The oncoprotein HBXIP suppresses gluconeogenesis through modulating PCK1 to enhance the growth of hepatoma cells

ARTICLE IN PRESS Cancer Letters ■■ (2016) ■■–■■

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Q2 Original Articles

The oncoprotein HBXIP suppresses gluconeogenesis through modulating PCK1 to enhance the growth of hepatoma cells Q1 Hui Shi a, Runping Fang a, Yinghui Li a, Leilei Li a, Weiying Zhang a, Huawei Wang a,

Fuquan Chen b, Shuqin Zhang b, Xiaodong Zhang b,*, Lihong Ye a,* a b

State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071, China State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071, China

A R T I C L E

I N F O

Article history: Received 17 June 2016 Received in revised form 30 August 2016 Accepted 31 August 2016 Keywords: HBXIP PCK1 Gluconeogenesis FOXO1 PI3K/Akt HCC

A B S T R A C T

Hepatitis B X-interacting protein (HBXIP) as an oncoprotein plays crucial roles in the development of cancer, involving glucose metabolism reprogramming. In this study, we are interested in whether the oncoprotein HBXIP is involved in the modulation of gluconeogenesis in liver cancer. Here, we showed that the expression level of phosphoenolpyruvate carboxykinase (PCK1), a key enzyme of gluconeogenesis, was lower in clinical hepatocellular carcinoma (HCC) tissues than that in normal tissues. Mechanistically, HBXIP inhibited the expression of PCK1 through down-regulating transcription factor FOXO1 in hepatoma cells, and up-regulated miR-135a targeting the 3′UTR of FOXO1 mRNA in the cells. In addition, HBXIP increased the phosphorylation levels of FOXO1 protein by activating PI3K/Akt pathway, leading to the export of FOXO1 from nucleus to cytoplasm. Strikingly, over-expression of PCK1 could abolish the HBXIPpromoted growth of hepatoma cells in vitro and in vivo. Thus, we conclude that the oncoprotein HBXIP is able to depress the gluconeogenesis through suppressing PCK1 to promote hepatocarcinogenesis, involving miR-135a/FOXO1 axis and PI3K/Akt/p-FOXO1 pathway. Our finding provides new insights into the mechanism by which oncoprotein HBXIP modulates glucose metabolism reprogramming in HCC. © 2016 Elsevier Ireland Ltd. All rights reserved.

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Introduction Mammalian hepatitis B X-interacting protein (HBXIP, synonyms: LAMTOR5) as a conserved protein is widely expressed in tissues [1], which is originally identified by its interaction with the C terminus of hepatitis B virus X protein [2]. HBXIP can suppress apoptosis in a survivin-dependent manner [3]. In addition, HBXIP is required for amino acid sensing by the mTORC1 pathway as a regulator component [4]. Our group has reported that HBXIP plays critical roles in the development of breast cancer by acting as a coactivator of transcription factors, such as TFIID, c-Myc and E2F1 [5–8]. Recently, we have reported that HBXIP is highly expressed in the

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Abbreviations: cAMP, cyclic adenosine monophosphate; DAPI, 4′,6-diamidino2-phenylindole; FOXO1, forkhead box protein O1; G6P, glucose-6-phosphatas; HBXIP, hepatitis B X-interacting protein; HMP, hexose monophosphate pathway; IRE, insulin response element; IHC, immunohistochemistry; miRNA, microRNA; mRNA, messenger nucleolar RNA; PCK1, phosphoenolpyruvate carboxykinase; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1 alpha; PI3K, phosphatidylinositol 3-kinase; qRT-PCR, quantitative real-time quantitative polymerase chain reaction; UTR, untranslated region. * Corresponding authors. Fax: +86 22 23501385. E-mail addresses: [email protected] (X. Zhang); [email protected] (L. Ye).

66 clinical HCC tissues, in which the positive rate is 75.17% (112/ 67 149), and promote the development of HCC [9]. 68 Hepatocellular carcinoma (HCC) is the fifth most common cancer 69 worldwide and the third largest cause of cancer death globally [10]. 70 Liver has many unique metabolic functions including gluconeo71 genesis, glycogen synthesis and storage, as well as blood glucose 72 homeostasis. As HCC is highly proliferative, altered metabolic mecha73 nisms are required to support its vast demand for nutrients [11]. 74 It has been reported that the glucose metabolism reprogramming 75 contributes to the development of cancer [12–14]. Considerable effort 76 has been made in elucidating the mechanism and functional sig77 nificance of higher rates of glycolysis and production of lactate in malignant cells, commonly referred to as Warburg effect [15]. Glu- Q3 78 79 coneogenesis is the process of converting simple sugar precursor 80 (lactic acid, glycerin, amino acid, etc.) into sugar (glucose and gly81 cogen) [16]. However, whether gluconeogenic pathway is modulated 82 in the development of liver cancer, resulting in the alteration of levels 83 of glucose homeostasis, is poorly understood. It has been well docu84 mented that the key gluconeogenic enzymes such as PCK1, glucose85 6-phosphatase (G6PC), peroxisome proliferator-activated receptor 86 gamma (PPARγ), and coactivator 1 alpha (PGC-1α) are dramatical87 ly inhibited in liver cancer. The activation of IL-6-Stat3 signaling could 88 lead to the up-regulation of miR-23a expression, resulting in re89 duction in the expressions of G6PC and PGC-1α in HCC. Drastic 90 reduction of gluconeogenesis is likely to result in the accumula-

http://dx.doi.org/10.1016/j.canlet.2016.08.025 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Hui Shi, et al., The oncoprotein HBXIP suppresses gluconeogenesis through modulating PCK1 to enhance the growth of hepatoma cells, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.025

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tion of glucose-6-phosphate (G6P). Because G6P is utilized in the hexose monophosphate (HMP) shunt pathway for glucose metabolism, the tumors can metabolize excess G6P via this pathway and produce ribose-5-phosphate used in nucleotide synthesis [17]. However, the regulatory mechanism of PCK1 in HCC remains unclear. Recently, we have reported that HBXIP is able to induce aerobic glycolysis through suppressing SCO2 and PDHA1 in breast cancer [18]. However, the role of HBXIP in the regulation of gluconeogenesis of liver cancer is not well documented. The forkhead box O (FOXO) transcription factors are considered as tumor suppressors that limit cell proliferation and induce apoptosis [19]. FOXO1 is important for insulin-dependent regulation of hepatic gluconeogenesis [20,21]. When FOXO1 localizes to nucleus, it can bind to the promoter of PCK1 or G6PC through insulin response element (IRE) [22–24]. A major mechanism of regulation of FOXO1 consists of phosphorylation by Akt (also termed protein kinase B, PKB) following growth factor stimulation, leading to the inactivation of FOXO1 [25]. Interestingly, the phosphorylation of FOXO1 results in its export from the nucleus to the cytoplasm [26]. In addition, FOXO1 is regulated by post-transcriptional modifications, such as phosphorylation and acetylation, which affects its subcellular location, DNA-binding properties, and transcriptional activity [27–29]. In the present study, we investigated the role of HBXIP in modulation of gluconeogenesis of HCC progression. Intriguingly, we report that HBXIP suppresses gluconeogenesis through down-regulating PCK1 in HCC, involving miR-135a/FOXO1 axis and PI3K/Akt/pFOXO1 pathway, to promote the hepatocarcinogenesis. Our finding provides new insights into the mechanism by which aberrant glucose metabolism enhances hepatocarcinogenesis.

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Immunohistochemistry (IHC) assays

Materials and methods

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HCC tissue microarray (No. 03C03), containing duplicates of 192 cases of HCC tissues and 8 cases of normal liver tissues, was purchased from Xi’an Aomei Biotechnology (Xi’an, China). Immunohistochemistry assay was carried out as described previously [30]. The staining level of PCK1 was classified into three groups using a modified scoring method based on the intensity of staining (0 = negative; 1 = low; 2 = high) and the percentage of stained cells (0 = 0% stained; 1 = 1–49% stained; 2 = 50– 100% stained). A multiplied score (intensity score × percentage score) 0 was considered to be negative staining (−), 1 was considered to be low staining (+), 2 was considered to be moderate staining (++), and 4 was considered to be high staining (+++). For Ki67 staining in tumor xenograft, tumor slides (thickness, 5–10 μm) were fixed in 4% paraformaldehyde for 2 days. Sections were stained using primary antibody against Ki67 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

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Thirty HCC tissue samples and their corresponding adjacent non-tumor liver tissues were obtained from Tianjin First Center Hospital and Tianjin Tumor Hospital (Tianjin, China) after surgical resection. Informed consent was obtained from each patient and the study was approved by the Institutional Research Ethics Committee in Nankai University. The information of HCC patients is presented in Supplementary Table S1.

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The cell lines including HepG2, Huh7, 293T and Chang liver were maintained in Dulbecco’s Modified Eagle’s medium (Gibco, CA, USA). HepG2-HBXIP was a cell line that HepG2 established by stable transfection with pcDNA3.1-HBXIP plasmid [8]. All cell lines were supplemented with heat-inactivated 10% fetal bovine serum (FBS, Gibco, CA, USA), 100 U/mL penicillin and 100 mg/mL streptomycin, and grown at 5% CO2 and 37 °C. All transfections were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. All the experiments were conducted in the cells with about 80% convergence.

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HepG2-HBXIP cells were transfected with pcDNA3.1/zeo(+)-PCK1 plasmid provided by Dr. Shimin Zhao from FuDan University using Lipofectamine 2000 according

Patient samples

Cell culture and treatment

Stable transfection

to the manufacturer’s instructions. 48 hours after transfection, the cells were diluted 1:10 and cultured in a growth medium containing Zeocin (200 μg/mL, Invitrogen) for three weeks. Stable transfected clones were picked and maintained in the growth medium containing 100 μg/mL Zeocin for additional studies. Statistical analysis Each experiment was repeated at least three times. Statistical significance was assessed by comparing mean values (±SD) using a Student’s t-test and was assumed for P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) and not significant (NS). The correlation between PCK1 (or miR-135a) and HBXIP mRNA levels in clinical tumorous tissues was determined with Pearson′s correlation coefficient.

Results PCK1 is down-regulated in clinical HCC tissues and negatively correlated with HBXIP We examined the expression of PCK1, a gluconeogenesis key enzyme, in clinical HCC tissues. Immunohistochemistry (IHC) staining showed that the positive rate was 100% (8/8) in clinical peritumoral liver tissues, while the positive rate was 18.2% (54/ 192) in clinical HCC tissues using tissue microarrays (Fig. 1A, Supplementary Table S1). Heatmap analysis showed that the expression levels of PCK1 were negatively related to the grade of HCC in above tissues (Fig. 1B). Moreover, quantitative real-time PCR (qRTPCR) revealed that the mRNA levels of PCK1 were lower in HCC tissues relative to their adjacent non-tumorous liver tissues in 30 paired clinical HCC specimens (Fig. 1C). Interestingly, we observed that the expression levels of PCK1 were negatively associated with those of HBXIP in above clinical HCC samples (Fig. 1D), suggesting that HBXIP might regulate the expression of PCK1 in hepatoma cells. Thus, we conclude that PCK1 is down-regulated in clinical HCC tissues and negatively correlated with HBXIP.

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HBXIP down-regulates PCK1 and inhibits gluconeogenesis in hepatoma cells Next, we are interested in whether the oncoprotein HBXIP is involved in the modulation of gluconeogenesis of liver cancer. Interestingly, our data showed that the over-expression of HBXIP markedly down-regulated PCK1 in HepG2 and Huh7 cells at the levels of mRNA in a dose-dependent manner (Fig. 2A). Conversely, perturbation of HBXIP led to the elevation of PCK1 in HepG2HBXIP (stably expressing HBXIP) cells at the levels of mRNA (Fig. 2B). Meanwhile, we confirmed the effect of HBXIP on PCK1 at the protein levels (Fig. 2C,D), suggesting that HBXIP is able to down-regulate the expression of PCK1. The corresponding reduction of gluconeogenesis is reflected by the decrease of the glucose released from the cells [31]. Accordingly, we demonstrated that the suppression of PCK1 mediated by HBXIP resulted in the decrease of de novo glucose production in HepG2 and Huh7 cells (Fig. 2E,F). In contrast, a mild increase of glucose production was observed in HepG2-HBXIP cells treated with si-HBXIP in a dose-dependent manner (Fig. 2G), suggesting that HBXIP is able to depress gluconeogenesis in hepatoma cells. Overall, we conclude that HBXIP down-regulates PCK1, leading to the perturbation of gluconeogenesis in hepatoma cells.

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HBXIP inactivates the promoter of PCK1 through down-regulating transcriptional factor FOXO1 We have reported that HBXIP can regulate gene transcription [32,33]. To gain insights into the mechanism by which HBXIP downregulates PCK1, we constructed the promoter of PCK1. Luciferase reporter gene assays showed that over-expression of HBXIP dosedependently reduced the promoter activities of PCK1 in HepG2, Huh7, Chang liver and 293T cells, respectively. Conversely, the

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Fig. 1. PCK1 is down-regulated in clinical HCC tissues and negatively correlated with HBXIP. (A) The expression of PCK1 was detected by IHC staining in the peritumor tissues and HCC tissues using HCC tissues microarray. (B) Heatmap of the expression of PCK1 in HCC tissues. Number 0, 1, 2 or 3 represents the negative, low, moderate or high staining, respectively. (C) Relative mRNA levels of PCK1 were assessed by qRT-PCR in 30 paired clinical HCC tissues and corresponding non-tumorous tissues (**P < 0.01; Wilcoxon’s signed-rank test). (D) The correlation between HBXIP and PCK1 was examined by qRT-PCR in 30 cases of clinical human HCC issues (**P < 0.01, r = −0.6112, Pearson’s correlation coefficient).

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depletion of HBXIP dose-dependently elevated the promoter activities of PCK1 in HepG2-HBXIP and 293T cells (Fig. 3A, Supplementary Fig. S1A–C). To further elucidate the mechanism underlying that HBXIP inactivates the promoter of PCK1, we truncated the full-length region of PCK1 promoter to four fragments including −1651/+60 (pGL3-P1), −692/+60 (pGL3-P2), −434/+60 (pGL3P3) and −142/+60 (pGL3-P4) (Fig. 3B). Our results showed that HBXIP could remarkably decrease the promoter activities of fragment −434/ +60 (pGL3-P3) in a dose-dependent manner in HepG2 and 293T cells (Fig. 3B,C). However, the knockdown of HBXIP dose-dependently increased the promoter activities of pGL3-P3 in the cells (Fig. 3C and Supplementary Fig. S1D). Interestingly, we observed that the pGL3P3 contained two consensus sequences TGTTTTG (termed IRE1 and IRE2), which was the sequence of insulin response element (IRE) [34]. Therefore, we supposed that the IRE might be associated with the promoter activities of PCK1 mediated by HBXIP. Furthermore, we showed that HBXIP failed to work when IRE1 was mutated in above cells. However, the PCK1 promoter activities still could be inhibited by HBXIP when IRE2 was mutated in the cells, suggesting that IRE1 is required for HBXIP-mediated PCK1 repression (Fig. 3D and Supplementary Fig. S1E). It has been reported that transcriptional factor FOXO1 can regulate PCK1 and G6PC expressions through IRE and FOXO1 binds IRE1 with high affinity and IRE2 with low af-

finity [34,35]. Accordingly, we speculated that HBXIP might regulate PCK1 through FOXO1. We validated that the over-expression of FOXO1 could increase the promoter activities of PCK1 (pGL3-P3) in HepG2 cells. While HBXIP failed to work when FOXO1 was overexpressed in HepG2 cells (Fig. 3E). Moreover, the over-expression of FOXO1 could rescue the decreased protein levels of PCK1 mediated by HBXIP in the cells (Fig. 3F). In addition, HBXIP decreased the expression of FOXO1 at the protein levels in HepG2, Huh7 and Chang liver cells in a dose-dependent manner, and silencing of HBXIP increased the expression of FOXO1 in HepG2-HBXIP cells at the protein levels (Supplementary Fig. S1F–I), suggesting that HBXIP decreases the promoter activities of PCK1 through down-regulating transcriptional factor FOXO1. Thus, we conclude that HBXIP is capable of inhibiting PCK1 promoter through down-regulating transcriptional factor FOXO1 in hepatoma cells. HBXIP up-regulates miR-135a targeting the 3′UTR of FOXO1 mRNA to repress FOXO1 Next, we tried to identify the mechanism by which HBXIP decreased FOXO1. Interestingly, we observed that the over-expression of HBXIP failed to change the mRNA levels of FOXO1 (Supplementary Fig. S2A), implying that HBXIP down-regulates FOXO1 at post-

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Fig. 2. HBXIP down-regulates PCK1 and inhibits gluconeogenesis in hepatoma cells. (A) The expression levels of PCK1 were detected by RT-PCR in HepG2 and Huh7 cells transiently transfected with pCMV or pCMV-HBXIP. (B) The expression levels of PCK1 were examined by RT-PCR in HepG2-HBXIP cells transfected with si-control or siHBXIP. (C) The expression levels of PCK1 were assessed by Western blot analysis in HepG2 and Huh7 cells transiently transfected with pCMV or pCMV-HBXIP. (D) The expression levels of PCK1 were detected by Western blot analysis in HepG2-HBXIP cells transfected with si-control or si-HBXIP. (E, F) The secreted glucose levels in the glucose-free medium of HepG2 and Huh7 cells which were transiently transfected with pCMV or with pCMV-HBXIP were measured by colorimetric glucose assay kit. (G) The secreted glucose levels in the glucose-free medium of HepG2-HBXIP cells which were transiently transfected with si-Control or si-HBXIP were measured by colorimetric glucose assay kit.

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transcriptional level. It has been reported that miR-135a is able to target the 3′UTR of FOXO1 mRNA in bladder cancer [36]. Accordingly, we validated that miR-135a was able to target the 3′UTR of FOXO1 mRNA in HepG2 cells (Supplementary Fig. S2B–D). Furthermore, we noticed that the expression of FOXO1 was decreased by miR-135a in HepG2 cells. Inversely, the expression of FOXO1 was increased by anti-miR-135a in HepG2-HBXIP cells (Supplementary Fig. S2E–G), supporting that miR-135a is able to down-regulate FOXO1 at post-transcriptional level. Moreover, the over-expression of HBXIP increased the expression of miR-135a in HepG2 and Huh7 cells. Conversely, the knockdown of HBXIP resulted in the decreased expression of miR-135a in HepG2-HBXIP cells in a dosedependent manner (Fig. 4A–C), suggesting that HBXIP is able to upregulate the expression of miR-135a. Moreover, qRT-PCR analysis demonstrated that the expression levels of miR-135a were positively associated with those of HBXIP in 30 clinical HCC tissues

(Fig. 4D). Furthermore, the reduced expression levels of FOXO1 mediated by HBXIP could be reversed by treating with anti-miR135a (Fig. 4E). Interestingly, the decreased glucose production induced by HBXIP could be rescued in hepatoma cells through treating with anti-miR-135a or transfecting with pCMV-FOXO1 (Fig. 4F–H), suggesting that HBXIP modulates hepatic gluconeogenesis through miR-135a/FOXO1 axis. Together, we conclude that HBXIP is able to up-regulate miR-135a targeting the 3′UTR of FOXO1 mRNA to down-regulate FOXO1 in hepatoma cells. HBXIP promotes the nuclear export of FOXO1 through PI3K/Akt signaling It has been reported that phosphorylation of FOXO1 (Ser256) by Akt leads to cytoplasmic localization [37]. Previously, we reported that HBXIP could active Akt signaling in HepG2 cells [38]. There-

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Fig. 3. HBXIP inactivates the promoter of PCK1 through down-regulating transcriptional factor FOXO1. (A) The promoter activities of PCK1 in HepG2 cells and HepG2HBXIP cells were measured by luciferase reporter gene assays, respectively. (B) The activities of four fragments of PCK1 promoter in HepG2 cells were measured by luciferase reporter gene assays. (C) The promoter activities of PCK1 (PGL3-P3) in HepG2 cells and HepG2-HBXIP cells were measured by luciferase reporter gene assays. (D) The activities of wild-type or mutant PCK1 promoters in HepG2 cells were measured by luciferase reporter gene assays. (E) The promoter activities of PCK1 in HepG2 cells were measured by luciferase reporter gene assays. (F) The expression levels of PCK1 were detected in HepG2 cells by Western blot analysis. The intensity for each band was densitometrically quantified. The value under each lane indicates the relative amounts of protein relative to control group. The value was obtained by the intensity ratio between the target protein and β-actin band in each lane. Protein bands were quantified using Quantity One software (Bio-Rad). Statistically significant differences are indicated: *P < 0.05, **P < 0.01, ***P < 0.001versus control, Student’s t test.

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fore, we speculated that HBXIP might influence the intracellular localization of FOXO1 to regulate the expression of PCK1. Interestingly, immunofluorescence staining showed that cotransfection of DsRed-fused HBXIP and GFP-fused FOXO1 resulted in the nuclear exclusion of FOXO1 in HepG2 cells (Fig. 5A). As shown in Fig. 5B, HBXIP leads to a decrease of FOXO1 in nucleus and a corresponding increase of FOXO1 in the cytoplasm, indicating that HBXIP increases the nuclear export of FOXO1. As expected, the overexpression of HBXIP resulted in the increase of p-Akt and p-FOXO1 levels (Ser 256) in HepG2 and Huh7 cells (Fig. 5C and Supplementary Fig. S3A). However, the elevation of p-Akt and p-FOXO1 levels induced by HBXIP could be abolished when PI3K/Akt signaling

pathway was inhibited by wortmannin, an inhibitor of PI3K (Fig. 5C). Moreover, HBXIP siRNA resulted in decrease of p-Akt and p-FOXO1 levels in HepG2-HBXIP cells (Fig. 5D), suggesting that HBXIPinduced trafficking of FOXO1 from nucleus to cytoplasm is dependent on Akt-mediated phosphorylation of FOXO1. In addition, we observed that the decrease of glucose production mediated by HBXIP could be significantly rescued by wortmannin in HepG2 cells and Huh7 cells (Fig. 5E and Supplementary Fig. S3B), suggesting that HBXIP modulates hepatic gluconeogenesis through PI3K/Akt/pFOXO1 signaling pathway. Thus, we conclude that HBXIP increases the phosphorylation levels of FOXO1 by activating PI3K/Akt pathway to promote the nuclear export of FOXO1.

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Fig. 4. HBXIP up-regulates miR-135a targeting the 3’UTR of FOXO1 mRNA to repress FOXO1. (A, B) The relative expression levels of miR-135a were detected in HepG2 and Huh7 cells transfected with pCMV or pCMV-HBXIP by qRT-PCR analysis. (C) The relative expression levels of miR-135a were detected in HepG2-HBXIP cells transfected with si-Control or si-HBXIP by qRT-PCR analysis. (D) The correlation between HBXIP and miR-135a was detected by qRT-PCR in 30 cases of clinical human HCC tissues (**P < 0.01, r = 0.6863, Pearson’s correlation coefficient). (E) The expression levels of FOXO1 were detected by Western blot analysis in HepG2 and Huh7 cells transfected with pCMVHBXIP and treated with anti-miR-135a. (F, G) The secreted glucose levels in the glucose-free medium of HepG2 and Huh7 cells which were over-expressed with pCMVHBXIP and treated with anti-miR-135a were measured by colorimetric glucose assay kit. (H) The secreted glucose levels in the glucose-free medium of HepG2 cells which were over-expressed with pCMV-HBXIP and pCMV-FOXO1 were measured by colorimetric glucose assay kit. Statistically significant differences are indicated: *P < 0.05, **P < 0.01, versus control, Student’s t test.

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Reduction of PCK1 mediated by HBXIP promotes the growth of hepatoma cells Next, we evaluated the effect of HBXIP-decreased PCK1 on the growth of hepatoma cells in vitro and in vivo. Ectopic expression of HBXIP significantly promoted the colony formation efficiency of HepG2 cells. However, the promotion could be dramatically abrogated by over-expression of PCK1 in the cells (Fig. 6A,B). Moreover, tumor xenograft model showed that stable over-expression of HBXIP remarkably increased the tumor growth, but the over-expression of PCK1 blocked this enhancement of tumor in mice (Fig. 6C–E). Additionally, immunohistochemical staining displayed that the overexpression of PCK1 reduced the HBXIP-elevated expression of Ki67, a cell proliferation marker, in the tumor tissues from mice (Fig. 6F). Furthermore, we validated the effect of HBXIP on PCK1 in xeno-

graft tumor tissues by Western blot analysis (Fig. 6G). Collectively, we conclude that the reduction of PCK1 mediated by HBXIP promotes the growth of hepatoma cells in vitro and in vivo.

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Discussion It is well known that the glucose metabolism reprogramming is a hallmark of cancer [15]. The potential role of gluconeogenesis during transformation of normal cells to cancer cells, particularly in liver cancer, has not been documented, although there has been considerable progress in understanding the functional significance and probable mechanisms of Warburg effect [39,40]. Recently, our group has reported that HBXIP induces glucose metabolism reprogramming through down-regulating SCO2 and PDHA1 in breast

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Fig. 5. HBXIP promotes the nuclear export of FOXO1 through PI3K/Akt signaling. (A) GFP (green) and DsRed (red) were visualized using immunofluorescence staining in HepG2 cells transfected with ectopic GFP-FOXO1, DsRed or DsRed-HBXIP, respectively. We counted 1000 cells each group and quantified the results. N, nucleus; C, cytoplasm; N + C, nucleus and cytoplasm. DAPI staining (blue) was included to visualize the nucleus. The scale bar represents 10 μm for immunofluorescence. (B) Nuclear versus cytoplasmic localization of Flag-FOXO1 after cotransfection of pcDNA-HBXIP and flag-tagged pCMV-FOXO1 in HepG2 cells. Subcellular fractions were analyzed by Western blot analysis at 48 h after transfection with pCMV or pCMV-HBXIP in the presence of MG132 (10 μM, for 12 h). (C) After transfection with HBXIP, the levels of p-Akt, Akt, p-FOXO1 and FOXO1 were detected in HepG2 cells treated with wortmannin (a PI3K inhibitor). (D) The levels of p-Akt, Akt, p-FOXO1 and FOXO1 were detected by Western blot analysis in HepG2-HBXIP cells transfected with si-control or si-HBXIP. (E) The secreted glucose levels in the glucose-free medium of HepG2 cells which were overexpressed with pCMV-HBXIP and treated with wortmannin were measured by colorimetric glucose assay kit. Statistically significant differences are indicated: *P < 0.05, **P < 0.01, versus control, Student’s t test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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cancer cells [18]. In this study, we are interested in whether HBXIP is involved in the regulation of gluconeogenesis in liver cancer. PCK1 is the first key enzyme in the gluconeogenesis pathway [41]. Therefore, we need to evaluate the expression of PCK1 in HCC tissues. Interestingly, we observed that the expression levels of PCK1 were lower in HCC tissues relative to those in their peritumoral liver tissues. Thus, we supposed that HBXIP might down-regulate PCK1 in hepatoma cells. As expected, we validated the event. Next, we try to identify the mechanism. Many transcription factors are involved in PCK1 gene transcription, such as ATF, FOXO1 and SREBP-1 [16,42,43]. We have reported that HBXIP is involved in the regulation of the gene transcription [6–8,33]. Therefore, we speculated that HBXIP might be associated with the promoter activity of PCK1. Intriguingly, we identified that HBXIP suppressed the promoter activities of PCK1 in the cells. Previous studies showed that PCK1 promoter contained the sequence TGTTTTG (IRE) [34]. Our results showed that the suppressed activities of PCK1 promoter by HBXIP could be abolished when the IREs were mutated. It has been reported that FOXO1 is able to regulate the gene transcription through Q4 binding IRE [27]. Accordingly, we examined whether FOXO1 was involved in the regulation of PCK1 by HBXIP. As expected, we demonstrated that HBXIP depressed the promoter activities of PCK1 through down-regulating FOXO1.

MiRNAs are the single-stranded RNA molecules that inhibit gene expression by binding to the 3′UTR of a target mRNA [44,45]. Recently, miRNAs, such as miR-29a-c, miR-23a, and miR-33, have attracted attention as regulators of glucose metabolism [17,46,47]. The role of miR-135a in different tumors is more complicated. It has been reported that miR-135a can act as a suppressor in tumors, such as gastric cancer, prostate cancer and lung cancer [48–50]. But miR135a acts as an enhancer in some other tumors, like liver cancer, glioma, bladder cancer and cervical cancer [36,51–54]. Interestingly, we found that HBXIP governed the expression of FOXO1 through up-regulation of miR-135a in hepatoma cells. Our findings are consistent with above reports. Taken together, we conclude that HBXIP down-regulates FOXO1 through increasing miR-135a in hepatoma cells. Given that FOXO1 is controlled by post-translational modifications, such as phosphorylation and acetylation [55–57], we try to explore the other mechanisms by which HBXIP regulates FOXO1. It has been reported that activation of the PI3K/Akt signaling pathway leads to FOXO1 phosphorylation, which results in dysregulation of Cyclin D1, Cyclin D2, p21 and CDK4 [58]. HBXIP up-regulated CyclinD1 via activating PI3K/Akt [38]. In this study, we found that HBXIP resulted in the nuclear exclusion of FOXO1 in hepatoma cells, which could be blocked by wortmannin. It suggests that HBXIP in-

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Fig. 6. Reduction of PCK1 mediated by HBXIP promotes the growth of hepatoma cells. (A, B) Clonogenicity of HepG2 cells transfected with indicated plasmids was examined by monolayer colony formation assay. (C) The growth curve of tumors in nude mice transplanted with MCF-7 cells was shown. (D) The tumors in each group were shown. (E) The average tumor weight in each group was shown. (F) Ki67 expression in tumor tissues from mice was detected by IHC assays. (G) The levels of HBXIP and PCK1 were examined by Western blot analysis in the tumor tissues from mice. Statistically significant differences are indicated: **P < 0.01, versus control, Student’s t test.

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hibits FOXO1 nuclear localization through activation of PI3K/Akt signaling in the cells. It has been reported that p21, p27 and Cyclin D1 are dysregulated in miR-135a over-expressing bladder cancer cells [36], indicating that the effect of HBXIP on FOXO1 might result from the synergistic regulation of miR-135a and PI3K/Akt signaling. In this study we identified the pivotal role of gluconeogenesis mediated by HBXIP in the development of HCC. It has been reported that the depression of gluconeogenesis enhanced nucleotide synthesis, leading to the development of cancer [17]. The enhanced production of this key building block of nucleic acids is probably an important means of meeting the basic requirement for rapid cell division and growth of tumors. The increased utilization of glycolytic and HMP shunt pathways in HCC depending on the extent of accumulation of G6P due to block in gluconeogenesis may

contribute to the survival of the tumor cells under hypoxic conditions. Functionally, we explored the significance that the reduction of PCK1 mediated by HBXIP promotes the growth of hepatoma cells in vitro and in vivo. Thus, we conclude that the oncoprotein HBXIP is able to depress the gluconeogenesis through suppressing PCK1 to promote hepatocarcinogenesis, involving miR-135a/FOXO1 axis and PI3K/Akt/p-FOXO1 pathway. Therapeutically, HBXIP may serve as a target in hepatocarcinogenesis. Taken together, in this study we present a model that the oncoprotein HBXIP enhances hepatocarcinogenesis through depressing gluconeogenesis which leads to the accumulation of nucleotide synthesis, involving miR-135a/FOXO1 axis and PI3K/ Akt pathway. In this model, HBXIP is able to down-regulate PCK1 in hepatoma cells. Mechanistically, HBXIP is capable of down-

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regulating FOXO1, which is responsible for the activation of PCK1 promoter in the cells. HBXIP increases the expression of miR-135a targeting the 3’UTR of FOXO1 mRNA in hepatoma cells, leading to the down-regulation of FOXO1. In addition, HBXIP enhances the nuclear exclusion of FOXO1 through activating PI3K/Akt signaling. Our finding provides new insights into the mechanism of glucose metabolism reprogramming mediated by HBXIP in hepatocellular carcinoma.

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Conflict of interest The authors declare no conflict of interest.

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Acknowledgments This work was supported by the grants of the National Basic Research Program of China (973 Program, Nos. 2015CB553905, 2015CB553703), the National Natural Scientific Foundation of China (Nos. 81272218, 31470756, 31670771), and Tianjin Natural Scientific Foundation (No. 14JCZDJC32800). We thank Dr. Shimin Zhao of FuDan University for providing the PCK1 plasmids.

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Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2016.08.025.

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