The Combination of Alisertib, an Investigational Aurora kinase A inhibitor, and Docetaxel Promotes Cell Death and Reduces Tumor Growth in Pre-Clinical Cell Models of Upper Gastrointestinal Adenocarcinomas

Abstract

Background

Upper gastrointestinal adenocarcinomas (UGCs) respond poorly to current chemotherapeutic regimes. We and others have previously reported frequent AURKA gene amplification and mRNA and protein overexpression in UGCs. This study aimed to determine the therapeutic potential of Alisertib (MLN8237) alone and in combination with Docetaxel in UGCs.

Methods

Following treatment with Alisertib and/or Docetaxel, clonogenic cell survival, cell cycle analyses, Western blotting and tumor xenograft growth assays were carried out to measure cell survival, cell cycle progression, apoptotic protein expression and tumor xenograft volumes, respectively.

Results

Using AGS, FLO-1 and OE33 UGC cell lines, with constitutive AURKA overexpression and variable-p53 status, we observed significantly enhanced inhibition of cancer cell survival with Alisertib and Docetaxel combination (p<0.001), as compared to single agent treatments. Cell cycle analyses after 48hr of treatment with Alisertib demonstrated a significant increase in the percentage of polyploidy in UGC cells (p<0.01) that was further enhanced by Docetaxel (p<0.001). Additionally, an increase in the percentage of cells in the sub-G1-phase that was observed with Alisertib (p<0.01) was significantly enhanced with the combination (p<0.001). Western blot analysis demonstrated higher induction of cleaved caspase 3 protein expression with the combined treatment, as compared to single agent treatments. In addition, FLO-1 and OE33 cell xenograft models demonstrated an enhanced anti-tumor activity for the Alisertib and Docetaxel combination, as compared to single agent treatments (p<0.001).

Conclusion

This study demonstrates that Alisertib, combined with Docetaxel, can mediate a better therapeutic outcome in UGCs cell lines.

Introduction

Upper gastrointestinal adenocarcinomas (UGC i.e. adenocarcinomas of stomach and esophagus) are associated with poor patient survival rate due to inherent resistance to current therapeutic regimens ^{1, 2}. Global epidemiological data indicate that approximately 1.4 million new cases of UGC are diagnosed annually resulting in about 1.1 million deaths ³. Over the past several decades the incidence rates for distal gastric cancers have declined, however, incidence rates for adenocarcinoma of the gastric cardia, gastro-esophageal (GE) junction and esophagus continue to rise ^{3, 4}.

Multiple pro-survival and drug resistance genes such as the epidermal growth factor receptor (EGFR), ERBB2/HER-2, K-Ras and Aurora kinase A (AURKA) mediate oncogenic signaling pathways in gastric and esophageal adenocarcinomas ^5–9. Despite several adjuvant and neoadjuvant chemotherapeutic treatment strategies, survival rate for patients with UGC have shown only marginal improvements¹⁰. Therefore, laboratory investigations and preclinical studies specifically aimed at developing novel targeted therapies and chemotherapeutic combinations with potent anti-tumor activity are desperately needed to treat UGC.

We and others have previously reported amplification of chromosomal region 20q13 in UGC ^{11, 12}. The 20q13 chromosomal region harbors the Aurora kinase A (AURKA) gene, which is frequently amplified and/or overexpressed in several malignancies including bladder, breast, colon, liver, ovaries, pancreas, stomach and esophagus ^{8, 13}. AURKA is a key cell cycle regulator critical for mitotic events ^{14, 15}. However, when overexpressed, AURKA is a bona fide oncogene resulting in genetic instability, dedifferentiated morphology and poor prognosis in UGCs ^{6, 16}. Overexpression of AURKA promotes cancer cell growth and resistance to chemotherapy by upregulating oncogenic signaling pathways and suppressing cell death mechanisms, respectively ¹³. Overexpression of AURKA induces growth- and survival-promoting oncogenic signaling pathways such as PI3K/AKT and β-catenin in UGC cancer cells ¹³. The DCF (Docetaxel, Cisplatin and 5-Fluorouracil) chemotherapy regimen is one of the most efficacious chemotherapeutic regimens against advanced gastric cancer ¹⁷. Interestingly, AURKA overexpression has been reported to mediate resistance against Taxol and Cisplatin induced cell death ^{18, 19}. The cell death mechanisms, regulated by p53 family of pro-apoptotic proteins, are activated in cancer cells following treatment with chemotherapeutic agents. However, mutations in the p53 function are frequently observed in UGCs. In p53-mutant cancers, p73 can mediate p53 like apoptotic functions and activate apoptotic pathways following treatment with chemotherapeutic agents ²⁰. Interestingly, overexpression of AURKA in cancer has been shown to suppress p53 and p73 protein expression and function ^{21, 22}. These findings suggest that p53-mutant UGC with a constitutively higher level of AURKA expression could respond poorly to chemotherapy. Therefore, given the poor response of UGCs to current therapeutic regimens; development of novel therapeutic strategies, that take into account the molecular make-up of tumors to activate cell death response, are critically needed to combat UGCs.

Alisertib (MLN8237) is an investigational small molecule inhibitor developed by Millennium Pharmaceuticals, Inc. that has been shown to selectively inhibit AURKA and thereby induce cell cycle arrest, aneuploidy, polyploidy, mitotic catastrophe, and cell death ^{8, 23}. Currently, Alisertib is being tested in various Phase I, II and III clinical trials for advanced solid tumors and hematological malignancies (http://clinicaltrials.gov/). In addition, multiple clinical trials have indicated that Docetaxel and its combinations with Cisplatin and 5-Fluorouracil (DCF) have significant anti-tumor activity in UGC ¹⁷. Docetaxel is a microtubule polymerizing chemotherapeutic agent which disrupts microtubule dynamics by binding to the β-subunit of tubulin and promoting its polymerization ²⁴. Consequently, Docetaxel causes cell cycle arrest in mitosis and subsequently induces apoptosis. In this study, we investigated the potential therapeutic benefit of Alisertib alone and in combination with Docetaxel, using in vitro and in vivo cell models of UGC with variable-p53 status. We hypothesized that the Alisertib and Docetaxel combination treatment will cause enhanced cell cycle arrest resulting in polyploidy and subsequent induction of apoptosis in UGC cancer cells, irrespective of their p53 status.

Materials and Methods

Cell culture and pharmacologic reagents

AGS gastric adenocarcinoma cell line (p53 wild type) and FLO-1/OE33 (p53 mutant) esophageal adenocarcinoma cell lines were maintained as a monolayer culture in DMEM (Gibco, CA) cell culture medium supplemented with 10 % (v/v) fetal bovine serum or FBS (Gibco, CA) ²⁵. All cell lines were evaluated weekly to ascertain conformity to the appropriate in vitro morphological characteristics ²⁶. MLN8237 (Alisertib) was provided by Millennium Pharmaceuticals, Inc. and Alisertib stock solutions for in vitro and in vivo studies were prepared according to our previously reported methods ⁸. Docetaxel (Sanofi Aventis, Bridgewater, NJ) stock solution (11.6mM) prepared in 13% ethanol (v/v) was provided by TVC Outpatient Pharmacy, Vanderbilt University Medical Center.

Clonogenic cell survival assay

AGS and FLO-1/OE33 cells were seeded at 5000 and 10,000 cells/well, respectively, in a six well plate overnight and treated with various concentrations of Alisertib (0.25, 0.5, 1.0, 2.0 and 5.0μM) for 24hr. Following treatment, UGC cell survival was determined according to our previously reported protocol ⁸. Briefly, following treatment, cells were incubated in drug free cell culture medium for ten days. Subsequently, cells were fixed with 2% Paraformaldehyde solution, stained with crystal violet dye solution and cell survival was quantified by measuring the dye signal in each well with ImageJ analysis software (NIH, MD). Additionally, we selected an Alisertib dose close to IC-50 (0.5μM) and treated the cells with Alisertib (0.5μM) and/or Docetaxel (0.5, 1.0 or 5.0nM) for 24hr.

Cell cycle analysis

AGS and FLO-1 cells were treated with the Alisertib (0.1μM) and/or Docetaxel (0.5nM) in cell culture medium (2.5% FBS) for 24hr and 48hr, respectively. OE33 cells were treated with Alisertib (0.5μM) and/or Docetaxel (1.0nM) in cell culture medium (2.5% FBS) for 24hr and 48hr, respectively. Following treatment, supernatant media was collected and adherent cells were trypsinized. The supernatant and trypsinized cells were centrifuged together at 2000×g, 4°C for 10min. The cells were re-suspended in 1ml Propidium Iodide (PI) solution (PI 50μg/ml and RNase 1μg/ml in 1x Phosphate Buffered Saline) and incubated at room temperature in the dark for 30min. Subsequently, the cells were analyzed with BD LSR III flow cytometry (BD Biosciences, San Jose, CA) and the data were processed with BD FACS Diva software.

Western blot analysis

AGS, FLO-1 and OE33 cancer cells were plated overnight at 30% confluence in cell culture medium (10% FBS). The AGS cells were treated with Alisertib (0.25μM) and/or Docetaxel (1.0nM), FLO-1 cells were treated with the Alisertib (0.1μM) and/or Docetaxel (0.5nM) and OE33 cells were treated with Alisertib (0.5μM) and/or Docetaxel (1.0nM), respectively, for 48hr in cell culture medium supplemented with 2.5% FBS. Following treatment, cell lysates were prepared and evaluated for total and phosphorylated proteins; p-AURKA (Thr 288), AURKA, Cleaved Caspase 3 and β-Actin (Cell Signaling, MA), according to standard protocols ²⁷.

In vivo tumor xenograft inhibition

Four million FLO-1 or OE33 cells suspended in 200μl of DMEM matrigel mixture (50% DMEM supplemented with 10% FBS and 50% matrigel) were injected into the flank regions of female athymic nude - Foxn1 nu/nu mice (Harlan Laboratories Inc., IN). The tumors were allowed to grow until 200mm³ in size before starting the treatment with a daily Alisertib (30mg/kg, orally) and/or once per week Docetaxel (10mg/kg, via I.P. injection) for three weeks. Tumor xenografts were measured every alternate day and tumor size was calculated according to the following formula: T_vol = L × W² × 0.5 where T_vol is tumor volume, L is tumor length and W is tumor width²³.

Immunohistochemistry

After 21 days of animal treatment, the tumors were isolated and Immunohistochemistry was carried out to measure Ki-67 and cleaved caspase 3 protein expression levels as previously reported ⁸. Protein expression were scored using composite expression score (CES) that was determined utilizing the formula: CES = 4(Intensity − 1) + Frequency, as described earlier; intensity (scale 0–3) and frequency (scale 0–4)²⁸.

Statistical analysis

Data are presented as means ± standard error of mean. All in vitro experiments were performed in triplicates. One-way analysis of variance (ANOVA) with Tu-Key post hoc analysis was used to show statistical difference between control groups and treatment groups at the treatment end points. Two-way ANOVA with Bonferroni post hoc analysis was used to show statistical difference between various treatment groups and cell cycle stages. For tumor xenograft data, two-way ANOVA (time point matched) analysis with Bonferroni post-test was used to compare the “mean tumor size” of a treatment group at any given treatment day with the “mean tumor size” of another other treatment groups at the corresponding treatment day. All above statistical analyses were carried out using GraphPad Prism 5 software (GraphPad Software Inc., CA). The p values of ≤0.05 were considered statistically significant and are marked in the Figures: * = p<0.05 and ** = p<0.01.

Results

Alisertib significantly enhanced Docetaxel –mediated inhibition of cell survival

Both FLO1 and OE33 cell lines show gene amplification and overexpression of AURKA at the mRNA and protein level ^{8, 29}. Similarly, the AGS cells showed an increase in AURKA DNA copy number (2.26 fold) and mRNA level (4.98 fold) (data not shown). Therefore, these cell models mimic the in vivo data of overexpression of AURKA in primary UGCs ³⁰. The clonogenic cell survival assay data indicated that Alisertib (0.5μM) or Docetaxel (1.0nM) single agent treatments decreased the % survival of AGS cells (Alisertib 0.5μM: 45.5±4.6, p<0.01 and Docetaxel 1.0nM: 53.6±1.8, p<0.01) (Figure 1A), FLO-1 cells (Alisertib 0.5μM: 45.5±4.6, p<0.01 and Docetaxel 1.0nM: 70.1±5.6, p<0.05) (Figure 1B), and OE33 cells (Alisertib 0.5μM: 45.5±4.6, p<0.01 and Docetaxel 1.0nM: 32.4±3.5, p<0.01) (Figure 1C). The treatment with the Alisertib (0.5μM) and Docetaxel (1.0nM) combination led to a significantly enhanced inhibition of % survival in AGS cells (Alisertib 0.5μM + Docetaxel 1.0nM: 5.5±0.7, p<0.01) (Figure 1A), FLO-1 (Alisertib 0.5μM + Docetaxel 1.0nM: 4.5±0.5, p<0.01) (Figure 1B) and OE33 (Alisertib 0.5μM + Docetaxel 1.0nM: 13.0±1.8, p<0.01) (Figure 1C) cells. These results suggest that the combination of Alisertib with Docetaxel may have a significantly higher inhibitory effect on UGC cell survival.

Figure 1. Alisertib/MLN8237 and Docetaxel combination treatment significantly inhibits cell survival.

The cell survival assay data indicates significant inhibition of AGS (A), FLO-1 (B) and OE33 (C) cell survival after treatment with Alisertib (MLN) and Docetaxel combination. The AGS, FLO-1 and OE33 cells were treated with Alisertib (0.5μM) and/or Docetaxel (0.5nM, 1.0nM & 5.0nM) for 24hr and incubated in drug free medium for 10 days. Alisertib (0.5μM) and Docetaxel (1.0nM) combination treatment significantly suppressed survival of cells. CV - Control Vehicle; MLN 0.5 - MLN8237/Alisertib 0.5μM; DOCE 0.5 - Docetaxel 0.5nM; DOCE 1.0 - Docetaxel 1.0nM, DOCE 5.0 - Docetaxel 5.0nM,* p<0.05 and ** p<0.01.

Alisertib enhanced Docetaxel induced polyploidy and apoptosis in UGC cells

Using AGS, FLO-1 and OE33 cell lines as in vitro models of UGC to study the effect of Alisertib and Docetaxel on cell cycle progression, the treatment with the Alisertib alone or in combination with Docetaxel for 24hr significantly reduced the percentage of cells in G1-phase and S-phase and induced a significant delay in the transition from G2- to M-phase in AGS and OE33 cells (Figure 2A and 3C). The 24hr treatment with Alisertib alone had a similar effect on G1-phase, S-phase and G2-to M-phase transition in FLO-1 cells, however, this effect was significantly more pronounced after 24hr treatment with Alisertib and Docetaxel in combination (Figure 3A). In addition, treatment with Alisertib alone for 24hr significantly increased the percentage of polyploid cells, which was further enhanced by Alisertib and Docetaxel combination treatment in AGS and FLO-1 cells (Figure 2A and Figure 3A). At 48hr time point, FLO-1 cells with Alisertib or Docetaxel single agent treatments demonstrated an increase in the percentage of cells in the sub-G1-phase (p<0.05)(Figure 3B). This effect was significantly enhanced in FLO-1cells that received the combined treatment (p<0.01) (Figure 3B). The cell cycle analyses indicate that FLO-1 cells are more sensitive to Alisertib and/or Docetaxel treatments, as evidenced by an increase in sub-G1-phase cells after 24hr and 48hr treatment. In addition, 48hr treatment with Alisertib and Docetaxel enhances polyploidy in AGS and OE33 UGC cells. These finding are in agreement with previously published reports which indicate that Docetaxel induced microtubule stabilization impairs mitosis, generating aneuploid and tetraploid cells that subsequently undergo apoptotic cell death ^{24, 31, 32}. Our data suggests that the combination treatment promotes polyploidy early on and depending on the cell line; polyploidy likely leads to cell death (sub-G1) either early on (FLO-1 cells) or at later time points (AGS and OE33). These findings provide a plausible explanation and support for the clonogenic cell survival assay results that measured long-term cell viability. We also observed that the treatment with Alisertib or Docetaxel alone induced the expression of cleaved caspase 3, a common apoptosis marker; a finding that was significantly enhanced after combined treatment with Alisertib and Docetaxel for 48hr. (Figure 4). These results support our notion that Alisertib can significantly enhance Docetaxel-induced apoptosis in UGCs.

Figure 2. Alisertib/MLN8237 and Docetaxel combination treatment enhances polyploidy and alters cell cycle progression.

AGS cells were treated with Alisertib (0.1μM) and/or Docetaxel (0.5nM) for 24hr and 48hr, respectively, and cell cycle progression was analyzed with flow cytometry. After 24hr (A) and 48hr (B) of treatment, Alisertib (0.1μM) in combination with Docetaxel (0.5nM) significantly enhanced polyploidy in AGS cells. CV - Control Vehicle; MLN - MLN8237/Alisertib; DOCE - Docetaxel, * p<0.05 and ** p<0.01.

Figure 3. Alisertib/MLN8237 and Docetaxel combination treatment enhances polyploidy and alters cell cycle progression.

A–B) FLO-1 cells were treated with Alisertib (0.1μM) and/or Docetaxel (0.5nM) in cell culture medium (2.5% FBS) for 24hr and 48hr, respectively, and cell cycle progression was analyzed with flow cytometry. (A) After 24hr of treatment, Alisertib in combination with Docetaxel induces G2-M-phase arrest, suppresses G1-phase and enhances apoptosis (sub-G1) in FLO-1 UGC cells. (B) After 48hr of treatment, Alisertib and Docetaxel combination treatment significantly increases the percentage of sub-G1-phase cells in FLO-1 UGC cells. C–D) OE33 cells were treated with Alisertib and/or Docetaxel for 24hr and 48hr, respectively, and cell cycle progression was analyzed with flow cytometry. (C) After 24hr of treatment, Alisertib in combination with Docetaxel induces G2-M-phase arrest and suppresses G1 and S-phases, respectively in OE33 UGC cells. (D) After 48hr of treatment, Alisertib and Docetaxel in combination enhances polyploidy in OE33 cells. CV - Control Vehicle; MLN -MLN8237/Alisertib; DOCE - Docetaxel, * p<0.05 and ** p<0.01.

Figure 4. Alisertib/MLN8237 and Docetaxel combination treatment significantly enhances apoptotic marker expression.

The AGS (A), FLO-1 (B), and OE33 (C) cells were treated with Alisertib and/or Docetaxel for 48hr. Alisertib and Docetaxel combination treatment significantly enhanced expression of cleaved caspase 3 in AGS, FLO-1 and OE33 UGC cells. CV - Control Vehicle; MLN - MLN8237/Alisertib; DOCE – Docetaxel.

Alisertib and Docetaxel combination treatment exhibits enhanced anti-tumor activity in vivo

The aforementioned in vitro results prompted us to determine the anti-tumor activity of Alisertib and/or Docetaxel treatments in UGC xenograft mouse models. The in vivo anti-tumor activity analysis demonstrated that treatment with the Alisertib or Docetaxel alone can significantly reduce the % tumor volume of FLO-1 (Alisertib: 76.6±6.2, p<0.01 and Docetaxel: 128.73±15.1, p<0.01) and OE33 (Alisertib: 101.4±5.6, p<0.01 and Docetaxel: 44.1±3.1, p<0.01) cells. In comparison to the single agent treatments, the combination treatment with the Alisertib and Docetaxel led to enhanced reduction of % tumor volume (FLO-1: 12.6±1.7, p<0.01, and OE33: 12.9±1.0, p<0.01). A summary of these results is shown in Figures 5A and 5B.

Figure 5. Alisertib/MLN8237 and Docetaxel combination treatment exhibits enhanced anti-tumor activity in vivo.

FLO-1 and OE33 tumor xenografts were treated with Alisertib (30mg/kg) and/or Docetaxel (10mg/kg) for 21 days and tumor size was measured every alternate day. (A & B) The data indicates that MLN8237 (30mg/kg) and Docetaxel (10mg/kg) combination treatment has significantly enhanced anti-tumor activity against FLO-1 and OE33 tumor xenografts. MLN - MLN8237/Alisertib; DOCE – Docetaxel, * p<0.05 and ** p<0.01.

In addition, the immunohistochemical analysis of FLO-1 and OE33 tumor xenografts after treatment with Alisertib and/or Docetaxel (day 21) demonstrated a reduction in the number of cells positive for Ki67 and an increase in the number of cells with positive cleaved caspase 3; data are shown for FLO-1 (Figure 6), similar results were obtained for OE33. In concordance with the tumor growth results (Figure 5), these immunostaining patterns were more significant with the combined treatment (p<0.01) than with the single treatments (p<.05) (Figure 6). Therefore, the in vivo data indicate enhanced anti-tumor activity of Alisertib and Docetaxel combination in UGC tumor xenograft models.

Figure 6. Alisertib/MLN8237 and Docetaxel combination treatment suppresses proliferation and enhances apoptotic marker expression in FLO-1 tumor xenografts.

FLO-1 tumor xenografts were treated with Alisertib (30mg/kg) and/or Docetaxel (10mg/kg) for 21 days. Subsequently, tumors were isolated and immunohistochemical analyses were done to measure Ki-67 and cleaved caspase 3 expression. (A) The data indicates that Alisertib (30mg/kg) and Docetaxel (10mg/kg) combination treatment significantly inhibits Ki-67 expression in FLO-1 tumor xenografts. (B) Combination treatment with Alisertib (30mg/kg) and Docetaxel (10mg/kg) exhibits enhanced cleaved caspase 3 protein expression in FLO-1 tumor xenografts. MLN - MLN8237/Alisertib; DOCE – Docetaxel, * p<0.05 and ** p<0.01.

Discussion

Despite novel therapeutic advancements, improvement in the survival rate of patients suffering from UGC has been marginal suggesting the presence of unique active intrinsic mechanisms that impart resistance to chemotherapeutic agents in UGCs ^{33, 34}. AURKA is frequently overexpressed and/or amplified in various cancers including UGCs ^{8, 13}. Recent reports suggest that AURKA can induce chemotherapeutic resistance and regulate several key signaling pathways in cancer cells, implying its role as a central node in cancer cell signaling ¹³. Docetaxel has been shown to have significant in vitro and in vivo anti-tumor activity against a variety of UGC cell lines ³⁵.

In this study, we have determined the therapeutic response of recently developed AURKA selective inhibitor Alisertib, as a single agent and in combination with Docetaxel. Although, previous in vitro studies with Aurora kinase inhibitors have shown anti-tumor activity in combination with other chemotherapeutic agents such as Cisplatin, Docetaxel, Nilotinib and Vorinostat ^{8, 36–38}. Alisertib, an investigational small molecule AURKA inhibitor currently in clinical development, has not been tested in combination with Docetaxel in gastrointestinal cancer models. In this regards, our results in the current study suggest a potential therapeutic benefit of Alisertib and Docetaxel combination.

The P53 gene is frequently mutated in various cancers where P53-mutant tumors exhibit inherent resistance to several chemotherapeutic drugs ³⁹. This is of particular significance in UGC therapeutics because in addition to AURKA overexpression, high frequency of defective p53 signaling due to mutation or deletion, is observed in UGCs presenting a formidable clinical challenge ⁴⁰. In this study, we have utilized p53-mutant (FLO-1 and OE33) and wild-type (AGS) cell lines and obtained similar results. Of note, both in vitro and in vivo models, suggest a promising therapeutic potential for the Alisertib and Docetaxel combination as indicated by suppressed cell survival in vitro (p<0.001) and significant regression of tumor growth in vivo (p<0.001). Based on our findings, we suggest that the AURKA-targeted therapy alone and in combination with Docetaxel is effective independent of the p53 status. Our data indicates that the therapeutic effect is largely due to induction of aberrant mitosis leading to polyploidy and subsequently apoptosis. However, others factors such as suppressed proliferation and non-apoptotic forms of cell death may also be occurring. These findings are timely given the fact that Alisertib is being actively tested in various Phase I, II and III clinical trials (http://clinicaltrials.gov/).

AURKA is a serine/threonine kinase that facilitates accurate mitosis by regulating vital cell cycle events during various stages of mitosis ^{14, 15}. AURKA inhibition has been shown to induce aneuploidy, polyploidy and mitotic catastrophe in cells ³⁶. Following treatment with Alisertib, we observed a significant increase in the percentage of cells with polyploidy. This suggests that mitotic catastrophe remains as one of the predominant functions of Alisertib, a finding that is in agreement with previously published data ^{8, 36}. Docetaxel based combination chemotherapeutic regimens like DCF (Docetaxel, Cisplatin and 5-Fluorouracil) regimen are widely used for the treatment of advanced gastric cancers ¹⁷. Docetaxel induced defects in spindle formation and function (tubulin polymerization) activates spindle assembly checkpoint (SAC) and subsequently results in aneuploidy, polyploidy and/or apoptosis ²⁴. Interestingly, it has been previously reported that AURKA overexpression can over-ride SAC and impart resistance to Taxol in HEK-293 cells, suggesting that AURKA overexpression is critical for resistance to cell cycle inhibitors ¹⁹. Treatment with Docetaxel increased the percentage of cells in sub-G1-phase indicating late stage cell death which conforms to the fact that Docetaxel induced spindle defects activate SAC which subsequently activates apoptotic pathways ²⁴. The treatment with Alisertib alone and in combination with Docetaxel induced G2-M-phase arrest in vitro. AURKA is known to regulate G2- to M-phase transitions and its inhibition should result in G2-M-phase arrest ⁴¹. At low treatment concentrations, Docetaxel-induced SAC is transient and weak, an effect that can be easily overridden by overexpressed AURKA, as has been previously reported in HeLa cells ¹⁹. However, Alisertib mediated specific inhibition of AURKA could sensitize AURKA overexpressing cells to Docetaxel resulting in prolonged activation of SAC that is subsequently translated into accelerated mitotic slippage, polyploidy and cell death ³¹. Following a short 48hr treatment, our results demonstrated an increase in the percentage of polyploid cells in AGS and OE33 cells whereas an increase in apoptotic cells was observed in FLO-1 cells. These data suggests that FLO-1 cells are relatively more sensitive to AURKA inhibition as compared to AGS and OE33 cells when treated with Alisertib alone, an effect that is further enhanced by Docetaxel. Although, treatment of AGS and OE33 cells with Docetaxel alone for 48hr increased the percentage of apoptotic sub-G1-phase cells, an even higher percentage of polyploid cells were always observed after treatment with Alisertib and Docetaxel combination. It is well documented that combination chemotherapy is a common and effective therapeutic approach in the treatment of UGCs ¹⁷. In our case, the Alisertib and Docetaxel combination treatment induced a higher percentage of polyploidy will that will subsequently result in increased apoptosis as indicated by significantly enhanced expression of cleaved caspase 3 protein levels in AGS, FLO-1 and OE33 cells. In addition, compared to single agent treatments, combination treatment significantly reduced Ki-67 and enhanced cleaved caspase 3 protein levels in tumor xenografts, providing additional evidence of improved anti-tumor activity of this regimen.

In conclusion, the combination of Alisertib with Docetaxel results in significantly enhanced anti-tumor activity in cell line models, possibly mediated by apoptotic pathways induced after activation of SAC. As clinical trials for Alisertib are being actively conducted, the findings in the current study provide a credible rationale for evaluating AURKA targeted therapy in combination with Docetaxel as a therapeutic approach for the treatment of UGCs.