Molecular targeting therapy has recently shown hope as an approach to treatment of breast cancer; in part due to the success of treatment with Trastuzumab (Herceptin), a humanized monoclonal antibody against human epidermal receptor 2 (HER2/neu).1, 2 Gefitinib (ZD1839, Iressa), a known selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), is used worldwide, mainly for treatment of advanced non-small cell lung cancer. EGFR is also expressed in several types of breast cancer cell lines and plays major roles in their proliferation and malignancy.1, 3 However, the efficacy of gefitinib as a treatment for breast cancer remains controversial.4, 5 Some phase I and II clinical trials have reported that gefitinib monotherapy does not show any benefit for the treatment of advanced breast cancer patients.5, 6 In addition, although some studies of combination therapies involving gefitinib and other cytotoxic drugs have been reported,7, 8, 9, 10, 11 the clinical benefits remain unclear, and little information has been obtained. On the other hand, taxane shows excellent efficacy for breast cancer either as a monotherapy or in combination with other cytotoxic drugs, such as anthracycline agents. However, some patients, especially heavily pretreated patients, are resistant to taxane. Several reports have shown that gefitinib accelerates the activity of some cytotoxic drugs and may be able to reverse drug-resistance.7, 8, 9, 10 To obtain more preclinical information about combination therapies with gefitinib and other cytotoxic drugs, we examined the tumor inhibitory effect of a combination treatment with gefitinib and taxane on breast cancer cell lines overexpressing EGFR both in vitro and in vivo. Although a similar study on a gefitinib and taxane combination was reported by Caldiello et al. in 2002,7 we investigated the mechanism of the effect of the combination in more detail and from a different viewpoint. This study aimed to elucidate the basis of the combination with a molecular targeting therapy, such as EGFR TKI, and other cytotoxic drugs; and to develop the possibility of new 2nd and 3rd line therapies for heavily pretreated, drug resistant breast cancer patients. The breast cancer cell lines used in this study were MDA-MB-231 and MCF7/ADR. MDA-MB-231 cells overexpress EGFR, while MCF7/ADR cells are multidrug-resistant, hormone independent, and overexpress both EGFR and HER2/neu. These cell lines are known to have more malignant potency than other general breast cancer cell lines. For this reason, we thought they were suitable for the aims of this study. Overall, as well as the combination effect, we explored a potential clinical approach for using combination therapy with gefitinib and taxane as a new breast cancer treatment.
Some kinds of breast cancer cell lines, similar to several types of solid tumors, express epidermal growth factor receptor (EGFR). However, gefitinib, an EGFR tyrosine kinase inhibitor, is not effective for all these cell lines. Similarly, taxane is effective for many of the cell lines, although some, such as the multidrug-resistant MCF7/ADR cell line, show taxane-resistance. Here, we examined the growth inhibitory effect of combination treatment with gefitinib and taxane on the breast cancer cell lines MDA-MB-231 (EGFR-positive) and MCF7/ADR (EGFR- and HER2-positive). To estimate the combined effect, a Combination Index was calculated for each cell line. The combination of gefitinib and taxane showed a strong synergistic effect on MCF7/ADR cells, but an invitro additive-antagonistic effect on MDA-MB-231 cells. Similarly, the combination treatment showed a significantly increased tumor inhibitory effect on MCF7/ADR xenografts, but not on MDA-MB-231 xenografts. Regarding the mechanism of the synergistic effect, Western blotting analysis revealed that taxane activated the EGFR-Akt pathway in MCF7/ADR cells but not in MDA-MB-231. To determine the optimal sequential administration of gefitinib and taxane for MCF7/ADR cells, we used flow cytometry to analyze the cell cycle and apoptosis; finding that taxane treatment followed by gefitinib produced a higher rate of G2 arrest and apoptosis than gefitinib treatment followed by taxane. These results suggest gefitinib overcomes the drug-resistance of these cells, thereby increasing the effects of taxane on MCF7/ADR cells. Further, activation of the EGFR-Akt pathway by taxane is related to this synergistic effect. © 2006 Wiley-Liss, Inc.
Material and Methods
Gefitinib (ZD1839, Iressa) was kindly provided by Astra-Zeneca (Cheshire, UK). It was dissolved in dimethyl sulfoxide (DMSO) for the in vitro study and suspended in 1% Tween 80 for the in vivo study. Docetaxel or Paclitaxel was used as taxane in this study. Docetaxel (Taxotere) was purchased from Aventis Pharma (Tokyo, Japan). Paclitaxel was obtained from Bristol Pharmaceuticals (Tokyo, Japan).
The human breast cancer cell line MDA-MB-231 was obtained from ATCC (Rockville, MD). The MCF7/ADR cell line was kindly provided by Prof. K.H. Cowan (National Cancer Institute (NCI), National Institute of Health, Bethesda, MD). Cells were maintained in RPMI-1640 medium (Sigma–Aldrich, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere of 95% air/5% CO2 at 37°C.
Female BALB/c nu/nu mice (6 weeks-old) were purchased from Charles River Japan (Shiga, Japan) and maintained at our animal laboratory under specific pathogen-free (SPF) conditions.
Xenografts created using MDA-MB-231 or MCF7/ADR cells were subcutaneously transplanted into 6-week-old BALB/c nu/nu mice. After 2 weeks, administration of gefitinib alone, docetaxel alone or a combination of gefitinib + docetaxel was initated. Gefitinib (100 mg/kg) was administered orally on days 1–5 every week, while docetaxel (20 mg/kg) was injected intraperitoneally on day 1 every week. From 3 weeks after the transplantation, tumor volumes were measured every 5 days. The measurements were continued for 4 weeks for the MCF7/ADR xenografts and about 7 weeks for the MDA-MB-231 xenografts. A total of 8 mice were used in each experiment, and all experiments were independently carried out 4 times. Statistical analysis between monotherapy and combination therapy was performed.
Evaluation of the growth inhibitory and combination effects
Initially, we observed the growth inhibitory effects of gefitinib or taxane monotherapy, and calculated the ED50 (50% effective dose) values. Next, we determined the combination molar ratios for gefitinib and taxane on the basis of these results. The experimental protocol was as follows. MDA-MB-231 and MCF7/ADR cells were seeded in 96-well plates at 5,000 cells/well. After 24 hr, gefitinib alone, docetaxel alone, paclitaxel alone or combinations of the drugs at constant ratios were administered and the cells were incubated in 5% CO2 at 37°C for 72 hr. The combination molar ratios were as follows:
The growth inhibitory effect was evaluated using a MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. A 20 μl aliquot of MTS solution was added in each well, and incubated for 1–4 hr at 37°C in a humidified 5% CO2 atmosphere. The absorbance of each well was measured at 490 nm using a 96-well microplate reader (ImmunoMini NJ-2300) and used to calculate the effective dose. Each experiment was performed in quadruplicate wells for each drug concentration and independently carried out 3–4 times. The combination effect was evaluated by the Combination Index (CI), which was calculated using Calcusyn software (Biosoft, Cambridge, UK). The definition of CI is as follows:
where (Dx)1 is the dose of Drug 1 required to produce an X% effect alone, (D)1 is the dose of Drug 1 required to produce the same X% effect in combination with Drug 2, (Dx)2 is the dose of Drug 2 required produce an X% effect alone, and (D)2 is the dose of Drug 2 required to produce the same X% effect in combination with Drug 1.
The combination effects were defined as follows: CI < 1: synergistic effect; CI = 1: additive effect; CI > 1: antagonistic effect.
Immunoprecipitation and Western blotting
To estimate whether exposure to taxane leads to activation of the EGFR-Akt pathway, the ED50 or ED90 (90% effective dose) of taxane was added to subconfluent cells that had been serum-starved for 24 hr. After 6 hr, the cells were lysed in lysis buffer (50 mM HEPES pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM PMSF, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 2 mM Na3VO4, 1 mM NaF, 10 mM β-GP) and the proteins were extracted. At the same time, to estimate whether the activated EGFR-Akt pathway is downregulated by gefitinib, ED50 of taxane and 3 μM gefitinib were added in combination, and 6 hr later proteins were extracted in the same way, electrophoretically separated in 6% or 10% polyacrylamide gels (TEFCO, Tokyo, Japan) and then transferred to PVDF membranes (Amersham Biosciences, Piscataway, NJ). The membranes were incubated with rabbit monoclonal antibodies against phospho-EGFR (Tyr1068; Cell Signaling Technology, Beverly, MA), EGFR (Cell Signaling Technology), HER2/ErbB2 (Cell Signaling Technology) or phospho-Akt (Ser473; Cell Signaling Technology), or a rabbit polyclonal antibody against Akt1/2 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by a horseradish peroxidase-conjugated secondary antibody. The immunoreactive proteins were visualized by chemiluminescence (ECL-plus; Amersham Biosciences).
Cell cycle and apoptosis analysis after sequential exposure
The ED50 values of gefitinib, docetaxel and paclitaxel were calculated using the MTS assay. MCF7/ADR cells were sequentially exposed to gefitinib followed by taxane or taxane followed by gefitinib at the ED50 concentrations for 24 hr. Next, the cells were centrifuged and 70% ethanol was added to each cell pellet, followed by fixation at −20°C overnight. The following day, cells were stained with propidium iodide (PI) and analyzed by flow cytometry (FACSCalibur; BD Biosciences, Akasaka, Japan) to assess the cell cycle. For the apoptosis analysis, cells were sequentially exposed to gefitinib followed by taxane or taxane followed by gefitinib at the ED50 concentrations for 48 hr, stained with Annexin V/PI (BD Biosciences) and analyzed by flow cytometry. Each set of the experiments was independently carried out 3 times. Statistical assays were performed for the rate of apoptosis and G2 arrest between gefitinib → taxane and taxane → gefitinib.
The statistical analysis for the xenograft study was performed using the Statview4.5 software program (Abacus Concepts, Berkeley, CA). Repeated-measures one-way ANOVA followed by Fisher's PLSD was used for multiple comparisons. For cell cycle and apoptosis analysis, student-t test was performed using the JMP 5.0 software. Values of p < 0.05 were considered statistically significant.
Initially, we observed expressions of EGFR and HER2/neu on the two cell lines. Western blot analyses revealed that both cell lines overexpressed EGFR, and that MCF7/ADR cells also overexpressed HER2. The level of EGFR overexpression appeared to be higher in MDA-MB-231 than in MCF7/ADR cells (Fig. 1).
Next, we examined the growth inhibitory effect of combination treatment with gefitinib (100 mg/kg orally on days 1–5 every week) and docetaxel (20 mg/kg intraperitoneally on day 1 every week) on xenografts composed of MDA-MB-231 or MCF7/ADR cells (Figs. 2a and 2b). At the time of the transplantation, the average volume of the xenografts was about 0.25 mm3. After 2 weeks, prior to the onset of the combination treatment, the average volume had increased to about 50–60 mm3. Compared with the monotherapy treatments, the combination of gefitinib + docetaxel significantly suppressed the growth of MCF7/ADR xenografts (p < 0.001) (Fig. 2a), but the effect on MDA-MB-231 xenografts was not significant (p = 0.2021) (Fig. 2b).
In vitro, the growth inhibitory effects of monotherapy and combination therapy with the 2 drugs were estimated by the MTS assay. The ED50 value and the range of doses used in this assay are shown in Table I. The gefitinib + taxane combination showed a strong synergistic effect (CI < 1) in MCF7/ADR cells (Figs. 3a and 3b), but an additive-antagonistic effect (CI ≥ 1) in MDA-MB-231 cells (Figs. 3c and 3d); There was no clear difference in the combination effect between gefitinib + docetaxel and gefitinib + paclitaxel. These results suggest that the gefitinib + taxane combination is very effective for MCF7/ADR cells, but not for MDA-MB-231 cells, consistent with the results of the vivo study.I
|Gefitinib (μM):Docetaxel (nM)||1:30|
|Gefitinib (μM):Paclitaxel (nM)||1:60|
|Gefitinib (μM):Docetaxel (nM)||1:2|
|Gefitinib (μM):Paclitaxel (nM)||1:2.6|
On the basis of these results, we examined the mechanism of the synergistic effect. As shown in Figure 4, 6 hr taxane exposure led to increased phosphorylation of EGFR or activation of the EGFR-Akt pathway in MCF7/ADR cells, which showed the synergistic effect, and also revealed that activated EGFR was downregulated by 3 μM gefitinib. However in MDA-MB-231 cells, which did not show the synergistic effect, these phenomena were not observed. (Figs. 4a and 4b). These results suggest that the synergistic effect of the gefitinib + taxane combination therapy is associated with the activation of the EGFR-Akt pathway by taxane.
Assuming that this combination therapy will be applied to clinical cases in the future, we investigated the optimal sequential administration. In MCF7/ADR cells, the rate of apoptosis and the cell cycle were compared between gefitinib followed by taxane and taxane followed by gefitinib, using flow cytometric analyses. As shown in Figure 5, the rates of apoptosis were 12% for gefitinib followed by docetaxel and 10% for gefitinib followed by paclitaxel (Fig. 5). Contrarily, the rates were 17% for docetaxel followed by gefitinib and 22% for paclitaxel followed by gefitinib (Fig. 5). Therefore, the apoptosis rate of taxane treatment followed by gefitinib was about 2-fold higher than the reverse. Significant differences were seen between both. The results of the cell cycle analysis are summarized in Figure 6. The rate of G2 arrested phase cells was 30% for gefitinib followed by docetaxel, compared with 45% for docetaxel followed by taxane. Similarly, the rates of G2 arrest phase cells for gefitinib followed by paclitaxel and paclitaxel followed by gefitinib were 34% and 54%, respectively (Fig. 6). Significant difference was shown in both cases (p = 0.0395, 0.046 respectively). According to these results, we suggest that taxane treatment followed by gefitinib administration accelerates apoptosis and G2 arrest more effectively than gefitinib treatment followed by taxane administration.
As shown in this study, both MCF7/ADR and MDA-MB-231 were resistant to gefitinib monotherapy despite their overexpression of EGFR. However, the combination effect with taxane was contrastive between the 2 cell lines, showing a strong synergistic effect for MCF7/ADR cells and an additive-antagonistic effect for MDA-MB-231 cells (Fig. 3, Table I). To clarify the mechanism giving rise to these results, we noted the phosphorylation of EGFR, and the activation of the EGFR-Akt pathway.10, 12 Western blot analyses revealed that 6 hr exposure to taxane leads to dose dependent phosphorylation of EGFR or the activation of the EGFR-Akt pathway in MCF7/ADR cells, showing the synergistic effect; but not in MDA-MB-231 cells, which showed an additive/antagonistic effect. EGFR phosphorylation is known to be promoted by cellular stresses that threaten cell survival, such as irradiation or exposure to anticancer drugs.3, 11 This phenomenon plays an important role in the antiapoptotic response.3, 11 An interesting point is that gefitinib leads cancer cells toward apoptosis by blocking these activated EGFR-Akt pathways. Consequently, it is thought that cancer cells in which the EGFR-Akt pathway is not activated by taxane, such as MDA-MB-231 cells, do not show a synergistic effect. This supports the result of Campiglio et al.13, who showed that functional EGFR is necessary for the efficacy of gefitinib. Figure 4 lane 1 shows that there is no activated EGFR in a basal condition. This is well-grounded for resistance in gefitinib monotherapy.13 It remains unclear why taxane does not lead to EGFR phosphorylation in MDA-MB-231 cells, in contrast to the case for MCF7/ADR cells. However, since a genetic analysis was not performed in the present study, there may be a difference in the EGFR gene structure between the 2 cell types.14, 15 Campiglio et al. reported that HER2 overexpression as well as EGFR was also related to sensitivity to gefitinib.13 HER2 overexpression is observed in MCF7/ADR cells, but not in MDA-MB-231 cells. Therefore, it might be associated with the synergistic effect on MCF7/ADR cells. We sought to examine the possible changes in HER2 autophosphorylation in MCF7/ADR cells following exposure to taxane, but no apparent changes were observed (data not shown). Therefore, it is unclear at present whether HER2 takes part in the synergistic effect on MCF7/ADR cells.
It is known that the extent of EGFR overexpression is not correlated with sensitivity to gefitinib,13 as was also demonstrated in this study. But wild-type MCF7 cells, which do not overexpress either EGFR or HER2, are more resistant to gefitinib and its combination effect with taxane is strongly antagonistic (data not shown). Considering this result, it is thought that the expression of EGFR is a minimum requirement for the exertion of the combination effect.
Another possible important mechanism is participation of the ATP-binding cassette (ABC) transporter protein. Moreover, Kitazaki et al. reported that gefitinib directly inhibits the function of p-glycoprotein in MCF7/ADR cells and reverses its multidrug-resistance.19, 20, 21, 22 Indeed, these 2 mechanisms appear to be complimentarily related to the synergistic effect.
In the xenograft study, the combination therapy was more effective than monotherapy with each drug. For MCF7/ADR cells, the tumor inhibitory effect was significantly increased by the combination therapy, whereas the change in MDA-MB-231 cells was not significant (Fig. 2). These results are consistent with the results of the in vitro study.
Recently, Morelli et al. reported the sequence-dependent effect of a cytotoxic drug and EGFR inhibitor.23 Consistent with their reports, the apoptosis analysis using flow cytometry revealed that taxane followed by gefitinib was more effective than gefitinib followed by taxane. The earlier-mentioned Western blot analyses further support these results, since taxane exposure leads to activation of the EGFR-Akt pathway, and the subsequent administration of gefitinib would block this pathway; a mechanism that would lead to a stronger combination effect for taxane followed by gefitinib. However, a direct comparison of concurrent administration with sequential administration seems to be difficult because the ED50 values differ between the combination and mono-therapies, and the rate of apoptosis is low in a concurrent therapy such as gefitinib followed by taxane (data not shown).
Several reports focused on preclinical studies have shown the efficacy of combinations of gefitinib with other cytotoxic drugs, such as anthracycline agents, platinum or 5-FU.8, 9 For breast cancer in particular, Giardiello et al. reported that gefitinib enhances the taxane activity on bcl-2-overexpressing MCF7/ADR cells.7 They demonstrated the combination effect of gefitinib and taxane,7 but it remains unclear whether the effect is an additive or synergistic one. They also noted the relation of bcl-2 expression and the combination effect7, while our study focused on whether EGFR phosphorylation is related to it. Besides breast cancer cells, Koizumi et al. reported a synergistic effect of combination therapy with gefitinib and CPT-11 for colorectal cancer.10 Moreover, they revealed an interaction between the synergistic effect and phosphorylation of EGFR following exposure to CPT-11.10 Regarding combinations with molecular targeting agents, Warburton et al. studied a combination therapy of gefitinib and trastuzumab, but did not find any beneficial effect.24 Contrarily, Normanno reported the efficacy of this combination;25 however, the efficacy or not of the effect remains open to debate.
Recently, other preclinical studies have reported that gefitinib particularly exerts its growth inhibitory effect on ER-positive tamoxifen-resistant EGFR-overexpressing breast cancer cell lines, based on the evidence that increased expression of EGFR could lead to acquired anti-hormone-resistance and that gefitinib could block such evolution.26, 27, 28, 29, 30 MCF7/ADR and MDA-MB-231 cells are known to be ER-negative anti-hormone-resistant cell lines. Therefore, our present results do not appear to be associated with an anti-hormone-resistance mechanism. Moreover, it has been reported that specific mutations in the EGFR gene are correlated with the response to gefitinib.14, 15 It will be interesting to clarify whether EGFR mutations affect the combination effect observed in the present study.
Baselga et al. in respect to a clinical trial, reported a phase II study of gefitinib monotherapy in patients with advanced breast cancer. According to their results, no complete or partial responses have been observed.5 In respect to combination therapies of gefitinib and other cytotoxic drugs, such as anthracycline drugs or platinum, a phase I/II study completed by Fountzilas et al. found that a combination therapy of gefitinib, paclitaxel and carboplatin showed no acceleration of efficacy.31
Taking these reports together, combinations of gefitinib and other drugs such as taxane are generally favorable at preclinical studies, but their efficacies have not been demonstrated in clinical trials. The efficacy of the gefitinib and taxane combination therapy reported here remains unclear; as our present results revealed, this combination is not effective for all breast cancer cell lines that overexpress EGFR. Considering its application to clinical situations in the future, it is thought that identification of the breast cancer subgroups that show susceptibility to this treatment will be important. To achieve this, urgent attention needs to be given to identifying biomarkers that can predict the efficacy of the combination treatment.