Introduction
Inflammatory breast cancer (IBC) is a rare and highly metastatic variant of breast cancer with the poorest survival of all types of breast cancer[
1,
2]. IBC has shown the capacity to spread early, primarily through lymphatic channels and secondarily through blood vessels causing the typical inflammatory clinical signs. Characteristic clinical symptoms are rapid onset and progression of breast enlargement with overlying skin changes, such as diffuse erythema, edema or peau d’orange, tenderness, hardening, and warmth; a tumor mass may or may not be present[
3,
4]. IBC primarily affects younger women under the age of 50 at diagnosis, and is difficult to be detected as most patients do not present with a lump, but rather occurs as tumor emboli. At the time of diagnosis, most patients have lymph node metastases, and 30% of the patients have distal metastases including brain, bones, visceral organs and soft tissue with variable frequency, in contrast to 5% of patients with non-IBC[
5]. The lower survival rate of IBC patients may be due to the highly metastatic nature of the disease[
6].
Primary treatment of patients with IBC is typically multimodal involving neoadjuvant combination chemotherapy followed by surgery, adjuvant chemotherapy, or radiotherapy[
5]. The HER family has an important role in driving breast cancer. Epidermal growth factor receptor (EGFR) overexpression has been demonstrated as prognostic factors in IBC. Overexpression of epidermal growth factor receptor 2 (HER2) occurs during the stage of advanced tumor but whether the overexpression has a prognostic role in IBC has yet to be established[
7,
8]. Anti-HER2 therapies have shown benefit in IBC patients with HER2 amplification, which accounts for approximately 40% of IBC[
9]. However, therapeutic options for patients with ER-negative and HER2 non-amplified IBC are very limited. IBCs are predominantly basal-like or triple-negative (TN) as characterized by the estrogen receptor (ER)-negative, progesterone receptor (PgR)-negative and HER2 non-amplified status[
10]. EGFR is overexpressed in 30% of IBCs and 50% of TNIBCs[
2,
11]. IBC patients with EGFR-positive tumors have a lower overall survival rate than patients with EGFR-negative tumors, and EGFR overexpression in IBC is frequently associated with an increased risk of recurrence[
9]. EGFR overexpression is also correlated with large tumor size, aggressiveness and poor prognosis[
12,
13]. Thus, EGFR could be a potential therapeutic target in IBC and, in particular, in patients with EGFR-overexpressed IBC that currently has very limited treatment options.
Currently there are few human IBC cell lines available for studying this complex disease. Although available cell lines were derived from IBC patients, the molecular signatures among IBC cell lines are very distinct. SUM149 was developed from the primary tumor of IBC patient, but
in vivo xenograft model are unable to recapitulate the tumor emboli that are the signature of IBC in humans. We have recently developed a new IBC cell line, FC-IBC-02 that was derived from the pleural effusion fluid of a woman with secondary metastatic IBC[
14,
15]. FC-IBC-02 cells form tumor spheroids in suspension culture, a characteristic of cancer stem cells, and recapitulate the tumor emboli
in vivo xenograft models. SUM149 and FC-IBC-02 could be different representative models for studying the biology of IBC, both SUM149 and FC-IBC-02 cell lines are basal-like and ER/Pgr(-), EGFR-overexpressed and HER2 non-amplified.
AZD8931 was developed with the hypothesis that combined inhibition of EGFR, HER2, and HER3-mediated signaling may be more effective for clinical cancer treatment[
16]. Pharmacological profiling has shown that AZD8931 is a novel, equipotent, reversible small-molecule ATP competitive inhibitor of EGFR, HER2, and HER3 signaling. Previous results showed that AZD8931 was significantly more potent against EGFR, HER2 and HER3 signaling than other EGFR inhibitors such as lapatinib or gefitinib
in vitro. AZD8931 has shown greater antitumor activity in a range of xenografted models compared with lapatinib or gefitinib[
16]. In the present study, we examined the effects of AZD8931 on cell growth and apoptotic cell death of human IBC cells
in vitro. Further, we investigated the antitumor activity of AZD8931 alone or in combination with paclitaxel in EGFR-overexpressed and HER2 non-amplified IBC models.
Methods and materials
Reagents and cell culture
AZD8931 was synthesized and generously provided by AstraZeneca[
16]. SUM149 were obtained from Dr. Stephen Ethier (Kramanos Institute, MI, USA) and are commercially available (Asterand, Detroit, MI). SUM149 cells were cultured in Ham’s F-12 media supplemented with 10% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 5 μg/ml insulin and antibiotic-antimycotic. The FC-IBC-02 tumor cells were derived from primary human breast cancer cells isolated from pleural effusion of an IBC patient[
14,
15]. Human samples used in this study were acquired with approval of the Fox Chase Cancer Center’s Institutional Review Board. Importantly, written informed consent was obtained from each participant. FC-IBC-02 cells were cultured in DMEM/F12 media with 10% FBS and 1% L-glutamine and antibiotic-antimycotic.
Antibodies and immunoblot
Following treatment with AZD8931 at the indicated concentration and time points, immunoblotting was performed as previously described[
15]. In brief, cells were lysed in 1× lysis buffer (Cell signaling), and then the supernatant was collected by centrifuging at 10,000 rpm for 10 min at 4°C. Protein concentration was determined using the BCA protein assay reagent kit (Pierce, Rockford, IL). Equal amounts of protein from cell lysates were resolved by SDS-PAGE electrophoresis. The membranes were incubated at 4°C overnight with the following antibodies: mouse anti-EGFR (1:1000; Cell Signaling), rabbit anti-AKT and rabbit anti-phospho-AKT (1:1000; Cell Signaling), mouse anti-β-actin (1:5,000; Santa Cruz). After incubation with anti-mouse IgG horseradish peroxidase conjugated secondary antibody (1:5,000; Amersham Pharmacia Biotech), immunoreactive proteins were visualized by the enhanced chemiluminescence reagents.
Cell proliferation and apoptotic assay
SUM149 and FC-IBC-02 cells (2 × 103) were seeded in triplicate in a 96-well plate and cultured overnight. Cells were treated with AZD8931 at indicated concentration for 72 hrs. Cell proliferation was monitored at the indicated times, absorbance at 490 nm was measured using a microplate reader using the MTS assay (CellTiter 96 AQueous One Solution cell proliferation assay, Promega) according to the manufacturer’s instruction.
Apoptotic cells were measured by Annexin V staining. Cells (1 × 105) were treated with 1 μM AZD8931 for 48 and 72 hrs. Cells were harvested and labeled with Annexin V-PE and 7-amino-actinomycin D (7-AAD) (Guava Technologies Inc, Burlingame, CA) according to the manufacturer’s instructions. The samples were then analyzed by Guava system on a GuavaPC personal flow cytometer (Guava Technologies).
In vivo xenograft studies
The protocol was approved by FCCC institutional animal care and use committee (IACUC). SUM149 and FC-IBC-02 (3 × 106) cells were suspended in 200 μL of 1:1 ratio of phosphate-buffered saline/matrigel (BD Biosciences) and orthotopically injected into the mammary fat pads of six week old female C.B-17 severe combined immunodeficient (SCID) mice. Tumor volume was calculated from the formula TV = L*W*H*0.5236 where L, W, and H are the tumor dimensions in three perpendicular dimensions by caliper measurement. When tumor volumes were approximately 50 mm3 for SUM149 cells or 80 mm3 for FC-IBC-02 cells, the mice were randomly allocated into four groups (5 mice per group) and treatments were initiated. AZD8931 was suspended in a 1% (v/v) solution of polyoxyethylenesorbitan monooleate (Tween 80) in deionized water and given once daily by oral gavage at 25 mg/kg for 4 weeks. Paclitaxel solution was diluted in saline and given twice weekly by subcutaneously injection at 10 mg/kg. The control-group received 1% Tween 80 vehicle treatment. Mice were sacrificed at 33 days (SUM149) or 26 days (FC-IBC-02) post treatments. Tumors were surgically removed and weighed.
VeraTag analysis and immunohistochemical staining
Formalin fixed paraffin embedded sections of tumors from control animals were subjected to VeraTag™ analysis. A pair of antibodies, one conjugated to biotin and the other to a fluorescent molecule (VeraTag) suitable for analysis by capillary electrophoresis, bind to distinct epitopes on HER2, HER3 or PI3K. The VeraTag molecules are attached to the antibodies via photo-cleavable linkers. Methylene blue, conjugated to streptavidin, binds to the biotin-labeled antibody and is photo-activated by red-light. The released singlet oxygen, as a result of methylene blue catalyzed photosensitization, cleaves VeraTag molecules in close proximity to the antibody-biotin-streptavidin complex.
Tumor-bearing mice were treated with AZD8931 at 50 mg/kg/day for 4 days. Tumors were removed and fixed at 4 hrs after fourth dose. Formalin-fixed paraffin-embedded tumors were cut onto glass slides and processed for immunohistochemical (IHC) staining as previously described[
16]. In brief, antigen retrieval was performed on formalin-fixed, paraffin-embedded tumor sections and the following primary antibodies were used: total EGFR (DAKO PharmDx), total HER2 (DAKO Herceptest), total HER3 (CST clone D43D4), phospho-EGFR (Epitomics #1139-1), phospho-HER2 (CST #2243), phospho-HER3 (CST #4791), A polymer detection system (DAKO Envision + K4007) was used for secondary detection and sections were counterstained with Carazzi’s hematoxylin. Semiquantitative scoring was carried out by light microscopy by a pathologist (CW) for immunohistochemical brown staining on a four point scale (0+, none; 1+, weak; 2+, moderate; 3+, strong) and for percentage (%) distribution, to calculate an H-Score (sum of 1 x% 1+, 2 x% 2+, and 3 x% 3+). Cytoplasmic and membrane staining was recorded.
Statistical analysis
Quantitative data were expressed as mean ± SD. Analysis of variance (ANOVA) with Student’s t test was used to determine the significant differences among experimental groups, and P < 0.05 was considered significant.
Discussion
In this study, we have shown that AZD8931 significantly suppressed IBC cell growth in vitro and tumor growth in vivo in two IBC cell lines including a new cell line-FC-IBC-02 derived from pleural effusion of an IBC patient. AZD8931 could have the potential to increase the antitumor activity when used in combination with chemotherapy.
EGFR can be overexpressed in all subtypes of breast cancer, but it is more frequently overexpressed in basal-like and triple-negative breast cancer including IBC[
17‐
19]. A recent study showed that TNIBC is associated with poor overall survival and high locoregional relapse[
20]. EGFR-positive IBC was associated with a significantly worse survival rate and increased risk of recurrence than EGFR-negative IBC[
7,
8]. There are several specific inhibitors of EGFR including gefitinib, erlotinib and cetuximab, and others have been studied for the treatment of breast cancer including IBC in clinical trials[
21], but results so far remain controversial and disappointing. However, EGFR remains an important target for developing novel therapeutics because the options for TNIBC treatment are very limited.
Previous studies have shown that AZD8931 was significantly more potent in inhibiting cell growth
in vitro and tumor growth
in vivo across different cell line models including one human breast cancer cell line as compared with gefitinib or lapatinib[
16]. AZD8931 also significantly affected EGFR, HER2, and HER3 phosphorylation and downstream signaling pathways, apoptosis, and proliferation. In the present study, we extended the previous study to further evaluate the antitumor activity of AZD8931 alone or in combination with paclitaxel in preclinical models of EGFR-overexpressed and HER2 non-amplified IBC. The SUM149 cell line expresses high levels of EGFR and is considered a representative IBC preclinical model, in spite of the fact it was developed from patients with primary disease, who had not yet received neoadjuvant therapy. The newly developed FC-IBC-02 cell line is a more representative model for the IBC studies, particularly for evaluating progression and metastasis, since the cell line has been developed from a patient with advanced IBC. FC-IBC-02 cells formed tumor spheroids and were able to develop tumor with the presence of tumor emboli and metastasis in SCID mice[
14,
15]. FC-IBC-02 cells expressed a high level of EGFR and relatively low levels of total HER2 and HER2-HER3 heterodimers making an ideal model to evaluate EGFR-targeting therapies. As expected, AZD8931 significantly inhibited cell proliferation
in vitro and tumor growth of IBC cells
in vivo in orthotropic xenografted models. Since FC-IBC-02 cells also expressed an intermediate level of HER3, AZD8931 could have potential to inhibit tumor growth through inactivation of HER2/HER3 and its downstream pathway. Previous study has shown that AZD8931 caused a significant increase of apoptotic protein expression in xenografted tumors[
16]. Here, we showed a significant induction of apoptotic cell death following AZD8931 treatment
in vitro in IBC cells.
Most significantly, in current IBC models, we showed that AZD8931 monotherapy significantly inhibited tumor growth and the combination of paclitaxel + AZD8931 resulted in the highest levels of tumor growth inhibition
in vivo in both cell lines (Figure
4). The most common treatment for IBC is multimodal involving neoadjuvant combination chemotherapy followed by surgery, adjuvant chemotherapy, or radiotherapy[
5]. Conventional chemotherapy regimens are not sufficient for the treatment of IBC, particularly for TNIBC. Targeted therapy against HER2 is one promising strategy for the treatment of IBC patients with HER2 amplification. Several EGFR targeted therapies including small molecule inhibitors and anti-EGFR antibodies have been evaluated in preclinical and clinical studies[
21‐
25]. Patients with EGFR expressing tumors did not respond to EGFR-targeted therapy, which suggests that EGFR expression alone does not indicate tumor cell growth dependence on the EGFR pathway. One study indicated that the significant interactions between EGFR and other alternative signaling pathway kinases, such as c-MET and IGF-1R are linked to resistance to targeted therapies[
26]. Thus, future studies are warranted to consider combining of EGFR-targeted therapy with drugs targeting other alternate signaling pathways to improve efficacy. Several antibodies targeting EGFR have also been investigated for their efficacy in patients with TNBC, some results have showed the clinical benefit in combination with chemotherapy drugs for patients with TNBC[
27,
28].
Metastasis is the primary cause of breast cancer mortality. IBC is characterized by locally advanced disease and high rates of metastasis even after multimodality treatments[
29]. In IBC, inflammation is associated with the invasion of aggregates of tumor cells defined as tumor emboli, into the dermal lymphatics causing an obstruction of the lymph channels[
30]. Currently, the molecular pathways driving the early development of metastasis in IBC remain poorly characterized. EGFR family and its downstream signaling pathways are known to promote cell migration, angiogenesis, invasion, and metastasis[
22]. Previous studies have shown that the EGFR inhibitor erlotinib (Tarceva™) significantly inhibited cell motility, invasiveness, tumor growth, and spontaneous lung metastasis in EGFR-expressing IBC models[
31]. Further therapeutic studies are warranted to examine the effects of AZD8931 on the invasiveness and metastasis of IBC.
Competing interests
Teresa Klinowska, Emily Foster and Chris Womack are employees of and stockholders in AstraZeneca. All other authors declare that they have no competing interests.
Authors’ contributions
ZM performed the experiments, analyzed the data and wrote the manuscript. TK, EF and CW assisted with immunohistochemical staining, analysed the data, reviewed and finalized the manuscript. XD and SF assisted with in vivo experiments. MC conceived of the study and finalized the manuscript. All authors read and approved the final manuscript.