Introduction

Hepatocellular carcinoma (HCC) is the most prevalent form of the primary liver cancers, which in turn are the fifth most common cancers worldwide—contributing substantially to cancer mortality (1). HCC develops largely within an established background of chronic liver diseases, caused mainly by infections with hepatitis B and/or hepatitis C virus or alcoholic liver disease (2), but the molecular pathogenesis is not well understood. Although new therapies for chronic hepatitis have been established, the number of HCC patients has not declined (1). Treatment success of HCC is dependent on the tumor stage at the time of diagnosis, with good curability at early stages and poor prognosis at advanced stages with tumor recurrence and metastasis after surgery or ablation therapy (2,3). Various factors are involved in the regulation of metastasis, and epithelial-mesenchymal transition (EMT) is characteristic for the most aggressive metastatic cancer cells (4,5). Therefore, biomarkers suitable for diagnostic purposes and new targets for developing therapeutic strategies are needed.

Human augmenter of liver regeneration (ALR) is a member of the newly discovered ALR/Erv1 protein family with FAD-linked sulfhydryl oxidase activity catalyzing disulfide bond formation (6,7). ALR is expressed ubiquitously with the highest levels found in testis and liver (8–10). Alternative splicing generates (11) a 21/23 kDa ALR isoform predominantly localized to the mitochondrial innermembrane system (IMS). In concert with the redox-regulated receptor Mia40/TOM, ALR forms a disulfide relay system mediating the import of proteins of mitochondria into IMS (12). Furthermore, CXXC motif of ALR is essential for cell survival (13) and biogenesis of cytosolic Fe/S proteins (14). The short form of ALR (15 kDa) is found not only at extracellular sites but also at nuclear and cytosolic localizations participating in intracellular redox-dependent signaling pathways (15,16). ALR is a hepatotrophic factor stimulating proliferation of hepatocytes (17,18) and augmenting liver regeneration (15,16), thereby exerting beneficial effects in models of hepatic failure (19) and liver fibrosis (20).

Furthermore, enhanced ALR mRNA and protein expression is found in the liver of patients with cirrhosis, cholangio-cellular and hepatocellular carcinoma. Here, ALR is localized predominantly in hepatocytes and bile duct cells (10). However, it is not clear yet whether the cytosolic isoform of ALR plays a role in the development of HCC. The aim of this study was to investigate the functional role and clinical importance of the short cytosolic ALR isoform in hepatocancerogenesis.

Materials and Methods

Liver Samples

Tissues of 64 patients undergoing liver surgery at the University Medical Center Regensburg were collected, including 53 patients with primary HCCs, three patients with liver cirrhosis and eight patients with colorectal liver metastases serving as healthy liver tissues. Tissue samples were fixed in formalin and paraffin-embedded for immunohistochemical analysis. Experimental procedures were performed according to the guidelines of the charitable state controlled foundation HTCR (Human Tissue and Cell Research), with the informed patients’ consent (21) approved by the local ethical committee of the University of Regensburg.

Immunohistochemical Analysis of ALR Expression

Immunostaining was performed as described recently (10). Briefly, 2-mm sections of paraffin-embedded tissue were used, and unspecific binding was blocked by 30 min preincubation in phosphate buffered saline (PBS) with 3% human serum (Bio-Rad, Munich, Germany), sections were subsequently incubated with anti-ALR antibody (0.26 µg/mL) at 4°C overnight followed by incubation with secondary mouse antirabbit antibody (DAKO, Hamburg, Germany) for 1 h. Immunostaining was developed using APAAP complex (DAKO) and Fast Red Chromogen staining (Roche, Penzberg, Germany). Counterstaining was performed with hematoxylin (Vektor, Burlingame, Canada). Rabbit IgG (Sigma, Munich, Germany) was used as isotype control. To evaluate ALR immunostaining, the percentage of positive cells and intensity of staining was analyzed independently by two pathologists. The following scores were defined: ≤10% positive cells = 0; 10% to 25% positive cells = 1, 25% to 50 % positive cells = 2; 50% to 75 % positive cells = 3; and ε 75% positive cells = 4. Signal intensity was defined as 0 when no staining was detected, as 1 when staining was weak, as 2 when staining was moderate and as 3 when staining was strong. The expression level of ALR was calculated by multiplying the scores: 0 = negative, 1–4 = positive, 6–12 = highly positive.

Cell Culture and Stable Transfected Cells

Primary human hepatocytes (PHH) were isolated and cultured as described (22). The human hepatoma cell line HepG2 (clone HB-8065) was obtained from American Type Culture Collection, (ATCC, Manassas, VA, USA) and grown at 37°C, 5% CO2 in DMEM (BioWhittaker, Verviers, Belgium) supplemented with penicillin (100 units/mL), streptomycin (10 µg/mL) and 10% fetal calf serum. The human ALR cDNA encoding the short isoform was digested with BamHI and EcoRI and sub-cloned into the expression vector pcDNA3.1 (Invitrogen, Karlsruhe, Germany). HepG2 cells were transfected using siPORT XP-1 (Ambion, Austin, TX, USA) regarding to the manufacturer’s instructions. HepG2 cells stably expressing ALR were selected by 1 mg/mL G-418 (GIBCO BRL, Karlsruhe, Germany) over a period of 2 wks. Cells stably transfected with pcDNA3.1 served as controls (mock).

Immunocytochemical Analysis

Cells grown on BD BioCoat Collagen I culture slides (BD Biosciences, Heidelberg, Germanty) were fixed and incubated with anti-ALR (1:100) (10), anti-CDH1 (1:100) (Cell Signaling, Danvers, MA, USA), or anti-ZO-1 (1:100) antibody (Invitrogen). Cryosections (2 µm) from tumors derived from HepG2-ALR and HepG2 cells were stained using anti-CDH1 (1:100) or anti-ZO1 (1:100) antibody. Fluorescent signals were visualized by an Alexa Fluor 488 dye-conjugated goat antirabbit (IgG) antibody (Invitrogen).

Migration and Invasion Assays

Briefly, 0.5 × 105 cells were resuspended in RPMI medium containing 1% FCS and seeded on inserts with 8-mm filter pores, which were either uncoated (migration assay) or matrigel-coated (invasion assay) (Becton Dickinson Bioscience, Heidelberg, Germany). 10% FCS served as chemoattractant. After 48 h, cells were fixed and stained (Diff-Quick reagent, Dade Behring, Newark, NJ, USA) and migrated and invaded cells were counted.

Scratch Assay ‘Wound-Healing Assay’

Migration of cells was assayed by scratch assays. Cells were seeded in high density into six-well plates and scratched by a pipette tip in a definite array. Migration into this array was documented and measured after 48 h. Each analysis was performed in triplicate.

Nude Mouse Tumor Model

A model of inoculation of tumor cells into NMRI (nu/nu) mice to monitor tumor growth in vivo was performed as described previously (23). Ten-wk-old male athymic nude mice NMRI (nu/nu) from the universities breeding stock were used (mean body weight was 30 g). HepG2-ALR cells or mock-transfected HepG2 cells (1 × 106 cells per mouse) were injected subcutaneously into nude mice, and six mice per clone were used. After 21 d, the formed tumors were removed, snap frozen and stored at −80°C for subsequent analysis.

RT-PCR Analysis

Total RNA was purified using RNeasy Mini Kit (Qiagen, Hilden, Germany). One µg of total RNA was reverse transcribed using the Reverse-Transcription System (Promega, Madison, WI, USA). Transcript levels of ALR, MMP1, MMP3 and 18S rRNA as internal control were quantified using real-time PCR technology (Light-Cycler, Roche, Penzberg, Germany). Primers used to amplify ALR were: forward 5′-CAC AAT GAA GTG AAC CGC AAG-3′, reverse 5′-CAC CCA ACT GAG ACA CAA CAG-3′, MMP1: forward 5′-TCA CCA AGG TCT CTG AGG GTC AAG C-3′, reverse 5′-GGA TGC CAT CAA TGT CAT CCT GAG C-3′, 18S rRNA: forward 5′-GTA ACC CGT TGA ACC CCA TT-3′, reverse 5′-CCA TCC AAT CGG TAG TAG CG-3′, MMP3 primers were obtained from TIB Molbiol (Berlin, Germany). The PCR reaction was evaluated by melting curve analysis. PCR was performed in duplicate and three independent experiments were performed.

Western Blot Analysis

Protein lysates (20 µg/lane) were separated by SDS-PAGE under reducing conditions using 100 mM DTT. Proteins were transferred to PVDF membranes and incubated with anti-CDH1 (1:1000), anti-ZO-1 (1:250), anti-SNAIL (1:250) (Santa Cruz Biotechnology, Heidelberg, Germany), anti-ALR (1:440) or anti-β-actin (1:1000) (Santa Cruz Biotechnology) antibodies. Positive signals were developed using enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA).

Measurement of MMP1 and MMP3 Activity In Vitro

HepG2 and HepG2-ALR cells were cultured for 48 h and supernatants were collected. MMP1 and MMP3 activities were measured using the SensoLyte 520 MMP-1 Assay Kit and the SensoLyte 520 MMP-3 Assay Kit, respectively, following manufacturer’s instructions (AnaSpec, Fremont, CA, USA). Briefly, MMP activity in culture supernatants was determined using the fluorogenic MMP specific substrate 5-FAM/QXL520 FRET peptide. Assays were performed in black 96-well plates with a clear flat bottom (Corning Inc, Corning, NY, USA) at 37°C. The increase in fluorescence resulting from enzyme mediated cleavage of the substrates was measured at 490/520 nm (excitation/emission)

Statistical Analysis

Significant differences in paired and nonpaired samples were evaluated by using Wilcoxon signed-rank t test and Mann-Whitney U test, respectively. Clinicopathological parameters were calculated by using Pearson chi-square test.

All supplementary materials are available online at https://doi.org/www.molmed.org .

Results

Reexpression of the Short Isoform of ALR in HepG2 Cells

To gain more insight into the functional role of ALR in HCC, the short isoform of ALR (15 kDa) was reexpressed stably in the hepatoma cell line HepG2 (HepG2-ALR). RT-PCR and Western blot analysis revealed strong induction of ALR mRNA and protein in HepG2-ALR cell clones compared with mock transfected and nontransfected HepG2 cells, respectively (Figure 1A, B). All three isoforms (23 kDa, 21 kDa, 15 kDa) were detected in normal liver tissue whereas the 15 kDa ALR isoform was barely detected in HepG2 cells (Figure 1B). Since HepG2-ALR2 cells showed a more intense 15 kDa ALR expression than HepG2-ALR1, this clone termed HepG2-ALR was used in further studies. Mock transfected HepG2 cells were used as control. Immunocyto-chemical analysis identified a strongly enhanced expression of cytosolic ALR in HepG2-ALR compared with HepG2 cells (Figure 2A i and v).

Figure 1
figure 1

Stable reexpression of short isoform of ALR in HCC cells. HepG2 cells stably expressing the 15 kDa ALR isoform (HepG2-ALR1 and HepG2-ALR2). (A) RT-PCR analysis revealed strong ALR mRNA expression in both ALR expressing HepG2 cells compared with nontransfected HepG2 or mock-transfected cells (three independent experiments, mean ± SD). (B) Western blot analysis demonstrated reexpression of the 15 kDa isoform ALR in HepG2-ALR cell clones. Normal liver tissue served as a positive control. Expression of β-actin was used as loading control.

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Figure 2
figure 2

Expression of ALR in HCC cells affects markers of EMT. (A) Immunofluorescence analysis revealed strongly enhanced ALR expression in HepG2-ALR (i) compared with HepG2 (v) cells. HepG2-ALR cells demonstrated reexpression of EMT markers E-cadherin (ii) and ZO-1 (iii), whereas HepG2 cells showed no immunosignal (vi, vii). PHH were used a positive control for E-cadherin (iv) and ZO-1 (viii) immunostaining. (i, v-vii, magnification 400x; ii-iv, viii, magnification 650x). (B) Western blot analysis confirmed expression of E-cadherin and ZO-1 in HepG2-ALR compared with HepG2 cells. Furthermore, expression of SNAIL was not detected in HepG2-ALR, but was found in HepG2 cells. (C) RT-PCR and protein activity analysis of matrix metalloproteinases in HepG2-ALR compared with HepG2 cells revealed reduction of MMP1 and MMP3 mRNA expression and activity, respectively (three independent experiments, mean ± SD, *P < 0.05).

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Reduced Migration and Invasiveness of HCC Cells after ALR Reexpression

Impact of ALR expression on cellular function was studied by analyzing cell motility. By performing Boyden chamber and matrigel invasion assays, a significant reduction in migration and invasion of HepG2-ALR cells compared with control cells was found (Figure 3A). In addition, scratch-assays (wound-healing assays) displayed a clearly diminished migration capacity of ALR reexpressing HCC cells compared with control cells (Figure 3B). Furthermore, cell adherence of HepG2-ALR after seeding was much faster compared with HepG2 cells (data not shown). However, proliferation of HepG2-ALR and HepG2 cells after 72 h of cultivation was similar (Supplementary Figure S1A).

Figure 3
figure 3

Expression of ALR attenuates HCC cell motility. (A) HepG2-ALR cells showed a strong reduction of their migratory and invasive potential compared with HepG2 cells. Assays were performed in a modified Boyden chamber for 48 h (three independent experiments, mean ± SD). (B) Scratch assays revealed a diminished “wound healing” of HepG2-ALR cells after 48 h of culture (one out of three experiments is shown, magnification 200x).

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Reexpression of ALR Affects EMT Markers

Alterations in cell motility often are accompanied by changes in the expression of specific markers of EMT. Therefore, we performed immunocytochemistry and demonstrated a reexpression of the epithelial cell marker proteins E-cadherin (Figure 2A ii and vi) and tight-junction protein ZO-1 (Figure 2A iii and vii) in HepG2-ALR compared with control cells. Western blot analysis confirmed reexpression of E-cadherin and ZO-1 in HepG2-ALR, which were not detected in HepG2 cells (Figure 2B). Furthermore SNAIL, a negative regulator of E-cadherin, was expressed in HepG2 cells but was absent or in low abundance in ALR reexpressing HepG2 cells (Figure 2B). In addition, matrix metalloproteinases MMP1 and MMP3 mRNA expression and protein activity were found to be reduced significantly in HepG2-ALR compared with control-transfected HepG2 cells (Figure 2C).

ALR Expression Attenuates Tumorigenicity of HepG2 Cells In Vivo

Growth behavior of HepG2-ALR and mock-transfected HepG2 cells was analyzed in nude mice after subcutaneous injection of HepG2-ALR and control cells. One wk after injection, all mice developed tumors. Tumor size after 3 wks was similar for tumors derived from HepG2-ALR and mock-transfected HCC cells (Supplementary Figure S1). Immuno-fluorescence and Western blot analysis of in vivo grown tumors confirmed reexpression of the epithelial cell marker proteins E-cadherin and ZO-1, as well as reduction of SNAIL expression in tumors originated from HepG2-ALR compared with mock-transfected HepG2 cells (Figure 4A i, ii, v, vi; Figure 4B). Furthermore, immunohistochemistry demonstrated that tumors which originated from HepG2-ALR cells displayed a more epithelial-like cell growth and showed less necrotic area (Figure 4A iii) compared with HepG2 derived tumors with signs of necrosis (Figure 4A vii). In addition fewer polymorphisms and atypical mitotic figures were observed in HepG2-ALR (Figure 4A iv) than in mock-transfected HepG2 cell derived tumors (Figure 4A viii). Based on these findings and in-line with the in vitro results, we assume that diminished expression of 15 kDa isoform of ALR in HCC cells supports tumorigenicity and metastatic potential of HCC cells.

Figure 4
figure 4

Tumorigenicity of ALR reexpressing HCC cells in vivo. (A) Subcutaneously grown tumors in NMRI (nu/nu) mice were analyzed 3 wks after injection of 1 x 106 HepG2-ALR (i-iv) or mock transfected HepG2 cells (v-viii). HE staining of tumors derived from HepG2-ALR cells revealed more epithelial-like cell growth (iii arrow, iv) compared with tumors derived from mock transfected HepG2 cells demonstrating signs of necrotic areas (vii arrow head), cell polymorphism and atypical mitotic figures (viii arrow). Performing immunofluorescence analysis we demonstrated reexpression of EMT markers E-cadherin (i, v) and ZO-1 (ii, vi) in tumors derived from HepG2-ALR cells. (iii, vii, magnification 400x; i, ii, iv-vi, viii, magnification 650x). (B) Western blot analysis confirmed reexpression of EMT markers E-cadherin and ZO-1 as well as reduction of SNAIL expression in tumors originated from HepG2-ALR cells (1 out of 3 experiments).

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ALR Expression Correlates Inversely with Angioinvasive Activity of HCC

We have shown recently that ALR mRNA expression is enhanced in HCC tissues (10) and, therefore, we analyzed ALR protein expression by immunohis-tochemistry in a total series of 53 tissues of HCC patients including 41 HCC samples where the corresponding nontumorous tissue was available. ALR immunoreactivity (low and high) was found in all HCC tissues (n = 53) and surrounding cirrhotic (n = 25) tissues, whereas in 37.5% (6/16) of the nondiseased tissue samples ALR expression was not detected. ALR was expressed significantly higher in HCC compared with surrounding cirrhotic (P < 0.001) and nondiseased (P < 0.001) liver tissues. Clinico-pathophysiological characteristics and immunohistochemical results of the HCC tumor cohort are summarized in Table 1. Positive ALR immunostaining was found to be associated significantly with higher tumor stage (P = 0.024). ALR expression correlated inversely with tumor grading (P = 0.037) and the rate of histological angioinvasion (P < 0.001). Western blot analysis revealed that the short isoform of ALR was present predominantly in samples without histological angioinvasion (Figure 5). ALR expression did not correlate with gender, age, tumor size, proliferation index or serum tumor markers (Table 1).

Figure 5
figure 5

Expression of ALR in tissues of patients with hepatocellular carcinoma stratified for histological angioinvasion. Western blot analysis of hepatocellular carcinoma tissue samples (homogenates 25 µg of total protein extract) were performed. Short isoform of ALR (15 kDa) was expressed predominantly in HCC without histological angioinvasion (T1–;T2) compared with samples with histological angioinvasion (T3–;T4). Normal liver tissue served as control and expression of heatshock protein 70 (HSP70) was used as loading control.

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Table 1 ALR immunoreactivity (IR) in HCC tissues of 53 patients in relation to clinico-pathological characteristics.a
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Discussion

The three different isoforms of ALR with a molecular weight of about 15, 21 and 23 kDa were found to be expressed ubiquitously with the highest levels detected in liver, brain and testes (9,10,24). ALR and its homolog Erv1 (essential for respiratory and vegetative growth) are essential for survival of hepatocytes (13) and S. cerevisiae (25), respectively. Knockdown of full length ALR expression in hepatocytes by transfection with antisense oligonucleotides led to mitochondrial depletion of ALR, reduced ATP levels and apoptotic cell death (13). Silencing of 23 kDa ALR had almost no effect on hepatocyte cell viability but significantly inhibited hepatoma cell growth and survival (26). The full length 23 kDa isoform of ALR has a putative N-terminal mitochondrial targeting sequence (25) and Erv1 also encodes a leader sequence for mitochondrial import (27). ALR (23 kDa) located in the IMS was shown to be involved in export of Fe/S clusters from the mitochondrial matrix, contributing to biogenesis of Fe/S proteins and cellular iron homeostasis (14,28). Furthermore, ALR/Erv1 play key roles in generating disulfide bonds in the IMS, thereby facilitating import of proteins through the TOM complex and by Mia40, the latter, in its reduced form, is recycled by ALR (12,28). The 15 kDa ALR lacking N-terminal regions is neither involved in mitochondria associated function of full length ALR nor found in the IMS (28). The short isoform of ALR, which is located in the cytosol and released to the extracellular space was shown to act as hepatotrophic growth factor by stimulating hepatocyte proliferation thereby promoting liver regeneration (15). Recently, we reported that ALR induced c-myc expression and activated polyamine metabolism with both being critically important in cellular growth (17). Furthermore this isoform modulated hepatic detoxification by cross-linking growth signals to the regulation of hepatic metabolism (18).

The proliferation augmenting effect of ALR was attributed to its ability to activate EGF receptor phosphorylation and the subsequent stimulation of the MAPK cascade (29). Furthermore, it has been elucidated that intracellular ALR may activate transcription factors via a MAPK independent pathway. ALR was shown to bind to JAB1 (c-Jun activating domain-binding protein 1), a coactivator of transcription factor AP-1, and thereby increased AP-1 activity (30,31). Additionally, ALR was found to interact with macrophage migration inhibitory factor (MIF) and inhibit its effect on AP-1. MIF reduced AP-1 activity by preventing JAB1 to bind to AP-1. On the other hand, the augmenting effect of ALR on AP-1 was inhibited by MIF, indicating a balanced regulation of JAB1-induced AP-1 activity by ALR and MIF (32). Interestingly, it was demonstrated that MIF is expressed highly in HCC cells and stimulates tumor cell migration but not proliferation (33). Therefore, modulated cell migration of hepatoma cells upon ALR expression might be explained by an interaction of ALR and MIF as well as by altered JAB1 binding to AP–1. AP–1 has an indispensable role in activating MMP3 expression in hepatoma cells (34) and, further, it was shown that MMP3 activates SNAIL expression and subsequently downregulates E-cadherin (35). Therefore, we assume that ALR regulates AP-1 activity, which results in reduced MMP3 and SNAIL levels resulting in enhanced E-cadherin expression (35). On the other hand, it was reported that induction of EMT associated with loss of cell polarity, and promotion of invasive growth by AP-1 is attributed rather to c-fos than to c-jun expression (36), and therefore, further studies have to evaluate the role of ALR in AP-1 mediated EMT inhibition.

MMPs and EMT related genes are regulated by several important signal-transduction pathways such as MAPK, PI3K, Rho-GTPase as well as Smad cascades (37) and we speculate that MAPK/Erk activation through ALR regulates cell migration of HepG2. Additionally, it was reported that tumor promoter TPA (12-tetradecanoylphorbol-13-acetate) can trigger EMT like cell scattering and migration of HepG2 cells by activation of Erk and reactive oxygen species (ROS) (38). Furthermore, recent findings implicate that the cellular redox state may be sensed by binding of ALR to thioredoxin (TRX) based on their ability to form disulfides, subsequently regulating AP-1 and NFκ-B activity (39). Most likely, ALR, by transferring oxidizing equivalents and forming disulfides bonds, either modulates TRX-mediated transcription factor activity or directly acts as an antioxidative agent. A protective and antiapoptotic effect of ALR was reported in neuroblastoma (40) and hepatoma cells (26,41) after hydrogen peroxide treatment (40,41) or irradiation-induced oxidative stress (26). This was mainly due to reduction of mitochondria-mediated apoptosis characterized by increased mitochondrial membrane potential, reduced cytochrome c release and enhanced ATP levels (26,40,41). Therefore, we hypothesize that ALR may act as an antioxidant ameliorating oxidative stress, thereby regulating cellular ROS levels which consequently modulate redox-sensitive signaling pathways and hepatoma cell motility.

ALR expression was found to be increased in liver regeneration, in cirrhotic livers and HCCs. Although reports on ALR mRNA expression in HCCs vary (10,26), in the current study we confirmed our previously reported observation (10) that ALR protein expression is enhanced in HCC in a larger cohort of patients. Furthermore, an inverse correlation of ALR with tumor grading and angioinvasion was identified. The methods used were not suitable to discriminate between different ALR isoforms. Nevertheless, current in vitro data provide evidence that the short isoform of ALR regulates cell motility, a finding in accordance with increased expression of ALR most likely including higher abundance of the 15 kDa isoform in HCC tissues without angioinvasion. Recently it was reported that ALR synthesis and post-translational modification transport into and out of mitochondria as well as extracellular secretion (13) is a dynamic process. Therefore, it is assumed that the metastatic potential of HCC cells with enhanced growth activity and increased levels of ALR depends on the ratio of mitochondrial- and cytosolic-localized ALR. Cytosolic ALR might not protect from oxidative stress only, but also prevent EMT and maintain epithelial cell phenotype under conditions of cancerous growth as was shown in the current study.

In this report, we demonstrate that expression of 15 kDa ALR in HCC cells attenuates cell motility, supports and maintains epithelial cell growth both in vitro and in vivo and, therefore, abates the process of EMT. In addition, we found that ALR expression is inversely correlated with tumor grading and tumor angioinvasion. Therefore, we provide evidence that 15 kDa ALR may be considered as an antimetastatic protein in HCC having the potential to become a marker in HCC diagnosis and a therapeutic target.

Disclosures

The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.