HBx-mediated decrease of AIM2 contributes to hepatocellular carcinoma metastasis.

Tumor metastasis is responsible for the high mortality rates in patients with hepatocellular carcinoma (HCC). Absent in melanoma 2 (AIM2) has been implicated in inflammation and carcinogenesis, although its role in HCC metastasis remains unknown. In the present study, we show that AIM2 protein expression was noticeably reduced in HCC cell lines and clinical samples. A reduction in AIM2 was closely associated with higher serum AFP levels, vascular invasion, poor tumor differentiation, an incomplete tumor capsule and unfavorable postsurgical survival odds. In vitro studies demonstrated that AIM2 expression was modulated by hepatitis B virus X protein (HBx) at transcriptional and post-translational levels. HBx overexpression markedly blocked the expression of AIM2 at mRNA and protein levels by enhancing the stability of Enhancer of zeste homolog 2 (EZH2). Furthermore, HBx interacted with AIM2, resulting in an increase of AIM2 degradation via ubiquitination induction. Functionally, knockdown of AIM2 enhanced cell migration, formation of cell pseudopodium, wound healing and tumor metastasis, whereas reintroduction of AIM2 attenuated these functions. The loss of AIM2 induced the activation of epithelial-mesenchymal transition (EMT). Fibronectin 1 (FN1) was found to be a downstream effector of AIM2, with its expression reversely modulated by AIM2. Silencing of FN1 significantly halted cell migration induced by AIM2 depletion. These data demonstrate that HBx-induced loss of AIM2 is associated with poor outcomes and facilitates HCC metastasis by triggering the EMT process. The results of the present study therefore suggest that AIM2 is a potential prognostic biomarker in hepatitis B virus-related HCC, as well as a possible therapeutic target for tumor metastasis.

5‐aza‐2′‐deoxycytidine

absent in melanoma 2

chronic hepatitis B

chromosome immunoprecipitation

4′,6‐diamidino‐2‐phenylindole

epithelial‐mesenchymal transition

enhancer of zeste homolog 2

fibronectin 1

tri‐methylation of histone H3 lysine 27

hepatitis B virus

hepatitis B virus X protein

hepatocellular carcinoma

immunohistochemistry

matrix metalloproteinase

3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide

small interfering RNA

tissue microarray

tetramethylrhodamine

Introduction

Hepatocellular carcinoma (HCC), a chronic inflammation‐related cancer, is the second highest cause of cancer death globally as a result of tumor metastasis and early post‐surgery recurrence (El‐Serag, 2011; Galun, 2016; Villanueva and Llovet, 2014). Despite recent studies identifying many cancer‐related factors in HCC metastasis, the underlying mechanism of HCC progression remains largely unknown (Forner et al., 2012). Recent studies have proposed that inflammation not only comprises a malignant factor of tumorigenesis, but also is involved in the subsequent change of carcinogenesis that promotes tumor metastasis (Coffelt and de Visser, 2014). Hepatitis B virus (HBV) infection is a major cause of liver inflammation and accounts for half of HCC development (Chan et al., 2016). As a key oncogenic protein encoded by HBV, hepatitis B virus X protein (HBx) is essential in HBV‐related inflammation and tumorigenesis. HBx transactivates a series of genes involved in tumor growth and metastasis via epigenetic modulation and post‐transcriptional regulation (Decorsiere et al., 2016; Huang et al., 2013; Park et al., 2013). However, few studies have clarified the role of HBx in inflammasome‐mediated tumor progression.

Absent in melanoma 2 (AIM2), an essential inflammasome functioning in human innate immune system, was originally isolated from human melanoma cells (DeYoung et al., 1997). AIM2 is a member of the interferon‐inducible PYHIN (PYRIN and HIN domain‐containing) family proteins (also known as p200‐family proteins) and serves as the front line of defense against pathogen infection via caspase‐1 activation (Chuong et al., 2016; Gray et al., 2016; Man and Kanneganti, 2015; Rommereim and Subramanian, 2015). Aberration of AIM2 results in a large number of diseases (Choubey, 2016; Man et al., 2015). Previous studies have shown that AIM2 was differently expressed and functioned as both a tumor suppressor and oncogene in human cancers. AIM2 was overexpressed in nonsmall cell lung cancer and facilitated cell proliferation (Kong et al., 2015; Sorrentino et al., 2015). AIM2 restoration in colorectal cancer cells induced the expression of invasion‐associated genes such as VIM and MCAM (Patsos et al., 2010). On the other hand, silencing of AIM2 was associated with poor survival in colorectal cancer (Dihlmann et al., 2014), and promoted colon tumorigenesis via the activation of the Wnt and Akt pathways (Wilson et al., 2015). Excessive AIM2 expression inhibited cell proliferation and induced apoptosis in breast carcinoma (Chen et al., 2006; Liu et al., 2015b). Interestingly, AIM2 expression was elevated in chronic hepatitis B (CHB) patients (Han et al., 2015) and decreased in HCC patients (Ma et al., 2016). However, the clinical significance of AIM2 in HCC and the mechanism of its downregualtion have not been demonstrated.

In the present study, the expression and clinical significance of AIM2 in HCC was determined aiming to highlight the connection of HBx and AIM2. The biological function of AIM2 in HCC and the relevant mechanisms was investigated. The data obtained demonstrate that AIM2 was inhibited by HBx and exhibits anti‐metastatic activity towards HCC.

Materials and methods

Patients, tissue specimens and follow‐up

Archived paraffin‐embedded pathological specimens from 471 primary HCC patients were collected along with complete clinical and pathological data at Sun Yat‐sen University Cancer Center. The study was approved by the Institute Research Medical Ethics Committee. None of the patients had received radiotherapy or chemotherapy before surgery. All samples were anonymous.

Tissue microarray (TMA) construction and Immunohistochemistry (IHC) evaluation

HCC specimens were selected and re‐embedded into new paraffin blocks for TMA. The TMA blocks were cut into 4 μm sections and underwent IHC staining. Protein expression levels of AIM2 stained TMA slides were assessed by two independent pathologists (L‐LL and R‐ZL). The median of the IHC score was chosen as the cut‐off value for the high and low AIM2 groups.

Statistical analysis

Statistical analyses were performed using spss, version 19.0 (IBM Corp., Armonk, NY, USA). Student's t‐test, Pearson's χ2 test, Fisher's exact test, the Kaplan–Meier method and a multivariate Cox proportional hazards regression model were employed. P < 0.05 (two‐tailed) was considered statistically significant.

Cell lines and cell culture

Bel‐7402, SMMC‐7721, Huh7 and Bel‐7404 HCC cells were obtained from the Type Culture Collection Cell Bank, Chinese Academy of Science Committee (Shanghai, China) and routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA). All cells were maintained in a humidified incubator at 37 °C and 5% CO2.

Western blot and antibodies

Western blot was performed as described previously (Wang et al., 2015). The antibodies used in the present study were AIM2 (dilution 1 : 1000; Sigma, St Louis, MO, USA), EZH2 (dilution 1 : 2000; Cell Signaling Technology, Beverly, MA, USA), HA (dilution 1 : 1000; Santa Cruz Bitechnology, Santa Cruz, CA, USA), GFP (dilution 1 : 2000; Cell Signaling Technology), β‐actin (dilution 1 : 1000; Santa Cruz Biotechnology), H3K27me3 (dilution 1 : 1000; Affinity Bioreagents, Golden, CO, USA), FN1 (dilution 1 : 1000; Abcam, Cambridge, UK), E‐cadherin (dilution 1 : 1000; Abcam), N‐cadherin (dilution 1 : 1000; Santa Cruz Biotechnology) and vimentin (dilution 1 : 1000; Santa Cruz Biotechnology).

Quantitative real‐time RT‐PCR (qRT‐PCR)

Total RNA was extracted by Trizol reagent (BIOO Scientific Co., Austin, TX, USA). Reverse transcription and SYBR green‐based real‐time PCR were then carried out. Primers were: AIM2 forward: 5′‐TGGCAAAACGTCTTCAGGAGG‐3′ and reverse: 5′‐AGCTTGACTTAGTGGCTTTGG‐3′; 18S forward: 5′‐TGAGAAACGGCTACCACATCC‐3′ and reverse: 5′‐ACCAGACTTGCCCTCCAATG‐3′; HBx forward for C terminal: 5′‐GCACTTCGCTTCACCTCT‐3′ and reverse: 5′‐TATGCCTACAGCCTCCTA‐3′; HBx forward for N termina 5′‐TCCTTTGTTTACGTCCCGTC‐3′ and reverse: 5′‐ CGTTCCGACCGACCACGG‐3′; FN1 forward: 5′‐GGAGTTTCCTGAGGGTTT‐3′ and reverse: 5′‐GCAGAAGTGTTTGGGTGA‐3′.

Plasmid construction and transfection

The recombinant plasmids pcDNA 3.1/hygro(+) empty vector, as well as pcDNA 3.1/hygro(+)‐AIM2 and pcDNA 3.1/hygro(+)‐HA‐HBx, were confirmed by sequencing. The plasmids were transfected into HCC cell lines using Lipofectamine™ 2000 (Invitrogen, Life Technologies, Carlsbad, CA, USA) reagent.

RNA interference

Small interfering RNAs (siRNAs) were: AIM2 siRNA1: 5′‐GGAGAAAGUUGAUAAGCAA‐3′, siRNA2: 5′‐GAGAGUAAAUACAAGGAGA‐3′); FN1 siRNA1: 5′‐CCCGGTTGTTATGACAATGGA‐3′, siRNA2: 5′‐CUCUUGUGGCCACUUCUGATT‐3′; HBx siRNA1: 5′‐GCCACAACGUCUAUAUCAU‐3′, siRNA2: 5′‐GGCAGAGGAAGUCUUCUAA‐3′). All of these siRNAs were purchased from Shanghai GenePharma Co. Ltd (Shanghai, China). Transfection was performed using Lipofectamine™ RNAiMAX (Invitrogen, Life Technologies).

Migration and invasion assay

For this, 2 × 104 cells for the migration assay and 4 × 104 cells for the invasion assay were plated in the upper compartment of a transwell chamber (8 μm pore size; Millepore, Darmstadt, Germany) in serum‐free medium. The upper chamber for invasion assay was coated with 10% matrigel (BD Biosciences, San Jose, CA, USA). Fresh media containing 10% fetal bovine serum was placed in the lower chamber. After incubation for 24–48 h, cells on the lower membrane were fixed using 20% methanol and stained with 0.1% crystal violet. The experiments were performed in triplicate and repeated three times.

Wound‐healing assay

Cells (1 × 106 per well) were seeded into six‐well plates and transfected with pcDNA 3.1‐AIM2 or AIM2 siRNAs. When an appropriate confluence was reached, the cell layer was scratched with a sterile plastic tip and cultured for the indicated time with serum‐free medium. Images were captured at different time points under a microscope to assess the rate of gap closure.

3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) and colony formation assays

Next, 2–4 × 103 cells were seeded in 96‐well plates with 100 μL of medium and cultured for 5 days. MTT stock solution was added to each well by 10 μL·well−1 for 4 h at 37 °C. After the addition of dimethylsulfoxide (150 μL·well−1), A 490 was measured. For the colony formation assay, 500 cells were seeded into six‐well plates and incubated for 10–14 days. Colonies were fixed with methanol, stained with 0.1% crystal violet and counted.

Animal model

Five‐weak‐old male BALB/c nude mice were purchased from Vital River company (Beijing, China). For the orthotopic tumor implantation model, 1 × 106 Bel‐7402 or SMMC‐7721 cells were implanted into the left hepatic lobe. Six weeks later, the mice were killed to dissect the liver and lungs. For the caudal vein injection model, 1 × 106 Bel‐7402 or SMMC‐7721 cells were injected into the tail vein. After 40 days, metastatic lesions in the lung were counted. All animal studies were approved and performed by the animal institute of Sun Yat‐sen University Cancer Center in accordance with the protocols approved by the Medical Experimental Animal Care Commission of Sun Yat‐sen University Cancer Center.

Immunoflorescence and F‐actin staining

Cells were washed twice in PBS, then fixed in 3.7% formaldehyde and permeabilized with 0.1% Triton X‐100 both for 10 min at room temperature. After blocking in 1% bovine serum albumin in PBS for 30 min, the cells were incubated with diluted primary antibody, 4′,6‐diamidino‐2‐phenylindole (DAPI) or tetramethylrhodamine (TRITC)‐labeled phalloidin.

Dual‐luciferase report assay

For the dual‐luciferase report assay, HEK‐293T cells were transfected with AIM2 promoter plasmid vector pGL3‐WT and pGL3‐MUT separately and co‐transfected with pcDNA 3.1‐HA‐HBx plasmid. Renilla luciferase activity was employed as an internal control. The AIM2 cis‐regulatory elements were predicted via Genomatix (http://www.genomatix.de).

Chromosome immunoprecipitation (ChIP)

Bel‐7402 and SMMC‐7721 cells were subjected to ChIP in accordance with the manufacturer's instructions (Invitrogen, Life Technologies) with primers specific against the AIM2 promoter region. The AIM2 promoter primers used for ChIP were designed as: forward: 5′‐TAGAAGTCAGGGAGGAAAG‐3′ and reverse: 5′‐AGGGAAAGGAGGCAACT‐3′.

Results

AIM2 expression is decreased and correlated with metastatic features and poor outcomes

AIM2 expression in HCC cell lines and fresh tissue samples were detected by quantitative RT‐PCR and western blotting. The results showed that AIM2 mRNA and protein levels in most of HCC cell lines were lower than those in immortalized hepatic cell lines L‐02 and QSG‐7701 (Fig. 1A). In 59 pairs of HCC fresh tissues, AIM2 mRNA was reduced compared to matched nontumor tissues (Fig. 1B). Consistent with this, the AIM2 protein level was significantly decreased by 5.7‐fold in 14 HCC specimens (Fig. 1C).

Figure 1

AIM2 is down‐regulated in HCC and associated with disease progression. (A) The mRNA and protein expression of AIM2 in HCC and immortalized liver cell lines was examined by a quantitative RT‐PCR and western blotting. (B) mRNA expression of AIM2 in 54 pairs of HCC and corresponding adjacent liver tissues was determined. 18S RNA was used to normalize the fold change. (C) Expression profile of AIM2 protein in 14 paired HCC and adjacent nontumor tissues was detected by western blotting. The average level of AIM2 in tumor was calculated as the fold change according to nontumor tissues. (D) Expression of AIM2 was determined by a TMA‐based immunohistochemistry. Representative images of tumor (T) and nontumor (N) are shown. The IHC scores are shown and were analyzed statistically. (E) Correlation of AIM2 expression and overall and disease‐free survival was determined in a cohort of 471 patients by Kaplan–Meier analysis. (F1) Representative photomicrographs are shown to indicate the expression of AIM2 in non‐tumor (N), primary tumor (T) and metastasis (M) lesions. (F2) IHC staining of AIM2 was conducted in a cohort of 69 HCC patients with portal vein embolus. Quantitative data are presented as the mean ± SD. *P < 0.05; **P < 0.01.

To determine the clinical significance of AIM2 in HCC, we next examined the expression of AIM2 in a TMA cohort consisting of 471 patients with HCC. AIM2 was primarily located in the cytoplasm and significantly down‐regulated in HCC tissues (Fig. 1D). According to the median IHC score (8.53), HCC cases were divided into two groups: high AIM2 expression and low AIM2 expression. Low expression of AIM2 in tumor tissues was observed in 66.9% (315/471) of cases. Statistical analysis showed that low AIM2 expression was associated with a high serum AFP level, the presence of vascular invasion, poor tumor differentiation, incomplete tumor capsule and lymph node metastasis (Table S1). Kaplan–Meier analysis showed that patients with low AIM2 expression had a shorter overall and disease‐free survival (Fig. 1E). Stratified survival analysis further confirmed the prognostic value of AIM2 (Figs S1 and S2).

Because AIM2 expression was correlated with metastatic features such as vascular invasion and lymph node metastasis, another 69 HCC cases with portal vein embolus were recruited. Weaker immunoreactivity of AIM2 was depicted in tumor metastasis compared to the primary lesions (Fig. 1F1). The IHC score of AIM2 in embolus was significantly lower than that in primary tumors (Fig. 1F2).

AIM2 is downregulated by HBx

The results of previous studies suggested that AIM2 might affected by HBV infection (Pan et al., 2016). In our cohort, 66.7% (299/448) of cases with HBV infection had low AIM2 expression (Fig. 2A). AIM2 was down‐regulated at mRNA and protein levels in HBx‐expressing HepG2.2.15 cells compared to HepG2 cells without HBx expression (Fig. 2B). HBx expression was silenced by siRNA in stable cells (HepG2.2.15 and HepG2‐HBx) (Fig. 2C1). The expression of AIM2 was markedly induced in both cell lines. On the other hand, HBx was introduced into Bel‐7402 and SMMC‐7721 cell lines (Fig. 2C2). Overexpression of HBx decreased the expression of AIM2 mRNA and protein in a dose‐dependent manner. Deletion mutants of HBx were constructed to examine the effect of the N‐terminal and C‐terminal of HBx on the expression of AIM2 (Fig. 2D1). The results of quantitative RT‐PCR and western blotting confirmed that overexpression of mutant with an HBx C‐terminal (but not N‐terminal) was capable of down‐regulating AIM2 expression (Fig. 2D2). We further constructed another two HBx deletion mutants, ΔHBx134 and ΔHBx120, aiming to determine the dominant domain on AIM2 expression. ΔHBx134 with a truncated 20 amino acids at the C‐terminal retained the effect of HBx on AIM2, whereas ΔHBx120 with a truncated 34 amino acids at the C‐terminal lost the capability of downregulating AIM2, indicating that the domain of 120–134 amino acids of HBx is essential for the suppression of AIM2 (Fig. S3B,C).

Figure 2

AIM2 expression is modulated by HBx. (A) Representative IHC photomicrographs of AIM2 expression in HCC cases with or without HBV infection were presented. (B) HBx and AIM2 expression was measured in HepG2 and HepG2.2.15 cells by quantitative RT‐PCR and western blotting. (C1) HBx was silenced by siRNAs in HepG2.2.15 and HepG2‐HBx cells. The relative expression of AIM2 in cells treated with HBx siRNAs were determined. (C2) HBx was overexpressed by pcDNA3.1‐HBx‐HA plasmid in Bel‐7402 and SMMC‐7721 cells. AIM2 expression was examined in cells with pcDNA3.1‐HBx‐HA transfection. (D1) The structures of the full‐length HBx, as well as C‐terminal and N‐terminal deletion mutants, are shown. (D2) HBx and AIM2 expression was measured in Bel‐7402 and SMMC‐7721 cells with full‐length HBx or deletion mutant overexpression by quantitative RT‐PCR and western blotting. (E) AIM2 promoter activity was determined by dual‐luciferase assay in 293T cells with or without HBx expression. (F) The interaction of HBx to AIM2 promoter was confirmed by a ChIP assay in Bel‐7402 and SMMC‐7721 cells.

To determine whether HBx transcriptionally silenced AIM2 expression in HCC cells, luciferase constructed plasmids of AIM2 promoter were co‐transfected with HBx overexpression vector in 293T cells. Dual‐luciferase report assays showed that HBx transfection significantly inhibited the activity of full‐length AIM2 promoter but not that with deletion of the region from −2000 to −1447 (Fig. 2E). A ChIP assay was performed to demonstrate the interaction of HBx with the AIM2 promoter (Fig. 2F). These findings indicated that AIM2 is a direct target of HBx.

AIM2 repression by HBx requires EZH2

To investigate whether promoter methylation was involved in HBx‐mediated AIM2 down‐regulation, we treated Bel‐7402 and SMMC‐7721 cells with demethylase 5‐aza‐2′‐deoxycytidine (5‐Aza). Upon 5‐Aza treatment, AIM2 mRNA and protein levels were increased in a dose‐dependent manner (Fig. 3A). In HBx‐expressing cells, the expression of tri‐methylation of histone H3 lysine 27 (H3K27me3, an important mediator of DNA methylation) was elevated, whereas EZH2 (a histone‐lysine N‐methyltransferase enzyme) remained unchanged (Fig. 3B).

Figure 3

EZH2 is required for HBx‐induced AIM2 decrease. (A) AIM2 mRNA and protein expression levels were tested in Bel‐7402 and SMMC‐7721 cells treated with 5‐Aza for 24 h. (B) EZH2 and H3K27me3 levels were detected in Bel‐7402 and SMMC‐7721 cells transfected with HBx. (C) EZH2 was transiently knocked down by siRNAs in HBx‐expressing Bel‐7402 and SMMC‐7721 cells. AIM2 expression was determined. (D) EZH2 activity was blocked by its inhibitor, GSK‐126, in cells with or without the presence of HBx. The impact of EZH2 on AIM2 expression was checked. (E) The half‐life of EZH2 protein was detected in Bel‐7402 and SMMC‐7721 cells with or without HBx, using CHX (20 μg·mL −1). The degradation rates of EZH2 protein were calculated and indicated. (F) The interaction of EZH2 to AIM2 promoter was confirmed by a ChIP assay in Bel‐7402 and SMMC‐7721 cells.

EZH2, which is known to be activated during the formation of H3K27me3, was knocked down by siRNA to investigate the role of EZH2 in HBx‐mediated AIM2 silencing in HCC cells. The depletion of EZH2 in HCC cells had no impact on the expression of AIM2, although it abolished HBx‐induced suppression of AIM2 (Fig. 3C). Incubation of EZH2 inhibitor GSK‐126 resulted in a similar phenomenon (Fig. 3D).

Under normal circumstances, EZH2 was rapidly degraded. In cells with HBx overexpression, the half‐life of EZH2 protein was dramatically prolonged (Fig. 3E). Furthermore, in the presence of HBx, EZH2 was recruited to the promoter of AIM2 to block its expression (Fig. 3F). The data obtained indicated that EZH2 was required for HBx‐induced AIM2 down‐regulation.

AIM2 degradation is enhanced by HBx

Next, the interaction between HBx and AIM2 was determined. The results of the co‐immunoprecipitation assay showed that, in both HCC cell lines, full length and C‐terminal HBx directly bound to AIM2 (Fig. 4A). The co‐localization of HBx and AIM2 in the cytoplasm was observed by immunofluorescence staining (Fig. 4B).

Figure 4

HBx directly binds to AIM2 and increase its degradation by enhancing ubiquitination. (A) Co‐IP assays were performed to determine the interaction of HBx and AIM2. (B) Immunofluorescence staining indicating HA‐HBx (green) and AIM2 (red) together with DAPI (blue) showed the co‐localization of HBx and AIM2 in Bel‐7402 and SMMC‐7721 cells. (C) The half‐life of AIM2 protein was determined in Bel‐7402 and SMMC‐7721 cells with or without HBx. (D) Cells were pre‐incubated with MG‐132 (20 μm) for 12 h. AIM2 was immunoprecipitated by anti‐HA and immunoblotted by anti‐AIM2. The ubiquitination of AIM2 protein was detected after HBx overexpression. (E) Bel‐7402 and SMMC‐7721 cells were incubated with MG‐132 (20 μm) for 48 h. The AIM2 level in whole cell cysate was measured by western blotting.

Because HBx was demonstrated to alter the half‐life of EZH2 in the present study, the impact of HBx on AIM2 protein stability was tested. Under basal conditions, AIM2 possessed a half‐life of more than 24 h. In cells with HBx expression, however, AIM2 was quickly degraded within 6 h (Fig. 4C). Further investigation showed that HBx overexpression increased the ubiquitination of AIM2 protein (Fig. 4D). MG‐132, an inhibitor of ubiquitination, was used to examine the mechanism of HBx‐mediated degradation of AIM2. As expected, MG‐132 treatment was able to attenuate the AIM2 decrease induced by HBx in HCC cells (Fig. 4E).

Loss of AIM2 promotes HCC metastasis in vitro and in vivo

To explore the biological role of AIM2 in HCC, AIM2 was either silenced in Bel‐7402 and SMMC‐7721 cells or re‐expressed in Huh7 and Bel‐7404 cells (Fig. 5A,B). MTT and colony formation assays were performed to determine the effect of AIM2 in HCC cell proliferation. The results showed that there was no statistical difference in cell growth rate within the indicated cultured periods compared to the control groups (Fig. S4).

Figure 5

Loss of AIM2 promotes HCC cell migration and invasion in vitro. (A) AIM2 was silenced in Bel‐7402 and SMMC‐7721 cells by siRNAs. (B) AIM2 was exogenously expressed by tranfecting pcDNA3.1‐AIM2 plasmids in Huh7 and Bel‐7404 cells. Transwell assays detected the invasion and migration abilities in cells with AIM2 depletion (C) or overexpression (D). Representative images and the quantitative data of three randomly selected fields are shown. Wound‐healing assays demonstrated cell movement capacity in AIM2‐knockdown (E) of AIM2‐expressing (F) cells. Scale bar = 200 μm. Quantitative data are presented as the mean ± SD. *P < 0.05; **P < 0.01.

Based on the clinicopathological data indicating that AIM2 might participate in HCC progression, the impact of AIM2 in tumor metastasis was next examined. Transwell assays showed that the knockdown of AIM2 enhanced, whereas the exogenous expression of AIM2 reduced the ability of cell invasion and migration in HCC cells (Fig. 5C,D). Wound‐healing assays further demonstrated that AIM2‐depleted cells filled up the wound faster than cells with AIM2 expression (Fig. 5E). By contrast, re‐expression of AIM2 hindered cell movement (Fig. 5F).

Orthotopically implanted and caudal vein injection models were established to examine the effect of AIM2 on tumor metastasis in vivo. Six weeks after HCC cell transplantation, the body weights of all mice did not differ from each other. In the orthotopically implanted model, mice in the AIM2 deficient group developed more liver metastasis compared to the control group (Fig. 6A). Hematoxylion and eosin staining confirmed that tumor cells invaded into the liver (Fig. 6B). Metastatic foci were also depicted in lung sections (Fig. 6C). Statistical analysis indicated that the number of metastatic nodules in liver and lung in the AIM2 deficient group was significantly greater than that in the control groups (Fig. 6D). In a caudal vein injection model, tumors metastasized to the lung were more frequently found in the AIM2 deficient group (Fig. 6E,F). Collectively, the data suggest that a loss of AIM2 was capable of promoting HCC metastasis.

Figure 6

Loss of AIM2 promotes tumor metastasis in vivo. (A) Mice were killed 40 days after injection of HCC cells. The tumor nodules in livers in orthotopic implantation model are indicated by arrows. (B) Livers were sectioned and stained with hemotoxylin and eosin (HE). Representative images of metastatic nodules are shown. (C) The lungs of mice in the orthotopic implantation group were dissected. HE straining images of foci in lung sections are shown. (D) The number of lesions in liver and lung of each mouse was calculated and is indicated. (E) Representative HE images of lung metastasis in caudal vein injection model are shown. (F) The number of tumor foci in the lung were counted and compared statistically. Scale bar = 20 μm. Quantitative data are the mean ± SD. *P < 0.05; **P < 0.01.

AIM2 depletion triggers EMT by increasing the expression of FN1

To clarify the mechanism via which AIM2 was involved in HCC metastasis, we identifed the cell shape using phallotoxin staining to visualize cytoskeleton F‐actin. The formation of pseudopodium, which improved cell mobility, was increased in AIM2‐depleted cells but decreased in AIM2‐expressing cells (Fig. 7A). Western blotting and immunoflorescence staining showed that loss of AIM2 activated the EMT process by reducing the expression of epithelial marker E‐cadherin and increasing the expression of mesenchymal markers vimentin and N‐cadherin (Fig. 7B,C). Consistent with this, AIM2 overexpression up‐regulated E‐cadherin and down‐regulated vimentin and N‐cadherin (Fig. 7B,C).

Figure 7

Loss of AIM2 promotes the EMT process. (A) Bel‐7402 and SMMC‐7721 cells were introduced with AIM2 overexpression vector or siRNAs, and then stained with TRITC‐labeled phalloidin. F‐actin (red) was used to indicated the cell morphology. Scale bar = 20 μm. (B) The expression of EMT‐related markers, E‐cadherin, vimentin and N‐cadherin was determined by western blotting in cells with AIM2 overexpression or silencing. (C) Expression of EMT‐related markers were detected further by immunofluorescence staining. Scale bar = 20 μm.

To identify the downstream effector of AIM2, a gene microarray composed of 89 metastasis‐relating genes was carried out. Following the knockdown of AIM2 in Bel‐7402 cells, five genes were down‐regulated and six genes were up‐regulated (Fig. S5A). According to the confirmation shown in Fig. S5B, FN1 (a key mediator of EMT) was selected as a potential target of AIM2. The results of a quantitative PCR, western blotting and immunofluoresence showed that AIM2 inhibition increased, whereas overexpression of AIM2 decreased FN1 at both mRNA and protein levels (Fig. 8A,B).

Figure 8

AIM2 promotes EMT by targeting FN1. (A) FN1 expression was measured in Bel‐7402 and SMMC‐7721 cells treated with AIM2 siRNAs for 48 h. The effect of AIM2 overexpression on FN1 expression was examined in Huh7 and Bel‐7404 cells. (B) Immunofluorescence staining was performed to visualize the regulation of FN1 expression by AIM2. (C) FN1 expression was knocked down by its siRNAs. (D,E) Cells were subjected to Transwell assays for 48 h. Migrated and invaded cells were stained with 0.1% crystal violet and counted to detect migration (D) or invasion (E) abilities. (F) Schematic regulatory network of the HBx‐mediated AIM2 downregulation responsible for HCC metastasis.

To clarify the role of FN1 in AIM2‐mediated suppression of tumor metastasis, its expression was silenced by siRNAs (Fig. 8C). Rescue experiments using transwell migration and invasion assays demonstrated that the silencing of FN1 partially attenuated the cell migration and invasion induced by AIM2 depletion in both HCC cell lines (Fig. 8D,E). Taken together, these results indicate that a loss of AIM2 triggers the EMT process via modulation of FN1 expression.

Discussion

HCC is a lethal disease presenting a global challenge because of the high probability of tumor recurrence and metastasis (Nguyen and Massague, 2007). To date, knowledge of the underlying cellular and molecular pathways driving HCC progression is limited (Aravalli et al., 2008; Breuhahn et al., 2011). In the present study, we demonstrate that low AIM2 expression was significantly associated with malignant features and exerted pro‐metastatic activity towards HCC metastasis. The data represent a novel HBx/AIM2/FN1 signaling axis mediating HBV‐related HCC progression via the EMT process (Fig. 8F).

Dysregulation of AIM2 has been reported in human cancers, although its clinical significance has rarely been demonstrated. In the present study, AIM2 expression was decreased in a cohort of 471 patients with HCC. In line with our data, Ma et al. (2016) showed that AIM2 was down‐regulated in 113 HCC cases recruited from North China. Patients with low expression of AIM2 had a shorter life compared to those with high AIM2 expression. This might be a result of the observation that loss of AIM2 was frequently associated with a higher serum AFP level, vascular invasion, poor tumor differentiation and incomplete tumor capsule. Studies of AIM2 on colorectal cancer lead to a similar conclusion with respect to the lack of AIM2 expression being closely correlated with unfavorable outcomes (Dihlmann et al., 2014; Liu et al., 2015a). These findings suggest that AIM2 is a potential biomarker with respect to the clinical surveillance of tumor progression.

The fact of our finding that AIM2 expression was decreased in the portal vein embolus in a cohort of 69 HCC samples strongly suggests that AIM2 was involved in tumor metastasis (one of the tumor hallmarks). In the present study, our in vitro and in vivo data further confirmed the significance of AIM2 depletion in cell migration and tumor metastasis. The silencing of AIM2 triggered the EMT process via targeting of FN1. Blocking the expression of FN1 markedly abolished the cell migration induced by AIM2 depletion. During hepatocarcinogenesis, FN1 was up‐regulated to activate the EMT process, in turn by up‐regulating Snail, N‐cadherin, vimentin, matrix metalloproteinase (MMP)2 and phospho‐Smad2, as well as acquisition of cell migratory behavior (Wang et al., 2013). Supporting our indication, AIM2 has been showed to participate in melanoma and squamous cell carcinoma metastasis (de Koning et al., 2014). Similar to AIM2, another inflammasome, the NOD‐like receptor pyrin domain containing‐3 (NLRP3), inhibited lung cancer and colorectal cancer metastasis by suppressing natural killer cell‐mediated responses (Chow et al., 2012; Dupaul‐Chicoine et al., 2015).

As a well known inflammasome, AIM2 exerts its function particularly by recognizing double‐stranded DNA of bacteria or virus, such as orthopoxvirus vaccinia virus and Francisella tularensis virus (Man et al., 2016). AIM2 was also found to be activated when sensing the presence of HBV DNA in hepatocytes (Pan et al., 2016). Wu et al. (2013) reported that patients with acute hepatitis B had a higher expression of AIM2 in peripheral blood mononuclear cells compared to those with CHB. An elevated AIM2 level was also depicted in tissue biopsies of CHB and glomerulonephritis patients carrying a high plasma HBV‐viral load (Han et al., 2015; Ponomareva et al., 2013). However, the relationship between HBV and AIM2 in HCC remains elusive. Our data indicate that AIM2 expression was decreased in HCC patients with HBV infection compared to those without HBV infection. Further study demonstrated that AIM2 was down‐regulated by HBx, which is one of the seven viral proteins encoded by HBV. HBx has been well documented with respect to playing an oncogenic role in HCC development. There is evidence available indicating that HBx promoted HCC cell invasion and metastasis by down‐regulating Wnt‐5α and up‐regulating MMP10 (Li et al., 2016; Sze et al., 2013). HBx serves as a transcriptional regulator of many oncogenes and tumor suppressor genes. For example, HBx induced FoxM1 expression by transactivating its promoter activity (Xia et al., 2012) but repressed urokinase‐type plasminogen activator expression via inhibiting transcription activity of the promoter (Park et al., 2013). In the present study, we found that HBx is an upstream regulator of AIM2 via its C‐terminal as a result of being recruited to the promoter region of –2000 to –1447. Our data therefore demonstrate the mechanism that links HBV infection to AIM2 suppression in HCC progression (Pang et al., 2015).

Inflammation induced by infection may result in the alteration of important genes responsible for tumor metastasis. Protein encoded by Helicobacter pylori inhibited the p53 pathway by increasing the ubiquitination of p53 to promote cell migration in gastric cancer (Wei et al., 2010). The nuclear factor‐κB signaling pathway was activated by Epstein–Barr virus in nasopharyngeal carcinoma and contributed to tumor metastasis (Chung et al., 2013). In the present study, HBx directly interacted with AIM2 to enhance its ubiquitination, subsequently resulting in a rapid degradation of AIM2 protein. The stability of AIM2 protein can be modulated by infection with Pseudomonas aeruginosa in macrophages (Pang et al., 2015). Knockdown of TRIM11 led to a pro‐longed half‐life of exogenous AIM2 in 293T cells (Liu et al., 2016). These data may provide a post‐translational mechanism that bridges virus infection and tumor metastasis.

Promoter and histone methylation are critical events in the epigenetic silencing of genes. Our results showing that treatment of 5‐Aza induced the expression of AIM2 and also that HBx‐mediated suppression of AIM2 required EZH2 (a methyltrasferase that specifically catalyzes the formation of H3K27me3) indicated that histone methylation was attributed to the loss of AIM2. EZH2, co‐operating with HBx, has been implicated in the silencing of a large number of tumor suppressor genes. Hu et al. (2016) showed that HBx repressed p27Kip1 via the recruitment of EZH2 to form H3K27me3 in the p27Kip1 promoter. Fan et al. (2016) reported that HBx modulated the methyltransferase activity of EZH2. In the present study, knockdown of EZH2 mostly abolished HBx‐induced AIM2 suppression, suggesting the essential role of EZH2 in the epigenetic regulation of AIM2 by HBx.

In summary, we have found that AIM2, as a target of HBx, was frequently decreased in HBV‐associated HCC tissues. Low AIM2 expression was significantly associated with a poor overall survival and a high metastasis tendency. Loss of AIM2 facilitated HCC cell migration and invasion via activation of the EMT process. Collectively, our findings provide in‐depth insights for the understanding of HCC metastasis and the newly identified HBx/AIM2/FN1 axis also represents a new potential therapeutic target for HCC.

Author contributions

S‐LC and J‐PY were responsible for the conception and design of the study. S‐LC, L‐LL, C‐HW, HW and S‐HC were responsible for the generation, collection, assembly, analysis of data. S‐XL, R‐ZL and XY were responsible for the scoring and evaluation of IHC stained slides. S‐LC, CZZ, DX and J‐PY were responsible for the drafting and revision of the manuscript. All authors approved the final version of the manuscript submitted for publication.

Fig. S1. Stratified analysis showing the correlation of AIM2 expression and overall survival in the indicated groups.

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Fig. S2. Stratified analysis showing the correlation of AIM2 expression and disease‐free survival in the indicated groups.

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Fig. S3. HBx deletion mutants differently affect AIM2 expression.

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Fig. S4. AIM2 has no impact on HCC cell proliferation.

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Fig. S5. AIM2 affects cell migration and invasion by targeting FN1.

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Table S1. Correlation of clinicopathological parameters and AIM2 expression.

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