Of ten human ovarian tumour cell lines examined in this study, SW626, PA1, ES2, Caov3 and Ovcar-3 were purchased from American Type Culture Collection (Manassas, VA, USA) and SKOV3, Cov318, A2780, A2780^CIS and A2780^ADR were purchased from the European Collection of Cell Culture (Porton Down, UK). Caov3, SKOV3, Cov318, PA1 and ES2 were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma, UK), A2780, A2780^CIS, A2780^ADR and Ovcar-3 in Roswell Park Memorial Institute-1640 medium (RPMI-1640) (Sigma), and SW626 cells in Liebovitz's L-15 medium (L-15). All media were supplemented with 10% fetal calf serum (FCS) (Sigma) and the antibiotics penicillin, streptomycin, neomycin. All RPMI and Liebovitz medium were also supplemented with glutamine (Sigma). Drug-resistant cell lines A2780^CIS and A2780^ADR were also supplemented with cisplatin (1 μM, 2–3 passages) and doxorubicin (100 nM, once a week) respectively to maintain their resistant phenotype.

The IGF-1R TKI NVP-AEW541, the reversible EGFR TKI erlotinib (OSI-774), and irreversible pan-HER family blocker afatinib (BIBW2992) were kindly provided by Novartis (Switzerland), OSI Pharmaceuticals (Farmingdale, NY, USA), Boehringer Ingelheim (Austria) respectively. Sapitinib (AZD8931), lapatinib (GW-572016 ditosylate), canertinib (CI-1033), imatinib, dasatinib and neratinib (HKI-272) were purchased from Selleckhem (Suffolk, UK), crizotinib and gefitinib (Tocris, Bristol/Macclesfield, UK). The cytotoxic drugs paclitaxel, doxorubicin and cisplatin and the mouse anti-EGFR mAb F4 were purchased from Sigma-Aldrich (Dorset, UK). The mouse monoclonal antibodies against HER-3 (MAB3481), HER-4 (MAB11311), IGF-IR (MAB391), ALK (MAB77491), and HGF R/c-MET (MAB3583) were purchased from R&D Systems (Oxford, UK) and those against CD24 (55426), CD44 (555476), CD117/c-Kit (555713) and P-glycoprotein (557001) were purchased from Becton-Dickinson Ltd., (Oxford, UK) respectively. The primary in-house mouse antibodies HM50.67A and HM43.16B were raised against the external domain of HER-2 and EGFR respectively (30). The mouse anti-IGF-IR and the rabbit anti-pIGF-1R antibodies were purchased from Insight Biotechnology (Middlesex, UK). The mouse phosphor-Ty-100 and the rabbits anti-phosphor-EGFR, HER-2, phosphor-HER-2, HER-3, phosphor-HER-3, phosphor-HER-4, HER-4, MAPK, phosphor-MAPK, Akt, phosphor-Akt, Met, phospho-Met, Stat-3, phosphor-stat-3, Stat-5, phospho-stat-5, β-actin, and CD44⁺ were all purchased from Cell Signaling Technology (Hitchin, UK). The anti-mouse IgG FITC conjugated STAR9B was from Serotec Ltd. (Oxford, UK), and the alkaline phosphatase conjugated goat anti-mouse IgG and anti-rabbit IgG were from Fisher Scientific Ltd. (Loughborough, UK). The donkey anti-rabbit IgG IRDye 680RD and goat anti-mouse IgG IRDye 800CW were purchased from LI-COR Ltd. (Cambridge, UK).

The cell surface expression of the HER family members, IGF-IR, ALK, c-MET, putative CSC and P-glycoprotein markers was determined using flow cytometry, as described previously (31). Briefly, ~1×10⁶ cells in 1 ml of 2% FBS medium were incubated in the presence of primary mouse antibody or control medium for 1 h by rotation at 4°C. The tumour cells were then washed three times by centrifugation, and then incubated in the presence of the FITC-conjugated rabbit anti-mouse IgG secondary antibody for 1 h by rotation at 4°C. Cells were again washed three times by centrifugation, and the cell pellet was finally re-suspended in FACS Flow buffer (Becton-Dickinson). A minimum of 10,000 events were recorded through excitation with an argon laser at 488 nm and data were analysed using the FITC detector (at 525 nm) of BD FacsCalibur flow cytometer (Becton-Dickinson), and mean fluorescence intensity values were calculated using CellQuest pro software (Becton-Dickinson).

The effect of the various agents on the growth of human ovarian cancer cell lines was determined using the sulforhodamine B (SRB) colourimetric assay (Sigma-Aldrich), as described previously (31). Briefly, tumour cells were seeded at a density of 5,000 cells per well in 100 μl of growth medium supplemented with 2% FBS in a 96-well plate. Following 4-h incubation at 37°C, 100 μl aliquots of doubling dilutions of the drugs or control medium (i.e., without drug) were added to triplicate wells. Cells in the 96-well plates were then incubated at 37°C until cells in the wells containing the control medium were almost confluent. Tumour cells were then fixed with 10% trichloroacetic acid (TCA) for 1 h and were gently washed with tap water, air-dried for ~24 h, and then stained with 0.04% sulforhodamine B (SRB) in 1% acetic acid for further 1 h. Each plate was then washed with 1% acetic acid, air dried overnight and the SRB stain was solubilized by adding 100 μl of 10 mM Tris-base per well. The absorbance of each well was measured at 565 nm using the Epoch plate reader (Thermo Fisher, UK). To determine the initial number of cells prior to treatment, an extra plate of cells was set up and processed under the same condition after 4-h incubation at 37°C without the drugs. Growth as percentage of growth of control was determined through the following formula: % Growth = B−A/C−A × 100, where A = A565 nm before treatment, B = A565 nm after treatment with drugs, and C = A565 nm after medium (32). The IC₅₀ value of each agent was determined using Gen5 software (BioTek, UK).

The effect of two agents, when used in combination, on the growth of ovarian cells was determined, using the combination index (CI) as described previously (32). Briefly, the drugs for each combination were mixed at 8X their IC₅₀ value followed by 8 doubling dilutions (32). Data analysis was then performed using CalcuSyn software (Biosoft, UK). The combination index values <0.9 indicates synergistic effect, while CI values between 0.90–1.10 indicates an additive effect and CI values >1.1 denotes antagonistic effect.

Tumour cells were grown to near confluency in 6-well culture plates containing 5 ml of 10% FBS growth medium. Cells were washed once with 5 ml of 0.5% FCS/medium and then incubated in 5 ml of fresh 0.5% medium containing no drug (control), a TKI, or a cytotoxic drug for 24 h at 37°C, prior to treatment with the HER ligand heregulin (20 nM) (R&D Systems) for 15 min at 37°C. Tumour cells were then lysed in 400 μl of lysis buffer (Invitrogen, Paisley, UK) containing the cocktail protease inhibitor (Sigma-Aldrich). Cell lysates were heated to 75°C for 10 min, then 30 μl of protein samples (30 μg) were separated on 4–12% Bis-Tris gels (Invitrogen) and transferred onto polyvinylidene difluoride (PVDF) membranes using the XCell II Mini-Cell Blot Module kit (Invitrogen). PVDF membranes were probed with antibodies using SNAP i.d systems (Millipore, Watford, UK). Signal for all were detected using either the Western Breeze chemiluminescence kit (alkaline-phosphatase conjugated secondary antibody, Invitrogen) and visualized by the G-box imaging system (Syngene, Cambridge UK) or a fluorescence conjugated secondary antibody (LI-COR Ltd) and visualized by the accompanying software, LI-COR Image Studio.

The effect of various agents on the cell cycle distribution of ovarian cancer cell lines was determined using flow cytometry as described previously (32). Approximately 2.5×10⁵ cells were seeded into 25 cm² culture flasks containing 10 ml of 2% FBS growth medium plus no drug (control medium) or an agent. Once the cells containing medium only were almost confluent in the control flasks, the cells were harvested and pooled together with the cell supernatant, and washed three times with cold PBS. The final cell pellet was re-suspended in 200 μl of cold PBS, and permeabilised by the addition of cold 70% ethanol overnight at 4°C. The tumour cells were then washed once with PBS and incubated with 0.5 ml of PI/RNAse mix (Becton-Dickinson) for ~35 min at room temperature. The PI-stained tumour cells were then excited at 488 nm using the FL-3 detector (620 nM) of a BD FACsCalibur flow cytometer (Becton-Dickinson), and analysed using CellQuest Pro software (Becton-Dickinson).

The cell migration assay was conducted using the IncuCyte Clear View 96-well cell migration plate according to the manufacturer's instructions (Essen Bioscience Ltd., Hertfordshire, UK). Briefly, ~1×10³ tumour cells plus the treatment in total volume of 60 μl 0.5% FBS medium were added into Clear View 96-well insert. Each cell plate was then left to settle at the room temperature for 15 min followed by incubation for a further 30 min at 37°C. Then 200 μl of medium containing 10% FBS (chemoattractant) was added to the lower chamber. The cell plate was then placed onto the IncuCyte Zoom^® instrument and were left for 15 min at 37°C to settle. After careful removal of any condensation on the lid or bottom of the reservoir, each plate was returned into IncuCyte Zoom instrument with a 10× objective using the IncuCyte™ chemotaxis system. Chamber wells were analysed every 3 h using the IncuCyte chemotaxis software.

Linear regression was used to analyse the relationship between the expression of the HER family receptors in response to treatment with afatinib, erlotinib, crizotinib, NVP-AEW541 and cytotoxic drugs. A one-way analysis of variance (ANOVA) test was used to compare mean values between more than two groups. Data are presented as mean ± SD. P<0.05 was considered statistically significant.

The cell surface expression of HER-family members (EGFR/HER-1, HER-2/neu, HER-3 and HER-4) was determined by FACS analysis. Of the 10 ovarian cell lines examined, SW626, ES2, SKOV3, and Caov3 were moderately positive for the EGFR with the mean fluorescence intensity (MFI) values of 10, 15, 15 and 40, and respectively (Table I). Of the seven HER-2 positive cancer cell lines, HER-2 overexpression was only detected in SKOV3 cells (MFI=574) (Table I). SKOV3 cells were also found to be positive for the expression of IGF-IR and c-MET. In contrast, all ovarian tumour cell lines had undetectable levels of both HER-3 and HER-4. The highest level of IGF-IR, ALK and c-MET were present in A2780^CIS (MFI=13), Cov318 (MFI=11), and Cov318 (MFI=24) (Table I). Of the putative cancer stem cell markers, 5 cell lines were CD24-positive, with the highest level of CD24 expression in SKOV3 cells (MFI=440), and 6 cells lines were CD44-positive with the highest level of CD44 in ES2 (MFI=1,105). Finally, none of the ovarian cancer cell lines had overexpression of P-glycoprotein and all were found to be CD117/c-Kit-negative (Table I).

Of the TKIs, the two reversible EGFR TKI gefitinib and erlotinib inhibited the growth of 6 and all 10 ovarian cancer cell lines with IC₅₀ values ranging from 1.82 nM (Caov3) to 5.5 μM (A2780), and 152 nM (Caov3) to 9.6 μM (A2780^ADR) respectively (Table I). The reversible dual EGFR/HER-2 lapatinib inhibited the growth of 9 cancer cell lines, with IC₅₀ values ranging from 1.42 nM (SKOV3) to 5.28 μM (A2780). At cutoff value of 10 μM, the reversible EGFR/HER-2/HER-3 TKI sapitinib was less effective than the irreversible pan-HER family blockers (i.e., canertinib, neratinib and afatinib) at inhibiting the growth of ovarian cancer cells, and its IC₅₀ values were >10 μM for three ovarian cancer cell lines ES2, Cov318 and Ovcar-3 (Table II and Fig. 1A). Canertinib, neratinib and afatinib were the best cytotoxic HER TKIs and they inhibited the growth of all ovarian cancer cells with IC₅₀ values ranging from 57.6 nM (Caov3) to 3.55 μM (SW626), 1.38 nM (Cov318) to 927 nM (SW626), and 83 nM (Caov3) to 1.4 μM (ES2) respectively (Table II and Fig. 1A). At nM concentration, the IGF-IR TKI NVP-AEW541 was only effective at inhibiting the growth in vitro of PA1 cells (IC₅₀=79.3 nM). The ALK/c-MET inhibitor crizotinib inhibited the growth of all ovarian cancer cell lines with IC₅₀ values ranging from 162 nM (PA1) to 2.7 μM (A2780^ADR). Interestingly, the majority of ovarian cancer cells were highly sensitive to treatment with dasatinib with IC₅₀ values of <26 nM, but all were relatively resistant to treatment with imatinib (i.e., IC₅₀ value >10 μM, Table II and Fig. 1B). Of the three cytotoxic drugs used in this study, paclitaxel was the most effective agent at inhibiting the proliferation of ovarian cancer cell lines [IC₅₀ range: 102 pM (Caov3) to 278 nM (Ovcar-3)], followed by doxorubicin. Interestingly, the great majority of ovarian cancer cells were relatively resistant to treatment with cisplatin and had an IC₅₀ >20 μM (Table II and Fig. 1B).

We examined the effect of treatment with various agents on the cell cycle distribution of ovarian cancer cells. As shown by flow cytometry, SKOV3 cells have the highest level of HER-2 expression, low levels of EGFR, IGF-IR and c-MET expression, and overexpress the two putative ovarian cancer stem cell markers (Table I). As a result, SKOV3 cells were selected for comparing the effect of various forms of inhibitors (i.e., reversible EGFR, irreversible pan-HER inhibitor, IGF-IR TKI, C-MET-TKI) with cytotoxic drugs on cell cycle distribution. Treatment of SKOV3 cells with both the cytotoxic drugs paclitaxel and doxorubicin increased the proportion of apoptotic/dead cells in sub-G₁ phase (i.e., from 2 to 31 and 16%) and this was accompanied by a significant increase in the proportion of cells in G₂-M phase (i.e., from 8 to 28 and 54%) and a slight increase in S phase of the cell cycle (i.e., from 7 to 13 and 12%) respectively (Table III). Treatment of SKOV3 cells with various TKIs (erlotinib, afatinib, crizotinib, NVP-AEW541) also increased the proportion of cells in sub-G₁ phase, but this was accompanied by a reduction in the proportion of cells in S phase of the cell cycle (Table III).

In some studies, IGF-IR cross-talk with HER family members was associated with resistance to treatment with the HER inhibitors (please see Discussion e.g., refs. 58–60). As a result, we next examined the effect of IGF-IR TKI in combination with other agents on the growth of three human ovarian cancer cell lines. When NVP-AEW541 was combined with the reversible EGFR TKI erlotinib, there was an antagonistic response in all three ovarian cancer cell lines (SKOV3, Caov3 and ES2). When combined with afatinib there was a synergistic effect only in the HER-2 overexpressing SKOV3 cells (Table IV). Next, we examined the effect of NVP-AEW541 in combination with crizotinib and this combination resulted in the synergistic growth inhibition of both SKOV3 and Caov3 cells, but it was antagonistic in ES2 cells. Finally, when NVP-AEW541 was used in combination paclitaxel, this resulted in the synergistic or additive growth inhibition of all three ovarian cancer cells (Table IV).

Linear regression analysis was carried out for determining the association between expression of growth factor receptors and sensitivity to the tyrosine kinase inhibitors as well as the cytotoxic drugs. There was no significant association between the expression of EGFR, HER-2, IGF-IR, c-MET, ALK and putative CSC markers and the response to treatment with the irreversible HER TKIs, dasatinib, NVP-AEW541, and crizotinib. However, a significant association was found between the co-expression of CD24/HER-2, HER2/CD24/CD44, and EGFR/HER2/CD24 and response to treatment with gefitinib and lapatinib, and the co-expression of EGFR/HER2/CD24 and response to sapitinib (Table V). In addition, the co-expression of CD44 with EGFR, HER2, CD24, EGFR/CD24, HER2/CD24, EGFR/HER2, or EGFR/HER2/CD24 was associated with better response to treatment with paclitaxel.

Next, we investigated the effect of various forms of the HER inhibitors on the basal phosphorylation of the HER family members and the downstream cell signaling molecules in SKOV3 cells. With the exceptions of treatment with gefitinib and sapitinib, all other HER TKIs induced complete inhibition of phosphorylated EGFR, HER-2 and AKT and MAPK (Fig. 2A). We also compared the effect of afatinib, erlotinib with those of NVP-AEW541, crizotinib and three cytotoxic drugs on heregulin-induced cell signaling. Of these, only afatinib was an effective agent in inhibiting both the constitutive phosphorylation and heregulin-induced phosphorylation of HER family members, c-MET, IGF-IR as well as MAPK and AKT (Fig. 2B). Moreover, treatment with both afatinib and erlotinib were accompanied by a reduction in the expression of CD44 in such cells (Fig. 2B).

Finally, we investigated the migration of SKOV3 cells and whether treatment with various TKIs had any effect on the migration ability of these cells. Of these, only treatment with afatinib (p=0.027), neratinib (p=0.011) and dasatinib (p=0.004) produced a significant reduction in migration of SKOV3 cells (Fig. 3). Interestingly, treatment with sapitinib was accompanied by an increase in the migration of SKOV3 cells (Fig. 3).