Aurora kinase B inhibitor barasertib (AZD1152) inhibits glucose metabolism in gastric cancer cells
Jian Hea,b,*, Zihao Qia,*, Xiaofei Zhanga,*, Yufei Yangc, Fei Liuc, Guangfa Zhaob,d and Ziliang Wanga,b,d

Barasertib is a highly selective Aurora kinase B (AURKB) inhibitor and has been widely applied in a variety of cancer cells to investigate the regulatory function of AURKB. However, the effect of barasertib on glucose metabolism in gastric cancer (GC) remains illustrated. Here, barasertib was identified to effectively reduce glucose uptake and lactate production in GC cells in a dose-dependent and time-dependent manner. The expression levels of GLUT1, LDHA and HK2 were decreased by barasertib treatment of GC cells. Furthermore, we found that barasertib induced the expression of ribosomal protein S7 (RPS7), as a tumor suppressor, to regulate glucose metabolism. Silencing of RPS7 rescued the effects of barasertib on glucose metabolism in GC cells. Overexpression of RPS7 suppressed the promoter activity of C-Myc, which has been identified as an important regulator of glucose metabolism in cancer cells. The clinical data showed that the expression level of AURKB in GC patients’ sera and tissues were positively correlated with those of C-Myc, GLUT1 and LDHA, but negatively with that of RPS7. Therefore, these findings

provide new evidence that barasertib regulates GC cell glucose metabolism by inducing the RPS7/C-Myc signal pathway, and have important implications for the development of therapeutic approaches using AURKB as a target protein to prevent tumor recurrence. Anti-Cancer Drugs 30:19–26 Copyright © 2018 Wolters Kluwer
Health, Inc. All rights reserved.
Anti-Cancer Drugs 2019, 30:19–26

Keywords: barasertib, C-Myc, gastric cancer, glucose metabolism, ribosomal protein S7

aCancer Institute, bDepartment of Medical Oncology, cDepartment of Gynecological Oncology, Fudan University Shanghai Cancer Center and
dDepartment of Oncology, Shanghai Medical College, Fudan University, Shanghai,
China
Correspondence to Ziliang Wang, PhD, Cancer Institute, Fudan University Shanghai Cancer Center, 270 Dong’an Road, Shanghai 200032, China Tel: + 86 213 477 7310; e-mail: [email protected]
*Jian He, Zihao Qi and Xiaofei Zhang contributed equally to the writing of this article.

Received 12 May 2018 Revised form accepted 19 July 2018

Introduction
Gastric cancer (GC) remains one of the most common cancers, ranking as the second leading cause of cancer-related death worldwide [1]. Furthermore, GC seems much more prevalent in East Asian countries for almost half of the incidence observed in these regions [2]. The difficulty of early diagnosis of the disease and the che-moresistance during cancer therapy leads to poor prog-nosis [3]. Hence, exploring the internal mechanism during the carcinogenesis and progression of GC seemed even more urgent.
Human aurora kinase family, including Aurora kinase A (AURKA), Aurora kinase B (AURKB) and Aurora kinase C (AURKC), has been reported to be highly expressed in several cancer types and plays critical roles in cancer cell mitosis [4]. AURKB mainly exerts its functions on regulating chromatin modification and suppressing the completion of cytokinesis [5,6]. Overexpression of AURKB may contribute to chromosome instability [7], indicating that AURKB could be a perfect molecular target for anticancer therapy [8].
Barasertib (AZD1152) is a selective molecule inhibitor of AURKB and has been implicated in clinical evaluation [9–11]. For example, barasertib inhibited human small

cell lung cancer cell growth in-vitro and in-vivo sensi-tively [11]. Barasertib treatment played a significant role in acute promyelocytic leukemia through activating G2/M arrest and resulted in endomitosis and polyploidy in NB4 cells [9].
Ribosomal protein subunit S7 (RPS7) is a cardinal structural component of the ribosome, and its major functions rely on sensing cellular stress and DNA damage [12]. RPS7 also took part in p53 stabilization through interacting with MDM2, and subsequently modulated the transacti-vation function of p53 [13,14]. Our previous studies had shown that RPS7 served as a tumor suppressor during ovarian cancer tumorigenesis and metastasis by PI3K/AKT and MAPK signaling cascades [15]. It was also found that RPS7 also played critical roles in glucose metabolism and regulated HIF-1α expression to modulate the glycolysis and lactate production in colorectal cancer cells [16].
Energy metabolism reprogram is one of the hallmarks of cancer [17], and tumor cells enhanced their metabolic rates, glucose uptake and lactate production for amplifying proliferation [18,19]. Growing evidence showed that glucose metabolism had great importance in the ability of cancer cells to survive chemotherapy. By weakening cancer cells with glucose restriction before chemotherapy, cancer cells

0959-4973 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/CAD.0000000000000684

become more sensitive to therapy [20]. For example, it has been reported that overexpression of hexokinase II was associated with resistance to rituximab and chemotherapy agents in aggressive lymphoma, and this enzyme isoform has been identified as a potential therapeutic target [21]. Several crucial molecules were involved in the control of glycolysis during cancer progression [22], including C-Myc, HIF-1α and the members of the sirtuin family.
To evaluate the function and mechanism of barasertib on glucose metabolism in GC cells, we tested the inhibitory ratio of barasertib on GC cells and the effect of barasertib on glucose uptake and lactate production changes in GC cells. We found that barasertib inhibited glucose uptake and lactate production in GC cells through inducing the RPS7/C-Myc signaling pathway.

Materials and methods
Cell lines and cell culture
Human GC cells, HGC27 and MGC803, were purchased from American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium [HyClone (Logan, Utah, USA), Thermo Scientific (Logan, Utah, USA)] supplemented with 10% fetal bovine serum [Gibco (Grand Island, New York, USA), Life Technologies (Carlsbad, California, USA)], and 100 µ/ml streptomycin (Biowest). All cells were incubated at 37°C under a humidified atmosphere with 5% CO2.

Inhibitor
Barasertib (AZD1152-HQPA; Selleck Chem) was prepared in DMSO at a stock concentration of 10 μmol/l and stored at –20°C.

Plasmid construction and viral infection
Recombinant plasmid, containing full length of human cDNA sequences of RPS7, was purchased from Vigene Biosciences (Jinan, China). The cDNA sequences of RPS7 were inserted into lentivirus vector pCDH-CMV-MCS-EF1-PURO to generate recombinant plasmid pCDH/RPS7 OE.
The DNA oligonucleotides designed to generate short hairpin RNA (shRNA1 and shRNA2) against the open reading frame of RPS7 mRNA (positions 168–190 and 295–317) were 5′-TCGGAAAGCTATCATAATCTT TG-3′ and 5′-AGAATTCTGC CTAAGCCAACTCG-3′.
The recombinant plasmid, pLKO/shPRS7, was generated according to the previously reported method [15]. The control vector was similarly constructed by directly inserting oligonucleotides encoding short hairpin RNA against green fluorescence protein mRNA (shGFP) into the pLKO.1 vector.
Lentivirus carrying RPS7 cDNA or shRNA was generated and harvested as described previously [15]. Briefly, the cells were infected twice for a total of 4 days (2 days for each infection), and the positive clones were selected

with puromycin (200 ng/ml) for 7–10 days. Control cell lines were generated by infection with viruses containing the empty vector following the same protocol.

Cell counting kit-8 proliferation assay
The inhibitory effect of barasertib on HGC27 and MGC803 was evaluated by cell counting kit-8 (Dojindo, Japan) and performed according to the manufacturer’s protocol. The cells were seeded in 96-well plates (2 × 103 cells per well) and incubated with a series of concentrations of 1.25, 2.5, 5, 10, 20, 40, and 80 nmol/l of barasertib for 24, 48 and 72 h, respectively. Thereafter, 10 μl cell counting kit-8 solution was added to each well, and the plates were incubated at 37°C in 5% CO2 for 1 h. The absorbance of each sample was measured at a wavelength of 450 nm using a microplate reader. The inhibition ratio was calculated with the following equation: Inhibition ratio = (ODDMSO−ODbarasertib)/ (ODDMSO − ODblank) × 100%. Following this, the IC50 of barasertib in the two GC cells were utilized to perform synergistic effect assay.

Colony-forming assay
Aliquots of 1 × 103 cells/well were seeded in six-well plates and incubated for at least 1 week. All the cell lines used for colony formation were stable cell lines, which were selected by puromycin. The cells were then stained with gentian violet (Solarbio, Shanghai, China), and the colonies (foci containing > 50 cells) were counted. The assay was independently repeated three times.

RNA isolation and real-time PCR
The cells were treated with 0, 5, and 10 nmol/l Barasertib for 24 h. Total RNA was isolated from tissue samples, serum or cell lines with Trizol reagent (Invitrogen, Carlsbad, California, USA), and all RNA was reversely transcribed into cDNA, using the ExScript RT-PCR kit (TaKaRa, Dalian, China), according to the manu-facturer’s instructions. Primer sequences for RPS7 were 5′-GTCGTCTTTATCGCTCAGAG-3′ (forward) and
5′-TGTCAGAGTACGGCTCCTG-3′ (reverse). Primer sequences for Aur B were 5′-CGCAGAGAGATCGAA ATCCAG-3′ (forward) and 5′-AGATCCTCCTCCG GTCATAAAA-3′ (reverse). Primer sequences for C-Myc were 5′-AATAGA GCTGCTTCGCCTAGA-3′ (forward) and 5′-GAGGTGGTTCATACTG AGCAAG-3′
(reverse). Primer sequences for GLUT1 were 5′-CAGTT TGGCTACA ACACTGGAG-3′ (forward) and 5′-GCCCCCAACAGAAAAGATGG-3′ (reverse). Primer sequences for LDHA were 5′-TGGAGATTCCAGTGT GCCTGTATGG-3′ (forward) and 5′-CACCTCATAAG SACTCTCAACCACC-3′ (reverse).
All amplifications and detections were carried out in the Applied Biosystems Prism 7900 system (Applied Biosystems, Foster City, California, USA) using the ExScript Sybr green QPCR kit (TaKaRa; Dalian, China) and the following program: 95°C for 10 s, one cycle;

95°C for 5 s, 62°C for 31 s, 40 cycles; followed by a 30-min melting curve collection to verify the primer dimers. Three independent experiments were performed. Statistical analysis was performed using the 2—DDCt relative quantification method.

Glycolysis analysis
Glucose Uptake Colorimetric Assay Kit (Biovision, Milpitas, California, USA) and Lactate Colorimetric Assay Kit (Biovision, Milpitas, California, USA) were purchased to examine the glycolysis process in GC cells according to the manufacturer’s protocol. Three independent experi-ments were performed.

Western blot analysis
Western blot analysis was performed to determine the expression levels of various proteins in cells. The cells were harvested, washed with cold 1 × PBS, and lysed with RIPA lysis buffer (Beyotime, Shanghai, China) for 30 min; they were then centrifuged at 12 000g for 15 min at 4°C. The total protein concentration was determined by BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts (30 μg/load) of protein samples were subjected to SDS-PAGE electrophoresis and transferred on to polyvinylidene fluoride membranes (Millipore, Billerica, Massachusetts, USA). The blots were blocked in 10% non-fat milk, and incubated with primary antibodies, followed by incubation with secondary antibodies conjugated with horseradish peroxidase. The protein bands were developed with the chemiluminescent reagents (Millipore, Billerica, Massachusetts, USA). Antibodies to Aurora B, GLUTl, LDHA, HK2, RPS7, and C-Myc were purchased from Proteintech (Deansgate, Manchester, USA). The antibody to β-actin was obtained from Sigma-Aldrich (St. Louis, Missouri, USA).

Luciferase reporter assay
Human C-Myc gene promoter sequences were inserted into a pGL3 basic vector as pGL3-C-Myc-promoter. One hundred nanogram of constructed plasmid and 5 ng renilla luciferase control plasmid were transfected into cells expressing HGC27/RPS7 cDNA and MGC803/ RPS7 cDNA in six-well plates. Dual luciferase assay kit (Promega, Madison, Wisconsin, USA) was used for the detection of luciferase activities at 48 h after transfection. Reporter luciferase activities were normalized on the basis of Renilla luciferase, and then rescaled to vector control signals equal to unit 1. All experiments were repeated at least three times. Data represent mean fold change (± SE; n = 3) relative to the control.

Tissue and serum samples
Ethical approval for the study was obtained from the Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center (no. 050432-4-1212B). Fresh tis-sues from 45 GC patients who had undergone surgery

at Fudan University Shanghai Cancer Center between June 2016 and January 2017 and 22 age-matched and sex-matched healthy individuals enrolled as a control group were included for RNA preparation. We also col-lected 80 blood samples in patients with GC before sur-gery. Meanwhile, 35 age-matched and sex-matched healthy individuals were enrolled as a control group. These healthy individuals underwent medical examination to exclude the evidence of tumor and other metabolism-associated diseases. All sera were collected using standard procedures.

Statistical analysis
The SPSS software (version X; IBM, Armonk, New York, USA) was used for statistical analysis. The Student’s t-test or analysis of variance was used to compare quantitative data. P value of less than 0.05 (two tailed) was considered to be statistically significant.

Results
Barasertib inhibits glucose metabolism in gastric cancer cells
To investigate the inhibition ratio of barasertib on GC
cells, we treated the GC cell lines HGC27 and MGC803 with a series of concentrations of barasertib for 24, 48 and 72 h. As shown in Fig. 1a, barasertib inhibited cell growth of HGC27 and MGC803 cells in a dose-dependent and time-dependent manner. We further detected the IC50 values of barasertib on HGC27 and MGC803 cells and found that the IC50 values of barasertib on HGC27 cells were 22.40 nmol/l at 24 h, 9.55 nmol/l at 48 h and
3.98 nmol/l at 72 h, and the IC50 values of barasertib on MGC803 cells were 10.47 nmol/l at 24 h, 5.01 nmol/l at 48 h and 2.82 nmol/l at 72 h. Thereafter, we checked the inhibition effect of barasertib on the expression level of AURKB in HGC27 and MGC803 cells, and found that barasertib inhibited the expression of AURKB in a dose-dependent manner in both HGC27 and MGC803 cells (Fig. 1b). Next, we performed colony formation assay to determine the cytotoxic effect of barasertib on HGC27 and MGC803 cells and found that the number of colonies formed by HGC27 and MGC803 cells was decreased by the treatment of barasertib in a dose-dependent manner (Fig. 1c). To verify the function of barasertib on glucose metabolism, we performed glycolysis analysis and found that barasertib abrogated the ability of GC cells to take in glucose and produce lactate, suggesting that barasertib alleviated the glucose metabolism in GC cells (Fig. 1d and e). Furthermore, we found that the treatment of GC cells with barasertib reduced the expression levels of the key glycolytic enzymes, including GLUT1, LDHA and HK2, in a dose-dependent manner (Fig. 1f).

Barasertib inhibits glucose metabolism by inducing the expression of ribosomal protein S7 in gastric cancer cells RPS7 has been identified to inhibit the glucose metabo-lism in colon carcinoma [23]. To examine whether

Fig. 1

Barasertib inhibits glucose metabolism in gastric cancer cells. (a) Barasertib inhibited HGC27 and MGC803 cells, in a dose-dependent and time-dependent manner, which was detected by cell counting kit-8 assay. (b) Barasertib inhibited AURKB, in a dose-dependent manner, which was detected by western blotting assay. (c) Barasertib inhibited colony formation ability of HGC27 and MGC803 cells in a dose-dependent manner.
*P < 0.05,**P < 0.01, error bars = 95% confidence intervals. (d, e) Barasertib inhibited gastric cancer cell glucose uptake and lactate production in a dose-dependent manner. *P < 0.05, **P < 0.01, error bars = 95% confidence intervals. (f) Barasertib inhibited the key genes in glycolysis, including GLUT1, LDHA and HK2, in a dose-dependent manner, which was detected by western blotting assay. β-Actin was used as the loading control.

Barasertib in GC glucose metabolism He et al. 23

barasertib regulated glucose metabolism through RPS7 in GC cells, we detected the mRNA and protein expression level of RPS7 in GC cells treated by barasertib. We found that barasertib induced the expression of RPS7 in a dose-dependent manner (Fig. 2a and b). To further verify that RPS7 might be a potential downstream target of barasertib, we knocked down the expression of RPS7 in HGC27 or MGC803 cells with shRNAs and found that expression levels of RPS7 were silenced in HGC27 or MGC803 cells (Fig. 2c). Subsequently, we found that the induction of RPS7 shRNA into HGC27 or MGC803 cells rescued the inhibition effects of barasertib on colony formation ability, glucose uptake capacity and lactate production (Fig. 3a–c). Furthermore, we established that the silencing of RPS7 by shRNA1 and shRNA2 recovered the expression of C-Myc, GLUT1 and LDHB suppressed by barasertib (Fig. 3d). The above results indicated that the antimetabolic effects of barasertib in GC cells might be achieved by inducing the expression of RPS7.

Ribosomal protein S7 suppresses the C-Myc promoter activity
C-Myc is an important transcript factor to regulate key
glycolytic enzymes [23–25]. We inferred that RPS7 might regulate glucose metabolism in GC cells by suppressing the expression of C-Myc. Therefore, we overexpressed RPS7 in HGC27 and MGC803 cells, and then found that the mRNA and protein expression levels of C-Myc were alleviated (Fig. 4a and b). Furthermore, we found that the promoter activity of C-Myc was decreased by inducing RPS7 cDNA into HGC27 and MGC803 cells detected by

luciferase reporter assay (Fig. 4c). These data indicated that RPS7 inhibited the expression of C-Myc through suppressing its promoter activity.

Correlations between the clinical pathologic characteristics and the expression of Aurora B, ribosomal protein S7, C-Myc, GLUT1 and LDHB in gastric cancer patients
We analyzed the associations among mRNA expression
levels of Aurora B, RPS7, C-Myc, GLUT1 and LDHB in GC patients’ tissues and serums. As shown in Fig. 5a, the expression of mRNAs isolated from GC tissues of Aurora B, C-Myc, GLUT1 and LDHB was higher than those isolated from normal controls, while RPS7 mRNA level revealed the opposite. The results of the detection of gene expression levels of Aurora B, C-Myc, GLUT1 and LDHB in the serum of GC patients and normal controls showed that the expression of RPS7 had a negative relationship with that of Aurora B, C-Myc, GLUT1 or LDHB (Fig. 5b).

Discussion
AURKB has been reported to have a crucial role in mitotic progression and could be regulated by several molecules to affect cell cycle, cellular senescence and cytotoxicity of eribulin and paclitaxel [23–25]. Thus, several inhibitors targeting AURKB had been chosen for anticancer therapy [4]. Barasertib, also named as AZD 1152, is a small molecule inhibitor of AURKB. It has been reported that barasertib could induce cell growth arrest and apoptosis in acute leukemia cells [9,26]. In addition, it appeared that small cell lung cancer cells with

Fig. 2

Barasertib activates ribosomal protein S7 (RPS7) to inhibit glycolysis. (a, b) Induction of RPS7 by treatment with barasertib was detected by qRT-PCR and western blotting assay in HGC27 and MGC803 cells.*P < 0.05,**P < 0.01, error bars = 95% confidence intervals. (c) Silencing of RPS7 by shRNA1 and shRNA2 was detected by western blotting assay.

24 Anti-Cancer Drugs 2019, Vol 30 No 1

Fig. 3

Ribosomal protein S7 (RPS7) rescues the inhibition effects of barasertib on HGC27 and MGC803 cells. (a–c) Silencing of RPS7 rescued the effect of barasertib on colony formation ability, glucose uptake capacity and lactate production of gastric cancer cells. *P < 0.05, error bars = 95% confidence intervals. (d) Silencing of RPS7 rescued the inhibition effect of barasertib, on the expression of C-Myc, GLUT1 and LDHB, which was detected by western blotting assay.

C-Myc overexpression were more sensitive to barasertib treatment compared with small cell lung cancer cells with low C-Myc expression [11].
In this study, we found that the AURKB inhibitor, bar-asertib, inhibited the glycolysis in GC cells through activating RPS7, which attenuated the C-Myc promoter

activity directly. Thus, our results uncovered an under-lying mechanism that Barasertib suppressed glycolysis of GC cells through RPS7-suppressed C-Myc.
Altered energy metabolism revealed to be the new emerging hallmark of cancer [17] and targeted to specific metabolic enzymes has provided new ideas for cancer

Barasertib in GC glucose metabolism He et al. 25

Fig. 4

Ribosomal protein S7 (RPS7) suppresses the C-Myc promoter activity. (a, b) The expression level of C-Myc was inhibited by RPS7 overexpression detected by qRT-PCR and western blotting assay in HGC27 and MGC803 cells. *P < 0.05, error bars = 95% confidence intervals. (c) The promoter activity of C-Myc was inhibited by RPS7 overexpression and was detected by dual-luciferase reporter assays in HGC27 and MGC803 cells.
*P < 0.05, **P < 0.01, error bars = 95% confidence intervals.

Fig. 5

Expression analyses of AURKB, ribosomal protein S7 (RPS7), C-Myc, GLUT1 or LDHB expression in gastric cancer patients. (a) Detection of expression levels of AURKB, RPS7, C-Myc, GLUT1 or LDHB in gastric cancer tissues and normal controls by qRT-PCR. *P < 0.05, error bars = 95% confidence intervals. (b) Detection of expression levels of AURKB, RPS7, C-Myc, GLUT1 or LDHB in gastric cancer serum and in normal controls by qRT-PCR. *P < 0.05, error bars = 95% confidence intervals.

therapy [27]. For a long time, researchers have carried out several studies on AURKB, but there is still a lack of research on the correlation between AURKB and

glycolysis. Now, we have focused on the metabolic manifestation affected by AURKB, to explore new functions of AURKB. In this study, we found that the AURKB inhibitor, barasertib, could inhibit glucose metabolism in GC cells, indicating that AURKB also plays the cardinal role in regulating glucose metabolism.
Ribosomal proteins play cardinal roles in maintaining the function of ribosome, which controls translating mRNA into proteins [28]. Thus, malfunction of the ribosomal proteins leads to disarranged protein translation, subsequently leading to cancer development [29,30]. RPS7 functions as a tumor suppressor gene mainly through binding to MDM2 and abrogating MDM2-mediated p53 ubiquitination, thus activating p53 activities [14]. Several studies have shown that RPS7 could induce cell apoptosis and suppress cell proliferation [14,15]. RPS7 had been reported to suppress glycolysis in colorectal cancer cells [16]. In our study, we found that the AURKB inhibitor, barasertib, suppressed glucose uptake capacity and lactate production through attenuating RPS7 expression. Furthermore, RPS7 could bind to and suppress the C-Myc promoter. Therefore, our study first uncovered that AURKB regulated glycolysis through the RPS7/C-Myc signaling pathway. Furthermore, the clinical data confirmed the correlation among AURKB, RPS7, C-Myc, GLUT1 and LDHB, and indicated the important role of AURKB/RPS7/C-Myc signaling pathway in the regulation of glycolysis in GC cells.

26 Anti-Cancer Drugs 2019, Vol 30 No 1

Conclusion
We demonstrate that barasertib decreases glycolysis in GC cells through the induction of a signal pathway. Our results not only provide new insight into the underlying mechanism of barasertib-mediated glycolysis, but also offer important implications for the development of therapeutic approaches using AURKB as a target protein, to prevent tumorgenesis in various cancers, including GC.
Acknowledgements
Z. Wang was supported in this work by National Nature Science Young Foundation of China (81502235).

Conflicts of interest
There are no conflicts of interest.

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