1 Introduction
Nasopharyngeal carcinoma (NPC) is a malignant tumor originated from the mucosal lining of nasopharyngeal tissues. In the nasopharynx, tumors often appear in the pharyngeal recess [
1]. The geographical distribution of NPC is extremely uneven, with more than 70% of new cases occurring in East and South-east Asia. NPC is also one of the most frequent malignant tumors in southern China, with an incidence of 20/100 000–30/100 000 [
2]. Radiotherapy is the mainstay treatment modality for early staged NPC patients; locally advanced disease requires more than radiotherapy, and concurrent chemoradiotherapy with adjuvant chemotherapy or induction chemotherapy followed by concurrent chemoradiation is recommended for stage II–IVA NPC patients by National Comprehensive Cancer Network (NCCN) Guidelines [
3]. Chemotherapy regimens vary from study to study, and cisplatin-based regimens are commonly the first choices. Despite responding to the initial treatment, many patients with NPC have failed anticancer treatment because they cannot tolerate the side effects of chemoradiotherapy or develop resistance to chemoradiotherapy [
4]. Due to toxicity of chemotherapeutic drugs and resistance of patients to chemoradiotherapy, targeted therapy has been increasingly used in the treatment of many types of malignancies, including breast cancer, colorectal cancer, lung cancer and other solid tumors, as well as lymphoma and leukemia. However, due to insufficient understanding of molecular mechanisms, targeted therapies for NPC are relatively undeveloped. In the past 10 years, only a few targeted therapeutic drugs have been studied in clinical trials for NPC, but the anti-tumor effects of these drugs were not satisfactory. Therefore, further studies are expected.
The epidermal growth factor receptor (EGFR) family, also known as the ErbB family or the HER family, includes EGRF/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4. HER family regulates many critical cellular processes, such as cell proliferation, differentiation, survival, migration, and cell cycle [
5]. The abnormal activation of HER family kinase activity is closely related to the development of human malignancies [
6]. In NPC, HER family proteins have also been found to be overexpressed, especially EGFR/HER1 and HER2, and their overexpression has shown to be significantly associated with poor prognosis [
7–
12]. Therefore, HER family is considered to be one of the key targets in cancer treatment based on the observations that overexpression of HER family proteins is significantly associated with human tumor progression and increased invasive potential of cancer cells.
Current therapies targeting HER family mainly include small molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs). Compared with mAbs, ATP-targeted small molecule TKIs have obvious advantages because of their lower cost, higher oral bioavailability and the ability to target multiple signaling pathways [
13]. Furthermore, in view of the fact that many types of tumors express multiple HER proteins, it is becoming increasingly clear that inhibiting multiple HER proteins has better tumor growth inhibitory effects than inhibiting a single HER family member [
14]. Pan-HER inhibitors are a new class of irreversible TKIs that target non-overlapping epitopes on HER1/EGFR, HER2, HER3 and HER4. HM781-36B is a novel, small-molecular and irreversible TKI of EGFR, HER2, and HER4, and its pharmacokinetic properties are superior to the first pan-HER inhibitor afatinib [
15,
16]. Currently, HM781-36B is in phase I and II clinical trials of various solid tumors such as advanced breast cancer, non-small cell lung cancer, esophageal cancer and colorectal cancer, alone or in combination with chemotherapeutic agents. Previous studies have demonstrated that treatment with cisplatin could lead to the activation of the EGFR signaling [
17]. In this study, we investigated the expression of HER family proteins in human NPC tissues and their prognostic value in patients with NPC. In addition, we used HER kinases as targets for the treatment of NPC and explored the anti-tumor effects of the novel pan-HER inhibitor HM781-36B
in vitro and
in vivo, alone or in combination with cisplatin.
2 Materials and methods
2.1 Patient samples
Ethical approval was obtained from the Medical Ethics Committee (No. YB M-05-02) of Shanghai Outdo Biotech Co., Ltd., and the informed consent was obtained from all the patients. The NPC tissue microarray (Shanghai Outdo Biotech Co.), which contained the tumor tissues of 132 NPC patients who underwent surgical excision between January 2010 and October 2011, was analyzed. The clinicopathological characteristics of all the 132 patients were extracted. The pathological subtypes of NPC were classified according to the World Health Organization classification, and patients with NPC were staged according to the 8th edition of International Union Against Cancer/American Joint Committee on Cancer (UICC/AJCC) TNM (tumor-node-metastasis) staging system.
2.2 Cell lines and cell culture
The human NPC cell lines C666-1, CNE-1, CNE-2, HNE-1, and HONE-1 were purchased from the American Type Culture Collection (ATCC). C666-1, HNE-1, and HONE-1 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotics (penicillin/streptomycin). CNE-1 and CNE-2 cell lines were cultured in RPMI-1640 supplemented with 10% FBS (Gibco) and 1% antibiotics (penicillin/streptomycin). All cells were maintained in humidified incubators at 37 °C in 5% atmospheric CO2.
2.3 Reagents
The pan-HER inhibitor HM781-36B was obtained from Selleck. For in vitro experiments, HM781-36B was dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich) to an initial concentration of 40 mmol/L and stored at −80 °C. Subsequently, the stock solution was diluted to appropriate concentrations in the relevant assay medium, and 0.1% DMSO (v/v) was used as a vehicle control. For in vivo studies, HM781-36B was dissolved in 10% (v/v) N-methyl pyrrolidone (NMP), 10% (v/v) Solutol and 80% (v/v) double distilled water and used at a concentration of 1 mg/kg or 0.5 mg/kg. Cisplatin was purchased from MedChemExpress, dissolved in double distilled water and prepared when used.
2.4 Cell viability assay
Cell viability was analyzed using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay. Cells were seeded into 96-well plates at 3000–5000 cells/well in 100 μL of medium. After treatment with different concentrations of HM781-36B for 24, 48, or 72 h, we added 10% (v/v) CCK-8 regent into every well according to the manufacturer’s protocol. Subsequently, the cells were further incubated in dark for 1–2 h at 37 °C. The optical density (OD) at 450 nm for each well was measured using a microplate reader. All concentrations were replicated in at least three wells. The half inhibitory concentration (IC50) values were calculated by GraphPad Prism 7 software.
2.5 Apoptosis analysis
Cells were allowed to attach overnight in 6-well plates and treated with HM781-36B or/and cisplatin for 48 h. Then, we collected the cells and supernatant, and washed them twice with precooled PBS. Subsequently, the cells were labeled with propidium iodide (PI) and annexin V (annexin V-FITC apoptosis detection kit, BD Biosciences, CA, USA) according to the manufacturer’s instructions and analyzed by a NovoCyte Flow Cytometer (ACEA Biosciences, China).
2.6 Cell cycle analysis
Cells were allowed to attach overnight in 6-well plates and treated with HM781-36B for 48 h. Cells were collected, washed twice with precooled PBS, fixed with precooled 70% ethanol, and then incubated in dark with PI and RNase (Sigma, Missouri, USA). Flow cytometry analysis was carried out to detect the distribution of cells in different cell cycle phases.
2.7 Wound healing assay
Cells were seeded in 6-well plates and allowed to grow to 80%–90% confluence. A cell-free wound area was scratched using a sterile plastic pipette tip, and the debris was removed by rinsing with PBS. Then, cells were exposed to specified treatments, including HM781-36B and/or cisplatin, and 0.1% DMSO was used as a control. To evaluate wound closure, images were taken with an inverted microscope (Olympus) at 100× magnification at 0, 12 and 24 h after wound formation.
2.8 Colony formation assay
To evaluate the clonogenic capacity, 500 cells/well were seeded in 6-well plates and incubated for 12 hours. Cells were then treated for 48 h with different concentrations of HM781-36B and/or cisplatin, washed with PBS, and incubated with complete medium. After 10 to 14 days of incubation, cells were fixed with 4% polyoxymethylene for 10 min and stained with 0.5% crystal violet for 10 min. The cells were photographed after washing 3 times with PBS and then the colonies were counted by eyes.
2.9 Western blot analysis
Cells of control group (treated with 0.1% DMSO) and drug-treated group were harvested, and tumor tissues of vehicle and experimental groups were cut up. Then, cells and tissues were lysed in a radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) with protease inhibitor cocktail (Sigma-Aldrich, USA). The protein concentration was measured by bicinchoninic acid (BCA) protein assay kit (Thermo Fisher, USA). Equal amounts of protein were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Bio-Rad, USA). Proteins separated by SDS-PAGE gel were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA), and the membranes were blocked with 5% skim milk for two hours and incubated with primary antibodies overnight at 4 °C followed by incubation with appropriate secondary antibodies for an hour at 37 °C.
Enhanced chemiluminescence detection kit (Thermo scientific, USA) was used to visualize the protein bands. The primary antibodies were shown as following: β-actin, GAPDH, signal transducer and activator of transcription 3 (Stat3), Bax and p-HER1 (Y1068) antibodies were provided by HuaAn Biotechnology Co., Ltd., Hangzhou, China; extracellular signal-regulated kinase (Erk), p-Erk (Thr202/Try204), p-Stat3 (Tyr705), procaspase-3, Bcl-2, cleaved caspase-3, Mcl-1, p-HER2 (Tyr1221/1222) and p-HER4 (Tyr1284) antibodies were provided by Cell Signaling Technology; V-akt murine thymoma viral oncogene homolog (Akt), EGFR/HER1, HER2, HER3, and HER4 antibodies were provided by Abcam; p-Akt (Ser473) antibody was provided by Life Technologies.
2.10 In vivo studies
All protocols for animal experiments were approved by the State Key Laboratory of Biotherapy Animal Care and Use Committee of Sichuan University, China. The experimental animals female BALB/c nude mice (5–6 weeks) used in this study were purchased from Beijing HFK bioscience Co., Ltd. Before the injection of cancer cells, the mice were allowed to acclimatize to local conditions for 1 week. NPC cells were injected subcutaneously into female BALB/c nude mice (1 × 107 cells per mouse). Approximately 4 days after injection of NPC cells, in addition to the control group, tumor-bearing mice were also randomized into treatment groups (10 mice per group) to receive vehicle control, HM781-36B, cisplatin or a combination of HM781-36B and cisplatin. Animal were given vehicle or HM781-36B (1 mg/kg or 0.5 mg/kg) by oral gavage once daily for 3 weeks. A dose of 5 mg/kg of cisplatin was administered intraperitoneally once a week for 3 weeks.
From the day of administration, body weight and tumor volume were recorded every three days. Tumor volume was calculated as the following formula: tumor length × tumor width2/2. After 3 weeks of treatment, mice were sacrificed by cervical dislocation, and subcutaneous tumors of nude mice were isolated and weighed. Those subcutaneous tumors were used for flow cytometry, hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) staining, and immunofluorescence staining. Vital organs of mice, including the heart, liver, spleen, lung and kidney were harvested for HE staining.
2.11 Haematoxylin and eosin staining
Mouse subcutaneous tumor tissues and vital organs, including the heart, liver, spleen, lung and kidney, were fixed with 4% formaldehyde, embedded in paraffin, and then sectioned with a microtome to obtain 5 μm-thick paraffin tissue sections. Thereafter, paraffin tissue sections received deparaffinization and rehydration, and then were stained with hematoxylin and eosin.
2.12 Immunohistochemistry staining
Subcutaneous tumor tissues were fixed with 4% formaldehyde, embedded in paraffin, and then sectioned with a microtome to obtain 4 μm-thick paraffin tissue sections. The slices were deparaffinized and hydrated, and 3% H2O2 was used for blocking the endogenous peroxidase activity. Sections were immersed in sodium citrate buffer (10 mmol/L, pH 6.0) and heat-treated in an autoclave under saturated conditions for 3 min for antigen retrieval. Sections were blocked with normal goat serum. Next, sections were incubated with primary antibodies overnight at 4 °C. The next day, sections were incubated with biotinylated secondary antibodies at 37 °C. Afterward, sections were incubated with a third antibody (SAB solution), visualized with diaminobenzidine (DAB) and counterstained with hematoxylin. All sections were evaluated and quantified by two independent pathologists using the Aperio imaging system (Leica).
2.13 Immunofluorescence analysis
Immunofluorescence analysis was performed using frozen sections. Tumor tissues embedded in optimum cutting temperature (OCT) compound were stored at −80 °C and sliced with a microtome to obtain 5 μm-thick tissue sections. The sections were fixed with precooled acetone for 20 min and permeabilized in 1% Triton X-100 for 10 min at room temperature. Next, the sections were blocked with normal goat serum for 20 min. After incubating with primary antibodies overnight at 4 °C, sections were incubated with fluorescent-labeled secondary antibodies for 40 min at 37 °C. Finally, the tissues were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and photographed using a fluorescence microscope (Leica Microsystems, Germany).
2.14 TUNEL staining
After exposure of NPC cells to HM781-36B for 48 hours in vitro, these samples were further processed for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining by using TUNEL apoptosis detection kit (Promega, USA) according to the manufacturer’s protocol. Subcutaneous tumor tissues were fixed with 4% formaldehyde, embedded in paraffin, and then sectioned with a microtome to obtain 4 μm-thick paraffin tissue sections. The slices were deparaffinized and hydrated, and then stained with TUNEL by using TUNEL apoptosis detection kit (Promega, USA) according to the manufacturer’s protocol. The sections were observed and photographed using a fluorescence microscope (Leica Microsystems, Germany).
2.15 Serological biochemical analysis
The blood of mice in each group (n = 3–5) was collected and centrifuged to obtain serum. Then, serum biochemical analysis was performed using an automatic analyzer (Hitachi High-Tech Company, Minato-ku, Tokyo, Japan). Albumin (ALB), amylase (AMY), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), direct bilirubin (DBIL), cholesterol (CHOL), creatine kinase-myocardial band isoenzyme (CK-MB), creatinine (CRE), glucose (Glc), high-density lipoprotein (HDL), lactate dehydrogenase (LDH), total protein (TP), triglyceride (TG), and uric acid (UA) were tested to compare the control and experimental groups.
2.16 Statistical analysis
The statistical significance of results was assessed using Student’s t-test and ANOVA analysis. Data were recorded as mean ± SD. Survival was analyzed using Kaplan–Meier method. All the statistical analyses were performed using GraphPad Prism 7.0 or IBM SPSS Statistics 17.0. Difference with a P value < 0.05 was considered statistically significant.
3 Results
3.1 Expression of HER family proteins in NPC tissues and cell lines
A total of 132 patients with NPC were identified and analyzed in this study. The average age of the patients was 65 years (range 37–84 years) at the time of diagnosis. Twenty-one patients (15.9%) were younger than 60 years while 111 patients (84.1%) were older than 60 years. The baseline characteristics of the patients were shown in the Table 1. During a median follow-up time of 72 months (range 32–86 months), 22 patients (16.7%) died.
The expressions of HER family proteins were analyzed using immunohistochemical staining. According to the results of immunohistochemical staining, we analyzed the proportion of positive staining cells (1%–25% = 1, 26%–50% = 2, 51%–75% = 3, or 76%–100% = 4) and the positive staining intensity (negative = 0, weak = 1, moderate = 2, or strong = 3). The total score was the multiplication of staining positive rate score and staining intensity score; score 0–5 was low expression and 6–12 was high expression. The typical pictures of high and low expressions of HER family proteins in NPC tissues were shown in Fig.1. Of the entire patients, 102 (77.3%) had high expression of EGFR/HER1, 88 (66.7%) had high expression of HER2, 42 (31.8%) had high expression of HER3, and 91 (68.9%) had high expression of HER4.
The prognostic significance of HER family proteins in patients with NPC was analyzed using Kaplan–Meier method. It was found that the high levels of HER1 (P = 0.001, P = 0.006, respectively), HER2 (P = 0.020, P = 0.014, respectively) and HER3 (P = 0.038, P = 0.008, respectively) were significantly associated with unfavorable progression-free survival (PFS) and overall survival (OS) (Fig.1–1C). Furthermore, the results of Kaplan–Meier analysis indicated that high expression of HER4 was significantly associated with worse OS (P = 0.001), but was not significantly associated with PFS (P = 0.119) (Fig.1).
The expression of HER family proteins in a panel of 5 human NCP cell lines was detected by flow cytometry. The results showed that HER1 and HER2 were expressed to different degrees in all 5 cell lines (Fig.1).
3.2 HM781-36B alone or in combination with cisplatin inhibits NPC cell proliferation and colony formation
We compared the anti-tumor activity of HM781-36B with other HER family targeted drugs, including lapatinib, afatinib, dacomitinib, and neratinib. The IC50 levels of HM781-36B, lapatinib, afatinib, dacomitinib, and neratinib for C666-1 were 0.8613, 2.19, 2.04, 5.56, and 9.44 μmol/L, respectively (Fig. S1). Therefore, C666-1 NPC cells were more sensitive to HM781-36B when compared to other HER family TKIs.
To further explore the anti-proliferative activity of HM781-36B, 5 NPC cell lines, including C666-1, CNE-1, CNE-2, HNE-1, and HONE-1, were incubated with increasing concentrations of HM781-36B or vehicle (0.1% DMSO) for 24, 48 or 72 h. As shown in Fig.2, HM781-36B inhibited the proliferation of NPC cells in a dose- and time-dependent manner. When exposed to HM781-36B for 24 h, the IC50 of C666-1, CNE-1, CNE-2, HNE-1, and HONE-1 cells were 14.61, 13.78, 19.11, 6.386, and 3.23 μmol/L, respectively; when exposed to HM781-36B for 48 h, the IC50 were 2.451, 3.289, 5.032, 2.997, and 1.204 μmol/L, respectively; when exposed to HM781-36B for 72 h, the IC50 were 1.35, 1.234, 2.754, 2.194, and 1.09 μmol/L, respectively. The results of colony formation assays indicated HM781-36B inhibited the colony formation of NPC cells (Fig.2).
Previous studies have demonstrated that treatment with cisplatin could lead to the activation of the EGFR signaling [
17]. Therefore, we examined whether targeting EGFR family with HM781-36B could sensitize NPC cells to cisplatin. We combined HM781-36B with cisplatin to investigate whether they have a synergistic antitumor effect. As a result, the combination of HM781-36B and cisplatin induced synergistic cell death of the two tested NPC cell lines (C666-1 and CNE-1), with a combination index (CI) of < 1, suggesting a synergistic effect (Fig.2). Furthermore, the results of colony formation assays indicated that compared with monotherapy, HM781-36B combined with cisplatin had a stronger effect on inhibiting colony formation of NPC cells (Fig.2). Taken together, these results suggested that HM781-36B could inhibit the proliferation of NPC cells and it could exert synergistic effect with cisplatin in inhibiting the proliferation of NPC cells.
3.3 HM781-36B alone or in combination with cisplatin induces apoptosis and cell cycle arrest
The apoptotic effect of HM781-36B was measured by flow cytometry using the annexin V/PI dual-labeling tool. As shown in Fig.3, after treatment with HM781-36B for 48 h, a dose-dependent increase in C666-1 and CNE-1 cells apoptosis rate was observed. After treatment with HM781-36B, the levels of Mcl-1 and Bcl-2 proteins were decreased, while the levels of Bax protein were increased (Fig.3). These observations suggested that HM781-36B can induce apoptosis in NPC cells. As shown in Fig.3, when HM781-36B was used in combination with cisplatin, the rate of apoptotic cells was significantly increased compared with monotherapy (Fig.3). Previous studies have indicated that activated EGFR signaling pathways can lead to G1/S cell cycle progression [
18]. In our study, after treatment with HM781-36B, there was a considerable dose-dependent increase in the G1 phase fraction for C666-1 cells (Fig.3). Taken together, these observations suggested that HM781-36B can induce cell apoptosis and cell cycle arrest at G1 phase, and HM781-36B and cisplatin have synergistic effects on promoting cell apoptosis.
3.4 HM781-36B suppresses NPC cell migration
To evaluate the effects of HM781-36B on the migration of NPC cells, we performed wound healing assays. C666-1 and CNE-1 cells were incubated with different concentrations of HM781-36B immediately after scratching. As shown in Fig.4 and Fig. S2A, the scratches healed significantly more slowly when cells were treated with HM781-36B. Furthermore, compared with control, cisplatin treatment, HM781-36B treatment, and HM781-36B combined with cisplatin treatment led to significantly reduced migration capacity (Fig.4 and Fig. S2B). Therefore, HM781-36B alone or in combination with cisplatin could effectively inhibit the migration of NPC cells.
3.5 HM781-36B suppresses activation of HER family proteins and their downstream signaling
To assess the effect of HM781-36B on HER family proteins and their downstream signaling pathways, C666-1 and CNE-1 cells incubated with various doses of HM781-36B for a period of 48 h were evaluated by Western blot analysis. As shown in Fig.4, the expression of pEGFR and pHER2 was decreased in a dose-dependent manner after HM781-36B treatment, while the levels of EGFR/HER1 and HER2 remained unchanged significantly in response to treatment with HM781-36B.
Subsequently, we investigated the effect of HM781-36B on downstream signaling of HER family. Previous studies have indicated that activated EGFR could contribute to the activation of many pro-oncogenic signaling pathways, including the MAPK, PI3K-AKT, SRC, PLC-γ1-PKC, and JAK-STAT pathways [
18]. These signaling pathways then influence many biological activities that are beneficial to cancer progression, such as promoting cell proliferation, differentiation, and migration, as well as inhibiting apoptosis [
18]. After treatment with HM781-36B for 48 h, the levels of AKT, ERK, and STAT3 proteins expressed by NPC cells did not change significantly, but their phosphorylated proteins p-AKT, p-ERK, and p-STAT3 were significantly reduced in a dose-dependent manner (Fig.4). These results indicated that HM781-36B inhibited the activation of HER family and its downstream signaling in NPC cells.
3.6 HM781-36B exerts anti-tumor effects in vivo
We established NPC subcutaneously implanted xenograft models using C666-1 and CNE-1 cells to evaluate the effects of HM781-36B on the growth of NPC in vivo. Mice bearing tumors were randomized into control, vehicle, and HM781-36B (1 mg/kg) groups (Fig. S3A and Fig. S4A). Tumor volume change curves were shown in Fig. S3B and Fig. S4B. The results showed that after treatment with HM781-36B for 3 weeks, the tumor weight and tumor volume of C666-1 and CNE-1 xenograft mice in the HM781-36B group significantly decreased than those in the control and the vehicle groups (P < 0.0001) (Fig. S3C and S3D, Fig. S4C and S4D). Furthermore, the body weight of mice in the HM781-36B group was reduced compared to the control and the vehicle groups (Fig. S3E and Fig. S4E). Overall, HM781-36B exhibits anti-tumor activity against NPC in vivo.
3.7 HM781-36B enhances the antitumor effects of cisplatin in NPC xenograft models
We also evaluated whether HM781-36B and cisplatin interact synergistically in inhibiting the growth of NPC using C666-1 and CNE-1 xenograft models. On the basis of previous studies that evaluated the anti-tumor effect of HM781-36B combined with 5-FU in gastric cancer xenograft models, a dose of 0.5 mg/kg HM781-36B was selected in our study [
16]. Cisplatin was administered through intraperitoneal injection once a week (5 mg/kg). Mice bearing tumors were randomized into control, vehicle, HM781-36B, cisplatin, and HM781-36B plus cisplatin groups. After treatment for 3 weeks, the growth of tumors in mice treated with HM781-36B alone or in combination with cisplatin was significantly inhibited compared with mice in the control and the vehicle groups (Fig.5 and 5D). In both the C666-1 and CNE-1 xenograft models, the volume of subcutaneous tumors in the HM781-36B plus cisplatin group was noticeably smaller than that in the HM781-36B or cisplatin groups (Fig.5 and 5E); tumor weight in mice receiving coadministration of HM781-36B and cisplatin was significantly less than that of mice receiving HM781-36B or cisplatin only (Fig.5 and 5F).
3.8 HM781-36B in combination with cisplatin inhibits HER protein activation in NPC tissues
To assess the effect exerted by HM781-36B and cisplatin on the activation of HER family proteins and their downstream signaling pathways, we performed Western blot to detect the expression of related proteins in NPC tissues after treatment. As shown in Fig.5, compared with the vehicle control, the expressions of p-HER1 and p-HER2 proteins in HM781-36B and HM781-36B plus cisplatin groups decreased, while the levels of EGFR/HER1 and HER2 proteins remained unchanged. Furthermore, the levels of p-HER1 and p-HER2 in the HM781-36B plus cisplatin group were the lowest. Subsequently, we also investigated the effect of HM781-36B and cisplatin on downstream signaling of HER family and found that HM781-36B, either independently or in combination with cisplatin, inhibited the activation of AKT, ERK, and STAT3. In the CNE-1 xenograft models, the levels of p-AKT and p-ERK of subcutaneous tumors in the HM781-36B plus cisplatin group were further decreased compared with those in the HM781-36B group (Fig.5). These results indicated that HM781-36B can inhibit the activation of HER family proteins and their downstream signaling pathways in vivo, and the combination therapy of HM781-36B and cisplatin may have a stronger inhibitory effect.
3.9 HM781-36B in combination with cisplatin inhibits cell proliferation and induces cell apoptosis
To evaluate the effect of HM781-36B and cisplatin on cell proliferation and apoptosis in vivo, we conducted ICH staining for Ki-67, TUNEL staining, and HE staining. As shown in Fig.6, the percentage of Ki-67-positive cells in the HM781-36B and cisplatin groups was significantly reduced than that in the vehicle group, and the percentage of Ki-67-positive cells in the HM781-36B plus cisplatin group was the lowest. The results of TUNEL staining showed that the proportion of TUNEL-positive cells in tumor tissues increased after treatment with HM781-36B, cisplatin, or HM781-36B plus cisplatin (Fig.6). Notably, the proportion of TUNEL-positive cells in the HM781-36B plus cisplatin group was significantly higher than that in the HM781-36B and the cisplatin groups. There were a lot of dead cells in drug-treated tumor tissues but few dead cells in vehicle-treated tumor tissues, as determined by the results of histopathological examination of tumor tissues stained with H&E (Fig.6). Tumor isolated from mice in the HM781-36B plus cisplatin group exhibited larger areas of dead cells than HM781-36B and cisplatin monotherapy groups. Taken together, the results suggested that HM781-36B and cisplatin have synergistic effects on inhibiting cell proliferation and promoting cell apoptosis.
3.10 Effect of HM781-36B and cisplatin on tumor microenvironment
To further explore the antitumor mechanism of HM781-36B and cisplatin, we measured the tumor microenvironment after drug treatment using flow cytometry. As shown in Fig.7, although the proportion of M2 macrophages remained unchanged after cisplatin treatment, the proportion of M2 macrophages in the HM781-36B combined with cisplatin group was significantly lower. Immunofluorescent staining of F4/80 and CD206 also confirmed the results (Fig.7). The proportion of DCs in tumor tissues of mice treated with HM781-36B was not statistically different from that in the control group, and the proportion of DCs in the combined treatment group was significantly higher than that in the control group (Fig.7). Furthermore, we found that the percentage of MDSCs was significantly lower in the HM781-36B treatment group compared with control group; after cisplatin treatment, the proportion of MDSCs in tumor tissues was significantly higher than that in the control group. The proportion of MDSCs in the combination treatment group was not significantly different from that in the control group (Fig.7).
3.11 Assessment of safety and toxicity of HM781-36B in mice
To preliminarily investigate the safety of HM781-36B in vivo, we observed the body weight, appearance, and fecal and urinary excretions of mice. The body weight of mice decreased after treatment with HM781-36B and cisplatin (Fig. S5), and skin ulceration, diarrhea, and toxic death were not observed. Serological biochemical analysis was performed in order to further evaluate the in vivo safety of HM781-36B, and we found no significant difference in biochemical indexes including AST, ALT, TG, and UA, between the control and the experimental groups (Figs. S6 and S7). HE staining of heart, liver, spleens, lung, and kidney was also used to evaluate the toxicities. It was found that when cisplatin and HM781-36B were used alone or in combination, there were no obvious toxicity and side effects on the heart, liver, spleen, lung, and kidney of mice (Fig. S7).
4 Discussion
There has been substantial advancement in the treatment strategies for NPC, including surgical and non-surgical methods [
19]. On the one hand, despite receiving aggressive treatment, local and distant progression may occur in non-metastatic and metastatic patients [
1]. On the other hand, radiotherapy and chemoradiotherapy inevitably lead to acute and late toxicity, which may occur months or even years after treatment [
1]. Due to resistance of patients to chemoradiotherapy and low toxicity of targeted therapy, targeted therapy has been increasingly used in the treatment of many types of malignancies, but targeted therapy for NPC is relatively undeveloped. In this study, we provided available data for the first time to demonstrate the antitumor effect of the pan-HER inhibitor HM781-36B
in vitro and
in vivo, and clarified its mechanisms of action in NPC cell lines.
The EGFR/HER1 is the first tyrosine-kinase receptor found to be directly related to human cancer [
20]. In NPC, EGFR/HER1 is commonly expressed. A previous study which enrolled 78 patients have reported that 94% of the patients expressed EGFR/HER1, and found a correlation between positive EGFR/HER1 expression and poor outcomes [
21]. As another important member of the EGFR/HER family, HER2 overexpression in cancer cells has been demonstrated to promote cell growth and increase tumorigenicity [
20]. HER2 has also reported to be involved in NPC progression, and its overexpression is associated with low survival rates and advanced tumor stages [
11]. Here, we confirmed the prognostic or predictive significance of EGFR and HER2 in patients with NPC. As a heterodimerization partner of HER2, HER3 is closely related to tumor growth and drug resistance [
12,
22]. Here, the result of survival analysis suggested that HER3 was a potential biomarker to predict the survival of NPC patients. Although the role of HER4 in tumor is controversial, in this study, associations between high expression of HER4 and poor OS were observed, but there was no significant correlation between HER4 expression and PFS [
23,
24].
The HER signaling pathway plays an important role in the progression of human cancer and its members are a group of anti-cancer targets with great clinical potential [
25]. Small-molecule TKIs and humanized or chimeric mAbs are two major classes of drugs that interfere with HER signaling, some of which are in clinical application. TKIs target the kinase domain to inhibit autophosphorylation, while mAbs directed against the extracellular domain of the receptors [
26]. mAbs targeting HER1 or HER2, such as cetuximab, trastuzumab, and pertuzumab, have been approved by the FDA for the treatment of several types of tumors [
27]. In addition, TKIs targeting single or multiple members of HER family have been shown to be useful in tumor therapy [
28]. Targeting HER pathways at multiple points is more effective than single-receptor inhibitors in inhibiting tumor growth, because many cancers express multiple HER family proteins [
14].
In this study, we evaluated the antitumor activity of the novel pan-HER inhibitor HM781-36B in NPC for the first time, and HM781-36B showed dramatic growth inhibitory effects on NPC cells
in vivo and
in vitro. HM781-36B inhibited proliferation and migration, promoted apoptosis, and block the cell cycle of NPC cells
in vitro;
in vivo, HM781-36B suppressed tumor growth in tumor xenograft models without obvious toxicities. Similarly, previous studies have reported that HM781-36B also showed anti-tumor effect in malignancies such as colorectal cancer and gastric cancer [
13,
16]. As previous studies reported, HM781-36B exerted synergistic effects with cytotoxic agents on cancer cells [
16,
29]. In this study, we preliminarily demonstrated that HM781-36B exerted synergistic effects with cisplatin on inhibiting proliferation, colony formation and migration, and promoting the apoptosis of NPC cells. In NPC xenograft models of C666-1 and CNE-1 in nude mice, HM781-36B and cisplatin synergistically inhibited the growth of NPC.
We conducted preliminary investigations into the antitumor mechanisms of HM781-36B. First, downregulating the activity of HER family proteins and their downstream signaling pathways is one of the most important mechanisms to explain the antitumor effect of HM781-36B [
15]. The results of Western blot indicated that after treatment with HM781-36B, the activation of EGFR/HER1, HER2, AKT, ERK, and STAT3 in NPC cells was downregulated in a dose-dependent manner. Similarly, in xenograft models of NPC, the levels of p-EGFR/HER1 and p-HER2 in tumor tissues of the HM781-36B group were decreased than those in the vehicle group. Furthermore, when HM781-36B was used in combination with cisplatin, the levels of p-EGFR/HER1 and p-HER2 in tumor tissues were further decreased. In view of the important role of HER family and its downstream signaling in tumor development, the downregulation of HER family activity can be one of the mechanisms explaining the synergistic effect of HM781-36B and cisplatin. Secondly, HM781-36B alone or in combination with cisplatin inhibits NPC cell proliferation and migration, and induces apoptosis.
Tumor-associated macrophages are recruited as monocytes from the peripheral blood; M2 tumor-associated macrophages are critical for tumor invasion and metastasis, and are responsible for tumor recurrence after chemotherapy [
30,
31]. Although the proportion of M2 tumor-associated macrophages in the cisplatin group was not significantly different from that in the control group, the proportion of M2 tumor-associated macrophages was significantly reduced in the HM781-36B and the HM781-36B plus cisplatin groups. As the most powerful antigen-presenting cells, DCs play a central role in initiating antigen-specific immunity and are considered to be a critical factor in antitumor immunity [
32,
33]. HM781-36B had no effect on the proportion of DCs in tumor microenvironment, but the proportion of DCs increased significantly when combined with cisplatin. MDSCs inhibit innate and adaptive antitumor immunity, which is an obstacle to cancer immunotherapy [
34]. The proportion of MDSCs in tumor microenvironment increased after treatment with cisplatin. MDSCs have been reported to play a role in the development of chemoresistance [
35]. Here, we found that after HM781-36B combined with cisplatin treatment, the proportion of MDSCs was not statistically different from the control group. Taken together, we believe that regulating tumor microenvironment is one of the most important mechanisms for the synergistic antitumor effect of HM781-36B and cisplatin.
The expression and prognostic value of HER family in NPC have been previously reported. In this study, we provided available data for the first time to demonstrate the antitumor effect of the novel pan-HER inhibitor HM781-36B in NPC, alone or in combination with cisplatin in vitro and in vivo. In addition to downregulate the activity of HER family proteins and their downstream signaling pathways, we also explained the antitumor mechanisms of HM781-36B in terms of regulating the immune microenvironment. However, the specific mechanisms underlying the altered tumor microenvironment after combination therapy remain to be elucidated. The synergistic effect of HM781-36B and cisplatin in the treatment of NPC remains to be further confirmed by clinical studies.
In conclusion, we showed the prognostic value of HER family proteins in NPC patients. Furthermore, we demonstrated the potential effects of HM781-36B alone or in combination with cisplatin in the treatment of NPC, providing a new idea for the comprehensive treatment of NPC.
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