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Oncol Ther DOI 10.1007/s40487-016-0031-1 ORIGINAL RESEARCH A Combination of Two Receptor Tyrosine Kinase Inhibitors, Canertinib and PHA665752 Compromises Ovarian Cancer Cell Growth in 3D Cell Models Wafaa Hassan . Kenny Chitcholtan . Peter Sykes . Ashley Garrill Received: July 12, 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com ABSTRACT EGFR/Her-2 inhibitor (canertinib) and a c-Met inhibitor (PHA665752) in ovarian cancer cell Introduction: Advanced ovarian cancer is often lines in 3D cell aggregates. a fatal disease as chemotherapeutic drugs have Methods: OVCAR-5 and SKOV-3 ovarian cancer limited effectiveness. Better targeted therapy is cell lines were cultured on a non-adherent needed to improve the survival and quality of surface to produce 3D cell clusters and life for these women. Receptor tyrosine kinases aggregates. Cells were exposed to canertinib including EGFR, Her-2 and c-Met are associated and PHA665752, both individually and in with a poor prognosis in ovarian cancer. combination, for 48 h. The effect on growth, Therefore, the co-activation of these receptors metabolism and the expression/ may be crucial for growth promoting activity. phosphorylation of selective signaling proteins In this study, we explored the effect of associated with EGFR, Her-2 and c-Met were combining two small molecule inhibitors that investigated. target the EGFR/Her-2 and c-Met receptor Results: The single drug treatments tyrosine kinases in two ovarian cancer cell significantly decreased cell growth and altered lines. The aim of this study was to investigate the expression of signaling proteins in the combined inhibition activity of a dual OVCAR-5 and SKOV-3 cell lines. The Enhanced content To view enhanced content for this combination treatment showed greater article go to http://www.medengine.com/Redeem/ reduction of cell numbers for both cell lines. EE76F06032E5AE54#/EsmModal. Total expression and phosphorylation of W. Hassan Á A. Garrill signaling proteins were further reduced in the School of Biological Sciences, University of combination drug treatments, compared to the Canterbury, Private Bag, 4800 Christchurch, New Zealand single inhibitor treatments. Conclusion: Our findings suggest that the K. Chitcholtan (&) Á P. Sykes Department of Obstetrics and Gynaecology, concurrent targeting of more than one University of Otago, Christchurch Women’s receptor tyrosine kinase may be useful in Hospital, 2 Riccarton Avenue, Christchurch, New Zealand developing more effective targeted drug e-mail: [email protected] Oncol Ther regimens for patients, who have EGFR, Her-2 signaling proteins. In this study, we explore and c-Met positive ovarian cancer cells. the possibility that targeting of multiple receptor tyrosine kinases will lead to greater Keywords: Canertinib; c-MET; Cell clusters; effects on tumor growth potential. EGFR; Ovarian cancer; PHA665752; Tyrosine Some advanced ovarian cancer cells have kinase inhibitors been shown to possess high levels of the receptors EGFR and Her-2 [6]. It is the activation of these receptors that may promote INTRODUCTION the growth of malignant cells, and the over expression of these receptors has been Ovarian cancer is the leading cause of death from associated with a poor prognosis and invasive gynecological cancers in developed countries. phenotypic changes [7, 8]. There is evidence to The majority of ovarian cancer patients are suggest that EGFR/Her-2 expressing malignant diagnosed with an advanced stage of the cells may not be sensitive to the EGFFR and disease, and as such they typically have a 5-year Her-2 inhibitors as ovarian cancer cells are able survival rate lower than 30% [1]. For the majority to use alternate a receptor tyrosine kinase for of women, cytotoxic chemotherapeutics have growth promotion [9]. The c-Met is also a limited efficacy. Therefore, there is an urgent receptor tyrosine kinase, which has elevated need for alternative anticancer agents that have levels in ovarian cancer cells [10]. The c-Met the potential to control tumor growth, improve receptors are activated when they are bound by the quality of life, and increase the longevity of the hepatocyte growth factor, HGF, which is patients. elevated in the ascitic fluid of women with A number of targeted anticancer drugs have advanced stages of ovarian cancer [11]. The been studied; among these drugs are small EGFR, Her-2 and c-Met receptors share common molecule inhibitors of receptor tyrosine downstream signaling molecules that can kinases. A subset of cancer cells is dependent promote cell survival, motility, and invasion on the over activation of epidermal growth and cross-communication between these three factor receptor proteins including EGFR and receptors may promote cancer progression and Her-2. Therefore, an inhibitor that specifically resistance to therapy [12]. Thus, c-Met could be blocks these receptors may have clinical an additional potential target for cancer activity. Specific inhibitors of EGFR and Her-2 treatment and raises the strategic possibility of including gefitinib, erlotinib, lapatinib, and a combined targets of receptor tyrosine kinases monoclonal antibody Herceptin have been concurrently. approved by the FDA to treat different types of Candidate molecules for such combined cancers that include breast cancer, colon cancer strategies are canertinib and PHA665752. and non-small cell lung cancer, NSCLC [2, 3]. Canertinib, also known as CI-1033, is an The EGFR and Her-2 inhibitors as a irreversible inhibitor of receptor tyrosine monotherapy show minimal clinical benefit in kinases, binding to the ATP-binding site. It has ovarian cancer trails [4, 5]. The lack of clinical shown potent effects against several cancer cell efficacy of these inhibitors is perhaps due to lines targeting EGFR and Her-2. Clinical trials of activation of multiple receptor tyrosine kinases canertinib have been conducted against a and the activation of several down stream number of tumor types, including breast Oncol Ther cancer [13], NSCLC [3], and advanced ovarian (GIBCOÒ, Life Technologies, New Zealand) at a cancer [5]. However, canertinib alone showed working concentration of 100 units/ml modest activities in clinical studies with ovarian penicillin, 100 lg/ml streptomycin, 2 mM cancer patients [5]. PHA665752 is a reversible glutaMAXTM (GIBCOÒ, Life Technologies, New inhibitor of c-Met, which at nano-molar Zealand), and 2 lg/ml FungizoneÒ (Life concentrations has shown in vivo anti-tumor Technologies, New Zealand). The final activity [14]. concentration of glucose in the media was Advanced ovarian cancer patients often have 5.5 mM. The respective supplemented media is ascitic fluid in the abdominal cavity. In the henceforth referred to as working media. Cells fluid, ovarian malignant cells form 3D were maintained at 37 °C in a humidified 5% structures; these microscopic clusters and CO2 atmosphere. This article does not contain aggregates are a major source of secondary any studies with human or animal subjects growth potential [15, 16]. These clusters and performed by any of the authors. aggregates then deposit on the peritoneal lining and the surface of internal organs to continue Generation of 3D Cell Aggregates their growth. Therefore, it is appropriate to use cell clusters and aggregates as an in vitro cell Twelve or twenty-four welled culture plates model to replicate the behavior of ovarian were coated with 24 mg/ml poly cancer cells. We hypothesized that floating cell (2-hydroxyethylmethacrylate) (poly-HEMA) clusters and aggregates ovarian cancer cells may (Sigma, New Zealand), a polymer that use the concurrent activation of EGFR, Her-2 prevents cells from adhering to the surface of and c-Met to sustain growth and cell survival. culture wells. The poly-HEMA was dissolved in Combined inhibition of these receptors will 95% ethanol and heated to approximately produce a greater anti-growth effect in ovarian 70 °C until completely dissolved. The cell cancer cells. The aim of the present study was to culture plates were coated with 300 lL test the effect of such combined inhibitors on poly-HEMA solution and placed overnight on 3D cell cultures of two ovarian cancer cell lines, an orbital shaker at 37 °C and then left to cool OVCAR-5- and SKOV-3. down prior the cell culture. Plates were washed with PBS pH 7.4. Cell monolayers METHODS were incubated with diluted 19 Trypsin–EDTA (Thermo Fisher Scientific, New Zealand) for The human ovarian adenocarcinoma cell lines, 20–30 min to detach cells from the flask. Cell OVCAR-5 and SKOV-3 were used. Both cell lines numbers were determined with a were obtained from Dr. Judith Mckenzie, hemocytometer to ensure that each well Haematology Research group, University of contained approximately 100,000 cells with a Otago, Christchurch, New Zealand. OVCAR-5 total volume of 1 ml working media. Cells and SKOV-3 cells were maintained in DMEM/ were then incubated at 37 °C in a humidified Ò F12 or MEM media (GIBCO , Life Technologies, 5% CO2 atmosphere for 6 days to obtain New Zealand), respectively, both supplemented cellular aggregates. Media were removed and Ò with 5% fetal bovine serum (FBS) (GIBCO , Life replaced with 1 ml of fresh media every Technologies, New Zealand), PenStrep second day. Oncol Ther Treatments with Canertinib single cell suspensions, and cells were counted and PHA665752, HGF and EGF using a hemocytometer to assess cell viability. Canertinib (CI-1033) was purchased from LC Immunofluorescent Detection laboratories (Massachusetts, USA) and of Receptors PHA665752 was obtained from Sigma (Auckland, New Zealand). Both compounds For immunofluorescent imaging, OVCAR-5 and were dissolved in 100% DMSO. Final DMSO SKOV-3 cells were cultured for 6 days to volumes were normalized for all experiments generate cell clusters and aggregates. The and were less than 0.5% (v/v). Cells were then media were refreshed every second day. Cell synchronized in media without FBS for 24 h, clusters and aggregates were harvested and fixed before they were exposed to the inhibitors and with ice-cold 50% (v/v) acetone/methanol growth factors.

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