Identification and evaluation of novel drug combinations of Aurora kinase inhibitor CCT137690 for enhanced efficacy in oral cancer cells

ABSTRACT

Oral cancer is the most prevalent subtype of head and neck cancers and arises mainly from squamous cells of the oral cavity. Patients with advanced metastatic disease have poor overall survival resulting primarily from limited treatment options. Recent advances in the understanding of molecular basis of oral tumorigenesis provide an opportunity for identification and validation of new drug targets. The deregulated expression of the Aurora family of mitotic kinases, for example, has been associated with pathogenesis and poor prognosis in oral cancer. Here, we have evaluated the efficacy of the pan-Aurora inhibitor (CCT137690) alone and in combination with different chemotherapeutic and targeted drugs to identify its synergistic partners in oral cancer cell lines (ORL-48 and ORL-115). CCT137690 effectively inhibits Aurora kinases in both the cell lines and displays potent antiproliferative activity towards them. Prolonged treatment of these cells with CCT137690 results in abrogated mitotic spindle formation, misaligned chromosome attachment and polyploidy that ultimately leads to apoptotic cell death. We further identified that inhibitors of EGFR (gefitinib) and PI3-kinase (pictilisib) synergize with CCT137690 to inhibit the proliferation of the oral cancer cell lines. Moreover, we demonstrate that polyethylene glycol-based nanocapsules harboring combinations of CCT137690 with gefitinib or pictilisib inhibit the growth of oral cancer cell lines in 3D spheroid cultures and induce apoptosis that is comparable to free drug combinations. In conclusion, we have demonstrated the in vitro efficacy of CCT137690 in oral cancer cell lines, identified novel drug combinations with CCT137690 and synthesized nanocapsules containing these drug combinations for co-administration.

Introduction

Oral cancer is the most prevalent subtype of head and neck cancers with high incidence among men in South-Central Asia and Central and Eastern Europe [1]. Most oral cancers are squamous cell carcinomas that originate from the mucosal lining of the oral cavity [2]. Recent genomic characterization has provided a comprehensive mutational profile of oral cancers, and identified potential therapeutic targets [3]. Many of these targets are amplified receptor tyrosine kinases (including EGFR, FGFR1, and ERBB2), mutated oncogenic components of proliferation and survival pathways (HRAS, PIK3CA), amplified cell cycle regulators (CCND1) or inactivated tumour suppressor genes (PTEN, NF1, TP53, and CDKN2A) [3,4]. Mutations in TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA have also been identified in oral cancer cell lines derived from patients of South Asian origin [5].

Overexpression of aurora kinases (Aurora A and B) is also associated with squamous cell carcinomas of the head and neck (SCCHN) [6–8]. These structurally related serine/threonine kinases function in regulating progression through mitosis. Aurora A is localized at the centrosome and adjoining microtubules where it controls mitotic entry, centrosome maturation and separation, spindle assembly and bipolar spindle formation [9]. Aurora B, on the contrary is the catalytic component of chromosomal passenger complex (CPC) and changes localization as cells progress through mitosis [10]. Aurora B is involved in chromosome condensation, chromosome orientation, spindle assembly checkpoint (error correction) and cytokinesis [11]. Deregulated expression of Aurora kinases is associated with many cancer types including oral cancer. Aurora kinase A, for example, is frequently overexpressed at mRNA and protein levels in HNSCC and its overexpression correlates with advanced metastatic disease and poor prognosis [8,12,13]. Similarly, AURKA amplification is reported in oral squamous carcinomas and its overexpression augments HRAS-mediated transformation [14]. Overexpression and activation of Aurora B is also associated with poor prognosis in OSCC [8,15].

Several small molecule inhibitors that target both Aurora A and B kinases (pan-Aurora inhibitors such as CCT137690, PHA-739358, AT9283 and AMG900) or are selective towards Aurora A (e.g. MLN8237) or Aurora B (e.g. AZD1152) have been discovered [16–19]. Only a few studies however, have evaluated their efficacy in oral squamous cell carcinomas. Pan-aurora inhibitor VX680, for example, inhibits growth and induces apoptotic cell death in an oral squamous cell carcinoma cell line [18]. Similarly, reversine causes mitotic arrest, suppresses growth and induces autophagy in two different OSCC cell lines [19]. Alisertib (MLN8237), a second generation Aurora A selective inhibitor, suppresses the growth of OSCC cells both in vitro and in vivo [20]. Moreover, Alisertib inhibits the growth of primary cells isolated from OSCC patients’ tumors in vitro [20]. The clinical evaluation of Alisertib as a single agent, however, shows a partial response in only 9% head and neck cancer patients [21]. To enhance their efficacy in these cancers, combinations of Aurora kinase inhibitors with chemotherapeutic or targeted drugs have been proposed [8]. Synergistic effects of Aurora A inhibition through knockdown or Alisertib treatment have been achieved with paclitaxel [22] and cetuximab [23], respectively in HNSCC cell lines.

Despite the identification of new drug combinations, the differential pharmacokinetics, stability and biodistribution of chemically diverse drugs can hamper their co-administration in the clinic [24]. Nanoparticles provide an opportunity to increase the therapeutic index and targeted delivery of chemotherapeutic drugs and various nanomedicines have been approved by FDA for treatment of different cancers [25]. The controlled loading of several drugs into nanoparticle formulations at particular ratios can help overcome diverse pharmacokinetic behaviors associated with chemically unique drugs. Recently approved treatment (Vyxeos) for high-risk Acute Myeloid Leukemia, for example, is a lipid based nanoparticle formulation containing combination of cytarabine and daunorubicin at 5:1 ratio [26]. There are, however, few reports of nanoparticle formulations with targeted drugs or their combinations. Nanoparticles containing an Aurora B kinase inhibitor, AZD2811 for example, have shown better biodistribution in tumors and extended release of the encapsulated drug which result in improved efficacy and tolerability [27].

CCT137690, a pan-Aurora inhibitor [28], inhibits the proliferation of cancer cell lines of different origins including colorectal, ovarian, neuroblastoma and leukemia [29,30]. Here we demonstrate that CCT137690 has potent antiproliferative activity in two oral cancer cell lines, ORL-48 and ORL-115. It inhibits Aurora kinases in both cell lines and thereby causes aberrant mitosis, endoreduplication and apoptotic cell death. We have also identified that CCT137690 synergizes with gefitinib and pictilisib (GDC-0941) in both the oral cancer cell lines. Furthermore, we demonstrate that polyethylene glycol-based nanocapsule formulations containing combinations of CCT137690 with gefitinib or pictilisib, potently inhibit the growth of oral cancer cell lines both in 3D spheroid cultures.

Materials and methods

Cell culture

Oral cancer cell lines, ORL-115 and ORL-48 were provided by Dr. Cheong Sok Ching of the Cancer Research Initiatives Foundation, Malaysia. Both cell lines were cultured in the DMEM/F12 media (Gibco) supplemented with 10% FBS (Gibco), 2µg/ml hydrocortisone and 1x Antibiotic-Antimycotic (Gibco). Cells were grown in the T75 flasks (SPL) in humidified incubators at 37°C and 5% CO₂.

Immunofluorescence

Cells seeded on poly-L-Lysine coated coverslips were treated with different concentrations of the inhibitors as indicated. Treated cells were then fixed in ice-cold methanol, washed with PBS and blocked with 2% BSA (in PBS). The cells were then incubated with primary antibodies diluted in 2% BSA for 1 h at room temperature; Antibodies for phospho-Aurora A (Cell Signaling), alpha-tubulin and pericentrin (MerckMillipore) were used. Next, the cells were washed with PBS and incubated with the fluorescently labeled secondary antibodies for 1 h in dark at room temperature; anti-mouse-568 (red) and anti-rabbit-488 (green) antibodies were used. Cells were then washed with PBS and stained with DAPI. The coverslips were mounted in Vectashield onto slides and images were taken with Nikon confocal microscope.

Fluorescence-activated cell sorting (FACS)

For cell cycle analysis, cells were treated for 24 and 48 h with DMSO and indicated concentrations of CCT137690. Following treatment, cells were collected by trypsinization and washed with PBS containing 1% FBS. Cells were then fixed with 70% ice-cold ethanol and stained with a solution containing propidium iodide and RNase for 30 min at 37°C. Analysis of cell cycle profile was carried out using BD FACSCalibur. For Annexin V/PI staining, cells treated with CCT137690 were collected by trypsinization, washed with PBS and stained with FITC-labelled Annexin V/PI kit according to manufacturer’s instructions (Santa Cruz). Induction of apoptosis was measured using BD FACSCalibur.

Assay for cell viability

The inhibition of cell proliferation by CCT137690 in ORL-48 and ORL-115 cell lines was measured by Sulphorhodamine B (SRB) assay as described elsewhere [31]. Briefly, ORL-48 and ORL-115 cells were split in 96 well plates and treated with nine different concentrations of CCT137690 for 72 h. Next, the cells were fixed with 3% trichloroacetic acid, washed with water and air dried. Plates were then stained with 100µl/well of 0.06% SRB solution for 30 minutes. Plates were next washed with 1% acetic acid, air dried and the SRB dissolved in 100 µl of 10mM Tris pH 10.5. Finally, absorbance was measured at 490nm using a microplate reader and GI₅₀ values were calculated using GraphPad Prism.

Western blot analysis

Cells were lysed in lysis buffer containing 50mM NaCl, 20mM Tris pH7.5, 1mM EDTA, 1% Triton X100 and 50mM NaF supplemented with protease and phosphatase inhibitors. Protein concentration was measured using Bradford reagent and samples were prepared with equal amounts of proteins. Proteins were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skimmed milk and incubated overnight with cleaved-PARP (Cell Signaling) phospho-Histone H3 MerckMillipore), histone H3 (Abcam), and anti-alpha-tubulin (Santa Cruz) antibodies. Following incubation with the primary antibodies, blots were washed with PBST and incubated with HRP-labeled secondary antibodies for 1 h. Blots were washed again with PBST and developed using ECL reagent on BioRad ChemiDoc system.

Formation and analysis of 3D spheroids

Matrigel was diluted in F12/DMEM medium on ice to make final concentrations of 2% and 5%. 96-well plates were centrifuged upside down at 1500 rpm to remove debris and coated with 1.5% agarose. Next, 3000 cells were seeded in 100µl per well in 96-well plates. 100 µl of Matrigel solutions (described above) were then added per well to make final Matrigel concentrations of 2.5% and 1% for ORL-48 and ORL-115, respectively. Plates were immediately centrifuged at 1500 rpm and incubated at 37⁰C for 4 days. Images were captured at 4, 7, 10, 12 and 14 days and 50% of medium was replenished with fresh F12/DMEM.

Imaging of Spheroids was performed with an inverted light microscope. Open source tools ReViSP, AnaSP and ImageJ were used to measure different morphological parameters of spheroids such as area, volume, perimeter, diameter and sphericity.

Results

CCT137690 mediated inhibition of Aurora kinases stops proliferation in oral cancer cells

The anti-proliferative effect of CCT137690 was investigated in two oral cancer cell lines, ORL-48 and ORL-115, that were derived from patients of South Asian origin [32]. CCT137690 inhibited proliferation of both the cell lines with GI₅₀ values of 0.81 µM and 0.84 µM in ORL-48 and ORL-115, respectively (Figure 1(a)). Next, we validated CCT137690-meditated inhibition of Aurora kinases in these cells by determining histone H3 phosphorylation at S10 (an Aurora B substrate) and autophosphorylation of Aurora A at T288. Phosphorylation of histone H3 was completely inhibited upon treatment of nocodazole-arrested ORL-48 and ORL-115 cells with different concentrations of CCT137690 (Figure 1(b)). Similarly, treatment with CCT137690 completely inhibited the phosphorylation of endogenous Aurora A at T288 in both the ORL-115 (top) and ORL-48 (bottom) cells, as demonstrated by immunofluorescence of mitotic cells (Figure 1(c)).

Figure 1.

Inhibition of Aurora A and B kinases in oral cancer cell lines. (a) GI₅₀ values of CCT137690 in ORL-48 and ORL-115 cell lines. (b) Oral cancer cells were treated with 50ng/ml nocodazole for 24 h followed by treatment with CCT137690 for 2 h. Inhibition of Aurora B kinase was determined by immunoblotting using phospho-histone H3 (S10)-specific antibodies, while total histone H3 served as a loading control. (c) Oral cancer cells ORL-115 (top) and ORL-48 (bottom) were treated with 1µM CCT137690 for 4 h and then analyzed by immunofluorescence with phospho Aurora A (T288; Green), alpha-tubulin (Red) and DAPI (blue) .

CCT137690-treated oral cancer cells exhibit aberrant mitosis and apoptotic cell death

Aurora kinases play important roles in different phases of mitosis and selective inhibition of Aurora isoforms leads to specific phenotypes such as monopolar spindle formation in case of Aurora A inhibition or misalignment of chromosomes and endoreduplication when Aurora B function is blocked. However, when both kinases are inhibited simultaneously, the Aurora B phenotype is dominant [30,33]. Treatment of ORL-115 cells with 0.5µM and 1µM concentrations of CCT137690 for 24 and 48 h resulted in polyploidy (4N and 8N DNA content at 24 and 48 h and 16N at 48 h), indicating endoreduplication (Figure 2(a)). The endoreduplication was further confirmed by an increase in the size of ORL-48 and ORL-115 cells treated with CCT137690 for 48 h (Figure 2(b)). CCT137690-treated cells were more than double in total and nuclear size compared to the DMSO-treated cells. Treatment of both ORL-48 and ORL-115 cells with 1 µM CCT137690 for 24 and 48 h also resulted in multipolar spindle formation and distortion of chromosome alignment during metaphase (Figure 2(c)).

Figure 2.

CCT137690 induces polyploidy and multipolar spindle formation in oral cancer cell lines. (a) ORL-115 cells were treated with indicated concentrations of CCT137690 for 24 and 48 h followed by analysis with flow cytometry. (b) ORL-115 and ORL-48 cells were treated with 1µM CCT137690 for 48 h and analyzed by immunofluorescence with alpha-tubulin (Red), pericentrin (Green; in the case of c) and DAPI (Blue). Scale bar in B indicates the length of 50 µm.

Endoreduplication of cells due to failed cytokinesis may lead to the mitotic catastrophe which ultimately induces apoptotic cell death through the mitochondrial pathway [34]. Therefore, we next determined the CCT137690-induced apoptosis in oral cancer cells through flow cytometry following Annexin V/PI staining. Treatment of both ORL-48 and ORL-115 cells with 0.5 µM CCT137690 for 24 and 48 h resulted in increased apoptosis at both the time points (Figure 3(a)). Similarly, a robust increase in apoptosis following CCT137690 treatment for 24 and 48 h in both the cell lines was also determined through increased caspase 3-mediated cleavage of poly (ADP-ribose) polymerase (PARP). Expression of alpha-tubulin was used as a loading control (Figure 3(b)).

Figure 3.

Treatment with CCT137690 induces apoptosis in oral cancer cells. (a) ORL-48 and ORL-115 cells were treated with 0.5µM of CCT137690 for 48 h, stained with Annexin V and Propidium Iodide and analyzed through flow cytometry. (b) Oral cancer cells were treated with two concentrations of CCT137690 for 24 and 48 h and cell lysates were analyzed by immunoblotting using specific antibodies for cleaved-PARP and alpha-tubulin (loading control) .

Inhibitors of EGFR and PI3K synergize with CCT137690 in oral cancer cells

In order to identify drugs that synergize with CCT137690 in oral cancer cell lines, we evaluated CCT137690 in combination with several other inhibitors including gefitinib (EGFR), AZD6244 (MEK), paclitaxel (microtubule stabilizing agent), vincristine (microtubule depolymerizing agent), GDC-0068 (pan-AKT) and pictilisib (GDC-0941; PI3K). CCT137690 was found to be highly synergistic with gefitinib and pictilisib as determined by the combination index (CI) values which were below 0.8 at most of the effective dose concentrations (ED 50, 75, 90 and 95) in both ORL-48 and ORL-115 cells (Table 1).

Table 1.

Combinations of CCT137690 with gefitinib and pictilisib exhibit synergism in oral cancer cells, (a) ORL-48, (b) ORL-115. Combination index values were calculated by compusyn software as described in supplmentary methods, <0.8 = synergism, >1 = antagonism.

Drug Combinations in ORL-48	CI values at
	ED50	ED75	ED90	ED95
(a)
CCT137690 + Gefitinib	0.770	0.663	0.690	0.818
CCT137690  + AZD6244	0.171	0.310	1.000	2.303
CCT137690 + Paclitaxel	593	347	203	247
CCT137690 + Vincristine	0.679	0.561	0.467	0.415
CCT137690 + GDC0068	1.424	1.234	1.103	1.047
CCT137690 + Pictilisib	0.385	0.375	0.381	0.395
(b)
CCT137690 + Gefitinib	0.440	0.434	0.450	0.484
CCT137690 + AZD6244	0.145	0.171	0.697	1.969
CCT137690 + Paclitaxel	1.557	374	898	374
CCT137690 + Vincristine	1.366	1.619	1.970	2.268
CCT137690 + GDC0068	0.736	0.433	0.424	0.479
CCT137690 + Pictilisib	0.197	0.181	0.171	0.168

Synthesis of drug loaded (L-P-NCps) and empty (E-P-NCps) PEG nanocapsules

We, next synthesized drug loaded (L-P-NCps; CCT137690 in combination with gefitinib or pictilisib) and empty (E-P-NCps) polyethylene glycol (PEG) nanocapsules and determined their activity in oral cancer cells. L-P-NCps (~500 nm in diameter) were prepared through modified solvent diffusion method [35], followed by evaporation while exploiting the self-assembling property of polyethylene glycol (PEG) and Linoleic acid to form nanocapsules upon hydration (Figure 4). Hydrophobic drugs (CCT137690 with pictilisib and gefitinib) were entrapped to get two different types of nanocapsules, CCT137690 & pictilisib loaded PEG nanocapsules (CG-L-P-NCps) and CCT137690 & gefitinib loaded PEG nanocapsules (CGe-L-P-NCps). Empty nanocapsules (E-P-NCps) were synthesized without any drug and used as a control. SDS was used to improve the electrostatic stability of these NCps. The formation of NCps was confirmed by observing a change in the hydrodynamic diameter of L-P-NCps measured by dynamic light scattering (Zetasizer Nano ZSP). The detailed synthesis and evaluation of NCps is described in the supplementary information.

Figure 4.

Synthetic scheme for Nanocapsules. (1–3) Involves emulsification by mixing an aqueous solution of PEG with solutions of CCT137690 and gefitinib or CCT137690 and pictilisib in linoleic acid followed by their dispersion in water (4). It resulted in the formation of drug loaded L-P-NCps with a hydrophobic core and hydrophilic surface (diameter ~500 nm) as shown in SEM micrographs (5) .

Nanocapsules harboring drug combinations induce apoptosis in oral cancer cells

Both types of loaded nanocapsules, CG-L-P-NCps and CGe-L-P-NCps, induced apoptotic cell death in ORL-48 and ORL-115 cells following 24 h treatment as indicated by increased levels of cleaved-PARP (Figure 5). The apoptotic cell death induced by both CG-L-P-NCps and CGe-L-P-NCps (as determined by cleaved-PARP levels) was higher compared to the one induced by the drugs individually. It was, however, comparable to the apoptosis induction in cells treated with combinations of CCT137690 with gefitinib or pictilisib, indicating that the nanocapsules are acting as carriers of drug combinations and have a similar potency to the drug combinations alone at the concentration used.

Figure 5.

Nanocapsules and Drug mediated induction of apoptosis in Oral cancer cell lines. ORL-48 (top) and ORL-115 (bottom) were treated with indicated combinations of CCT137690, pictilisib and gefitinib in the form of drugs or nanoparticles for 24 h and then cell lysates were analyzed by immunoblotting with cleaved PARP and alpha-tubulin (loading control) .

E-P-NCps: Empty nano-capsules; CGe-L-P-NCps: Nano-capsules containing 1.6 µM gefitinib and 1.29 µM CCT137690; CG-L-P-NCps: Nano-capsules containing 1.6 µM pictilisib and 1.46 µM CCT137690

Nanocapsules inhibit oral cancer cells in 3D spheroids:

The three dimensional (3D) culture of tumor cells provides a superior in-vitro model for drug evaluation than the two dimensional (2D) monolayer culture that fails to mimic the in-vivo tumor microenvironment [36]. We, therefore, optimized the growth of 3D spheroids of oral cancer cells in Matrigel and evaluated the effect of our drug loaded nanocapsules on their growth (Supplementary Figure S2). ORL-48 cells formed irregular spheroids in the presence of 2.5% Matrigel at day 4 (Figure 6(a)). When treated with increasing concentrations of L-P-NCps for 72h (day 4 to day 7), the growth of these spheroids (at day 14), was significantly inhibited with increasing concentrations of the nanocapsules. Volumes of spheroids were reduced by up to 97% and 98% at the highest concentrations of CGe-L-P-NCps and CG-L-P-NCps, respectively (Figure 6(a)). Similar inhibition was also observed in spheroids treated with the drug combinations without nanocapsules, where volumes were reduced up to 91% and 94% for combinations of CCT137690 with pictilisib and gefitinib, respectively (Supplementary Figure S3A). ORL-115 cells formed smooth and spherical spheroids in the presence of 1% Matrigel at day 4. Treatment with the CGe-L-P-NCps and CG-L-P-NCps for 72h, inhibited their growth, with 64% and 77% reduction in their volumes at the highest concentrations, respectively (Figure 6(b)). However, for their corresponding drug combinations without nanocapsules, we observed 92% and 85% volume reduction in CCT137690 in combination with pictilisib and gefitinib, respectively (Supplementary Figure S3B). Other spheroid parameters including size, diameter, perimeter and area also exhibited a significant reduction as compared to the control at higher concentrations of nanocapsules and their corresponding free drug combinations (Supplementary Figure S3).

Figure 6.

Nanocapsules based formulations of drugs inhibit the growth of oral cancer cells in 3D spheroid cultures. (a) ORL-48 and (b) ORL-115 spheroids treated with increasing concentrations of indicated drugs containing nanocapsules, CG-LP-NCps (above) and CGe-LP-NCps (below). Images shown above were taken at 14th day and their parameters were determined as described in methods section.

Discussion

Oral squamous cell carcinoma has a disproportionately high incidence in developing countries in South-Central Asia including Pakistan, where it is the second most common type of cancer [1,37]. In terms of treatment, surgery is considered the best available option for early-stage OSCC patients but at later stages, radiotherapy in combination with chemotherapy is routinely used [38]. Despite the use of such combination therapies, the five years survival rate remains low with a high risk of recurrence [39]. Identification and validation of new drug targets, therefore, can provide novel therapeutic options for improved treatment with better prognosis in OSCC. One of such targets is Aurora family of kinases, which play key roles in regulating different stages of mitosis [8]. Their overexpression or amplification in oral cancers promotes tumor progression and is associated with poor prognosis [6,14,15]. Here we have evaluated the efficacy of the pan-aurora kinase inhibitor, CCT137690 in oral cancer cell lines and identified drugs (gefitinib and pictilisib) that are synergistic with CCT137690 in the cell lines. Furthermore, we have synthesized nanocapsules containing combinations of these drugs with CCT137690, characterized them and determined their activity in 3D spheroid cultures of both the oral cancer cell lines.

CCT137690 is a potent pan-Aurora inhibitor that inhibits proliferation and induces apoptosis in different cancer cell lines [29,30]. In vivo studies have shown significant efficacy of CCT137690 in MYCN-driven neuroblastoma model [30] and xenograft models of colorectal cancer [28] and FLT3-driven Acute Myeloid Leukemia [29]. In the present study, CCT137690 potently inhibited the viability of oral cancer cell lines with GI₅₀ values of 0.81 µM and 0.84 µM in ORL-48 and ORL-115 cell lines, respectively. Activation of both Aurora A and B kinases was also completely inhibited by CCT137690 in both the cell lines as determined by inhibition of autophosphorylation of Aurora A at T288 and histone H3 phosphorylation at S10, two biomarkers routinely used to assess cellular inhibition of Aurora A and Aurora B, respectively [16,30,40] (Figure 1). Consistent with the inhibition of Aurora kinases, CCT137690-treated oral cancer cells displayed phenotypes associated with pan-Aurora inhibition including induction of polyploidy (8N and 16N DNA content) and aberrant mitosis (Figure 2). The former results from endoreduplication due to failed cytokinesis (a consequence of Aurora B inhibition), while the latter results from failed mitotic spindle formation and misalignment of chromosomes (due to Aurora A and B inhibition, respectively). This aberrant mitosis and induction of polyploidy ultimately lead to apoptotic cell death as determined by Annexin V/PI staining and PARP-cleavage (Figure 3).

Administration of drugs in combination not only enhances their efficacy but also helps avoid and overcome drug resistance [16,40]. In order to identify drugs that synergistically enhance the efficacy of CCT137690 in oral cancer cells, we evaluated CCT137690 in combination with six different chemotherapeutic and targeted drugs in ORL-48 and ORL-115 cell lines. Inhibitors of EGFR (gefitinib) and PI3 kinase (pictilisib) exhibited strong synergistic interaction with CCT137690 in both the cell lines (Table 1). This is in accordance with the recent studies demonstrating that Aurora A kinase up-regulation plays a key role in generating resistance against EGFR inhibitors in lung adenocarcinoma cells [41] and simultaneous inhibition of EGFR and Aurora kinases confers enhanced therapeutic effect in SCCHN cells [23]. Moreover, both ORL-48 and ORL-115 cell lines were recently found to be sensitive to EGFR inhibitors erlotinib and afatinib due to polymorphism in periplakin gene [42] explaining the synergistic effect of gefitinib with CCT137690. Similarly, PI3 kinase inhibitors have been shown to be synergistic with aurora kinase inhibitors in SCCHN cells [43].

One of the challenges associated with using drugs in combinations is the difference in their properties including distinct chemical structures, biodistribution, and systemic clearance that may require repeated administration at different time points [44,45]. Owing to these shortcomings, usage of several potent drug combinations is limited in the clinic, despite exhibiting intriguing in vitro efficacy. Amongst the therapeutic strategies to reduce the problems associated with drug combinations, nanoparticle-based drug formulations have emerged as an effective method that confers several advantages including reduced cytotoxicity, controlled drug release, synchronized pharmacokinetics and enhanced bioavailability [46]. Following the identification of drugs synergistic with CCT137690, we synthesized polyethylene glycol based nanocapsules harboring these drug combinations (Figure 4), characterized them (Supplementary Table 1 and Supplementary Figure S1) and evaluated their efficacy in 2D monolayer and 3D spheroid cultures of oral cancer cell lines. The nanocapsules formulations for both the drug combinations inhibited the growth of oral cancer cell lines in 3D cultures, which better mimic tumor pathophysiology. Similarly, treatment of both cell lines with nanocapsules of the two drug combinations resulted in apoptotic cell death comparable to that induced by the free drug combinations (Figure 5). Although the induction of apoptosis at drug concentrations used for nanocapsules is comparable to the free drug combinations at the same ratio (Figure 5), they were slightly less efficacious in 3D spheroids for ORL-115 cell line at lower concentrations compared to the free drug combinations (Figure 6 and Supplementary Figure S3). Most nanoparticle formulations that target cancer have focused on the efficient delivery and reduced cytotoxicity of chemotherapeutic drugs and there are at least six lipid-based nanoparticle formulations of routinely used chemotherapeutic drugs that have been approved for the treatment of different types of cancers [44]. There are, however, few reports of delivery of targeted drugs in nanoparticle formulations. Accurin polymeric nanoparticles of Aurora B inhibitor AZD2811, for example, demonstrate improved efficacy, reduced toxicity with infrequent dosing schedules in preclinical models [27] and are being evaluated in phase I clinical trial [47]. Similarly, micellear gold nanoparticles containing dual tyrosine kinase inhibitor ZD6474 demonstrated targeted and sustained release in the acidic tumor environment, which resulted in efficacy in a xenograft model of triple negative breast cancer [48].

Taken together, we have demonstrated the efficacy of the pan-Aurora inhibitor CCT137690 in oral cancer cell lines, identified two drugs that are synergistic with CCT137690 and have presented nanocapsules formulation for the co-delivery of CCT137690 in combination with these drugs.

Funding Statement

This work was supported by the Higher Education Commission, Pakistan [NRPU 5909].

Acknowledgments

We thank Lahore University of Management Sciences for the startup grant and Higher Education Commission of Pakistan for National Research Program for Universities grant (NRPU 5909) to support this work. We would like to thank Dr. Safee Ullah Chaudhary for carefully reading the mansucript and providing valuable input.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.