Introduction
Human epidermal growth factor receptor 3 (HER3) is a single membrane-spanning receptor that exerts its function through heterodimerization with other HER family receptors [
1-
3]. Activation of HER3-containing heterodimers leads to HER3 phosphorylation and subsequent activation of signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt and RAF/MEK/extracellular signal-regulated protein kinase (ERK) pathways, that drive tumor cell proliferation and promote survival [
2,
4]. Increasing evidence has identified HER3 as one of the most potent oncogenic factors in promoting breast cancer tumorigenesis [
5]. In animal models of breast cancer driven by HER2, HER3 expression and phosphorylation are upregulated [
6,
7]. Knockout of HER3 impairs the ability of HER2 to induce tumor formation in mouse mammary tumor virus-HER2 (MMTV-HER2)-driven mouse models [
8]. In addition, overexpression of HER3 has been shown to significantly enhance the invasiveness of breast cancer cells. Specifically, HER3 promotes invasion and metastasis through its ability to activate the PI3K pathway [
9]. The clinical impact of HER3 is indicated by the observation that increased HER3 expression and the detection HER2/HER3 dimers have prognostic significance in breast cancer [
10,
11].
Therapies targeting epidermal growth factor receptor (EGFR) and HER2 have been extensively developed. Unfortunately, clinical efficacy is not satisfactory as drug resistance reduces durable responses. Upregulation of HER3 has been found as one of the major mechanisms underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors (for example lapatinib, gefitinib, erlotinib) and to endocrine therapy in the treatment of breast cancer [
12-
14]. For example, it has been shown that the expression of HER3 ligand heregulin (HRG) as well as activation of HER3 signaling is involved in resistance to anti-estrogen therapies
in vitro and
in vivo [
15-
17]. It has been suggested that effective therapies against HER2 require simultaneous targeting of HER3 [
18]. Thus, mounting evidence highlights the importance of targeting HER3 to decrease breast cancer mortality [
3].
In this report, we engineered an image-based screening platform using membrane localized HER3-yellow fluorescent protein (YFP) to identify small molecules that promote HER3 internalization and degradation. Using this platform, we screened a library of Food and Drug Administration (FDA) and foreign regulatory agency-approved drugs, and identified that perhexiline, an anti-anginal drug that inhibits mitochondrial carnitine palmitoyltransferase I (CPT-1) [
19], promotes HER3 internalization and downregulation, inhibits signaling downstream of HER3, and inhibits cancer cell proliferation
in vitro and
in vivo.
Materials and methods
Reagents and antibodies
Perhexiline maleate salt was purchased from Sigma-Aldrich (St. Louis, MO, USA). Neuregulin was purchased from R&D Systems (Minneapolis, MN, USA). The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): HER2 (3B5), HER3 (C-17), and ubiquitin. The pAkt (Ser473), pERK1/2, total EGFR, and Alexa Fluor™ 488 Conjugate Flag antibodies were purchased from Cell Signaling (Beverly, MA, USA). The antibodies were used at a 1:500 dilution in Western blotting. LysoTracker™ Red DND-99 was purchased from Invitrogen (Grand Island, NY, USA).
Cell culture
HEK293 and U2OS cells were cultured in Minimum Essential Medium (MEM), SK-BR-3 cells were cultured in McCoy’s 5A medium, and MDA-MB-468 cells were cultured in Leibovitz’s L-15 medium. AU565 and BT474 cells were cultured in RPMI-1640 medium. All media were supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA, USA), 200 U/ml penicillin, and 50 ng/ml streptomycin (Invitrogen, Grand Island, NY, USA). Cells were grown at 37°C in 5% CO2 except for the MDA-MB-468 cells that were grown at 37°C without CO2. All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA).
Expression constructs
The cDNA clone of human HER3 (pCMV-sport6-ERBB3, IMAGE: 6147464) was obtained from the Mammalian Gene Collection (MGC) through Open Biosystems (GE Dharmacon, Lafayette, CO, USA). To generate the C-terminal YFP-tagged HER3 (HER3-YFP), the coding region of HER3 was amplified using polymerase chain reaction (PCR) and subcloned in-frame into pcDNA3.1-mYFP vector (a gift from Roger Y Tsien, University of California at San Diego, USA). The primers used for PCR amplification were 5′-GGGGTACCGGAGTCATGAGGGCGAACGACGCTC −3′ and 5′-ATAAGAATGCGGCCGCGTTCTCTGGGCATTAGCCTTGGG −3′, and the HER3 fragment was cloned into the Kpn I and Not I sites on the vector. In order to delete the nuclear localization sequence (NLS2, ‘RRRR’) in HER3, site-directed mutagenesis experiments were performed using HER3-YFP as the template, and the primers used were: 5′-GAGTATGAATACATGAACCACAGTCCACCTCATCCC −3′ and 5′-GGGATGAGGTGGACTGTGGTTCATGTATTCATACTC −3′. To generate the Flag-tagged HER3ΔNLS2 construct, the coding sequence was amplified by PCR (primers used were: 5′-GGGGTACCGAGGGCGAACGACGCTCTG-3′and 5′-GCTCTAGATTACGTTCTCTGGGCATTAGC-3′) and subcloned into the Kpn I and Xba I sites on the pFlag-CMV3 vector (Sigma-Aldrich, St Louis, MO, USA). All constructs were verified by sequencing.
Imaging-based primary screening assay
Primary screening assays were performed as previously described [
20,
21]. Briefly, U2OS cells stably expressing HER3ΔNLS2-YFP were treated with compounds from a library containing approximately 1,200 FDA and foreign regulatory agency-approved drugs and drug-like tool compounds (Prestwick Chemical Illkirch-Graffenstaden, France). Cells were incubated with each compound for 6 hours at 37°C prior to fixation in phosphate-buffered saline (PBS) containing 4% paraformaldehyde and 0.002% of the fluorescent nuclear stain DRAQ5. Plates were stored at 4°C until analysis on an ImageXpress Ultra high-throughput imaging system (Molecular Devices, Sunnyvale, CA, USA) equipped with a 488 nm argon laser for imaging GFP and a 568 nm krypton laser for imaging DRAQ5. All imaging data were verified by visual inspection and a Z′ factor of 0.44 was calculated for the robustness of the assay.
Immunofluorescence staining and imaging analysis
U2OS cells stably expressing HER3ΔNLS2-YFP plated on 35-mm, poly-D-lysine-coated, glass-bottom microwell dishes (MatTek Cultureware, Ashland, MA, USA) were treated with dimethyl sulfoxide (DMSO) or perhexiline for the indicated time at 37°C and followed by fixation with 4% paraformaldehyde. HEK293 cells grown in microwell dishes were transfected (Fugene6; Roche Diagnostics Corp., Indianapolis, IN, USA) with Flag-HER3ΔNLS2, and 24 hours post-transfection cells were incubated with Alexa Fluor™ 488 Conjugate Flag antibody in culture medium on ice for 30 minutes. After washing out unbound antibodies, cells were incubated with perhexiline or DMSO in culture medium at 37°C for 1 hour followed by fixation. To detect endogenous HER3 receptors, MDA-MB-468 cells were allowed to grow for 24 hours and then treated with DMSO or perhexiline for the indicated time at 37°C before fixation in 4% paraformaldehyde. Fixed cells were permeabilized and blocked in blocking buffer (5% bovine serum albumin (BSA) with 0.2% saponin in PBS) for 20 minutes at room temperature and washed in PBS. Where indicated, cells were incubated with HER3 antibody in blocking buffer for 1 hour at room temperature and subsequently incubated with the Alexa Fluor™ 488-conjugated goat anti-rabbit secondary antibody (Invitrogen, Grand Island, NY, USA) in blocking buffer for 1 hour at room temperature. The slides were mounted in mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA) and examined using a LSM 510-Meta confocal microscope (Carl Zeiss, Thornwood, NY, USA) equipped with 40× and 100× apo chromat objectives. YFP was excited using a 488-nm argon laser line. Images were processed using the LSM software Image Browser (Carl Zeiss, Thornwood, NY, USA).
Assay of HER3 degradation and ubiquitination
MDA-MB-468 or SK-BR-3 cells seeded into 6-well plates (1.5 × 105 cells/well) were allowed to grow for 24 hours in the complete growth medium. Cells were subsequently treated with DMSO or perhexiline (10 μM) for the indicated time. Cell lysates were prepared in 2 × SDS sample buffer and subjected to Western blotting analysis. For ubiquitination assays, cells cultured and treated as described were collected into glycerol lysis buffer (50 mM Hepes, 250 mM NaCl, 0.5% NP40, 10% glycerol, and 5 mM ethylenediamine tetraacetic acid). Cell lysates were incubated with agarose-conjugated anti-HER3 antibody overnight, washed three times with the glycerol lysis buffer, and subjected to Western blotting analysis.
Western blotting
The protein samples were subjected to SDS-PAGE using 4 to 12% Novex™ Tris-Glycine Gels (Invitrogen, Grand Island, NY, USA), transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) blocked with 5% nonfat milk powder in TBS-0.2% Tween-20 for 30 minutes, followed by incubation with primary antibodies and then horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA). The ECL signals were detected using SuperSignal substrate (Pierce Biotechnology, Rockford, IL, USA) and quantified using ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).
Cell proliferation assay
The breast cancer cell lines MDA-MB-468, SK-BR-3, AU565, and BT474 were used in the cell proliferation assay. The cells were plated at 3,000 cells per well into 96-well plates and treated with compounds for 72 hours, at which point the cell proliferation was measured using the colorimetric MTS assay (Promega, Madison, WI, USA). For the dose–response assays, the cells were treated with perhexiline ranging from 0.5 to 10 μM.
To examine the combinational effect of perhexiline and lapatinib, the cells were treated with perhexiline and lapatinib alone or in combination. For the MDA-MB-468 cells, the ratio of perhexiline to lapatinib was 1:1. For the SK-BR-3, AU565, and BT474 cells, the ratio was 20:1. Experiments were performed in three replicates and performed three times. Values were normalized as a percentage of DMSO-treated cells. The Combination Index (CI), which quantifies the degree of synergism in a drug combination, was obtained using the method of Chou and Martin in the software CompuSyn.
Testing the antitumor effect of perhexiline in vivo
All animal experiments were followed animal protocol A294-11-11, which was reviewed and approved by the Duke Institutional Animal Care and Use Committee. MDA-MB-468 tumor cells (1 × 106/mouse) were injected into the flank of SCID mice on day 0. On day 7, oral gavage treatment with perhexiline (0, 400 mg/kg) was initiated, and repeated for 5 days a week for 4 weeks. Tumor diameter was measured every 3 to 4 days. Each group consisted of five mice. Student’s t test was used to analyze differences in tumor volumes at each perhexiline concentration compared to vehicle control. Differences at P <0.05 were considered statistically significant.
Discussion
Collectively, we report the development of a novel approach to inhibit HER3 signaling via the application of an imaging-based HER3 internalization assay to identify small molecule inhibitors of HER3-mediated signaling. This robust assay allows high-throughput screening to discover both small molecules and antibodies targeting HER3. Moreover, it overcomes the barrier of developing HER3-targeted small molecule therapies imposed by the fact that HER3 lacks active kinase activity. To our knowledge, perhexiline is the first small molecule identified that promotes selective HER3 degradation and inhibition of HER3-mediated signaling by inducing receptor internalization. Consistent with this mechanism, perhexiline also inhibits breast cancer cell proliferation in vitro and tumor growth in vivo and synergizes with lapatinib to inhibit breast cancer cell growth.
Increasing evidence has identified HER3 as one of the most potent oncogenic factors in promoting breast cancer tumorigenesis [
5]. In animal models of breast cancer driven by HER2, HER3 expression and phosphorylation are upregulated [
6,
7]. In addition, overexpression of HER3 has been shown to significantly enhance the invasiveness of breast cancer cells. Specifically, HER3 promotes invasion and metastasis through its ability to activate the PI3K pathway [
9]. The clinical impact of HER3 is indicated by the observation that increased HER3 expression [
10] and the detection HER2/HER3 dimers [
11] have prognostic significance in breast cancer.
With the exception of HER3, the HER family of membrane receptors all have intrinsic tyrosine kinase activity. This kinase activity enabled successful small molecule drug discovery approaches by targeting the kinase enzyme activity with inhibitors that bind in the ATP active site. Given HER3 is not an active kinase enzyme, small molecule approaches to inhibit its signaling activity was not considered feasible. Pertuzumab, a monoclonal antibody that blocks dimerization of HER2 and HER3 receptors, was approved by the FDA for concurrent use in combination with trastuzumab and docetaxel [
25]. It provides a proof of concept of targeting HER3 as an effective therapy. However monoclonal antibodies against HER3 have not demonstrated clinical benefits as single agents based on published data as summarized in recent reviews [
26,
27]. Other approaches targeting HER3 are demanded in clinic.
Perhexiline is an anti-angina drug with the inhibitory properties against the enzyme mitochondrial CPT-1. The plasma concentration of perhexiline in humans within its therapeutic range is about 2 μM [
28]. Distribution studies indicate that perhexiline is preferentially distributed in tissues. For example in heart, the perhexiline concentration is at least 10-fold higher than in serum [
19,
29]. The distribution of perhexiline into tumors versus other tissues is unknown; the concentration of perhexiline in tumors needs to be determined.
The anticancer properties of perhexiline are not well known. It has been reported that perhexiline has an effect on adriamycin-resistant human breast cancer cells. Simultaneous exposure to tamoxifen or perhexiline decreased resistance to adriamycin in a clonogenic assay by an undefined mechanism [
30]. Here, we report the anti-breast cancer properties of perhexiline through HER3 degradation and HER3-mediated signaling inhibition. The synergistic inhibitory effect of perhexiline with lapatinib on tumor growth may provide immediate therapeutic benefits for breast cancer patients with drug resistance and metastasis.
In summary, our findings represent a proof of principle by demonstrating the ability of a small molecule to selectively downregulate HER3 but not other epidermal growth factor receptor (EGFR) family members and inhibit downstream signaling in breast cancer cells and tumors. As a clinically used drug, perhexiline may be administrated alone, or in combination with other existing therapies such as lapatinib, to overcome drug resistance and metastasis in cancer treatment. Perhexiline is a first-in-class small molecule that targets HER3. Optimization of its pharmaceutical properties could provide improved derivatives with greater benefit to breast cancer patients. Clinical evaluation of perhexiline and its improved derivatives may directly lead to a new therapy to treat breast cancer and other HER3-dependent cancers.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XR participated in the design of the study, data acquisition and analysis, and drafting the manuscript. JW participated in the design of the study, data acquisition and analysis, and drafting the manuscript. TO participated in the design of in vivo experiments, data acquisition, analysis and interpretation of data, and drafting the manuscript. RAM participated in the analysis and interpretation of data, assisted in conception of the mechanism studies, conducted synthesis and purity test agent, and assisted in drafting the manuscript. MAM participated in the data analysis and interpretation of data, and drafting the manuscript. LSB participated in conception, analysis and interpretation of data, and drafting the manuscript. HKL participated in the conception and design of study, analysis and interpretation of data, and drafting the manuscript. WC participated in the conception, design of the study, data acquisition, analysis and interpretation, and drafting the manuscript. All authors read and approved the final manuscript.