Ad-p53 enhances the sensitivity of triple-negative breast cancer MDA-MB-468 cells to the EGFR inhibitor gefitinib

Triple-negative breast cancer (TNBC) accounts for 20% of all molecular subtypes of breast cancer. Neither endocrine nor anti-HER2 molecular targeting treatment yield promising results. At present, epidermal growth factor receptor (EGFR) inhibitor, as a single agent, is unable to obtain encouraging results in the treatment of TNBC, even though most of these tumors overexpress EGFR. In the present study, we used recombinant human p53 adenovirus (Ad-p53) and EGFR inhibitor gefitinib to treat the TNBC cell line MDA-MB-468. The combined treatment of gefitinib and Ad-p53 synergistically inhibited the proliferation of MDA-MB-468 cells; it restrained colony formation, enhanced cellular apoptosis and arrested the cell cycle in vitro, and decreased tumor burden of xenografts in nude mice. Western blot analysis revealed that Ad-p53 and gefitinib in combination significantly downregulated the phosphorylation of protein kinase B (p-Akt) and upregulated caspase-9 and cleaved caspase-3, while there were minimal effects on the expression of extracellular signal-regulated kinase (ERK) and phosphorylation of ERK (p-ERK). These results suggest that Ad-p53 may block the PI3K/Akt pathway rather than the Raf/MEK/ERK pathway. Importantly, wild-type p53 was able to reverse the drug resistance of MDA-MB-468 cells to gefitinib through inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The apoptotic activity induced by this combined treatment may be regulated by caspase cascade-dependent activation.


Introduction
Triple-negative breast cancer (TNBC) is the most invasive subtype and accounts for 20% of all molecular subtypes of breast cancer (1). Owing to its lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2/neu), neither endocrine nor anti-HER2 molecular targeting treatment yield promising results, and standard chemotherapy is the backbone of systemic treatment. Over the past decade, preclinical findings have suggested various protein targets and pathways as possible TNBC treatments, such as growth factor receptors, proteins involved in cellular DNA repair capacities and epigenetic regulation (2). Nevertheless, these novel molecular targeting treatments have achieved little clinical progression and there are no effective therapeutic targets to date available against TNBC (1,3).
EGFR overexpression in breast cancer is correlated with large tumor size, more stem-cell like properties and poor prognosis (4). Its overexpression is present in more than 50% of TNBC cases, which is more frequent than in other subtypes (5). Gefitinib, approved for lung cancers, is a tyrosine kinase inhibitor (TKI) that targets the adenosine triphosphate binding site in the cytoplasmic domain of EGFR (6). Unfortunately, gefitinib has shown little efficacy in most clinical studies of breast cancer (7)(8)(9). The complex interplay downstream pathways of EGFR with other cellular components lead to their continued activation and insensitivity toward EGFR inhibitors (10). In this regard, many studies have reported that both the Raf/MEK/MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways together promote the resistance of cancer cells to gefitinib (11,12).
p53 is critical to the induction of cell cycle arrest, DNA repair, cellular apoptosis and senescence in response to a wide array of stimuli (13). Its mutation has been related to the poor prognosis and drug resistance of cancer cells (14,15). The p53 mutation is common in TNBC (16,17), and the mutant p53 endows tumor cells with invasive and metastatic abilities (15,18). Huang et al (19) reported that p53 regulates the sensitivity to EGFR inhibitors and induces apoptosis by modulating EGFR downstream signaling in lung cancer cells.

Ad-p53 enhances the sensitivity of triple-negative breast cancer MDA-MB-468 cells to the EGFR inhibitor gefitinib
Recombinant human p53 adenovirus (Ad-p53), a replication incompetent human type 5 adenovirus whose E1 region is replaced by an expression cassette containing the human wild-type p53 cDNA (20), was shown to restore p53 activity in p53-deficient hepatocytes, therefore inducing G 2 /M arrest and apoptosis. However, its effect was not apparent as a single agent treatment in breast cancer. In the present study, Ad-p53 was used in combination with gefitinib to treat a TNBC cell line in vitro and in vivo. A significant sensitivity toward gefitinib was observed after p53 activity was restored.
Cell culture. The human breast cancer cell line MDA-MB-468 was seeded in DMEM supplemented with 10% fBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine. Cells were grown in a humidified atmosphere of 5% CO 2 at 37˚C.
MTT assay. The MDA-MB-468 cell line was plated in 96-well plates in triplicate with 3x10 3 cells/well. Next, the cells were treated with Ad-p53 at a multiplicity of infection (MOI) of 100 for 24 h, while the vehicle-treated control cells were incubated with 0.5% of DMSO. Next, fixed-ratio concentrations (0, 1.25, 2.5, 5, 10 and 20 µM) of gefitinib were administered. After 48 h, 20 µl of 5 mg/ml MTT solution was added to each well for 4 h. MTT was carefully aspirated and replaced with 200 µl DMSO/well. Absorbance was measured on a Bio-Rad 680 microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm, and the results were reported relative to a reference wavelength of 630 nm. The inhibitory rate of growth was calculated according to the following equation: Growth inhibition rate = (1 -the mean OD of the samples/the mean OD of the controls) x 100%.
Clonogenic survival assay. MDA-MB-468 cells were plated in 6-well plates (300 cells/well) and treated with 3 µl of gefitinib and/or an MOI of 100 of Ad-p53 for 48 h. Then, the cells were washed with phosphate-buffered saline (PBS) and replaced with fresh medium. After 14 days, the cells were fixed with 70% ethanol and stained with crystal violet. The number of colonies (>50 cells) was counted under a microscope. Survival was expressed relative to the untreated controls.
Measurement of apoptotic cells. The cells were treated with either gefitinib (3 µM) or Ad-p53 (MOI of 100) alone or in combination for 48 h, and then washed twice with cold PBS. An indirect immunofluorescence assay was performed using the Annexin V-FITC apoptosis kit according to the manufacturer's instructions. The samples were assessed by flow cytometry (fACSCalibur; BD Biosciences) using CellQuest software within 1 h.
Analysis of the cell cycle distribution. MDA-MB-468 cells (1x10 6 ) were treated with gefitinib (3 µM) and/or Ad-p53 (MOI of 100) for 48 h and then fixed in 70% of ethanol. After being washed twice with PBS, the cells were stained with propidium iodide (PI) for 30 min. Flow cytometric analysis was performed on the fACSCalibur (BD, Bedford, MA, USA). ModifIT software (Topsham, ME, USA) was used to analyze the cell cycle distribution.
Western blot analysis. Cells were treated with gefitinib (3 µM) and/or Ad-p53 (MOI of 100), and then lysed in RIPA buffer (50 mM Tris pH 7.4, 0.15 M NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) with 1 mM PMSf after 48 h. Supernatants were recovered, and the total protein concentration was detected using the BCA protein assay kit. The proteins were applied to sodium dodecyl polyacrylamide gel electrophoresis, and electrophoretically transferred onto a PvDf membrane (Amersham Biosciences, Buckinghamshire, UK). Appropriate antibodies were applied to determine the membrane. The band density was normalized to GAPDH.
Effect of gefitinib and Ad-p53 on the growth of MDA-MB-468 xenografts. Sixteen healthy BALB/C female nude mice (4 weeks old) were obtained from the Beijing HfK Bioscience Company. All animals were housed and treated according to the guidelines outlined by the Institutional Animal Care and Use Committee of Shandong Cancer Hospital and maintained under a germ-free controlled environment. The mice were grown until 6 weeks before being subcutaneously injected in the right axillary with MDA-MB-468 cells (1x10 7 ). After the tumor xenograft had grown to 1 cm in diameter, the mice were randomized into four treatment groups: i) vehicle; ii) Ad-p53; iii) gefitinib; and iv) the combination. Ad-p53 (1x10 10 VP) was dissolved in 100 µl physiological saline. All were injected peri-/intratumorally. Gefitinib (100 mg/kg) was administered via oral gavage. The combined treatment was the same as the single agent treatment. Physiological saline was administered to the vehicle-treated group. The greatest longitudinal diameter (a) and the greatest transverse diameter (b) were measured on day 0, 3, 6, 9, 12 and 14. Tumor volume (Tv) was calculated by the following formula: TV = 1/2ab 2 . Tumor inhibition rate (TIR) = (average TV of the vehicle-treated group -average TV of the experimental group)/average TV of the vehicle-treated group x 100%.
Statistical analysis. Statistical analysis was carried out using SPSS 17.0 statistical software. Significant differences between two groups (Table I) were conducted by t-test. The analysis of variance (>2) was analyzed by one-way analysis of variance (ANOvA) to determine statistical significance. Tukey's multiple comparison was applied to compare two subsequent samples. All statistical tests were two-sided. P<0.05 was considered to indicate a statistically significant result.

Results
Ad-p53 enhances the cytotoxic effect of gefitinib on MDA-MB-468 cells. Gefitinib alone or combined with Ad-p53 inhibited the proliferation of MDA-MB-468 cells in a dosedependent manner (Fig. 1). Combined treatment of Ad-p53 with gefitinib synergistically inhibited the proliferation of the MDA-MB-468 cells with a lower IC 50 value of 4.3 µM; in comparison, cells that were not pretreated with Ad-p53 were relatively resistant to gefitinib with a higher IC 50 value of 8.5 µM (Table I). When extra wild-type p53 gene was integrated into the MDA-MB-468 cells, the cancer cells became more sensitive to gefitinib.
Combination of Ad-p53 and gefitinib inhibits clonogenic cell survival. A clonogenic assay was performed to further investigate the separate and combined effects of Ad-p53 and gefitinib on cell proliferation. Survival was expressed relative to the vehicle-treated cells. Ad-p53 or gefitinib could only slightly weaken colony formation. An approximate 64.4 and 48.5% clonogenic survival rate was detected when cells were treated with Ad-p53 and gefitinib, respectively. Nevertheless, when Ad-p53 and gefitinib were administered in combination, colony formation was significantly decreased with a lower clonogenic survival rate of 24.5% ( fig. 2).
p53 is required for gefitinib-induced cellular apoptosis and cell cycle arrest. MDA-MB-468 cells were treated with either drug alone or in combination with Ad-p53 for 48 h. According to flow cytometry, the apoptosis rate in the Ad-p53, gefitinib, combination and vehicle-treated group was 17.4, 20.5, 32.6 and 8.5%, respectively. Treatment with Ad-p53 or gefitinib alone slightly induced cellular apoptosis (fig. 3). The combined treatment increased cellular apoptosis to 4-fold when compared with the vehicle-treated control (Fig. 3). As shown in fig. 4, gefitinib induced G 2 /M phase arrest from 21.9   to 45.4% compared to the vehicle-treated groups; G 2 /M phase increased to no more than 31.5% after exposure to Ad-p53. In comparison, when the combined treatment was performed, G 2 /M arrest was enhanced evidently from 21.9 to 65.3%.
Effect of treatment with Ad-p53 and/or gefitinib on intracellular signaling. Ad-p53 or gefitinib, as a single agent, produced a slight reduction in the levels of p-Akt (S473) in the MDA-MB-468 cells ( fig. 5). Surprisingly, combined treatment synergistically produced a significant reduction in p-Akt (S473) (fig. 5). The expression levels of caspase-9 and cleaved caspase-3 were low in the vehicle-treated cells. In contrast, when Ad-p53 or gefitinib was used as a single agent, the levels of caspase-9 and cleaved caspase-3 increased (Fig. 5). Nevertheless, when they were administered in combination, the levels were markedly increased. However, none of the treatments led to an obvious change in ERK and p-ERK. These results suggest that Ad-p53 may enhance the sensitivity of MDA-MB-468 cells to gefitinib by blocking the PI3K/Akt pathway rather than the Raf/MEK/ERK pathway.

Efficacy of the treatments on the MDA-MB-468 xenografts in vivo.
Ad-p53 and gefitinib were administered to the MDA-MB-468 xenografts and tumor volume was measured periodically. Ad-p53 or gefitinib alone caused slight tumor volume shrinkage (Fig. 6). The tumor inhibition rate (TIR) in the Ad-p53, gefitinib and combination group was 35.7, 28.7 and 74.4%, respectively. The xenografts were significantly reduced in size when Ad-p53 and gefitinib were administered simultaneously (Fig. 6).

Discussion
p53 is a tumor suppressor gene known to be the most commonly altered gene in human cancer (21). Due to the aggressiveness  of p53 (23), and disrupting the interaction between EGFR and colony stimulating factor 1 receptor (CSf-1R) (24). All these approaches may remedy the defect of p53 dysfunction. The p53 mutation is kept at a low level in breast cancer, yet mutant p53 is more prevalent in TNBC (25). Perhaps this is the reason why TNBCs progress rapidly and have a poor prognosis. MDA-MB-468 cells harboring EGfR overexpression, as a gefitinib-resistant model, have been demonstrated in different studies (26,27). Small-molecule tyrosine kinase inhibitors (TKIs) against EGFR have been evaluated in breast cancer. Baselga et al (9) reported that gefitinib demonstrated minimal single-agent activity when treating metastatic breast cancer. Resistance to EGFR inhibitors present a huge obstacle to breast cancer patients (3). In the present study, in order for us to explore whether p53 increases the sensitivity of an EGFR inhibitor, Ad-p53 and EGfR TKI gefitinib were used to treat a TNBC cell line. Notably, the sensitivity of MDA-MB-468 cells to gefitinib was significantly increased when they were pretreated with Ad-p53. The cell proliferation assay indicated that when cells were treated with gefitinib, the IC 50 value of Ad-p53infected cells was almost half as much as the vehicle-treated cells (Fig. 1, Table I). Furthermore, the results of the in vitro experiments, such as the clonogenic and apoptosis assays and cell cycle distribution, revealed that p53 enhanced the sensitivity to gefitinib by inhibiting colony formation ( fig. 2), by regulating cellular apoptosis (Fig. 3) and by inducing cell cycle arrest ( fig. 4).
MDA-MB-468 cells pretreated with Ad-p53 showed enhanced sensitivity to gefitinib with downregulation of p-Akt, according to western blotting results, while ERK and p-ERK exhibited little or no change. Both the PI3K/Akt and Raf/MEK/ERK pathways are downstream of EGfR activation. The former can resist apoptosis, while the latter is involved mostly in anti-apoptosis as well as cell proliferation (28,29). MDA-MB-468 cells possess an elevated level of p-Akt, and their persistent activation of p-Akt is relevant to their resistance to EGFR inhibitors (12). Previous studies suggest that p53 may participate in the modulation of the PI3K/Akt and Raf/MEK/ ERK pathways in cancer cells (30)(31)(32). Our data, however, indicate that Ad-p53 may interfere with the PI3K/Akt pathway rather than the Raf/MEK/ERK pathway, leading to an increase in the sensitivity to gefitinib. Moreover, caspase-9 is a crucial component of the apoptosis pathway, and activated caspase-9 initiates the caspase cascade by driving the activity of downstream caspases such as caspase-3, -6 and -7. In the present study, caspase-9 and cleaved caspase-3 increased synergistically when MDA-MB-468 cells were exposed to both Ad-p53 and gefitinib in comparison to the single agent treatment, suggesting that caspase-mediated apoptosis was triggered in this TNBC treatment. Similarly, Chang et al (33) reported that gefitinib induced apoptosis via a p53-dependent pathway in a lung cancer cell model, which was accompanied by the upregulation of pro-apoptotic molecules (such as fas and PUMA) and the downregulation of anti-apoptotic molecules (such as XIAP and survivin). Therefore, the synergistic effect of the combined treatment could be attributed to the effect of gefitinib in triggering caspase-dependent apoptosis via inhibiting the PI3k/Akt pathway potentiated by Ad-p53. Recently, Yu et al (34) reported that caspase-dependent apoptosis and inactivation of the PI3K/ Akt pathway were the main apoptotic mechanisms of human gastric carcinoma AGS cells. Further studies of expanded TNBC cells should be conducted in order to obtain more detailed mechanisms related to the dysfunction of the PI3K/Akt pathway and caspase cascade activation.
Ad-p53 is effective for treating numerous malignancies, including colon, glioma, lung, ovarian and head and neck tumors (35)(36)(37)(38)(39). In the present study, the in vivo experiment was designed to mimic a clinical situation in order to document whether Ad-p53 and gefitinib together synergistically inhibit the growth of transplanted breast tumors in nude mice. According to our results, the combination of Ad-p53 and gefitinib significantly alleviated the bulk of the tumor burden in the nude mice. These results may pave the way for the clinical treatment of patients who are resistant to EGfR TKIs. Moreover, when these two agents are used together, they not only compensate for the shortcomings of one another, but also maximize their benefit. For example, their effective combination could potentially reduce EGFR inhibitor-related side-effects, while exhibiting better antitumor abilities, thus enhancing the quality of life of these patients. To further judge the clinical applicability of both agents, clinical trials should be conducted in follow-up studies.
In conclusion, finding an effective therapeutic regimen to cure TNBCs has become an urgent need; TNBCs are diagnosed in nearly 20% of breast cancer patients, most of whom are young and have a high rate of recurrence. In the present study, we demonstrated the feasibility of combining Ad-p53 and the EGfR inhibitor gefitinib to treat TNBC cells, which are relatively resistant to gefitinib. Wild-type p53 has a good application perspective for sensitizing EGFR inhibitors both in vitro and in vivo. This may stimulate researchers to restore wild-type p53 in order to enhance the effectiveness of EGFR targeted therapies.