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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The protumorigenic insulin-like growth factor (IGF)-II is highly expressed in a significant fraction of human hepatocellular carcinomas (HCC). However, a functional dissection that clarifies the contribution of IGF-II–binding receptors in tumor progression and a respective molecular characterization of IGF-II signaling has not been performed. Therefore, expression of IGF-II and its receptors IGF-receptor type I (IGF-IR) and insulin receptor (IR) was efficiently blocked using small interfering RNA (siRNA) in HCC cells. Despite functional IR-signaling, oncogenic IGF-II effects such as tumor cell viability, proliferation, and anti-apoptosis were solely transmitted by IGF-IR. Although IGF-II signaling was previously not described in the context of HCC cell migration, the IGF-II–dependent expression profile displayed a high percentage of genes involved in cell motility and adhesion. Indeed, IGF-II overexpression promoted HCC cell migration, especially in synergy with hepatocyte growth factor (HGF). The therapeutic relevance of IGF-II/IGF-IR signaling was tested in vitro and in a murine xenograft transplantation model using the IGF-IR inhibitor picropodophyllin (PPP). IGF-IR inhibition by small molecule treatment efficiently reduced IGF-II–dependent signaling and all protumorigenic properties of the IGF-II/IGF-IR pathway. Conclusion: In human HCC cells, IGF-IR but not IR is involved in oncogenic IGF-II signaling. Autocrine stimulation of IGF-II induces HCC motility by integration of paracrine signals for full malignant competence. Thus, activation of IGF-II/IGF-IR signaling is likely a progression switch selected by function that promotes tumor cell dissemination and aggressive tumor behavior. (HEPATOLOGY 2008.)

Insulin-like growth factor (IGF)-II is predominantly active in fetal liver under physiological conditions; however, its expression is strictly down-regulated shortly after birth. High-level expression of IGF-II is an important autocrine protumorigenic event in many human malignancies, such as pediatric tumors, breast and colon cancer, as well as hepatocellular carcinoma (HCC).1, 2 Indeed, IGF-II is elevated in approximately 40% of human HCCs,1 many HCC cell lines,3, 4 and different independent HCC animal models.5 Its overexpression is mainly regulated by complex transcriptional and posttranscriptional mechanisms involving promoter-specific genomic imprinting and regulation of protein bioavailability.6, 7 Binding of IGF-II to the tyrosine kinase IGF-I receptor (IGF-IR) as well as to the highly homologous insulin receptor isoform A (IR-A/INSR-A; lacking exon 11)8 has been linked to increased tumor cell mitosis and anti-apoptosis as well as improved angiogenesis. In addition, IGF and hepatocyte growth factor (HGF) signaling synergize in tumor progression, for example, in pancreatic carcinoma cells.9 Based on these results, IGF-II–induced signaling represents a challenging but promising therapeutic target structure in human hepatocarcinogenesis.

IGF signaling may be inhibited by different methodical approaches such as application of neutralizing antibodies against ligands and receptors, expression of dominant negative IGF-IR and IGF-binding proteins, gene-specific oligonucleotide-based inhibition of IGF-II and IGF-IR, as well as receptor tyrosine kinase (RTK) inhibitors. However, a comparative functional and molecular dissection of IGF-induced signaling constituents in human hepatocarcinogenesis has not been performed because of methodical limitations such as insufficient inhibitory efficiency of this maximally activated pathway or unspecific off-target effects. This is of special importance, because experimental strategies and specific therapeutic approaches rely on the comprehensive understanding of the signaling constituents to avoid unspecificity (for example, unwanted diabetogenic side effects after reduction of IR bioactivity) or insufficient specificity (for example, alternative protumorigenic signaling through IR-A).

Our analysis demonstrates that IGF-II/IGF-IR but not IGF-II/IR signaling regulated oncogenic properties in HCC cells. Integration of autocrine (IGF-II) and paracrine (HGF) derived signals determined a complete motile and therefore malignant phenotype in tumor cells. Indeed, an IGF-IR–specific RTK inhibitor reduced IGF-II–dependent tumor growth, clearly demonstrating the therapeutic relevance of this pathway in the treatment of human HCC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Cell Culture, Transfection, Reagents, and Sequences.

HCC cells were maintained as described.10 All RNA interference experiments were performed using two independent small interfering RNAs (siRNAs) in two HCC cell lines (HuH-7 and Hep3B); however, for clarity only one respective siRNA is shown. For optimized siRNA transfection, cells were seeded with fetal bovine serum 1 day before transfection. The cationic carrier (Oligofectamine; Invitrogen/Gibco, Karlsruhe, Germany) and siRNAs (final concentration, 5 nM) were diluted in OptiMEM (Invitrogen/Gibco), mixed, incubated at room temperature, distributed onto the cells covered with fresh medium, and transfected over night. Interferon independence after siRNA transfection was analyzed as published.11

For IGF-II stimulation and RTK-inhibitor treatment, cells were cultured under serum-free conditions for 12 hours. Media were exchanged 2 hours before stimulation to avoid accumulation and autocrine stimulation by IGF-II. For specific inhibition of IGF-IR signaling, picropodophyllin (PPP; Calbiochem/Merck Biosciences, Schwalbach/Ts., Germany) was applied in different concentrations (0.1, 1, 10 μM) for 1 hour. After stimulation with recombinant IGF-II (50 ng/mL; PAN, Aidenbach, Germany) for 10 minutes, protein fractions were collected. For functional analyses cells were cultured with fetal bovine serum and appropriate concentrations of PPP for 24 hours [fluorescence-activated cell sorting (FACS), proliferation, and migration assays] or 24, 48, and 72 hours (Methylthiazoletetrazolium [MTT] assay). Sequences of primers, probes, antibodies, and siRNAs used in this study are listed in Supplementary Table 1.

Real-time Polymerase Chain Reaction and Western Blot Analyses.

Sample preparations of total RNA for semiquantitative real-time polymerase chain reaction (PCR), protein isolations for western blot analyses, and densitometric quantification were performed as described.10 For protein enrichment and subsequent analysis of secreted IGF-II, cells were cultured under serum-free conditions without antibiotics overnight 3 days after siRNA transfection. After centrifugation (14,000g, 4°C, 5 minutes), the supernatant was mixed with 4 volumes of ice-cold acetone. After incubation at −20°C for 1 hour, the protein fraction was collected (2800g, 4°C, 30 minutes) and resuspended in protein lysis buffer.

Cell Viability, Proliferation, and Flow Cytometry.

Cell viability (MTT assay), proliferation (bromodeoxyuridine–enzyme-linked immunosorbent assay), and apoptosis (FACS) were analyzed as described.10 For cell cycle analyses of the murine tumor tissues, a Galaxy pro flow-cytometer (Partec, Münster, Germany) was used. For high-resolution flow cytometry, single cells were isolated from native sampled tissues using 2.1% citric acid/0.5% Tween 20 as published.10 Each histogram represents 30.000 to 100.000 cells analyzed for DNA index and cell cycle. Histogram analyses were performed with the Multicycle program (Phoenix Flow Systems, San Diego, CA).

To provide functional evidence for the specificity of siRNA-mediated knock-down of IGF-II, we performed a rescue experiment 2 days after siRNA treatment in 48-well dishes. Cell culture media of samples (siRNA-treated and controls) were replaced every 3 hours by the conditioned supernatant of HuH-7 cells, which secrete high amounts of IGF-II.4 After 60 hours (20 changes), tumor cell viability was measured.

Cell Motility Assays.

The scratch assay was performed as described, analyzing cell-free areas before and after HGF stimulation.10 For digital time-lapse microscopy, HCC cells were harvested 3 days after siRNA transfection and filtered through a sterile nylon mesh with a pore size of 40 μm (Becton Dickinson, Heidelberg, Germany). Gel preparation was performed as described with changes.12 Briefly, collagen gel (with or without PPP (2.5 μM)) was supported by laminin (20 μg/mL, BD Biosciences, Bedford, MA) and fibronectin (20 μ/mL, Sigma Aldrich, Deisenhofen, Germany). The number of migrating cells was measured and expressed as a percentage of the total cell fraction. Cell velocity of the 10 tumor cells showing the highest migratory activity was measured by “frame-to-frame” comparison using Capimage 8.02 software (Zeintl, Heidelberg, Germany).

Microarray Hybridizations and Data Analyses.

Microarray experiments were performed using Affymetrix Human Genome U133A 2.0 Arrays (Affymetrix, Santa Clara, CA) according to the standard protocol. Raw data were analyzed using the SAS software package Microarray Solution version 1.3 (SAS Institute, Cary, NC). Custom CDF, which contains an updated probe set definition, was applied to map the probes to genes.13 Analysis of differential gene expression was based on the log linear mixed model of perfect matches. A false discovery rate of α = 0.05 with Bonferroni correction for multiple testing was used to set the level of significance. The pathway analysis was performed by Fisher's exact test using SAS release 8.02. Microarray data are available through the NCBI Geo Database (record number: GSE8714). The data for IGF-II–regulated genes were filtered for genes whose expression significantly changed 1.2-fold in both and 1.5-fold in one array as compared with untransfected cells. Functional assignments of genes were performed using the GeneCards database (Weizmann Institute of Science, Rehovot, Israel) and PubMed literature search (NCBI, Bethesda, MD).

Murine Xenograft Transplantation Model.

In vivo experiments were performed according to the previously described protocol14 with minor changes. Huh-7 cells (2 × 106) in sterile phosphate-buffered saline were injected subcutaneously in the flanks of 8-week-old immunodeficient female nu/nu Balb/c mice. After 22 days, mice were treated twice daily with intraperitoneal injections of PPP (10 mg/kg/injection) in 10 μL dimethylsulfoxide:vegetable oil [10:1 (vol/vol)] for 5 days. Control animals were treated with the vehicle only. Tumor growth was documented daily using a vernier calliper. Solid tumors of PPP-treated and control mice were isolated, weighted, and processed for paraffin embedding and histological evaluation. Fresh-frozen tissue samples were processed for 3D-FACS analyses.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Protumorigenic IGF-II Signaling Is Transmitted Through IGF-IR But Not IR in HCC Cells.

It is unknown to which extent protumorigenic effects of IGF-II are transmitted through its known receptors IGF-IR and IR-A in HCC cells. Therefore, transient transfection of two independent gene-specific siRNAs for human IGF-II, IGF-IR, and IR were optimized for serum content of culture media, siRNA, and cationic carrier concentration in HuH-7 and Hep3B cells. For all target genes, a maximal inhibition efficiency of greater than 80% was detected at the transcript level (Fig. 1A), whereas no unspecific effects on minor IGF-I and insulin transcript levels in HCC cells were detected (data not shown). Densitometric signal quantification at the protein level showed reduction of greater than 92% for IGF-II, IGF-IR, and IR as compared with controls (Fig. 1B). Combined inhibition of IGF-IR and IR (both >90%) was achieved after equimolar transfection of both respective siRNAs. Unspecific interferon activation by siRNAs was excluded by the analysis of the phosphorylation status of STAT-1 as well as the expression of an interferon target gene (Supplementary Fig. 1).11

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Figure 1. Inhibition of IGF-II and IGF-IR reduces protumorigenic effects in HCC cells. Inhibitory efficiency of gene-specific siRNAs for IGF-II, IGF-IR, IR, and IGF-IR/IR after transient transfection in HCC cells (HuH-7) was determined by (A) semiquantitative real-time PCR and (B) western blot analyses in comparison with respective controls after 3 days. (C) Reconstitution/rescue experiment with conditioned IGF-II–containing media. Tumor cell viability was measured using an MTT assay. Biological effects of gene-specific inhibition on tumor cell viability (D; MTT assay), proliferation (E; bromodeoxyuridine–enzyme-linked immunosorbent assay), and apoptosis (F; FACS) was measured 5 days after siRNA transfection. (G) Phosphorylation/activation of IGF-IR and AKT after siRNA-mediated inhibition of IGF-II, IGF-IR, and IR. Data are presented as mean ± SD. *P < 0.05; **P < 0.01, ***P < 0.001; ns: not significant; #: control used for statistical calculation (Mann-Whitney U-test). Untreated cells were used for calibration.

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The functionality of siRNA-based inhibition was controlled by reconstitution experiments using conditioned cell culture media of HCC cells that secrete high amounts of IGF-II.4 Media replacement should rescue effects of IGF-II inhibition but fail to do so after receptor inhibition. As expected, a clear induction was observed by media replacement after IGF-II inhibition (with intact cellular signaling pathway), whereas no such effects were seen after IGF-IR or IGF-IR/IR inhibition (Fig. 1C). Only mild stimulatory effects were detected in nonsense siRNA-transfected or untreated cells. Surprisingly, no significant relative changes were observed in cells after IR inhibition compared to controls, suggesting IR independence after IGF-II stimulation. To check for intactness of IR signaling, we validated the expression of both IR isoforms as well as pathway activation after insulin stimulation (Supplementary Fig. 2).

Next we determined the functional consequences of siRNA-mediated inhibition of IGF-signaling components in HuH-7 cells. Tumor cell viability was significantly diminished 5 days after inhibition of IGF-II (−27%), IGF-IR (−35%), and combined treatment with IGF-IR and IR-specific siRNAs (−42%), respectively (Fig. 1D). Proliferation also declined in tumor cells after IGF-II (−26%), IGF-IR (−50%), and combined IGF-IR/IR inhibition (−47%; Fig. 1E). Equally, the apoptosis rate was increased in these cells as compared with controls (IGF-II: +67%, IGF-IR: +73%, IGF-IR/IR: +73%; Fig. 1F). Again, no significant biological effects were observed after IR inhibition alone or after combined receptor inhibition as compared with IGF-IR–specific inhibition alone. In addition, only IGF-II and IGF-IR reduction but not IR inhibition diminished phosphorylation of IGF-IR and AKT (Fig. 1G). All results were reproduced with an independent cell line (Hep3B: Supplementary Fig. 3).

In summary, highly efficient gene-specific inhibition of IGF-II and IGF-IR significantly impaired tumor cell biofunctionality based on decreased proliferation and increased apoptosis. IGF-II–induced effects were solely attributable to IGF-IR signaling, suggesting IR independence in HCC cells.

IGF-II–Induced Gene Regulation in HCC Cells.

To characterize the IGF-II–dependent pattern of gene expression attributable to maximal steady-state autocrine stimulation in hepatocarcinogenesis, we performed expression profiling of HCC cells (HuH-7) after siRNA-mediated IGF-II inhibition (Fig. 2A). We analyzed two siRNAs (each in duplicate) that efficiently reduced IGF-II expression (>80% inhibition on messenger RNA level). Overall, 61 genes were differentially regulated by both siRNAs according to our selection criteria [21 down-regulated (Supplementary Table 2), 40 up-regulated (Supplementary Table 3)]. Exemplarily, the expression of induced [for example, GLIPR1 (GLI pathogenesis-related 1), ANXA1 (annexin-1)] and reduced [e.g., S100P (migration inducing gene), bradykinin] genes was verified using real-time PCR (Fig. 2B and data not shown). IGF-II dependence of these genes was confirmed by continuous replacement of media (and therefore reduction of secreted IGF-II concentrations) for 12 hours, which equally leads to increased GLIPR1 and ANXA1 as well as reduced bradykinin expression as compared with cells with conditioned medium containing IGF-II (Fig. 2C).

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Figure 2. Identification and confirmation of IGF-II target genes. (A) Scheme illustrating the experimental setup for the identification of IGF-II target genes using oligonucleotide expression arrays after siRNA-mediated inhibition. (B) Confirmation of induced (for example, GLIPR1) and reduced (for example, S100P) genes after IGF-II inhibition by real-time PCR. Both siRNAs used for array experiments and PPP-treated cells (1 μM) were analyzed compared with respective controls (untreated and nonsense siRNA-transfected cells). (C) Independent confirmation of IGF-II–dependent gene expression (GLIPR1, ANXA1, and bradykinin) after continuous replacement of cell culture media for 12 hours (reduced IGF-II levels: IGF-II−) compared with HCC cells with conditioned media (autocrine IGF-II stimulation: IGF-II +). Untreated cells were used for calibration.

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After detailed annotation, 19 genes (31%) had previously been published in the context of hepatocarcinogenesis and 11 genes (18%) had already been shown to interact directly or indirectly with IGF signaling (Supplementary Tables 2, 3). In addition, nine genes (15%) were described to be involved in the regulation of mitosis, and five genes (8%) had previously been described in the context of apoptosis. Overall, 17 genes (28%) had been published in the context of cell adhesion and motility such as promotogenic (for example, S100P, galectin-3) and antimotogenic factors (for example, ANXA1, GLIPR1). This number was surprisingly high, given the fact that IGF-II has not been found to be a motogenic factor in HCC cells.

IGF-II Overexpression Stimulates HCC Cell Migration.

Induction of motogenesis would further support a functional relevance of IGF-II in HCC cell invasion and thus tumor progression. Based on the profiling data, we analyzed the impact of IGF-II signaling on HuH-7 cell invasion/velocity by highly sensitive time-lapse microscopy after siRNA-mediated inhibition of IGF-II and IGF-IR. Motility of HCC cells is generally low, but a fraction of motile cells (<10%) with significant velocity (approximately 6 μm/hour) was detected (Fig. 3A). The number of motile cells further declined after IGF-II (−44%) and IGF-IR (−89%) inhibition. Moreover, velocity was equally diminished after transfection of IGF-II (−26%) and IGF-IR (−18%) specific siRNA.

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Figure 3. IGF-II/IGF-IR signaling induces cell migration of HCC cells. (A) For time-lapse microscopy the number of motile HuH-7 cells (bars) and tumor cell velocity (open circles) was determined for 21 hours after siRNA transfection. (B) For scratch assays, tumor cell proliferation was blocked using mitomycin C, and migration was stimulated by motogenic HGF. Area closure in siRNA-treated cells and controls were digitally documented 18 hours after “wounding.” Representative “scratches” are shown. Bar: 100 μm. (C) Densitometric evaluation of phosphorylated/activated AKT after IGF-II, HGF, and IGF-II/HGF application for 5 minutes (10 ng/mL each). Signals were normalized for total AKT and actin expression. Data are presented as means ± SD. *P < 0.05; **P < 0.01, ***P < 0.001; ns: not significant; #: control utilized for statistical calculation (Mann-Whitney U-test). Untreated cells were used for calibration.

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Because a close cooperation between IGF/IGF-IR and HGF/c-MET signaling axes has been discussed9 and significant HGF concentrations are typically provided by the nontumorous (inflamed) liver, we tested whether both factors may cooperate as pro-motogenic cofactors in a two-dimensional scratch-migration assay (Fig. 3B). Application of HGF alone promoted liver tumor cell motility in this experimental setup.11 After “wounding,” and subsequent HGF stimulation, recovery of the scratched area was significantly lower in samples with diminished IGF-II (−53%) and IGF-IR (−77%) expression. Again, no changes in HCC cell motility were detected after IR-specific inhibition. Combined inhibition of IGF-IR and IR did not reduce migration (−68%) any further compared with IGF-IR inhibition. Thus, IGF-II and IGF-IR but not IR synergistically contribute to HCC tumor cell migration in concert with HGF/ MET signaling.

These data suggest that IGF-II/IGF-IR signaling alone provides a basal level of HCC cell motility; however, in combination with HGF it supports the complete motogenic properties of HCC cells. Indeed, only combinatory treatment of IGF-II and HGF leads to optimal phosphorylation of the downstream effector AKT (Fig. 3C). All results were reproduced with an independent HCC cell line (Hep3B: Supplementary Fig. 4).

Inhibition of IGF-(II) Signaling by a Selective Receptor-Tyrosine Kinase Inhibitor Reduces Motility and Tumor Growth.

Our findings suggest IGF-II/IGF-IR signaling as a potential target structure for inhibition of growth and dissemination of HCC cells. Because siRNA-mediated inhibition is not available for treatment of cancer, we analyzed small molecule–mediated IGF-IR inhibition. First, we stimulated different IGF-II–depleted HCC cell lines with IGF-II and tested a previously described IGF-IR–specific inhibitor (cyclolignan: picropodophyllin/PPP)14 for its ability to reduce IGF-II–mediated activation (Fig. 4A). IGF-II stimulation efficiently induced phosphorylation of AKT, whereas PPP application reduced its activation in a concentration-dependent manner. In addition, PPP treatment regulated the same target genes as observed after IGF-II inhibition in HCC cells, including GLIPR-1 and S100P (Fig. 2B). Thus, IGF-II–dependent activation of HCC cells can specifically be reduced by PPP.

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Figure 4. PPP reduces protumorigenic IGF-II effects in HCC cells. (A) HCC cell lines were treated with varying PPP concentrations (0.1, 1, 10 μM) under serum-free conditions for 1 hour. After IGF-II stimulation for 10 minutes, activation of the IGF-II/IGF-IR signaling pathway was determined using a phospho-specific antibody against AKT as compared with respective loading controls (total AKT and actin). (B) Relative viability (MTT-assay) of HCC cell lines was measured after application of PPP at different time points (24, 48, and 72 hours). Relative proliferation (C; BrdU-assay) and apoptotic index (D; FACS) of HCC cell lines were determined 24 hours after application of PPP. Results are presented as means ± SD. *P < 0.05; **P < 0.01, ***P < 0.001 (Mann-Whitney U-test). Untreated cells (C) were used for calibration.

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Whereas treatment of PPP in higher concentrations (1, 10 μM) resulted in a significant reduction of tumor cell viability after 48 to 72 hours, low concentrations (0.1 μM) induced minor effects in HuH-7 cells (Fig. 4B). As observed after siRNA-mediated inhibition of IGF-II and IGF-IR, a significant reduction of tumor cell proliferation (Fig. 4C) and induction of apoptosis (Fig. 4D) was detected 24 hours after application of PPP. Equally, in the presence of HGF, a significant inhibition of tumor cell migration (−75%) was obtained with increasing concentrations of PPP (Fig. 5). Basal tumor cell motility was completely lost after PPP application under HGF-free conditions (0% motility and 0% velocity; appropriate controls in Fig. 3A). Therefore, PPP efficiently blocks basal autocrine promigratory stimulation as well as co-motogenic effects of IGF-II in HCC cells without affecting other pathways. This lack of inhibitory effects on several RTKs has previously been demonstrated15; in addition, HGF-induced activation of MET in HCC cells was also unaffected by PPP (Supplementary Fig. 5).

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Figure 5. PPP reduces tumor cell motility in HCC cells. After inhibition of proliferation in HCC cells, migration was stimulated using the motogenic HGF. Cells were treated with PPP in varying concentrations (0.1, 1, 10 μM). Area closure was digitally documented 18 hours after “wounding.” Representative scratches are shown. Data are presented as means ± SD. ***P < 0.001 (Mann-Whitney U-test). Untreated cells were used for calibration. Bar: 100 μm.

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Because PPP reduced all progression-relevant cellular effects in HCC cells, we asked whether PPP reduces HCC growth in vivo using a heterotopic xenograft transplantation model. Tumor growth was significantly retarded in mice after PPP injection (Fig. 6A). Histological evaluation revealed significantly reduced areas of vital tumor mass in PPP-treated animals (35%) as compared with controls (69%, Fig. 6B). By 3D-FACS-analyses of all samples, two effects of PPP treatment were detected: the number of human aneuploid cells was reduced and tumor cell apoptosis and necrosis were increased (+61%; Fig. 6C). These data show that selective inhibition of IGF-IR by RTK-inhibitors reduces IGF-II–dependent HCC growth in vitro and in vivo.

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Figure 6. PPP reduces HCC tumor growth in immunodeficient mice. (A) After tumor establishment (day 22, arrow), mice (n = 3) were treated with PPP (10 mg/kg/12 hours) or the drug-free vehicle for 5 days (days 23-27). Tumor growth was measured daily. (B) The percentage of necrotic (arrows) and vital tumor tissue after PPP-free and drug-free solvent injection was analyzed by measurement of appropriate areas in all tumors. Bar: 50 μm (C) 3D-FACS-analyses discriminate murine (diploid) and human tumor (aneuploid) cell fraction allocated to DNA and protein content. The sub-G0/G1 population represents the apoptotic/necrotic fraction. Data are presented as means ± SD. *P < 0.05 (Mann-Whitney U-test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Excessive tumor cell–derived overexpression of IGF-II is a relevant protumorigenic mechanism in human hepatocarcinogenesis. IGF-II binds with lower affinity to its cognate receptor IGF-IR as compared with IGF-I; nevertheless, high-level expression of IGF-II in HCC cell lines efficiently stimulates tumor cells in an autocrine manner.4 Several experimental approaches for the functional characterization of IGF signaling in different cell types have been used; however, they have suffered from methodological limitations such as incomplete inhibition efficiency, toxicity, or IGF-independent off-target effects.4, 16, 17 Thus, for the functional dissection and molecular characterization of the IGF-II/IGF-IR/IR-signaling pathway, siRNA is currently the most specific, nontoxic, and effective method to inhibit expression in vitro.

IGF-II–induced receptor activation appears to be complex. In addition to IGF-IR, protumorigenic signaling through IR (especially IR-A) has been described in several human tumor cell types.18 Although only high-affinity binding to IGF-II (but not IGF-I) was shown for IR-A,15 biological effects due to IR-B (containing exon 11) in systems with high IGF-II levels cannot be excluded.19 Thus, the functional relevance of IGF-II/IR-signaling was tested in our HCC model systems by reducing both IR isoforms (IR-A: exon −11; IR-B: exon +11) using one siRNA. Surprisingly, inhibition of IR did not affect biofunctionality of IGF-II in HCC cells at all. These data strongly suggest IR-A/IR-B independence of all tested IGF-II–induced effects on HCC cells. This was further supported by IGF-II reconstitution/rescue experiments, which showed no differences in restoration of cell viability between IR inhibition and controls. In addition, only IGF-II and IGF-IR but not IR inhibition is associated with decreased phosphorylation of the tyrosine kinase SRC (data not shown), which typically initiates AKT phosphorylation and subsequent cell migration.20 However, what are the underlying mechanisms for IR independence? IR receptor isoform A and isoform B transcripts are present in both cell lines analyzed in this study. Additionally, insulin treatment of HuH-7 and Hep3B leads to the activation of this signaling axis. Therefore, IR pathway inactivation is unlikely, and other mechanisms might be responsible such as differential expression of receptor-interacting proteins in HCC cells as compared with other cell types. For instance, αVβ3-integrin, which physically associates with phosphorylated/activated IR, is expressed in low amounts in HCC cells.21, 22

Our results also suggest that at least the majority of IGF-IR–induced biological effects in HCC cells are due to IGF-II overexpression, because inhibition of IGF-II alone resulted in comparable extent of functional effects as observed after IGF-IR inhibition. Therefore, additional significant stimulation of the IGF-IR pathway by other protumorigenic factors such as IGF-I is unlikely in this experimental setup.

The role of IGF-II in HCC cell proliferation and anti-apoptosis is well established. Our data now demonstrate that inhibition of IGF-II and IGF-IR decreased HCC cell motility, pointing toward a relevant role of IGF-II in invasiveness and dissemination. This is in keeping with findings showing IGF-II–stimulated migration and invasion in breast, bladder, and ovarian tumor cells and melanoma cells.23, 24 However, IGF-II–induced motility in HCCs displays several peculiarities. Without further stimulation, the tumor cell motility is generally low; thus, IGF-II is unlikely to induce motility and the invasive switch of HCC cells alone. However, our data provide strong evidence for a synergy of IGF/IGF-IR and HGF/c-MET signaling in the regulation of motogenesis.9 Both pathways share molecular downstream effectors: IGF-IR and c-MET recruit in part identical adaptor proteins (for example, Gab-1) and activate the same cellular signaling pathways [for example, AKT and mitogen-activated protein kinase (MAPK) pathways]. Cooperation of both signaling axes in the efficient activation of the urokinase plasminogen activator/urokinase activator receptor (uPA/uPAR) pathway for migration and invasion of colon and pancreatic carcinoma cells has been discussed.9 C-MET is expressed on HCC cells; however, HGF is not secreted by tumor cells themselves but can be supplied by tumor-associated stromal cells and from the nontumerous liver. Because most HCC patients suffer from chronic liver disease, HGF concentration in sera and livers are highly elevated.25 Therefore, cooperative IGF-IR and c-MET signaling are likely to synergistically integrate tumor cell–derived autocrine and neighborhood-derived paracrine pathways for fully competent HCC cell invasiveness. This conclusion is further supported by the fact that high-level expression of IGF-II has been described to correlate with extrahepatic metastasis.26

The IGF-II/IGF-IR pathway represents a promising target structure because its signaling outcome is essential for proper embryogenesis but not for cellular functions in adults. Different approaches to the specific therapeutic modulation of this pathway have been developed, including expression of receptor antagonists, administration of neutralizing antibodies, or inhibition by oligonucleotides and siRNAs. All these strategies are difficult to deliver in humans and may face the challenge of extremely high IGF-II concentrations; however, small molecule components build up sufficient concentrations for the specific inhibition of RTKs in tumor patients. In vitro and in vivo data support the therapeutic potential of IGF-IR–specific inhibitors such as PPP in the treatment of human HCC.27, 28 PPP inhibits tumor growth in an IGF-IR–dependent manner without affecting other RTKs, such as fibroblast growth factor receptor, platelet-derived growth factor receptor, epidermal growth factor receptor, MET, or IR.14 Thus, the risk of unspecific cross-reactivity and especially diabetogenic side-effects is reduced in vivo. Indeed, glucose and albumin levels are unchanged in mice after PPP injection, suggesting that this inhibitor is well tolerated.29 In addition, only minor effects concerning the development of tumor cell resistance to PPP have been observed.30 All analyzed IGF-II–induced, tumor-relevant characteristics such as proliferation, anti-apoptosis, and cell motility were affected by PPP, suggesting its therapeutic potential in the treatment of progressed, invasive, and metastatic HCCs. Although unspecific side effects of small molecule components cannot be excluded completely,29, 31 our profiling data demonstrate good concordance between IGF-II inhibition and PPP treatment, suggesting that at least most of the biological anti-tumorous effects after PPP treatment are based on the inhibition IGF-II/IGF-IR signaling.

HCC tumor cell growth was reduced after 5 days of PPP injection, but no complete regression was observed in our xenograft experiments. This difference from previous data in other tumor models may be attributable to lower PPP concentrations in our experimental setup (10 mg/kg/12 hours) as compared with other experiments (20 mg/kg/12 hours),14, 29 which were chosen to reduce possible off-target effects. Previous studies have underlined the synergistic effects of IGF-IR inhibition in combination with the application of cytotoxic drugs and ionizing radiation.16 Therefore, additive or even synergistic anti-tumor effects after IGF-II/IGF-IR inhibition and treatment with chemical compounds are likely.

These and other data demonstrate that high-level expression of IGF-II in HCCs promotes the full program of malignant competence, including tumor growth, invasiveness, angiogenesis, and therapy resistance. IGF-II overexpression denominates a group of HCCs with fewer tumor-infiltrating lymphocytes and lower apoptosis rate.3 Its expression is highest in HCCs with poor differentiation, clearly demonstrating that high autocrine activation of IGF-IR by IGF-II represents a progression event in approximately 40% of human HCCs. Thus, there is strong evidence that IGF-II/IGF-IR signaling represents one of the decisive functionally selected progression switches in hepatocarcinogenesis.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supplementary material for this article can be found on the H EPATOLOGY Web site (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

FilenameFormatSizeDescription
hep22297-SupplementaryFig.1.eps3512K Supplementary Figure 1: Lack of an IFN-response after administration of IGF-II-, IGF-IR- and IR-specific siRNAs in HuH-7 cells.(A) RT-PCR analysis of oligoadenylate synthetase (OAS)-2 three days after transfection of different IGF-II specific siRNAs. 18S-rRNA served as a loading control. RNA (cDNA) isolated from a primary human liver tumor served as positive control (p.C.). Phosphorylation status of signal transducer and activator of transcription (STAT)-1 using western-blot analyses 3 days after IGF-II-specific siRNA transfection. Protein extract of IFN&&num;947;-treated HuH-7 cells served as positive control (p.C.; 600 U for 16 hrs). Actin was used as a loading control. (B) Phosphorylation status of STAT-1 by western-blot analyses 3 days after transfection of independent IGF-IR and IR-specific siRNAs. Protein extracts of IFN&&num;947;-treated HuH-7 cells served as positive control. Actin was used as a loading control. All experiments were reproduced with Hep3B showing equal results.
hep22297-SupplementaryFig.2.eps4024K Supplementary Figure 2: Intact IR-response in HCC cells.(A) The presence of both IR-isoforms (IR-A: lacking exon 11; IR-B with exon 11) in HuH-7 and Hep3B cells were determined using RT-PCR. Primers flanking exon 11 were used (expected size: IR-A: 68 bp; IR-B: 104 bp). (B) Phosphorylation of AKT was determined after treatment with insulin (100 nM) for 30 min in both HCC cell lines.
hep22297-SupplementaryFig.3.eps8228K Supplementary Figure 3: Key data showing that inhibition of IGF-II and IGF-IR reduces protumorigenic effects in Hep3B cells.Inhibitory efficiency of gene-specific siRNAs for IGF-II, IGF-IR, IR, and IGF-IR/IR after transient transfection in Hep3B cells was determined by (A) Western-Blot analyses in comparison to respective controls after 3 days. Tumor cell viability was measured using a MTT-assay. Biological effects of gene-specific inhibition on tumor cell viability (B; MTT-assay), proliferation (C; BrdU-ELISA), and apoptosis (D; FACS) was measured 5 days after siRNA transfection. (D) Phosphorylation/activation of IGF-IR and AKT after siRNA-mediated inhibition of IGF-II, IGF-IR, and IR. Data are presented as mean +/- SD. *P<0.05; **P<0.01, ***P<0.001; ns: not significant; #: control utilized for statistical calculation (Mann Whitney U-test). Untreated cells were used for calibration.
hep22297-SupplementaryFig.4.eps8364K Supplementary Figure 4: IGF-II/IGF-IR signaling induces cell migration of Hep3B cells.(A) For time-lapse microscopy the number of motile Hep3B cells (bars) and tumor cell velocity (open circles) was determined for 21 hrs after siRNA transfection. (B) Scratch assays: after HGF-stimulation area closure in siRNA-treated cells and controls were digitally documented 18 hrs after wounding. Representative scratches are shown. (C) Densitometric evaluation of phosphorylated/activated AKT after IGF-II, HGF, and IGF-II/HGF application in Hep3B cells for 5 minutes (10 ng/ml each). Signals were normalized for total AKT and actin expression. Data are presented as means +/- SD. *P<0.05; ns: not significant; #: control utilized for statistical calculation (Mann Whitney U-test). Untreated cells were used for calibration.
hep22297-SupplementaryFig.5.eps1072K Supplementary Figure 5: Effects of IGF-IR specific RTK-inhibitor PPP treatment on HGF-dependent MET phosphorylation.HGF treatment (20 ng/ml) of HCC cells induced phosphorylation of MET within 15 minutes. Pretreatment with PPP (0.1, 1, and 10 M) for 1 hr did not reduce HGF-dependent receptor activation. M marker lane.
hep22297-Supplementaryfigurelegends.rtf35K Supplementary Figure Legends
hep22297-SupplementaryTable1.doc389K Supplementary Table 1
hep22297-SupplementaryTable2.doc67K Supplementary Table 2
hep22297-SupplementaryTable3.doc99K Supplementary Table 3

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