Accepted Manuscript Title: Elevated glucose levels impair the WNT/␤-catenin pathway via the activation of the hexosamine biosynthesis pathway in endometrial cancer Author: Fuxing Zhou Junwei Huo Yu Liu Haixia Liu Gaowei Liu Ying Chen Biliang Chen PII: DOI: Reference:

S0960-0760(16)30030-9 http://dx.doi.org/doi:10.1016/j.jsbmb.2016.02.015 SBMB 4636

To appear in:

Journal of Steroid Biochemistry & Molecular Biology

Received date: Revised date: Accepted date:

14-7-2015 4-1-2016 18-2-2016

Please cite this article as: Fuxing Zhou, Junwei Huo, Yu Liu, Haixia Liu, Gaowei Liu, Ying Chen, Biliang Chen, Elevated glucose levels impair the WNT/rmbetacatenin pathway via the activation of the hexosamine biosynthesis pathway in endometrial cancer, Journal of Steroid Biochemistry and Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2016.02.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Elevated glucose levels impair the WNT/β-catenin pathway via the activation of the hexosamine biosynthesis pathway in endometrial cancer

Fuxing Zhou1, Junwei Huo2, Yu Liu1, Haixia Liu1, Gaowei Liu1, Ying Chen3, Biliang Chen1*

1Department

of Gynecology and Obstetrics, Xijing Hospital, The Fourth Military

Medical University, Xi'an Shaanxi, 710032, China; 2Department of Gynecology and Obstetrics, The First Hospital of Yulin, Yulin Shaanxi, 718000, China; 3Department of Natural Medicine, School of Pharmacy, The Fourth Military Medical University, Xi’an Shaanxi, 710032, China

∗ Corresponding author Email address: [email protected]

1

Graphical abstract

Highlights 

AN3CA and HEC-1-B cells showed high β-catenin expression under elevated glucose levels



Both cell lines also exhibited an increase in O-GlcNAcylation levels



β-catenin expression increases via activation of HBP in both cell lines.



Glucose-induced β-catenin elevation triggers the transcription of target genes

2

ABSTRACT Endometrial cancer (EC) is one of the most common gynecological malignancies in the world. Associations between fasting glucose levels (greater than 5.6 mmol/L) and the risk of cancer fatality have been reported. However, the underlying link between glucose metabolic disease and EC remains unclear. In the present study, we explored the influence of elevated glucose levels on the WNT/β-catenin pathway in EC. Previous studies have suggested that elevated concentrations of glucose can drive the hexosamine biosynthesis pathway (HBP) flux, thereby enhancing the O-GlcNAc modification of proteins. Here, we cultured EC cell lines, AN3CA and HEC-1-B, with various concentrations of glucose. Results showed that when treated with high levels of glucose, both lines showed increased expression of β-catenin and O-GlcNAcylation levels; however, these effects could be abolished by the HBP inhibitors, Azaserine and 6-Diazo-5-oxo-L-norleucine, and be restored by glucosamine. Moreover the AN3CA and HEC-1-B cells that were cultured with or without PUGNAc, an inhibitor of the O-GlcNAcase, showed that PUGNAc increased β-catenin levels. The results suggest that elevated glucose levels increase β-catenin expression via the activation of the HBP in EC cells. Subcellular fractionation experiments showed that AN3CA cells had a higher expression of intranuclear β-catenin in high glucose medium. Furthermore, TOP/FOP-Flash and RT-PCR results showed that glucose-induced increased expression of β-catenin triggered the transcription of target genes. In conclusion, elevated glucose levels, via HBP, increase the O-GlcNAcylation level, thereby inducing the over expression of β-catenin and subsequent transcription of the target genes in EC cells.

Keywords: Elevated glucose levels, WNT/β-catenin, O-GlcNAc, Endometrial cancer

3

1. INTRODUCTION

The WNT/β-catenin signaling pathway plays an essential role in the proliferation, differentiation, and migration of eukaryotic cells [1-3]. In the absence of stimulation, β-catenin, the key effector of this pathway, is continuously degraded by a destruction complex, which mainly comprises of Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3 (GSK-3). Axin, a tumor suppressor protein, acts as the scaffold and interacts with β-catenin, APC and GSK-3. APC is a large protein that interacts with both β-catenin and Axin, while GSK-3 can phosphorylate β-catenin, which is then ubiquitinated and degraded by the ubiquitin–proteasome system and is thus maintain at a relatively low level in the cytoplasm [4]. Unphosphorylated β-catenin is stabilized and transported to the nucleus where it activates the T cell-specific transcription factor/lymphoid enhancer-binding factor (TCF/LEF) to induce the cell development and the expression of specific target genes, including cyclin D1, Axin2 and c-Myc [4-8]. Various mutations in the destruction complex as well as in β-catenin can also activate this transcription process [9, 10]. These mutations protect β-catenin from being degraded and facilitate the accumulation and subsequent translocation of β-catenin into the nucleus where it triggers oncogenic gene transcription [4]. Such dysfunctions in this pathway are often observed in cases of hepatocellular carcinoma, colorectal cancer, ovarian cancer [11-13], and notably in nearly 40% of cases of EC [14]. EC is the most common cancer of the female genital tract in the United States with approximately 52,630 new cases and 8,590 deaths occurring in 2014 [15]. With economic development and westernization of lifestyles, the occurrence of EC has also risen rapidly in Asian countries [16]. Although it is widely known that EC is more prevalent among diabetic and/or obese women, little is known about the underlying mechanism [17-19]. Excessive exposure to carcinogens, including insulin, estrogens, insulin-like growth factors, leptin, and adiponectin, may contribute to the development of EC in diabetic and/or obese women [18, 20, 21]. However, elevated serum glucose levels, an early symptom of diabetes and obesity, may directly regulate 4

tumor-related signaling pathways, especially meeting the high glucose need of cancer cells [18, 22]. Previous reports suggest that the hexosamine biosynthesis pathway (HBP) might be involved in the regulation of β-catenin by glucose [23-25]. The HBP is involved in glucose metabolism, and its final product, UDP-GlcNAc (uridinediphosphate N-acetylglucosamine), is a nucleotide sugar substrate involved in multiple biological processes, including classical glycosylation and O-GlcNAcylation [26-31]. As a post-translational modification (PTM), O-GlcNAcylation reflects the cell’s glucose status and regulates a wide range of cellular functions [30]. Thus, unbalanced glucose metabolism can affect O-GlcNAcylation, consequently leading to cellular dysfunctions, such as cancers or other related diseases and pathologies [27, 29, 31]. Thus, in this study, we determined whether the elevated glucose levels significantly affected WNT/β-catenin signaling activity and investigated a potential mechanism involved in glucose-induced O-GlcNAcylation of β-catenin, which consequently led to the activation of the WNT signaling pathway in EC cells.

2. MATERIAL AND METHODS 2.1 Cell culture and transfection AN3CA and HEC-1-B cell lines were purchased from the Shanghai Cell Collection (Shanghai, China) and passaged 4–6 times prior to use in experiments. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) containing various concentrations of glucose (0, 5.5, or 25 mM/L). All of the media were supplemented with 10% fetal bovine serum (FBS, Gibco), 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. Cells were transfected with the TOP-Flash and FOP-Flash vectors using Lipofectamine 2000 (Invitrogen) reagent (2 μl) in 24-well plates with 0.2 μg of DNA for 24 h.

2.2 Coimmunoprecipitation Cells were washed with 10 ml of cold phosphate-buffered saline (PBS), then lysed on ice with Western and IP (Beyotime Institution of Biotechnology), 1 mM 5

phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors. The cell extracts were then centrifuged at 20,000 g for 10 min at 4°C. The supernatants were precleared with protein A/G (Santa Cruz) for 30 minutes at 4°C. The beads were discarded after a 1 min centrifugation at 1000 g, and the supernatants were incubated with 2 g of either mouse anti-O-GlcNAc (RL2, Abcam, ab2739, monoclonal) or rabbit anti-β-catenin (Abcam, ab32572, monoclonal) and rocked at 4°C overnight. Then, 20 µl of the resuspended volume of Protein A/G was added with incubation at 4° C on a rocker platform for 1 h. The beads were gently centrifuged for 1 min at 1000 g and the pellet was washed 4 times with 1 ml of western and IP lysis buffer. The Beads were resuspended in 2× sodium salt (SDS) loading buffer, heated to 100 °C for 10 min, and centrifuged for 1 min at 5,000 g. Supernatants were collected and then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting.

2.3 Western blot Cells were washed with 10 ml of cold phosphate-buffered saline (PBS) 3 times and lysed on ice with lysis buffer [50 mM Tris•HCl, 150 mM NaCl,

1% Triton X-100, 1%

sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors, pH 7.4]. Cell extracts were then centrifuged at 12,000 g for 20 minutes at 4°C. Subcellular fractionation was performed using the Nucleoprotein extraction kit (Solarbio). Equal amounts of protein samples were separated by 10% SDS-PAGE and electroblotted onto the PVDF membranes (Millipore). The membranes were blocked with 5% non-fatty-acid milk, followed by incubation with the following antibodies: anti-β-catenin (Abcam, ab32572, monoclonal, 1:2000), anti-O-GlcNAc (RL2, Abcam, ab2739, monoclonal, 1:1000), anti-H2B (Solarbio, A13347R, polyclonal, 1:500), and anti-GAPDH (Abcam, ab8245, monoclonal, 1:2000) overnight at 4°C. O-GlcNAc competition was performed using 1 M GlcNAc(SIGMA). The membranes were then incubated with anti-mouse (Cwbio, 1:5000) or anti-rabbit (Cwbio, 1:5000) secondary antibodies conjugated with horseradish peroxidase for 2 h at room temperature and visualized using ECLTM Western Blotting Detection Reagent (Bio-Rad). 6

2.4 Real-time PCR analysis Total RNA of each sample was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa). Real-time PCR was done with SYBR Premix Ex Ta (TaKaRa) on a MX 3000 instrument. The primers used in the present study were: human CyclinD1 (CCND1) forward, 5′–TTTGTTCAAGCAGCGAGTCC-3′and reverse,

5′-CTCCAAGCCGATATCCCTGC-3′;

TAACCCCTCAGAGCGATGGA-3′

and

human

Axin2 reverse,

forward,

5′5′-

AGTTCCTCTCAGCAATCGGC-3′.

2.5 Statistical analysis Results are presented as the mean + SEM, with the number of experiments indicated in the figure legend. Statistical significance was assessed using one-way ANOVA. p < 0.05 was considered significant.

3. RESULTS 3.1 β-catenin expression is associated with glucose concentration in EC cells. Higher O-GlcNAcylation and β-catenin expression levels were observed for 25 mM glucose than for 0 and 5.5 mM glucose (“high glucose effects”; Fig 1A and 1B). We also observed that 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of HBP, reversed these effects (Fig 1A and 1B). To validate the specificity of RL2, the antibody was co-incubated with 1 M GlcNAc, which completely prevented the detection of O-GlcNAc-modified proteins (Fig 1C).

3.2 Glucose modulates β-catenin via the hexosamine pathway. We explored the HBP using DON (Fig 1A) and Azaserine (Fig 2A), another inhibitor of the HBP rate-limiting enzyme GFAT (glutamine: fructose-6-phosphate amidotransferase). We found that DON and Azaserine reversed the “high glucose effects” on the expression of β-catenin in the AN3CA cell line. Additionally, GlcNH2 (glucosamine), which can directly enter the HBP downstream of GFAT, was able to replicate the “high glucose 7

effects” in low glucose medium (Fig 2C). Similar results were also observed in the HEC-1-B cell line (Fig 2B and 2D).

3.3 HBP enhances the expression of β-catenin by elevating its O-GlcNAcylation levels. UDP-GlcNAc, the main end product of HBP, is used for O-GlcNAcylation. Therefore, we hypothesized that the fate of β-catenin is linked to its post-translational modification via O-GlcNAcylation. PUGNAc is an inhibitor of O-GlcNAcase (OGA) that mediates the removal of O-GlcNAc from proteins. We cultured AN3CA and HEC-1B cells in 0 and 5.5 mM glucose with or without PUGNAc (50 μM). Results showed that PUGNAc elevated the expression of β-catenin as well as increased the overall level of O-GlcNAcylation (Fig 3A and 3B). These results indicate that O-GlcNAcylation is associated with the elevated expression of β-catenin. Accordingly, we examined whether β-catenin was highly O-GlcNAcylated in cells treated with high concentrations of glucose relative to cells treated with a lower concentration. We detected increased β-catenin O-GlcNAcylation in 5.5 mM glucose-treated cells (Figure 3C and 3D).

3.4 Glucose-induced β-catenin elevation enhances the activation of WNT signaling. To investigate whether the glucose-induced β-catenin elevation could activate the WNT signaling pathway, we used the TOP/FOP FLASH assay. As shown in Fig 4A, high glucose treatment enhances WNT reporter gene activation in AN3CA cells, and PUGNAc augments this effect. Meanwhile, these phenomena could be reversed by adding Azaserine and DON. Similar results were also observed in the HEC-1-B cell line (Fig 4B).

3.5 Glucose-induced elevation of β-catenin O-GlcNAcylation increases nuclear expression of β-catenin and activates downstream target genes. Since the functions of β-catenin depend on the amount of protein in the cell nucleus, subcellular fractionation experiments were performed. AN3CA and HEC-1-B cells were cultured in 0 mM, 5.5 mM or 25 mM glucose with or without Azaserine (50 μM). 8

It showed that higher glucose concentrations led to an increase in β-catenin in the nucleus and Azaserine reversed this phenomenon (Fig 5A and 5B). Consistent with this result, the expression of WNT target genes (Axin2 and CyclinD1) were increased following the increase of glucose concentration, which could be reversed by adding Azaserine and DON. Additionally, PUGNAc elevated the expression of Axin2 and CyclinD1 (Fig 5C and 5D).

4. DISCUSSION

Previous studies documented that more than one-third of EC patients have abnormal activation of the WNT/β-catenin pathway [14, 32, 33]. Oncogenic mutations in β-catenin, APC, and Axin were commonly observed in these cases, and they frequently led to β-catenin changes such as aberrant stabilization, translocation to the nucleus, and over activation [14, 34]. However, the pathogenesis of abnormal accumulation of β-catenin is complicated and involves a multitude of molecules [4, 14]. Some studies have shown that not only diabetes, but also pre-diabetes is a high risk factor for cancer and can increase the incidence and mortality of multiple tumors, including EC [17, 22, 35]. Hyperglycemia, one of the early symptoms of pre-diabetes, obesity, and diabetes, was found to be associated with cancer [17, 22, 36]. However, the relationship between glucose and EC is unclear.

In this study, we found that elevated glucose levels increased the expression of β-catenin via the activation of the HBP and augmented the O-GlcNAcylation level in EC cells (Fig 6). Glucose-induced activation of the HBP enhanced β-catenin stability through O-GlcNAcylation. Higher glucose concentration increased intranuclear β-catenin levels and triggered the transcription of the target genes. However, these stimulatory effects were abolished by adding Azaserine and DON. When cells were treated with PUGNAc the expression of β-catenin was increased. Olivier et al. [24, 37] reported that elevated glucose levels can stabilize β-catenin via HBP and 9

O-GlcNAcylation. Our results showed that β-catenin was highly expressed in EC cells treated with 25 mM glucose, revealing that the upregulated β-catenin was correlated with elevated glucose levels.

Tumor cells increase the utilization of glucose, which is essential for cell growth and proliferation through the Warburg effect [38, 39]. In cancer cells, glucose is mainly used for glycolysis, the HBP, and the pentose phosphate pathway (PPP) [26]. Interestingly, tumor cells increase the consumption of both glucose and glutamine [40], and which are used for the generation of UDP-GlcNAc, the final product of HBP. In addition, O-GlcNAcylation is a PTM of β-catenin that can enhance its stability [23-25, 41]. Consistent with previous results [24, 37], we found that AN3CA and HEC-1B cells highly expressed β-catenin and O-GlcNAcylation in mediums with high concentrations of glucose. The effects of glucose could be blocked by Azaserine and DON and replicated by low levels of glucosamine. We also used PUGNAc, an inhibitor of the O-GlcNAcase, to confirm the function of O-GlcNAcylation in stabilizing

β-catenin.

In

addition,

we

found

that

β-catenin

was

highly

O-GlcNAcylated in cells treated with high concentrations of glucose. Accordingly, we hypothesize that elevated glucose levels can increase the expression of β-catenin via the HBP and its own O-GlcNAcylation level.

Mutations in the WNT/β-catenin pathway lead to an accumulation of β-catenin and its subsequent nuclear translocation [11, 12]. Upon reaching the nucleus, β-catenin promotes cancer progression through the persistent interaction with its downstream targets such as the TCF/LEF factor [4, 10, 42]. In the present study, our results from the TOP/FOP-Flash assays showed an increase of transcriptional activity occurring with high-glucose treatment, which could be reversed by adding Azaserine and DON. Subcellular fractionation experiments showed that glucose induced the accumulation of β-catenin not only in the cytoplasm but also in the nucleus. Additionally, Axin2 and CyclinD1 expression increased following the increase of glucose concentration and the mitigation by Azaserine and DON. These results suggest that glucose-induced 10

high β-catenin expression causes translocation to the nucleus, thus activating WNT signaling and the downstream target genes in EC cells.

Therefore, it can be concluded that deregulation of glucose metabolism is involved in EC. Currently, millions of people have problems with glucose metabolism, particularly individuals with diabetes and obesity, and this number is expected to increase [43-45]. Thus, aberrant glucose metabolism may have a severe negative impact on the health of women, especially if they have the aforementioned lifestyle diseases. The early management of hyperglycemia could be an effective way to prevent EC. Furthermore, pharmacological modulation of O-GlcNAc cycling enzymes may be a valuable target for the therapy of EC patients with hyperglycemia. In summary, our study suggests that elevated glucose levels are correlated with EC through upregulation of the HBP and O-GlcNAcylation, which enhances the stability of β-catenin. Further studies are required to understand whether this mechanism regulating WNT/β-catenin signaling exists in normal cells or arises with the onset of EC.

11

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Fig 1. β-catenin is overexpressed in high glucose medium. AN3CA (A) and HEC-1-B (B) cells were maintained with 0, 5.5, or 25 mM glucose with or without DON (50 μM) for 24 hours, and the levels of β-catenin expression and O-GlcNAcylation were determined by western blot. (C) GlcNAc (1 M) competition during primary antibody incubation was used to validate the specificity of the RL2 antibody in AN3CA cells. Results are presented as means + SEM, n = 3 independent experiments. **P < 0.01, *P < 0.05

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Fig 2. Glucose regulates the expression of β-catenin via the HBP. AN3CA (A) and HEC-1-B (B) cells were cultured with 5.5 mM or 25 mM glucose with or without Azaserine (50 μM) for 24 hours. GlcNH2, a HBP activator, was tested at different indicated concentrations for 24 hours. AN3CA (C) and HEC-1-B (D) cell homogenates were immunoblotted with anti-β-catenin or anti-O-GlcNAc antibodies. Results are presented as means + SEM, n = 3 independent experiments. **P < 0.01, *P < 0.05

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Fig 3. O-GlcNAcylation levels of β-catenin are linked to its expression. AN3CA (A) and HEC-1-B (B) cells were cultured in DMEM with 0 and 5.5 mM glucose, and PUGNAc (50μM) treated for 24 hours. Cell extracts were immunoblotted with anti-β-catenin or anti-O-GlcNAc antibodies. AN3CA (C) and HEC-1-B (D) cells were cultured with 0 and 5.5 mM glucose. Cell lysates were used for immunoprecipitation with a monoclonal β-catenin antibody and immunoblotted against O-GlcNAc. Whole-cell lysates were probed for O-GlcNAc level and β-catenin. Results are presented as means + SEM, n = 3 independent experiments. **P < 0.01, *P < 0.05

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Fig 4. Glucose-induced β-catenin elevation enhances WNT signaling activation. AN3CA (A) and HEC-1-B (B) cells were transfected with TOP-Flash and FOP-Flash vector and then incubated in medium using different conditions of glucose concentrations (0, 5.5, or 25 mM). The GFAT inhibitor Azaserine, the HBP activator glucosamine, and the OGA inhibitor PUGNAc were tested at the indicated concentration. Cells were incubated 24 hours before measuring luciferase activity. Results are presented as means + SEM, n = 3 independent experiments. *P < 0.05 compared with the 0 mM glucose group; #P < 0.05 compared with 25 mM glucose group.

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Fig 5 High glucose concentration increases intranuclear β-catenin levels and triggers the transcription of target genes. Cells were treated with 0, 5.5, or 25 mM glucose with or without Azaserine (50 μM) for 24 hours. Nuclear and cytoplasmic fractions of AN3CA (A) and HEC-1-B (B) were analyzed by western blot. H2B (nuclear) and GAPDH (cytoplasmic) served as fractionation controls. CyclinD1 and Axin2 transcript levels in AN3CA (C) and HEC-1-B (D) were examined after cells were treated in different medium for 24 hours. Results are presented as means + SEM, n = 3 independent experiments. *P < 0.05 compared with 0 mM glucose group; #P < 0.05 compared with 25 mM glucose group.

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Fig 6 Elevated glucose levels increase the production of UDP-GlcNAc, the end product of the HBP, inducing aberrant stability of β-catenin. As a result, β-catenin translocates to the nucleus and activates the transcription factor TCF/LEF.

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