Background
More than 90% of tumors in the head and neck are squamous carcinomas (HNSCC) that arise in the paranasal sinuses, nasal cavity, oral cavity, pharynx, and larynx [
1]. About two-thirds of patients with HNSCC present with advanced-stage disease (stages III and IV), and the high rate of metastasis is highly associated with a poor 5-year survival rate [
2,
3]. Despite significant improvements in multiple-modality therapy with surgery, chemotherapy, and radiation, long-term survival rates in patients with advanced-stage HNSCC have not increased significantly in the past few decades [
4]. The poor clinical outcomes reveal an obvious and urgent need to develop more effective and tolerated treatments against HNSCC, especially for aggressive tumors.
Modern research is now focusing on seeking specific molecular targets involved in the development and procession of cancer in an attempt to develop more officious and selective treatments. Src, a member of Src family of non-receptor tyrosine kinases (SFKs), is often activated by direct or indirect interaction with receptor tyrosine kinases (RTK), such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and insulin-like growth factor 1 receptor (IGF-1R), as well as G-protein-coupled receptors (GPCRs), cytokines, integrins, and others [
5]. It appears that Src acts as a critical molecular switch in regulating signal transduction for many fundamental cellular processes by a diverse set of cell surface receptors in the context of a variety of cellular environments. Overexpression and hyperactivation of Src have been found in a wide variety of human cancers, including HNSCC [
5,
6]. Additionally, the extent of increased Src activity often correlates with malignant potential and patient survival [
7]. Multiple signaling pathways converge on Src activation to epithelial-mesenchymal transition (EMT) phenotypic features to promote tumor cell metastasis [
8]. In mice models of breast cancer, inhibition of Src kinase activity can improve survival through suppressing metastasis [
9]. In HNSCC, Src is activated following EGF stimulation and decreases cell migration and invasion in treatment with Src inhibitors [
10,
11]. The involvement of Src in tumor progression and metastasis has generated considerable interest in Src as a therapeutic strategy to treat metastatic disease.
Src-targeting agents, including dasatinib and saracatinib (AZD0530), are currently in clinical development for patients with solid tumors. Dasatinib, a potent oral tyrosine kinase inhibitor which targets Src and other several kinases [
7], has shown a marked efficacy in patients with chronic myeloid leukemia (CML) as first-line treatment [
12]. The capacity of dasatinib to block migration and invasion without affecting proliferation and survival is demonstrated in human melanoma cells [
13]. Dasatinib is also reported to suppress migration and invasion of HNSCC cells, coupled with the inhibition of Src and downstream mediators of cell adhesion, such as focal adhesion kinase (FAK) [
11]. Dasatinib as a single agent has modest clinical activity with liver failure on many types of solid tumors, including non-small cell lung cancer, prostate cancer, and breast cancer [
14]. Saracatinib, originally developed by AstraZeneca, is a novel anilinoquinazoline inhibiting deregulated elevated Src kinase activity in a wide range of cancer cells, such as colorectal, ovary, prostate, and breast cancer [
7,
15‐
17]. Several preclinical reports suggest that saracatinib has potent anti-migratory and anti-invasive effects in endocrine-resistant breast cancer cells [
18] and significantly suppressed the metastatic nature of bladder cancer in a murine model [
19]. Although saracatinib was evaluated in phase I/II clinical trials for advanced stage HNSCC and other various types of cancer [
20], the anticancer efficacy was not sufficiently promising to justify continued accrual to active trials. Therefore, developing a novel saracatinib-based strategy would open a new avenue for Src-targeted therapy.
Physicochemical and pharmacokinetic profiles of anticancer drugs render optimal delivery challenging. Moreover, distribution, biotransformation, and clearance of anticancer drugs in the body must be overcome to deliver therapeutic agents to tumor cells in vivo [
21]. Nanoparticles (NPs) have shown promise as both drug delivery vehicles and direct anticancer systems, based on the quantum properties and the ability to carry and absorption [
22]. Most solid tumors possess unique pathophysiological characteristics that are not observed in normal tissues or organs (e.g., extensive angiogenesis, low pH and hypoxia), which greatly increase production of a number of the tumor site-specific delivery of NPs. Numerous studies have shown that both tissue and cell distribution profiles of anticancer drugs can be controlled by their entrapment in NPs [
23,
24].
In the present study, we show that Src is one of the most targetable molecules involved in invasion and metastasis of HNSCC, and saracatinib can significantly suppress the invasive and metastatic phenotype through inhibiting Src kinase activity and its mediated metastatic signaling in HNSCC cells. We also designed and synthesized novel multifunctional NPs for selective release of saracatinib into head and neck tumor cells and evaluated the anti-tumor efficacy and efficiency of saracatinib-loaded NPs (Nano-sar) in mice. Our studies reveal that Nano-sar has superior anticancer effects than the free drug through suppressing head and neck tumor metastasis more efficiently. The tumor site-specific delivery of NPs, especially with the use of saracatinib, would be straightforwardly extended from HNSCC to other types of solid tumors.
Methods
Cell lines and standard assays
HNSCC cell lines HN6, HN8, and HN12 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum as previously described [
25,
26]. HN6 was derived from tongue squamous cell carcinoma. HN8 and HN12 were derived from the metastatic lymph node site from oral cavity and tongue squamous cell carcinoma, respectively. The cell passage number less than 10 was used for experiments. Cell proliferation was determined by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI), and invasion was determined by Transwells (BD biosciences, San Jose, CA) with 8-μm pore size filters covered with Matrigel. Transfection and infection, colony formation, and scratch-wound healing were carried out as previously described [
7,
27‐
29].
Constructs, reagents, and antibodies
pLKO.1 lentiviral vectors harboring short hairpin RNAs (shRNAs) targeting Src or green fluorescent protein (GFP) were obtained from Open Biosystems (Huntsville, AL). Saracatinib and dasatinib were purchased from Selleckchem (Houston, TX). For Western blot, antibodies that recognize p-Src (Tyr416) and Src were purchased from Cell Signaling Technology (Beverly, MA). β-Actin antibody was purchased from Sigma-Aldrich (St Louis, MO). Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (#9782) and Tight Junction Antibody Sampler Kit (#8683) were purchased from Cell Signaling Technology (Beverly, MA).
Western blot assay
The protein levels for the biomarkers were semi-quantified by Western blot analysis as previously described [
29,
30]. Electrophoresis was performed on 10% SDS-PAGE gel, and the proteins were transferred to nitrocellulose membrane. The membranes were incubated with the primary antibodies overnight at 4 °C and with secondary antibody for 1 h at room temperature. The antigen-antibody complexes were then visualized using Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA). The protein bands were quantified by densitometry analysis.
Solid-phase peptide synthesis
Synthesis of the peptide was carried out using the Fmoc strategy manually in a glass reaction vessel fitted with a sintered glass frit using 2-chlorotritylchloride. Coupling reactions were performed manually by using 2 equiv. of N-Fmoc-protected amino acid (relative to the resin loading) activated in situ with 2 equiv. of PyBOP and 4 equiv. of diisopropylethylamine (DIPEA) in DMF (10 mL/g resin). The coupling efficiency was assessed by Kaiser test. N-Fmoc protecting groups were removed by treatment with a piperidine/DMF solution (1:4) for 10 min (10 mL/g resin). The process was repeated three times and the completeness of deprotection verified by UV absorption of the piperidine washings at 301 nm. Synthetic linear peptides were recovered directly upon acid cleavage. Before cleavage, the resin was washed thoroughly with methylene chloride. The linear peptides were then released from the resin by treatments with a solution of acetic acid/trifluoroethanol/methylene chloride (1:1:8, 10 mL/mg resin, 2 × 30 min). Hexane (5–10 volumes) was added to the collected filtrates, and the crude peptides were isolated after concentration as white solids. The residue was dissolved in the minimum of methylene chloride, and diethyl ether was added to precipitate peptides. Then, they were triturated and washed three times with diethyl ether to obtain crude materials. Peptide was further purified by preparative high-performance liquid chromatography (HPLC) prior to conjugation.
Synthesis of the polymeric drug carrier
Linear-dendritic mPEG-BMA4 was synthesized according to a method in literature [
31,
32]. Under a nitrogen atmosphere, branched mPEG-BMA4 (1 equv. based on amino group), peptide (Ac-K(Boc) GFLG-OH, 1 equv.), HBTU (1 equv.), and HOBT (1 equv.) were added into a round flask and dissolved in anhydrous DMF. Then, DIPEA (2 equv.) was added dropwise under ice bath. The solution was stirred in ice bath for 30 min and at room temperature for 48 h. The solution was dialyzed against deionized water using dialysis membrane (MWCO = 2000). The final product was obtained via lyophilization.
Saracatinib-loading into NPs
Hydrophobic saracatinib was loaded into the NPs by the solvent evaporation method as described in literature [
33,
34]. Briefly, drug (1.0 mg) and amphiphilic polymer (10 mg) were first dissolved in anhydrous chloroform/methanol (1/1) in a 10 mL round bottom flask. The solvent mixture was evaporated under vacuum to form a thin film. PBS buffer (1 mL) was added to re-hydrate the thin film, followed by 30 min of sonication. The unloaded drug was removed by running the NP solutions through centrifugal filter devices (MWCO: 3.5 kDa, Microcon®). The saracatinib-loaded formulation on the filters were recovered with PBS.
Characterization of NPs
The amount of drug loaded in the NPs was analyzed on a HPLC system (Agilent 1200 LC, Santa Clara, CA). The drug loading was calculated according to the calibration curve between the HPLC area values and concentrations of drug standard. The loading efficiency was defined as the ratio of drug loaded into NPs to the initial drug content. The size and size distribution of Nano-sar were measured by dynamic light scattering (DLS) instrument for three times with an acquisition time of 30 s at room temperature.
Drug release study
The drug release from Nano-sar was carried out in the solution with or without cathepsin B (CTSB). Cysteine solution in McIlvaine’s buffer (10 mm) was added in equal volume of enzyme stock solution and pre-incubated at 37 °C for 5 min. NPs were incubated in the buffer at 37 °C for 48 h in the presence or absence of CTSB (100 nM, pH = 5.4). A drug release control study at physiological condition (without enzyme, pH 7.4) was also performed. At pre-determined time points, the samples were withdrawn and analyzed by reversed-phase HPLC (RP HPLC) with gradient elution.
Three-dimensional (3D) tumor spheroid invasion assay
The experiment was modified and carried out as previously described [
35,
36]. Briefly, 2 × 10
4 HN12 cells were incubated overnight to form 3D spheroid in hanging droplet in a well of an inverted round bottom 96-well plate. Then, 150 μl mixture of Matrigel: DMEM without serum (1:1 ratio) was added in the well and solidified at 37 °C, followed by adding 150 μl complete culture medium containing double doses of drugs. After 3 days, spheroids from different treatments were imaged under a microscope.
Animal study
Six-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA), and all animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Augusta University. To generate a metastasis model in NSG mice, 5 × 105 HN12 with a luciferase reporter gene were suspended in 100 μl of PBS/Matrigel (3:1) and injected into the right flank. When mean tumor volumes reached approximately 100 mm3, mice were randomized to receive equal volume treatment of vehicle (sterile saline), the free drug saracatinib (20 mg/kg), or Nano-sar (at dose 10 mg/kg) by tail vein administration every other day for a total of 12 days. Tumor growth was measured externally every 4 days using vernier calipers as length × width2 × 0.52. Mice were imaged for bioluminescent luciferase signal by an intraperitoneal injection of D-luciferin bioluminescent substrate (Sigma-Aldrich, St Louis, MO) using a Xenogen IVIS-200 In Vivo Imaging System (PerkinElmer, Waltham, MA). When the experiment was terminated, blood was collected via ocular vein for determination of serum Alanine Transaminase (ALT/GPT), Aspartate Transaminase (AST/GOP), and creatinine. ALT and AST were measured by EnzyChrom™ Alanine Transaminase Assay Kit and Aspartate Transaminase Assay kits (BioAssay System, Hayward, CA), respectively. Serum creatinine was measured by Creatinine Assay Kit (Cayman chemical, Ann Arbor, MI). The mice were then sacrificed, and the xenografts and the major organs (heart, intestine, liver, spleen, lung, and kidney) were removed for histopathological analysis with hematoxylin-and-eosin (H&E) staining.
Immunohistochemistry (IHC)
Paraffin-embedded xenografts were cut into 3 μm sections and mounted on slides, and IHC was performed as described previously [
7,
37]. Briefly, tissue sections were blocked in 10% of normal goat serum after antigen retrieval in hot citrate buffer and were incubated with the primary antibodies against p-Src, Vimentin, and Snail, respectively. Immuno-reactivity was visualized by using the DAB Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturers’ procedure, and images were reviewed and analyzed by a CCD camera (Olympus, Center Valley, PA). At least nine random microscopic fields were captured, and signal intensity was quantified using the Image pro-Plus6.0 software.
Statistical analysis
Treatment effects were evaluated using one-way ANOVA at each measurement time-point. To assess the longitudinal effect of treatment, a mixed model was employed to test the overall difference across all groups as well as between each pair of groups during the whole study period. Experiments shown are the means of multiple individual points from multiple experiments (± S.D.), and p < 0.05 was considered as statistically significant.
Discussion
Increased activity of Src is a frequent occurrence in HNSCC [
10,
11]. Src acts as an integrator of divergent signal transduction pathways and promotes numerous tumor-promoting activities, including tumorigenesis, invasion, and metastasis. Therefore, inhibitors targeting Src are considered as promising drugs for cancer therapy. In this study, we demonstrate that saracatinib can effectively suppress invasion and metastasis of HNSCC, at least in part, through blocking Src-dependent Vimentin/Snail signaling. Our findings also show, for the first time, that the efficiency of tumor-responsive nano-based drug delivery system largely improves effectiveness of saracatinib in suppressing metastasis of HNSCC without systemic toxicity.
EMT is a dynamic process that endows the incipient cancer cell with invasive and metastatic properties [
30]. Loss of E-cadherin-mediated cell-cell adhesion leading to detachment from neighbor epithelial cells and/or acquisition of some mesenchymal characteristics are key events of EMT [
30,
40]. Src is frequently hyperactivated in cancer cells, resulting in facilitating tumor progression towards metastasis by promoting EMT [
8]. For example, Src signaling has been shown to regulate E-cadherin associated EMT in pancreatic cancer cells [
41]. In contrast to these studies, knockdown of Src by shRNAs or inactivation of it by small molecule inhibitors in HNSCC cells cannot affect E-cadherin levels. Instead of this, it appears that invasion repression induced by loss of Src function is resulted from downregulation of mesenchymal markers Vimentin and Snail proteins. Interestingly, all three cell lines used in this study express E-cadherin, although they show mesenchymal morphology. The possible reason is that HNSCC involves transformation of the squamous epithelial lineage, which is histologically similar to the epidermis [
42]. Consistently, there were no changes in E-cadherin levels in epidermoid carcinoma A431 cells in the presence or absence of dasatinib [
43]. Nevertheless, Src-mediated EMT in HNSCC cells remains to be better defined.
Surprisingly, the protein levels of total Src were increased in HNSCC cells treated with Src inhibitors, dasatinib or saracatinib, although its phosphorylation was markedly inhibited. The similar results were also observed in other studies when HNSCC cells and other types of cancer cells were treated with Src inhibitors [
44‐
46]. Our data and previous studies suggest an unrecognized feedback mechanism for compensation of Src kinase inhibition with increased levels of Src protein expression, which maybe through downregulation of Src degradation or increase its transcription. However, the exact mechanism still needs to be deciphered.
Saracatinib, a highly selective small molecule, inhibits Src kinase activity by interfering with Src phosphorylation at tyrosine 419-human/423-mouse [
47]. Preclinical studies of head and neck tumor models showed that saracatinib treatment impaired perineural invasion and cervical lymph node metastasis [
48]. Here, we show a higher inhibitory rate of nano-based saracatinib in HNSCC metastasis compared with the free drug. The efficacy of Nano-sar seems to be on metastasis, which may be due to drug administration of a fixed dose within a short period of experimental time. Expansion of the time window for treatment of Nano-sar may achieve better therapeutic outcomes either on tumorigenesis or metastasis, resulting from the combined influence of Src inactivation and the tumor site-specific delivery of NPs. One of the major challenges for new therapeutics to enter the clinic remains improving their translational value to the clinical situation. We are aware that HNSCC rarely displays distant metastasis; rather, it invades and colonizes cervical lymph nodes in the clinical setting. The orthotopic mouse model of tongue tumors has been established in our group by sublingual injection of HN12 cells, but we are still facing the challenge to observe high rate of cervical metastasis in this model before tumor-bearing mice reach a moribund state. The flank model used in our study is not the best method to recapitulate HNSCC in mice; however, the analyses on it at least provide the proof of principle that the pharmacology and potency of Nano-sar is promising. Further exploration of this novel treatment in highly preclinical animal models of HNSCC is warranted.
Biocompatible and amphiphilic polymers are able to self-assemble to nanoscale formulations that possess ideal features for drug delivery, including prolonged blood circulation, high stability, and high accumulation in tumor tissues [
49,
50]. As such, NPs have been explored as one of the most promising drug vehicles in the development of drug delivery system to enhance drug efficacy as well as reduce systematic toxicity. Particularly, stimuli-responsive NPs that are sensitive to biological stimuli such as pH, temperature, redox potential, and enzymes have been extensively exploited for triggered drug release. Enzymes that express at relatively low level in normal tissue but frequently overexpressed in pathological tissues appear to be an ideal stimulus. Lysosomal enzyme of CTSB, an overexpressed and secreted enzyme in tumor endothelial and epithelial cells, is one of targets that are frequently used in the development of enzyme-triggered nanomedicine. The expression of CTSB has been reported to be increased along with the cancerization in oral squamous cell carcinoma [
38,
51], which is also positively associated with highly invasive and metastatic phenotypes [
52]. We collected 19 primary HNSCC tissues with paired adjacent normal tissues and determined the expression levels of CTSB by real-time RT-PCR. More than tenfold higher levels of CTSB were observed in HNSCC tissues compared with paired adjacent normal tissues (data not shown), providing a strong rational basis for the design of CTSB-sensitive NP for saracatinib delivery. Given that solid tumors have an acidic extracellular environment and an altered pH gradient across their cell compartments [
53,
54], the formulations of Nano-sar were designed to exploit the pH gradients that exist in tumor microenvironments. Therefore, Nano-sar can be selectively activated and release the loaded saracatinib into head and neck tumors in order to maintain effective drug levels at tumor tissues.