 and CDH2 (which encodes N-cadherin). Conversely, the anti-tumour activity of single-targeted ErbB agents including cetuximab (a ligand-blocking anti-EGFR mAb), transtuzumab (anti-HER2 mAb), H3.105.5 (anti-HER3 mAb) and erlotinib (EGFR small-molecule tyrosine kinase inhibitor) were marginal. Our results provide a rationale for further investigation on the therapeutic potential of dacomitinib in treatment of the chemoresistant EOC.

The ErbB family contributes to cisplatin resistance. In an attempt to examine possible correlation between the mRNA levels of the ErbB family and chemoresponsiveness, we found that higher expression of HRG1-α and HRG1-β are significantly associated with resistance to cisplatin by Pearson's correlation ( Fig. 2A). The correlation coefficient (r) between the expression of HRG1-α and HRG1-β and cisplatin IC 50 values is 0.9058 (P = 0.034) and 0.8997 (P = 0.037), respectively. In addition, our data demonstrated positive correlation between cisplatin resistance and higher expression of EGFR and HER2 ( Fig. 2A). We found no significant association  50 values and were collected from three independent experiments, each performed in triplicate. IC 50 is the concentration of drug that caused a 50% reduction in proliferation compared to the vehicle-treated cells.
between the ErbB family expression and resistance to carboplatin, paclitaxel, doxorubicin, gemcitabine and erlotinib ( Supplementary Fig. 2). Expression of both HRG1-α and HRG1-β has been observed in 30-83% of EOC cell lines and tumour samples 39 . After binding to HRG, HER3 heterodimerises with the other ErbB receptors, which activates down-stream pro-survival pathways 40 . HRG1-β binds to HER3 with more affinity and induces greater activation of the ErbB receptors than HRG1-α 41,42 . We therefore explored the effects of exogenous HRGβ-1 on proliferative response of the chemosensitive Caov4 cells to cisplatin. The resulting data show that pre-treatment with HRGβ-1 (10 ng/ mL) decreased cisplatin-induced cytotoxicity, a process abrogated when the cells were pre-treated with dacomitinib but not single-targeted ErbB inhibitors including cetuximab, erlotinib, trastuzumab and H3.105.5 (a ligand-blocking anti-HER3 antibody) (Fig. 2C).
We next compared the effects of the ErbB inhibitors on potentiation of the anti-tumour effects of cisplatin in the chemoresistant EOC cells. Combination of dacomitinib with cisplatin had a synergistic effect on growth inhibition. In comparison, erlotinib-cisplatin therapy was antagonistic (Fig. 2D,E; Supplementary Tables 1, 2). Altogether, these findings suggest that the ErbB family might contribute to cisplatin resistance and a pan-ErbB inhibition strategy is required to augment cisplatin efficacy in the chemoresistant EOC cells.

Dacomitinib inhibits cell viability and induces apoptosis.
MTT assay was performed to estimate anti-proliferative effects of the anti-ErbB agents on the chemoresistant EOC cells. Treatment with dacomitinib inhibited cell growth (Fig. 3A,B). Clonogenic capacity represents the renewal potential and a long-term response of cells after treatment. The results of a colony formation assay demonstrate that both trastuzumab and dacomitinib reduced clonogenic survival (Fig. 3C). In immortalized cells, detachment from the extracellular matrix induces anoikis, a special type of apoptosis 43 . Acquisition of resistance to anoikis is a prerequisite for EOC cells to survive in ascites before forming metastatic foci 44 . Our data show that dacomitinib diminished anoikis resistance (Fig. 3D). Moreover, dacomitinib but not the single-targeted agents induced apoptotic cell death, as demonstrated by Annexin V staining (Fig. 3E). These data suggest that dacomitinib had stronger anti-proliferative efficacy compared to the single-targeted ErbB inhibitors ( Fig. 3A-E). Dacomitinib inhibits PLK1-FOXM1 signalling. Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase which plays a central role in mitotic progression and its elevated expression in EOC correlates with histological grade 45 . PLK1 induces forkhead box protein M1 (FOXM1), a member of FOX family of transcription factors that regulates expression of a wide range of genes such as PLK1, survivin (encoded by BIRC5), cyclin B1 (encoded by CCNB1) and Aurora kinase B (encoded by AURKB) 46,47 . The FOXM1-target genes participate in different cellular functions including cell growth, metastatic dissemination and therapy resistance 48,49 .
PLK1 has been shown to mediate resistance to chemotherapeutics including cisplatin 50 . To determine if the ErbB family activates PLK1 in the EOC cells, the cells were serum-starved for 24 h and then treated with HRGβ-1 (10 ng/mL) for 30 min. Immunoblotting analysis showed that HRGβ-1 stimulation resulted in activation of PLK1 (Fig. 4A). This is in consistency with previous reports that the ErbB receptors activate the PLK1-FOXM1 axis 51,52 . We next sought if dacomitinib-induced sensitisation to cisplatin is through inhibition of PLK1 pathway. To achieve this, the cells were treated with cisplatin in combination with BI 2536, a highly selective PLK1 inhibitor. Our findings demonstrate that BI 2536 had synergistic activity with cisplatin on inhibition of cell growth (Fig. 4B,C; Table 2), suggesting that PLK1 blockade enhances sensitivity to cisplatin.
We therefore evaluated the effects of dacomitinib on PLK1-FOXM1 signalling. Our data show that dacomitinib, but not the single-targeted agents, inhibited p-PLK1 (Fig. 4D). Furthermore, dacomitinib reduced the mRNA and protein levels of FOXM1, survivin and Aurora kinase B. Conversely, the inhibitory effects of the single-targeted agents were marginal (Fig. 4D,E). Altogether, these data suggest that PLK1 blockade is a mechanism for dacomitinib-induced sensitisation to cisplatin and that a comprehensive ErbB inhibition strategy is required for blockade of PLK1-FOXM1 pathway and its down-stream targets. Dacomitinib reduces migration and invasion. Ovarian cancer metastasis includes tumour cells detachment from the primary site followed by their spread to the peritoneum and omentum 53 . The degree of peritoneal dissemination associates with poor prognosis 54 . Detachment of EOC cells from the primary site and their local invasion is driven by an epithelial-to-mesenchymal transition (EMT) 55 . EMT is triggered by down-regulation of cell adhesion molecules by transcriptional repressors ZEB1, ZEB2 and Snail 56 . It is thought that EMT contributes to loosening of intercellular adhesions and shedding of EOC cells into ascites 57 . We next determined the effects of dacomitinib on expression of the EMT markers ZEB1, ZEB2 and CDH2 (which encodes N-cadherin). The resulting data indicate that dacomitinib had stronger inhibitory effects on the expression of the EMT markers, as compared to the single-targeted agents (Fig. 5A). Moreover, these data show that dacomitinib hindered migration and invasion of the EOC cells through matrigel (Fig. 5B).

Discussion
There is evidence that the ErbB signalling network contributes to chemoresistance in EOC. EGFR drives resistance to cisplatin through induction of the anti-apoptotic protein Bcl-2 58 . HER2 promotes resistance to taxane chemotherapies and its depletion enhances chemosensitivity 59 . Moreover, activation of HER3 has been demonstrated to drive chemoresistance in EOC cells via activation of AKT pathway 60 . These findings suggest that the ErbB family is a potential therapeutic target in the chemoresistant EOC and its blockade might inhibit tumour growth and induce chemosensitisation 61 .
During ovarian carcinoma metastasis, epithelial cells lose E-cadherin-mediated cell-cell interactions, up-regulate N-cadherin and undergo EMT 62 . Evidence indicates that EMT correlates with a poor prognosis in EOC 63,64 . Moreover, EMT is thought to drive a chemoresistant behaviour 65,66 . Induction of EMT promotes peritoneal dissemination and reversing the EMTed phenotype is believed to be a novel strategy to hamper intraperitoneal metastasis 67 . Targeting signalling pathways contributing to EMT is a potential therapeutic approach in order to hinder invasiveness of EOC cells 68 . Both EGFR and HER2 downregulate E-cadherin expression, induce an EMTed phenotype and increase motility of EOC cells 69,70 . The results of the present study suggest that blocking the ErbB receptors by dacomitinib is an effective strategy in order to reduce the expression of the EMT markers and hamper invasive capability of the chemoresistant EOC cells.
Single-targeted ErbB agents have shown minimum response in chemoresistant ECO patients 26,31,71 . Compensatory activation of the other ErbB receptors sustains the activation of common downstream pro-survival pathways 72 . Targeting all the ErbB receptors is therefore a more effective treatment strategy, especially when resistance to a single-targeted ErbB agent has occurred 73 . For instance, breast cancer patients who experienced tumour progression after treatment with trastuzumab have demonstrated response to the dual EGFR and HER2 inhibitor lapatinib 74 . Furthermore, cetuximab-resistant colorectal and head and neck squamous cell carcinoma cells are sensitive to pan-ErbB inhibitors [75][76][77] . In consistency, our data show that the inhibitory effects of the single-targeted ErbB inhibitors on viability and invasiveness of the chemoresistant EOC cells were marginal. Conversely, dacomitinib exerted pronounced anti-tumour activity, suggesting that it may have potential for treatment of the EOC patients who ultimately have developed resistance after initial response.
Taken together, the results of the present study provide new insight into the mechanistic activity of dacomitinib through inhibition of the PLK1-FOXM1 axis (Fig. 6). These findings also indicate that dacomitinib-mediated   Table 2. Combination index (CI) and dose reduction index (DRI) of BI 2536 and cisplatin combination in OVCAR3, SKOV3 and A2780CP cells. DRI represents the order of magnitude of dose reduction that is allowed in combination for a given degree of effect as compared with the dose of each drug alone. "fa" denotes fraction affected.  Colony formation assay. Single-cell suspensions were seeded in 6-well plates at a density of 500 cells/well.
Once adhered, the cells were treated with the desired concentrations of the drugs for 48 h. The plates were further incubated for 10 d and colonies were stained with 0.5% crystal violet and counted under an inverted microscope. Plating efficiency (PE) and survival fraction (SF) were calculated using the following formula: PE = number of colonies/number of cells seeded; SF = number of colonies/number of cells seeded × PE and plotted graphically to obtain survival curves.
Anoikis resistance assay. For anoikis evaluation, 96-well plates were coated with poly-HEMA (20 mg/mL in 95% ethanol) and dried in a tissue culture hood. The cells were trypsinised into a single cell suspension and seeded in poly-HEMA-coated plates at a density of 5 × 10 3 cells/well. The cell suspension cultures were treated with the desired concentrations of the drugs for 48 h. Cell viability was estimated by MTT assay. Western blot analysis. The cells were lysed with RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) containing protease and phosphatase inhibitors (Roche Molecular Biochemicals). Protein concentration was determined using the Bradford assay. A total of 50 to 100 μg of protein were separated by SDS-PAGE and transferred onto PVDF membranes (Membrane Solutions, TX, USA). Membranes were blocked and blotted with the relevant antibodies. Horseradish peroxidase-conjugated secondary antibodies were detected with a BM chemiluminescence detection kit (Roche Molecular Biochemicals). β-actin was used as the loading control. All antibody dilutions were 1:500 except for the β-actin antibody, which was used at a dilution of 1:5000.
Annexin V/PI staining. Cells were stained with PI and FITC-conjugated Annexin V, as provided by Annexin-V-FLUOS Staining Kit (Roche Diagnostics) according to the manufacturer's instructions. The results were analysed using a Partec PAS III flow cytometer (Partec GmbH) and WindowsTM FloMax software (Partec).
Cell migration and invasion. Transwell cell migration and invasion assays were carried out as described earlier 80 . Statistical analysis. All data were evaluated in triplicate against vehicle-treated control cells and collected from three independent experiments. Data were graphed and analysed by GraphPad Prism Software 7.0a using one-way ANOVA and the unpaired two-tailed Student's t-test. All data are presented as mean ± standard deviation (SD).