Journal of Hepatology 44 (2006) 971–983 www.elsevier.com/locate/jhep
Aspartyl-(asparaginyl)-b-hydroxylase regulates hepatocellular carcinoma invasiveness Suzanne M. de la Monte1, Seishu Tamaki1, M. Chiara Cantarini2, Nedim Ince1, Marcus Wiedmann1, Jade J. Carter1, Stephanie A. Lahousse1, Sophia Califano1, Takashi Maeda1, Takato Ueno3, Antonia D’Errico4, Franco Trevisani2, Jack R. Wands1,* 1
Departments of Medicine and Pathology, Brown Medical School, Liver Research Center, Rhode Island Hospital, 55 Claverick Street, 4th Floor, Providence, RI 02903, USA 2 Department of Internal Medicine, Cardioangiology and Hepatology, University of Bologna, Bologna, Italy 3 Second Department of Medicine, Kurume University School of Medicine, Tokyo, Japan 4 Department of Oncology and Hematology, Pathology Unit, F. Addarii Institute of Oncology, University of Bologna, Bologna, Italy
Background/Aims: We measured aspartyl (asparaginyl)-b-hydroxylase (AAH) gene expression in human hepatocelluar carcinoma and surrounding uninvolved liver at both the mRNA and protein level and examined the regulation and function of this enzyme. Methods: Since growth of HCC is mediated by signaling through the insulin-receptor substrate, type 1 (IRS-1), we examined—if AAH is a downstream gene regulated by insulin and IGF-1 in HCC cells. In addition, IRS-1 regulation of AAH was examined in a transgenic (Tg) mouse model in which the human (h) IRS-1 gene was over-expressed in the liver, and an in vitro model in which a C-terminus truncated dominant-negative hIRS-1 cDNA (hIRS-DC) was overexpressed in FOCUS HCC cells. The direct effects of AAH on motility and invasiveness were examined in AAHtransfected HepG2 cells. Results: Insulin and IGF-1 stimulation increased AAH mRNA and protein expression and motility in FOCUS and Hep-G2 cells. These effects were mediated by signaling through the Erk MAPK and PI3 kinase-Akt pathways. Overexpression of hIRS-1 resulted in high levels of AAH in Tg mouse livers, while over-expression of hIRS-DC reduced AAH expression, motility, and invasiveness in FOCUS cells. Finally, over-expression of AAH significantly increased motility and invasiveness in HepG2 cells, whereas siRNA inhibition of AAH expression significantly reduced directional motility in FOCUS cells. Conclusions: The results suggest that enhanced AAH gene activity is a common feature of human HCC and growth factor signaling through IRS-1 regulates AAH expression and increases motility and invasion of HCC cells. Therefore, AAH may represent an important target for regulating tumor growth in vivo. q 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Hepatocellular carcinoma; Cell motility; Tumor cell invasion, Metastasis 1. Introduction Hepatocellular carcinomas (HCCs) are one of the most prevalent malignant neoplasms world-wide [1,2]. However, the molecular pathogenesis of these tumors is largely Received 30 March 2005; received in revised form 20 December 2005; accepted 25 January 2006; available online 10 March 2006 * Corresponding author. Tel.: C1 401 444 2795; fax: C1 401 444 2939. E-mail address:
[email protected] (J.R. Wands).
unknown. An approach to identify genes associated with transformation of hepatocytes to HCC was taken by generating several libraries of monoclonal antibodies to the FOCUS human HCC cell line and identifying overexpressed antigens that distinguish HCCs from normal hepatocytes [3,4]. Those studies led to the observation that HCCs exhibit over-expression of both the insulin receptor substrate-1 (IRS1) and aspartyl-asparaginyl-b-hydroxylase (AAH) [5–7]. IRS-1 is a w185 kD docking protein that transmits growth factor-stimulated mitogenic and metabolic signals
0168-8278/$32.00 q 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2006.01.038
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by interacting with downstream src-homology 2 domain (SH2)-containing molecules through specific tyrosyl phosphorylated motifs located within the C-terminus [8,9]. For example, the 897YVNI motif of IRS-1 binds to the growthfactor receptor-bound protein 2 (Grb2) adapter molecule, the 1180YIDL motif binds to Syp protein tyrosine phosphatase, and 613YMPM and 942YMKM motifs bind to the p85 subunit of phosphatidylinositol-3 (PI3) kinase [8,10–14]. Grb2 binds to PY-IRS-1 [14,15], leading to sequential activation of p21ras, mitogen-activated protein kinase kinase (MAPKK), and MAPK [15–18]. Erk MAPK activation contributes to growth factor-stimulated mitogenesis and gene expression [17,19]. The binding of PY-IRS-1 to p85 stimulates glucose uptake [20] and inhibits apoptosis by activating Akt/Protein kinase B (PKB) [21–23], and inhibiting glycogen synthase kinase 3b (GSK-3b) [24–26]. Experimental over-expression of IRS-1 causes transformation of NIH-3T3 cells manifested by increased proliferation, anchorage-independent growth, tumor formation in nude mice, and resistance to apoptosis [27]. In addition, overexpression of IRS-1 in transgenic (Tg) mouse livers results in increased cellular proliferation and constitutive activation of Erk-MAPK in hepatocytes [27,28]. Therefore, IRS-1 mediated signaling regulates hepatocyte growth, and constitutive activation of IRS-1 pathways contributes to the transformed phenotype. In the liver, insulin and IGF-1 stimulated signaling through IRS-1 regulates growth by activating the intrinsic receptor tyrosine kinases [29], which then phosphorylate IRS-1 [8,9]. Therefore, the increased proliferation of HCCs that over-express IRS-1 is likely mediated by increased responsiveness to insulin or IGF-1. The downstream genes involved in IRS-1-associated cellular transformation are largely unknown. However, we have obtained preliminary evidence that the human aspartyl (asparaginyl)-b-hydroxylase (AAH) gene [5], which was isolated from a FOCUS HCC library [7], can mediate IRS-1 transmitted signals. The w86 kD AAH transmembrane protein is encoded by a 2.2 kB mRNA transcript [5]. AAH is an a-ketoglutarate-dependent dioxygenase that hydroxylates Asparate and Asparagine residues in EGF-like domains [30,31] of proteins such as Notch and Notch homologs [32], which have known roles in cell migration [33–36]. AAH gene is over-expressed in infiltrating malignant neoplasms of liver, bile duct, mammary tissue, pancreas, nervous system, and colorectal origin [5,37–39], and abundantly expressed in trophoblastic cells, which are normally motile and invasive [5]. The high levels of AAH expression observed in infiltrating and metastasized malignant neoplastic cells and the abundant endogenous expression in trophoblastic cells led us to hypothesize that AAH may have a role in regulating invasive or metastatic spread of malignant neoplasms. In addition, since HCC growth is promoted by insulin or IGF-1 stimulation, and IRS-1 is over-expressed in a high percentage of HCCs, we postulated that AAH expression was also modulated by growth factors, and that AAH represents a downstream target of IRS-1-mediated signaling.
2. Materials and methods 2.1. In vitro models HepG2 and FOCUS human hepatocellular carcinoma (HCC) cells were studied because HepG2 cells were found to express relatively low levels of AAH, whereas FOCUS cells express relatively high levels of AAH [7]. Subconfluent cultures were serum-starved over night and then stimulated with insulin (20 nM), IGF-1 (25 nM), or epidermal growth factor (EGF; 10 ng/ml-negative control) for 24 h to examine the effects of growth factor stimulation on AAH expression and motility. AAH expression was measured using real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and motility or invasion was measured using established methods (see below). In addition, studies were performed to demonstrate that insulin or IGF-I stimulation activated Erk MAPK and Akt using Western blotting to detect increased levels of phospho-Erk and phospho-Akt relative to total Erk and Akt, respectively. To validate the effects of AAH over-expression on motility and invasiveness, HepG2 cells were transfected with the human AAH cDNA ligated into pcDNA3.1 (InVitrogen, Carlsbad, CA) in which expression was controlled by a CMV promoter [5,37]. Negative control cells were transfected with empty vector. Neomycin-resistant clones were used in the studies [40]. To further demonstrate the role of AAH in cell motility, AAH gene expression was silenced with small interfering (si) RNA molecules that targeted Exon 2 sequences of the AAH gene [ASPH NM_004318]. FOCUS cells were transfected with commercially generated Smartpool AAH siRNA molecules (Dharmacon, Inc., Chicago, IL) using the Amaxa electroporation apparatus (Amaxa, Inc., Gaithersburg, MD). As negative controls, FOCUS cells were transfected with scrambled RNA sequences, or they were subjected to the electroporation procedure with no siRNA molecules included in the reactions. AAH expression was examined by Western blotting, and directional motility was measured using the ATP luminescence based motility and invasion assay (see below) [41]. To determine if growth factor signaling through IRS molecules regulates AAH expression and motility, FOCUS cells were transfected with a dominant-negative mutated human (h) IRS-1 cDNA-pcDNA3.1 in which the nucleotides encoding the C-terminal functional domains were deleted (hIRS-1-DC) [42], rendering the molecule incapable of transmitting downstream signals through SH2 domain proteins [42]. Stable neomycinresistant clones (including cells transfected with non-recombinant plasmid) were analyzed for AAH, IRS-1, and hIRS1-DC expression by Western blot analysis or real-time RT-PCR. Directional motility and invasion assays (see below) were used to determine if intact signaling through IRS-1 was required to render HCC cells motile and invasive.
2.2. In vivo model to evaluate the potential link between IRS-1 and AAH expression Transgenic mice that over-express the hIRS-1 cDNA under the control of the mouse albumin promoter (Tg-hIRS-1) and non-transgenic (non-Tg) control littermates were used to assess the potential role of growth factor stimulated signaling through IRS-1 as a modulator of AAH expression in the liver. The Tg-hIRS-1 line was generated and maintained as described in a previous report [27]. The hepatic levels of AAH, IRS-1, and a-tubulin expression were measured by Western blot analysis with densitometry, and AAH mRNA levels were measured using real time quantitative RT-PCR. The liver proliferation indices were measured by bromodeoxyuridine (BrdU) and [43] thymidine incorporation assays [44]. Relative hepatic mass was determined from the ratio of liver weight to body weight for each animal.
2.3. mRNA studies mRNA levels were measured by real-time quantitative RT-PCR. Total RNA was isolated with TRIzol (Invitrogen, Carlsbad, CA) and 2 mg RNA samples were reverse transcribed using the AMV First Strand cDNA synthesis kit (Roche, Basel, Switzerland) and random oligodeoxynucleotide primers. PCR primer pairs were designed using MacVector 7.0 software (Accelrys, Inc., Oxford Molecular Ltd, Oxford, UK). PCR reactions contained 2.0 ng of RT product, 0.4 mM each of the forward and
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calculated for each assay, and results from identically treated cells were averaged.
Table 1 Real time PCR primer pairs Primer
Sequence
18S For. 18S Rev. AAH For. AAH Rev.
5 0 -GGACCAGAGCGAAAGCATTTGCC-3 0 5 0 -TCAATCTCGGGTGGCTGAACGC-3 0 5 0 -GGGAGATTTTATTTCCACCTGGG-3 0 5 0 -CCTTTGGCTTTATCCATCACTGC-3 0
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Amplicon size (bp) 50 256
For, forward; Rev, reverse; AAH, aspartyl (asparaginyl)-b-hydroxylase. reverse primers (Table 1), and SYBR Green I Master Mix (BioRad, Beverly, MA) in 25 ml volumes. The amplified signals were detected continuously with the BioRad iCycler iQ Multi-Color Real Time PCR Detection System (Hercules, CA). Cycle thresholds were determined using the iQ software. Relative mRNA abundance (nanogram) was calculated from the nanogram ratio of AAH mRNA to 18S rRNA. To generate PCR standards, the cDNA amplicons were cloned into the PCRII vector (Invitrogen, Carlsbad, CA) and sequenced. Serial dilutions of purified recombinant plasmid DNA were used in amplification reactions. The regression lines equations relating cycle threshold to nanogram of DNA were used to calculate transcript abundance. Relative transcript abundance was determined from the calculated nanogram ratios of specific mRNA to 18S [45,46].
2.4. Protein studies Western blot analysis was performed using clarified tissue homogenates prepared in radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris– HCl, pH 7.5, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 2 mM EGTA) containing protease (1 mM PMSF, 0.1 mM TPCK, 1 mg/ml aprotinin, 1 mg/ml pepstatin A, 0.5 mg/ml leupeptin, 1 mM NaF, 1 mM Na4P2O7) and phosphatase (2 mM Na3VO4) inhibitors. Protein concentrations were measured using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Sixty or 100 mg protein samples were analyzed by Western blotting [44], using the FB50 monoclonal antibody [5] to detect AAH, and polyclonal antibodies generated to the C-terminus or plextrin homology domains of IRS-I (Upstate Biotechnology, NY) to detect fulllength hIRS-1 or hIRS-1-DC, respectively. Immunoreactivity was revealed with horseradish peroxidase conjugated secondary antibody, SuperSignal (Pierce Chemical Company, Rockford, IL) enhanced chemiluminescence reagents. and the Kodak Digital Science Imaging Station (NEN Life Sciences, Boston, MA).
2.5. Motility and invasion assays Non-directional motility was measured using the phagokinetic track assay, which is based upon phagocytosis of gold particles in the paths of motile cells [47]. Phagocytosis of colloidal gold particles layered onto the coverglass results in plaque-like areas of clearance. Migration was allowed to proceed for 12 h at 37 8C, after which the cells fixed with 4% paraformaldehyde and stained with hematoxylin. Plaque size and density were determined by image analysis using the Kodak Digital Image System. Results are expressed as meanGSD of plaque area (mm2/h) [48]. Directional motility was measured using 8 mM pore Boyden chambertype culture inserts (Becton Dickinson, NJ, USA) seated in 24-well plates. One percent of FCS, 25 nM IGF-I, or 50 nM insulin was supplied as the trophic factor in the lower chamber. Migration was allowed to proceed for 1 or 2 h. Results were analyzed in one of two ways. After swabbing the upper surface to remove non-migrated cells, the membranes were stained with Crystal violet and the density of migrated adherent cells was determined. Alternatively, cell densities in the upper chamber (non-migrated), fixed to the undersurface of the membrane (migrated adherent), or distributed at the bottom of the chamber (migrated non-adherent) were determined using the ATP luminescence-based motility and invasion assay [41]. Invasion assays were identically constructed except the membranes were pre-coated with 0.5% Matrigel, and invasion was allowed to proceed for 12 h. After staining the membranes with Crystal violet, the number of invaded cells was enumerated by light microscopy. The percentages of motile cells were
2.6. Immunohistochemical staining studies Paraffin-embedded sections of liver (8 mM thick)—were stained with hematoxylin and eosin to delineate the regions that contained HCC or were tumor-free. Paraffin sections of liver containing HCC and adjacent tumorfree tissue within the same block were immunostained to detect AAH immunoreactivity using the 15C7 and FB50 monoclonal antibodies. Both antibodies were generated against recombinant human AAH protein. The FB50 antibody recognizes a region within the N-terminus of the protein (aa epitopte RRGQIKY), whereas 15C7 (aa epitope QNPVEQS) binds to the catalytic domain of AAH. As negative controls for the immunostaining reactions, either the primary antibody was omitted or the non-relevant monoclonal antibody to neurofilament was used in place of the relevant antibody. Immunoreactivity was detected with biotinylated secondary antibodies, avidin–biotin horseradish peroxidase complex (ABC) reagents, and diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA) [49]. The immunostained sections were lightly counterstained with hematoxylin and preserved with mounting medium and coverglass.
2.7. Source of human tissue Fresh frozen surgical biopsy (resection) specimens of HCC and tumorfree liver were obtained for diagnosis or treatment at the S. Orsola-Malpighi Hospital, Bologna, Italy. The diagnoses and staging of HCC were made using standard criteria. Human tissue samples obtained by surgical resection for diagnosis and treatment were collected and stored in the Pathology Unit at the F. Addarii Institute of Oncology of Bologna University, Italy. Paired fresh frozen samples of HCC and adjacent tumorfree tissue were stored at K80 8C and used for RNA and protein extraction. Formalin-fixed, paraffin-embedded tissue sections were used for immunohistochemical staining. The specimens were provided stripped of personal identifiers and use was approved by the Institutional IRB. Paired snap frozen tissue samples (w100 mg each) of HCC and adjacent HCC-free tissue were used to extract RNA for real time quantitative RT-PCR analysis of AAH gene expression. Formalin-fixed paraffin-embedded tissue blocks from the same cases were available for immunohistochemical staining. A total of eight paired samples were used in this study.
2.8. Source of reagents QuantiTect SYBR Green PCR Mix was obtained from (Qiagen, Inc., Valencia, CA). Antibodies to Erk (p44/42), phospho-Erk MAPK, Akt, phospho-Akt, a-tubulin, and a-actin were purchased from Cell Signaling (Beverly, MA). All other fine chemicals were purchased from either CalBiochem (Carlsbad, CA) or Sigma–Aldrich (St Louis, MO).
2.9. Statistical analysis The graphs depict meansGSD’s of results generated from individual experiments with 3–6 replicate assays, and all in vitro experimental results were reproduced at least three times. Between-group comparisons were made with Student’s t-tests.
3. Results 3.1. Growth factor modulation of AAH expression and motility in vitro Real time quantitative RT-PCR was used to measure AAH expression in different HCC cell lines. In all cases, the mean levels of AAH mRNA were significantly higher in the HCC cells compared with normal human adult liver tissue
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Fig. 1. AAH over-expression in hepatocellular carcinoma (HCC) cells. Total RNA was isolated from six different human HCC cell lines and normal human liver tissue obtained at biopsy. RNA was reverse transcribed using random oligodeoxynucleotide primers, and the cDNA templates were subjected to real time quantitative PCR to measure transcript abundance using the primers listed in Table 1. The levels of 18S ribosomal RNA were measured in the same samples in parallel reactions (see Table 1). The graphed data depict the meanGSD of results obtained from four replicate cultures. The results were analyzed statistically using ANOVA with the Fisher LSD post hoc significance test (*P!0.001 relative to all HCC cell lines).
obtained from biopsy (P!0.001) (Fig. 1). Therefore, AAH expression is up-regulated in HCC. Growth factor regulation of AAH expression was examined in FOCUS and HepG2 cells stimulated with insulin, IGF-1, EGF or vehicle for 24 h in serum-free medium. The real time quantitative RT-PCR studies demonstrated AAH mRNA transcripts in both unstimulated (control) and growth factor stimulated FOCUS and HepG2 cells. Real-time quantitative RT-PCR demonstrated higher levels of AAH with insulin or IGF-1 compared with EGF stimulation or vehicle treatment (P!0.005), and higher levels of insulin or IGF-1 stimulated AAH in FOCUS compared with HepG2 cells (P!0.01) (Fig. 2A). In contrast, EGF stimulation did not significantly increase AAH mRNA expression in either the FOCUS or the Hep-G2 cells relative to the corresponding vehicle-treated control cells, and consequently, the levels of AAH mRNA were significantly lower in EGF-stimulated compared with IGFI- or insulin-stimulated cells (Fig. 2A). Western blot analysis using the FB-50 antibody detected the expected w86 kD AAH protein in FOCUS and HepG2 cells (Fig. 2B). Relatively low levels of AAH protein were observed in vehicle-treated cells. IGF-I and insulin stimulation of FOCUS and HepG2 cells resulted in increased levels of AAH protein relative to the levels observed in corresponding vehicle-treated cells. In contrast to the results obtained by real-time RT-PCR, EGF stimulation resulted in modest increases in AAH expression relative to control (Fig. 2B). As a negative control, the blots were stripped and re-probed to detect a-actin. Those studies
demonstrated that a-actin immunoreactivity was not modulated by growth factor stimulation (Fig. 2B). The effects of insulin and IGF-1 stimulated AAH expression were next characterized in relation to directional cell motility using a luminescence-based assay. With vehicle supplied as the trophic factor, only 10–15% of the cells were directionally motile within the time period studied (data not shown). IGF-I and insulin stimulated directional motility above the background (vehicle-treated) levels in FOCUS and HepG2 (data not shown). Significantly higher total motility indices were measured in IGF-1 compared with insulin stimulated FOCUS (Fig. 2C) and HepG2 (data not shown) cells due to the higher percentages of motile adherent versus non-motile cells in the IGF-1stimulated cultures (Fig. 2C). In addition, corresponding with the higher levels of AAH protein expression, IGF-1 (Fig. 2D) or insulin-stimulated (data not shown) directional motility indices were significantly higher in FOCUS compared to HepG2 cells (P!0.001), due to the higher percentages of motile-adherent versus non-motile cells in the FOCUS cultures (P!0.01; Fig. 2D). The HepG2 cultures also had a slightly higher mean percentage of non-motile cells, but the difference relative to the FOCUS cells was not statistically significant. 3.2. Signaling mechanisms mediating insulin and IGF-1 stimulated AAH expression and motility Insulin and IGF-1 transmit signals downstream through IRS molecules, which interact with SH2-domain containing proteins leading to the activation of Erk MAPK and PI3 Kinase-Akt [50–52], both of which have known roles in cell motility [53–55]. Initial studies were used todemonstrate that insulin and IGF-I stimulation activated signaling through Erk MAPK and Akt in HCC cells. Subconfluent cultures of FOCUS and HepG2 cells were serum-starved over night, then stimulated for 0–60 min with 20 nM insulin or 25 nM IGF-I. The cells were harvested in RIPA buffer and analyzed by western blotting. The blots were first probed with antibodies to phospho-Erk MAPK or phosphoAkt, corresponding to the activated forms of these kinases. The blots were stripped and re-probed with antibodies to total Erk MAPK or total Akt. The Western blot signals were quantified by digital imaging using the Kodak Digital Science Imaging Station. The ratios of phospho-Erk or phospho-Akt/total Erk or Akt were calculated for each sample and representative results are depicted graphically (Fig. 3). The results demonstrated insulin- and IGF-Istimulated relative increases in Erk MAPK and Akt phosphorylation (Fig. 3). The peak period of Erk MAPK phosphorylation occurred 10–15 min after the addition of insulin or IGF-I to the cultures (Fig. 3A–D). Insulin and IGF-I stimulated Akt phosphorylation/activation was more abrupt and sustained compared with Erk-MAPK (Fig. 3E–H). In contrast, the levels of total Erk MAPK and total Akt did not vary substantially over the time period of
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Fig. 2. Insulin and IGF-1 stimulate AAH expression and motility. Subconfluent FOCUS and HepG2 cell cultures were serum-starved for 24 h, then stimulated with insulin (20 nM), IGF-1 (25 nM), or epidermal growth factor, EGF (25 ng/ml) for an additional 24 h. (A) AAH mRNA levels were measured using real time quantitative RT-PCR (see Section 2). AAH mRNA levels were normalized to 18S rRNA measured in the same samples. The graphs depict the meanGSD of results from six replicate assays (*P!0.001 relative to corresponding insulin or IGF-1-stimulated cells; DP!0.01 relative to corresponding FOCUS cells.). (B) AAH protein expression was examined in vehicle-treated control or growth factor stimulated FOCUS and HepG2 cells by Western blot analysis. The blot was stripped and re-probed with antibodies to b-actin. The positions of molecular weight standards are indicated at the left. (C) Directional motility was measured in FOCUS and HepG2 (data not shown) cells using the ATP luminescencebased motility/invasion assay with IGF-1 or insulin supplied as the trophic factor (see Section 2). The significantly higher motility indices (% total motile) measured in the IGF-1 relative to insulin-stimulated cultures were due lower percentages of motile-adherent cells (*P!0.001) relative to corresponding IGF-1 stimulated cells. Similar results were obtained with HepG2 cells (data not shown). (D) Comparison of IGF-1 stimulated directional motility in FOCUS and HepG2 cells. Directional motility was measured using the ATP luminescence-based motility/invasion assay with IGF-1 supplied as the trophic factor (see Section 2). The significantly higher mean motility index (% total motile) measured in FOCUS compared with HepG2 cells was due the higher mean percentage of motile-adherent versus non-motile cells in the FOCUS cultures (*P!0.001).
study (Fig. 3). These results demonstrate that both insulin and IGF-I effectively stimulate signaling through the Erk MAPK and Akt pathways. We next examined the effects of inhibiting insulin/IGF-I stimulated Erk MAPK and Akt on AAH expression and motility. The H-89 protein kinase A (PKA) inhibitor served as a negative control. After 24 h of serum starvation, FOCUS and HepG2 cells were treated with vehicle, or a chemical inhibitor of MAPKK (PD98059), PI3 kinase (LY294002), Akt, or PKA (H-89), and then stimulated with insulin or IGF-1 for 24 h (Table 2). Western blot analysis demonstrated dose-dependent inhibition of insulin or IGF-1 (data not shown) stimulated AAH expression in cells treated with PD98059 or LY294002 (Fig. 4A and B). Correspondingly, real-time RT-PCR demonstrated significantly reduced levels of AAH mRNA in cells treated with inhibitors of PD98059 or Akt inhibitor relative to vehicle or H-89-treated cells (Fig. 4C). In addition, treatment with PD98059 or Akt inhibitor significantly reduced IGF-1
stimulated directional motility (P!0.001) whereas H-89 had no significant effect on cell motility (Fig. 4D). 3.3. Functional link between IRS-1 and AAH expression in vivo Transgenic adult mice that over-express the human (h) IRS-1 in the liver (Tg-hIRS-1) have significantly increased (30%) mean hepatic mass/body weight ratio and higher hepatic proliferation indices as demonstrated with BrdU or [3H]-thymidine incorporation assays relative to non-Tg control littermates [28]. Western blot analysis demonstrated that over-expression of IRS-1 in the livers of Tg-hIRS-1 mice was associated with significantly increased levels of AAH protein (Fig. 5A and B) and mRNA (realtime quantitative RT-PCR; Fig. 5C) relative to non-Tg control littermates. Densitometric analysis of the autoradiographs revealed that AAH expression was increased by approximately 20-fold (P!0.001; Fig. 5B) due to over-expression
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Fig. 3. Insulin and IGF-I Stimulated Erk MAPK and Akt Phosphorylation in HCC Cells. Subconfluent FOCUS and HepG2 cell cultures were serumstarved for 24 h, then stimulated with insulin (20 nM) or IGF-1 (25 nM) for 0–60 min. Cell lysates were analyzed by Western blotting to detect changes in the relative levels of phospho (p) erk and pAkt in response to insulin or IGF-I stimulation. The blots were probed with phospho-specific antibodies that detect activated forms of Erk (A, C) or Akt (E, G), and then stripped and re-probed to detect total Erk or Akt. Immunoreactivity was detected with horseradish peroxidase conjugated secondary antibody, enhanced luminescence reagents, and digital imaging. Immunoreactivity was quantified using the Kodak Digital Science Imaging station (arbitrary densitometry units). The relative levels of phospho-Erk or phospho-Akt to total Erk or Akt were calculated (ratio of phospho/total) and the results are depicted graphically to the right of each panel of blots (B, D, F, H).
of IRS-1 in the liver. In contrast, the hepatic levels of a-tubulin were similar in the Tg-hIRS-1 and non-Tg groups (Fig. 5A). FOCUS cells have constitutively elevated levels of both IRS-1 (data not shown) and AAH expression
(Fig. 6A). In contrast, FOCUS cells that were stably transfected with hIRS1-DC, a dominant-negative mutant of the hIRS-1, had significantly reduced levels of AAH protein relative to mock transfected FOCUS cells (Fig. 6A). Western blot analysis confirmed the high levels of the
S.M. de la Monte et al. / Journal of Hepatology 44 (2006) 971–983 Table 2 Kinase inhibitors Inhibitor
Target
Concentration (mM)
PD98059 Akt inhibitor LY294002 H-89
Erk MAPK Akt PI3 Kinase PKA
20 8 10 2
Erk, extracellular signal-regulated kinase; MAPK, mitogen associated protein kinase; PKA, protein kinase A.
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truncated hIRS mutant protein in cells transfected with hIRS1-DC, and similar levels of full-length endogenously expressed IRS-1 and a-tubulin in all cells (Fig. 6A). The inhibitory effects of hIRS1-DC on cell migration were demonstrated using a directional motility assay with 1, 2, 3 or 5% fetal calf serum (FCS) supplied as the trophic factor. FCS was used in place of specific growth factors because cells transfected with hIRS1-DC were adversely affected by FCS withdrawal. With increasing concentrations of FCS, both the mock- and hIRS1-DC-transfected cells exhibited progressively higher motility indices (Fig. 6B). However, at
Fig. 4. Insulin-stimulated AAH expression and motility is mediated by signals transmitted through Erk MAPK and PI3 kinase-Akt. Subconfluent FOCUS and HepG2 cell cultures were serum-starved for 24 h, then stimulated with insulin (20 nM), IGF-1 (25 nM), or no growth factor (control) for an additional 24 h. Prior to adding the growth factors, the cells were treated with vehicle or a kinase inhibitor at the indicated concentrations (A, B) or as listed in Table 2 (C, D). The effect of inhibiting Erk MAPK with PD98059 (A) or PI3 kinase with LY294002 (B) on AAH protein expression was examined in HepG2 cells by Western blot analysis using the FB-50 monoclonal antibody. The Western blot signals were quantified by digital image densitometry and the results (meanGSD) from three experiments are depicted in the graphs below the autoradiographs (*P!0.001 relative to insulinstimulated cells; *P!0.01 relative to no inhibitor; DP!0.001 relative to insulin plus no inhibitor). Similar results were obtained using IGF-1 as the stimulus or with FOCUS cells in the experiments (data not shown). (C) The effect of inhibiting Erk MAPK or Akt on AAH mRNA expression was examined in FOCUS and HepG2 cells using real time quantitative RT-PCR (see Section 2). Cells treated with the H-89 inhibitor of PKA were used as a negative control. AAH mRNA levels were normalized to 18S rRNA measured in the same samples. The graphs depict the meanGSD of results from six replicate assays (*P!0.001 relative to corresponding insulin stimulated control cells). (D) The effect of inhibiting Erk MAPK or Akt on insulin and IGF-1 stimulated cell motility was examined in HepG2 cells using the ATP luminescence-based motility/invasion assay (see Section 2). Similar results were obtained using FOCUS cells, or with IGF-1 supplied as the trophic factor (data not shown). The graphs depict the meanGSD of results obtained from six replicate experiments (*P!0.001 relative to corresponding insulin stimulated control cultures). Statistical comparisons with untreated cultures were made using ANOVA with the Fisher LSD post-hoc test for significance.
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Fig. 5. Over-expression of IRS-1 in transgenic mouse livers results in increased AAH expression. Levels of IRS-1, AAH, and a-tubulin in livers of hIRS-1 Tg (NZ4) and non-Tg (NZ4) mice as measured by Western blot analysis. Proteins were extracted from liver tissue using RIPA buffer. Immunoreactivity was detected with polyclonal antibodies to IRS-1 or a-tubulin, or with monoclonal antibodies to murine AAH. Immunoreactivity was revealed with horseradish peroxidase conjugated secondary antibody, enhanced chemiluminescence reagents, and film autoradiography. Only a single immunoreactive protein was detected with each antibody. (A) Western immunoblots demonstrating expression of IRS-1 (w185 kD), murine AAH (w86 kD), and a-tubulin (w55 kD) in the same tissue homogenates. (B) Densitometric analysis of the above Western blot autoradiograph demonstrating the relative differences in the levels of AAH protein expression between hIRS-1 Tg and non-Tg mouse livers. (C) The levels of AAH mRNA were measured in 12 hIRS-1 Tg and nine non-Tg livers using real time quantitative RT-PCR with values normalized to 18S measured in the same samples (see Section 2). The graphed data reflect the meanGSD of AAH immunoreactivity (arbitrary units) or mRNA measured in each group. Intergroup comparisons were made using Student’s t-tests (*P!0.001).
each of the FCS concentrations tested, the hIRS1-DCtransfected cells had significantly lower motility indices relative to control cells (Fig. 6B). 3.4. Potential role of AAH over-expression in relation to motility and invasive growth of HCC cells HepG2 cells that were stably transfected with the AAH cDNA (pcDNA3-AAH) or empty vector (pcDNA3.1; mock transfected) were used to study the effects of AAH over-expression on motility and invasive growth. The pcDNA3-AAH transfected cells had significantly higher levels of AAH expression relative to the mock transfected cells, as demonstrated by Western blot analysis (Fig. 7A). Using the phagokinetic track assay, which
Fig. 6. Inhibition of AAH expression by over-expression of a dominantnegative mutant of IRS-1 that had the C-terminus deleted (IRS-DC). FOCUS cells were stably transfected with pcDNA3-IRS-DC or pcDNA3 (Mock; empty vector). Protein expression was detected by Western blot analysis of the cell lysates. (A) IRS-1 expression detected with antibodies generated to the plextrin homology domain located in the N-terminus of IRS-1 protein. The upper band (w185 kD) reflects endogenously expressed wildtype IRS-1 protein. The lower band (w90 kD) verifies specific expression of IRS-DC in the cells transfected with pcDNA3-IRS-DC. Analysis of the same lysates demonstrated sharply reduced levels of AAH expression in cells transfected with pcDNA3-IRS-DC relative to mock-transfected cells. The similar levels of a-tubulin in cells transfected with pcDNA3-IRS-DC or pcDNA3 reflects specificity of the affects shown above. (B) Effects of pcDNA3IRS-DC over-expression on directional motility of FOCUS cells. Directional motility was measured with Boyden chamber type cell culture inserts, and 1, 2, 3, or 5% FCS was supplied as the trophic factor in the well below. Migration was allowed to take place for 12 h, after which the cells remaining on the upper surface were removed with a cotton swab, and the cells that migrated to the undersurface were stained with Crystal violet, fixed, and counted. The graphed data represent the meanGSD of the number of cells that had migrated in six replicate cultures.
measures non-directional cell motility, mock transfected cells exhibited minimal motility as demonstrated by the relatively small areas of gold particle clearance. Overexpression of AAH increased HepG2 cell motility as demonstrated by the significantly larger areas of gold particle phagocytosis and clearance (Fig. 7B1, B2 and C) relative to control cells. In addition, HepG2 cells transfected with pcDNA3.1-hAAH had significantly higher invasive indices compared with mock-transfected (pcDNA3.1) cells (Fig. 7B3, B4 and D). These differences could not be attributed to AAH effects on DNA synthesis because proliferation of HepG2 cells requires 24–26 h of 5% FCS stimulation, and the motility and invasion assays were conducted within 12 h or less. FOCUS cells transfected with AAH siRNA had reduced levels of AAH expression relative to control, as demonstrated by Western blot analysis (Fig. 8A). In contrast, the expression levels of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were similar in the AAH siRNA transfected relative to control cells (Fig. 8B). The siRNA
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Fig. 7. AAH-over-expression increases the motility and invasive growth of HepG2 HCC cells. HepG2 HCC cells were stably transfected with the human (h) AAH cDNA ligated into the pcDNA3.1 vector in which gene expression was under the control of a CMV promoter. Control cells were transfected with non-recombinant pcDNA3.1 plasmid. Stably transfected cells were selected with G418. (A) Constitutive AAH over-expression (w86 kD band) was demonstrated by Western blot analysis of the cell lysates generated from two different clones. (B1, B2) AAH-associated increase in HCC cell motility demonstrated with the phagokinetic track assay. Cells were plated onto colloidal goldcoated surfaces. As the cells moved, the gold particles were phagocytized leaving a cleared (dark) area. The areas of clearance surrounding cell aggregates were measured by image analysis. The trophic factor was 1% FCS. Measurements were corrected for cell density (area cleared/cell). (C) The graph depicts the mean area of clearance/cellGSD (B3, B4) AAH over-expression increases HCC cell invasiveness. Invasion assays were constructed with Boyden chambertype inserts in which the membranes were coated with 0.5% Matrigel. 105 cells were seeded into the upper chamber in serum-free medium. Medium containing 1% FCS was supplied as the trophic factor in the well below. Invasive growth was allowed to proceed for 12 h. The nonmigrated cells were removed from the upper chamber with a cotton swab. The number of viable cells that migrated into the matrix or through the pores to the under surface or into the well below were stained with crystal violet and counted. (D) The graphed data reflect the mean number of invasive cells per culture (GSD). The between group differences were analyzed using Student’s t-tests (P!0.001).
silencing of AAH expression resulted in significantly reduced mean directional motility indices relative to cells transfected with scrambled RNA sequences or vehicle (Fig. 8C).
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Fig. 8. siRNA Silencing of AAH Inhibits Directional Motility in FOCUS Cells. FOCUS cells were transfected with siRNA targeting Exon 2 sequences of the AAH gene (si-AAH), or scrambled RNA duplex sequences (Scramb) using the Amaxa electroporation system. As an additional negative control, FOCUS cells were subjected to electroporation procedure without siRNA molecules included in the reaction (Control). Western blot analysis was performed using the (A) FB50 monoclonal antibody or (B) monoclonal antibodies to GAPDH as a negative control. (C) Directional motility was measured using the ATP luminescence-based motility/invasion assay with IGF-1 supplied as the trophic factor (see Section 2). Inter-group differences were analyzed statistically using ANOVA. Significant differences in mean motility index (% total motile) relative to the control cells are indicated by the P-values over the bars.
3.5. AAH over-expression demonstrated in human HCC tissue biopsy specimens Paired samples of HCC and adjacent HCC-free liver were used to measure AAH mRNA levels by real time quantitative RT-PCR. 18S ribosomal RNA levels measured in the same samples were used to calculate relative AAH mRNA abundance (see Section 2). Inter-group comparisons were made using paired t-tests. The results demonstrated higher levels of AAH mRNA in all 8 of the HCC relative to the adjacent tumor-free liver tissue (Fig. 9A). The levels of AAH mRNA in the HCC’s were generally at least 3-fold higher than in the adjacent HCC-free tissue. The calculated mean level of AAH mRNA was 7-fold higher in the HCC samples compared with the uninvolved liver (Fig. 9B; PZ0.00023). In contrast, 18S rRNA was similarly abundant in the HCC and adjacent tumor-free tissue (Fig. 9C), as was further demonstrated by the similar mean levels of 18S rRNA in the HCCs and corresponding adjacent tumor-free liver tissue (Fig. 8D). Immunohistochemical staining
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Fig. 9. AAH Over-Expression in Human Biopsy Samples of HCC. Paired samples of snap frozen human HCC and adjacent HCC-free tissue were analyzed to measure AAH mRNA levels using real time quantitative RT-PCR (see Section 2). The AAH mRNA levels were normalized to 18S rRNA measured in the same samples. (A) Relative AAH mRNA abundance demonstrated in eight cases of HCC and the paired adjacent HCC-free tissue samples. (B) Mean levels of AAH/18S in the HCC and adjacent HCC-free tissue samples. (C) 18S rRNA levels measured in the same 8 cases of HCC and paired adjacent HCC-free tissue samples. (D) Mean levels of 18S rRNA calculated for the HCC and adjacent HCC-free tissue samples. Statistical comparisons were made using paired t-tests. P-values are indicated above the bar.
studies demonstrated prominent AAH immunoreactivity in all 8 HCC, but not in the adjacent un-involved liver using the 15C7 (Fig. 10A and B) as well as the FB50 (Fig. 10C and D) monoclonal antibodies that were raised against the human recombinant protein [5]. In contrast, only very low levels or no immunoreactivity corresponding to AAH were detected in the HCC-free portions of the specimens.
4. Discussion AAH expression at both the mRNA and protein levels are strikingly increased in HCC compated to adjacent uninvolved liver and we explored the possible biologic consequence of this overexpression. It was determined that: [1] AAH expression and motility increase with insulin or IGF-1 stimulated signaling through IRS-1, and downstream through PI3 kinase-Akt or Erk MAPK; and [2] overexpression of AAH increases motility and invasiveness of HCC cells. AAH is over-expression in infiltrating and metastasized malignant neoplasms, including those of hepatic, biliary or pancreatic origin [5,37,56], and previous studies linked AAH over-expression to cellular transformation rather than proliferation per se [37]. These results, together with the finding that AAH is abundantly expressed
in trophoblastic cells [5], which are normally invasive, led us to hypothesize that AAH has a functional role in cell motility or invasiveness. In this regard, it is noteworthy that the consensus sequence for AAH b-hydroxylation is found in EGF-like domains of signaling molecules such as Notch [32], which have known roles in cell migration [36,57]. More important insulin or IGF-I stimulates AAH mRNA and protein expression, suggesting that the regulation may occur at the level of transcription although post transcriptional regulation undoubtedly occurs as well (unpublished observations). The finding that chemical inhibitors of either Akt or MAPKK significantly reduced IGF-1-stimulated AAH expression and motility suggests that IGF-1 mediates these effects by signaling through PI3 kinase/Akt and Erk MAPK, consistent with previous reports [58]. Erk MAPK signaling increases motility by activating Rac1 and RhoA GTPases, which promote membrane ruffling, actin–cytoskeleton reorganization, and formation of lamellopodia and filopodia [59]. Similarly, PI3 kinase-Akt regulates the assembly and re-organization of the actin cytoskeleton [60] and motility [59,61] by activating Rac/Cdc42 in response to growth factor stimulation [62]. The downstream effects of Rac1 on cell motility are mediated through Pak1 phosphorylation of LIM kinase [63,64], which phosphorylates targets such as cofilin [65,66], promotes actin
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Fig. 10. AAH Immunoreactivity Demonstrated in Human HCC Liver Biopsy Specimens. Formalin fixed, paraffin-embedded sections of HCC with adjacent HCC-free tissue were immunostained to detect AAH immunoreactivity using the 15C7 and FB50 monoclonal antibodies generated with recombinant AAH protein. Immunoreactivity was detected with biotinylated secondary antibody, avidin-biotin complex reagents, and diaminobenzidine (brown precipitate). Negative controls included sections immunostained with the primary antibody omitted or a non-relevant monoclonal antibody substituted for the 15C7 or FB50 antibodies. The sections were lightly counterstained with hematoxylin and preserved under coverglass. (A) HCC immunostained with the 15C7 antibody. (B) Adjacent HCC-free tissue in the same section as the tumor shown in Panel A. (C) HCC immunostained with the FB50 antibody. (D) Adjacent HCC-free tissue in the same section as the tumor shown in Panel B. Omission of the primary antibody or substitution of a non-relevant primary antibody did not produce specific immunostaining reactions (data not shown). (Original magnification, !320)
depolymerization, thereby allowing changes in cell shape and structure. Therefore, growth factor stimulated Rac1 function has a dynamic role in regulating cytoskeletal reorganization as is required for cell migration. Previous studies demonstrated that the livers of Tg-IRS1 mice exhibit constitutively increased levels of IRS-1 protein, PY-IRS-1, and Erk MAPK activity leading to enhanced hepatic growth [27,28]. To determine if AAH is regulated by growth factor signaling through IRS-1, AAH expression was examined the livers of adult transgenic mice in which hIRS-1 was over-expressed under the control of an albumin promoter [27,28]. We observed strikingly and selectively increased AAH expression in the hIRS-1 transgenic mouse livers relative to control littermate livers. Further studies demonstrated inhibition of AAH expression and motility in FOCUS cells that were stably transfected with a dominant negative hIRS-1 mutant (hIRS-1-DC) that inhibits interactions between IRS-1 and SH2-domain containing molecules required for downstream signaling through Erk MAPK and PI3 kinase-Akt [27]. Correspondingly, treatment of HCC cells with inhibitors of PI3 kinaseAkt or MAPKK also inhibited AAH expression and motility, emphasizing the importance of these signaling
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mechanisms as modulators of AAH gene expression and function in relation to motility. The finding of increased motility and invasiveness in HepG2 cells that were stably transfected with the AAH cDNA provides further evidence that AAH over-expression contributes to cellular transformation by enhancing cell motility and invasive growth. More important, the effects of AAH are direct since anti-sense constructs produced against the AUG start codon inhibits gene expression and tumor cell motitlity and migration [39], and correspondingly, FOCUS cells transfected with AAH siRNA had significantly lower mean directional motility indices relative to control cells. The mechanisms by which AAH over-expression promotes cell motility and invasiveness are not known. However, the effects may be mediated through AAH b-hydroxylation of EGF-like domains such as those present in Notch or Jagged, since mutation of the histidine residue required for enzymatic activity reverses the transformed phenotype induced by AAH expression including enhanced migration and invasion cells [37]. In order to access the biologic significance of a gene, it is essential to establish the relationship to the disease of interest. Evidence was presented that AAH is highly overexpress at the protein level by immunostaining and the gene is upregulated in HCC tumors compared to adjacent peritumoral regions by real time PCR. This transcriptional upregulation is controlled, in part, through a well known growth factor signal transduction cascade that has relevance to hepatic oncogenesis [42]. The function of this gene is to enhance tissue invasion and thus overexpression promotes a metastatic phenotype. Taken together, AAH appears to play a significant role in the pathogenesis of HCC and represents a novel biomarker for this devastating disease.
Acknowledgements Supported by Grants AA-02666, AA-02169, AA-11431, AA-12908, and CA-35711 from the National Institutes of Health.
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