Integration of Ras subeffector signaling in TGF-ß mediated late stage hepatocarcinogenesis

Alexandra N.M. Fischer 1, Blanca Herrera 2, Mario Mikula 1, Verena Proell 1, Eva Fuchs 1, Josef Gotzmann 3, Rolf Schulte-Hermann 1, Hartmut Beug 4 and Wolfgang Mikulits 1, *

1 Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria, 2 Department of Biochemistry and Molecular Biology I, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, Avda/Complutense s/n, 28040 Madrid, Spain, 3 Max F.Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Dr Bohr-Gasse 9, A-1030 Vienna, Austria and 4 Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria

* To whom correspondence should be addressed. Tel: +43 1 4277 65250; Fax: +43 1 4277 65239; Email: wolfgang.mikulits{at}meduniwien.ac.at


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Immortalized p19ARF null hepatocytes (MIM) feature a high degree of functional differentiation and are susceptible to transforming growth factor (TGF)-ß driven growth arrest and apoptosis. In contrast, polarized MIM hepatocytes expressing hyperactive Ha-Ras continue proliferation in cooperation with TGF-ß, and adopt an invasive phenotype by executing an epithelial to mesenchymal transition (EMT). In this study, we analyzed the involvement of Ras subeffectors in TGF-ß mediated hepatocellular EMT by employing MIM hepatocytes, which express Ras mutants allowing selective activation of either mitogen-activated protein kinase (MAPK) signaling (V12-S35) or phosphoinositide 3-OH (PI3)3 kinase (PI3K) signaling (V12-C40). We found that MAPK signaling in MIM-S35 hepatocytes was necessary and sufficient to promote resistance to TGF-ß mediated inhibition of proliferation in vitro and in vivo. MIM-S35 hepatocytes showed also PI3K activation during EMT, however, MAPK signaling on its own protected hepatocytes from apoptosis. Yet, MIM-C40 hepatocytes failed to form tumors and required additional MAPK stimulation to overcome TGF-ß mediated growth arrest. In vivo, the collaboration of MAPK signaling and TGF-ß activity drastically accelerated the cell-cycle progression of the hepatocytes, leading to vast tumor formation. From these data we conclude that MAPK is crucial for the cooperation with TGF-ß to regulate the proliferation as well as the survival of hepatocytes during EMT, and causes the fatal increase in hepatocellular tumor progression.

Abbreviations: EMT, epithelial to mesenchymal transition; GFP, green fluorescent protein; GSK, glycogen synthase kinase; HCC, hepatocellular carcinoma; IGF-II, insulin-like growth factor II; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; PI3K, phosphoinositide 3-OH (PI3)3 kinase; TGF, transforming growth factor


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Hepatocellular carcinomas (HCCs) arise from hepatocytes undergoing malignant transformation and dedifferentiation in response to various stimuli. Studies on human HCCs revealed various molecular events which may play crucial roles in human hepatocarcinogenesis. The most frequently occurring molecular alterations in HCCs are the overexpression and secretion of cytokines such as transforming growth factor (TGF)-ß1 (13) and the insulin-like growth factor-II (IGF-II) (4,5), the loss of tumor suppressors such as E-cadherin, retinoblastoma protein, p16INK4a and p53 (68), and the induction of the Wnt/ß-catenin signaling pathway through stabilization of cytoplasmic and nuclear ß-catenin (9,10). Owing to the absence of suitable model systems and corresponding molecular analyses, the particular contributions of these and other mechanisms to liver tumorigenesis as well as the crosstalk between the different pathways are still poorly understood.

Members of the TGF-ß superfamily of cytokines are crucially involved in a wide range of cellular processes such as proliferation, differentiation and death of various cell types. TGF-ß transduces signals via serine/threonine type I and II receptors and Smad downstream signaling mediators, which then regulate the expression of target genes (11,12). Upon activation of TGF-ß signaling, hepatocytes undergo cell-cycle arrest and apoptosis which has been suggested to remove excessive tissue mass in vivo (1315). This led to the idea that TGF-ß might act in a tumor-suppressive fashion by negatively regulating proliferation and thus, the turnover of the liver parenchyma. On the other hand, TGF-ß has tumor promoting effects on malignant cells by enhancing the motility and the invasiveness, and also modulate the tumor stroma by suppresing the immune surveillance and increasing angiogenesis (1618). Interestingly, although TGF-ß is not detectable in the hepatocytes of healthy adult livers, human HCC cells show significant intracellular expression (1) and secretion of TGF-ß1 protein, indicating an autocrine stimulatory mechanism of TGF-ß signaling (19). Accordingly, the increased levels of TGF-ß1 in the urine and serum of HCC patients are associated with poor prognosis (2022). Hence, it is important to distinguish between the tumor suppressive function of TGF-ß in normal hepatocytes and during neoplastic initiation, besides its promoting role in tumor progression (23).

TGF-ß can cooperate with Erk/MAPK (mitogen-activated protein kinase), c-Jun N-terminal kinase (JNK), p38MAPK, RhoA and PI3K signaling pathways in a tissue specific fashion and this might be important in TGF-ß mediated tumor promotion (2426). One key regulator in growth factor signaling cascades is the Ras protein, controlling several aspects of cell growth and differentiation while contributing to malignant transformation after constitutive activation (2729). Ras integrates signals from many receptors and activates multiple downstream effectors, including Raf which activates the Erk/MAPK pathway, the lipid kinase phosphoinositide 3-OH (PI3)3 kinase (PI3K) which stimulates PKB/Akt kinase to provide signals for cell survival, and the Ral guanine nucleotide dissociation stimulators which contribute along with PKB/Akt to the inhibition of Forkhead transcription factors (30).

In several tumor models, the cooperation of oncogenic Ras and TGF-ß induces progression to undifferentiated and invasive tumors which display migratory and fibroblastoid phenotypes (3134). This process referred to as epithelial to mesenchymal transition (EMT) is highly conserved and fundamental for governing morphogenesis in multicellular organisms. The phenotypical transition involves the loss of epithelial markers such as components of cell-to-cell contacts, and de novo expression of mesenchymal markers. In vivo, EMT occurs during embryonic development, during transient pathological events such as wound healing, but also during late stage tumor progression leading to invasion and metastasis (3537).

Even though it is well known that the collaboration of active Ras and TGF-ß induces EMT, the signaling pathways by which Ras contributes to EMT are controversially discussed (3537). While on the one hand PI3K-Akt signaling was suggested to be required for TGF-ß induced EMT and cell migration in mammary epithelial cells (26), others reported the implication of Raf-MEK-Erk/MAPK in TGF-ß mediated EMT of pancreatic cancer cells (38) and primary pig thyrocytes (39). EMT was also shown to be induced by TGF-ß alone in breast epithelial cells (40), and in MDCK cells, EMT and autocrine TGF-ß secretion were seen in response to sustained Raf activation alone without any exogenous TGF-ß stimulation (41). Based on the fact that the impact of PI3K and Erk/MAPK could differ in a cell type-specific manner, it is interesting to study the implication of PI3K and Erk/MAPK in particular in hepatocellular EMT.

In this study, we analyzed the TGF-ß1 and oncogenic Ha-Ras induced EMT in immortalized p19ARF null hepatocytes, called MIM-1-4, and investigated the involvement of Ras subeffectors in proliferation and survival during TGF-ß mediated liver tumor progression. This was accomplished by employing cells, which express Ras mutants selectively activating either Erk/MAPK (V12-S35) or PI3K signaling (V12-C40) in response to cognate signals, and by using corresponding pharmacological inhibitors. The results clearly demonstrate that Erk/MAPK activity is necessary and sufficient to protect hepatocytes against TGF-ß mediated cell-cycle arrest and apoptosis. Furthermore, Erk/MAPK signaling alone is able to provide tumorigenic potential to immortalized hepatocytes, leading to EMT in cooperation with TGF-ß, thereby strongly enhancing tumor growth in vivo.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
Immortalized p19ARF–/– hepatocytes, called MIM-1–4, were grown in RPMI 1640, 10% fetal calf serum (FCS), 40 ng/ml recombinant human TGF-{alpha} (Sigma, St Louis, USA), 30 ng/ml recombinant human IGF-II (Sigma, St Louis, USA), 1.4 nM insulin (Sigma, St Louis, USA) and antibiotics as described previously (42). MIM-R hepatocytes were generated by stable retroviral transmission of MIM-1-4 with oncogenic v-Ha-Ras and green fluorescent protein (GFP) as outlined recently (34). MIM-C40 and MIM-S35 were retrovirally transmitted with S35-V12–Ras and C40-V12–Ras (43), respectively, as described recently (44). MIM-C40 cells were propagated in RPMI 1640, 10% FCS and growth factors as described for MIM-1-4, whereas MIM-R and MIM-S35 were cultured without additional growth factor supply. All cells were kept at 37°C and 5% CO2 and routinely screened for the absence of mycoplasma. In kinetic studies, recombinant human TGF-ß1 (R&D Systems, Minneapolis, MN, USA) was used at a concentration of 2.5 ng/ml for the first 72 h. For long-term treatment of cells, TGF-ß1 was supplemented at a concentration of 1 ng/ml. The low molecular weight compounds PD184.352 (45), a gift of Boehringer Ingelheim Pharma KG, Biberach/Riss, Germany) and LY294.002 (Alexis Corporation, San Diego, CA, USA) were added to the culture medium at concentrations of 5 and 20 µM, respectively.

Confocal immunofluorescence microscopy
Cells were fixed and permeabilized as described recently (34). Primary antibodies were used at the following dilutions: anti-E-cadherin (Transduction Laboratories, Lexington, UK), 1:50; anti-ß-catenin (Transduction Laboratories, Lexington, UK), 1:40; anti-ZO-1 (Zymed Laboratories, South San Francisco, USA), 1:50; anti-plakoglobin (Transduction Laboratories,Lexington, UK), 1:100; anti-Smad2/3 (Transduction Laboratories, Lexington, UK), 1:100; anti-Smad4 (Santa Cruz, California, USA), 1:100. After application of cye-dye conjugated secondary antibodies (Jackson Laboratories, West-Grove, USA), imaging of cells was performed with a TCS-SP confocal microscope (Leica, Heidelberg, Germany).

Western blot analysis
The preparation of cellular extracts, separation of proteins by SDS–PAGE and immunoblotting were carried out essentially as described recently (34). The protein extract from 1 x 105 cells per sample was loaded onto gels and immunological detection of proteins was performed with the SuperSignal detection system (Pierce Chemical Company, Rockford, USA). The following primary antibodies were used at dilutions: anti-E-cadherin (Transduction Laboratories, Lexington, UK), 1:3000; anti-ß-catenin (Transduction Laboratories, Lexington, UK), 1:1000; anti-ZO-1 (Zymed Laboratories, South San Francisco, USA), 1:1500; anti-plakoglobin (Transduction Laboratories, Lexington, UK), 1:1000; anti-tubulin ß (Sigma, St Louis, USA), 1:20 000; anti-phospho-Erk/MAPK (Cell Signaling, Beverly, USA), 1:1500; anti-Erk/MAPK (Cell Signaling, Beverly, USA), 1:1500; anti-phospho-Akt (Cell Signaling, Beverly, USA), 1:1000; anti-Akt (Transduction Laboratories, Lexington, UK), 1:2500; secondary antibodies (Calbiochem, LaJolla, USA) were used at dilutions of 1:10 000.

Transient transfections and reporter gene assays
The cells were plated at a density of 5 x 104 cells per 12-well plate 1 day before transfection. Lipofectamine Plus was used for transient transfections as recommended by the manufacturer (Invitrogen, Carlsbad, USA). Smad3-dependent transactivation was determined by cotransfection of 1 µg of (CAGA)12-Luc, containing concatemeric Smad3 consensus binding sites (46), and 0.25 µg of ß-galactosidase reporter (47) per well. After cell lysis, the luciferase activity was determined using a Luminoskan (Labsystems, Farnborough, UK) as described previously (44). All assays were performed in triplicate and results represent the averages of two independent experiments after normalization to ß-galactosidase activities.

Proliferation kinetics
A total of 1 x 105 cells were seeded in duplicate on Petri dishes with the indicated culture conditions. The number of cells in the corresponding cell populations was determined periodically in a multichannel cell analyzer (CASY; Schärfe Systems, Reutlingen, Germany). Cumulative cell numbers were generated from the absolute cell counts and their calculated dilution factors (34). The proliferation kinetics were performed in triplicate of which one representative is shown.

Thymidine incorporation assay
The cells were seeded in triplicates at a density of 1 x 104 cells per 96-well. To monitor DNA synthesis rates of cell populations, the cells were incubated with the respective standard medium and pulsed with 1 µCi [3H]thymidine (ICN, Irvine, USA) for 2 h. Subsequently, the cells were harvested on glass-fiber filters (Packard, Meriden, USA) and the radioactive labeling was determined in a microplate scintillation counter (Packard, Meriden, USA). All values depicted correspond to counts (c.p.m.) representing the mean of triplicate measurements (48). Normalization was done by crystal violet staining of adherent cells (0.2% in 2% ethanol). After 20 min, plates were rinsed with tap water, allowed to dry and overlaid with 1% SDS to solubilize the cells. The absorbance was read photometrically at 560 nm and calculated as percent absorbance with respect to untreated control cells (49). Experiments were performed in triplicate and results represent the averages of two independent experiments.

Analysis of caspase 3 activity
Cells were scraped on ice in phosphate-buffered saline (PBS) and lysed in 5 mM Tris–HCl pH 8.0, 20 mM EDTA and 0.5% Triton X-100 at 4°C. After centrifugation at 13 000 g for 10 min, protein concentration of cell lysates was determined by using the Bio Rad protein assay kit (Bio Rad Laboratories, Richmond, CA). An aliquot 30 µg of protein per condition was mixed with 20 mmol/l HEPES pH 7.0, 10% glycerol, 2 mmol/l dithiotreitol and 20 mol/l Ac-DEVD-AMC substrate. After 2 h of incubation in the dark, enzymatic activity was measured in a Microplate Fluorescence Reader FL600 (Bio-Tek, Winooski, VT). A unit of caspase activity is defined as the amount of active enzyme necessary to produce an increase in one arbitrary unit in the luminescence spectrophotometer after 2 h. Final results are presented as units of caspase activity per microgram protein, as previously described (49,50). Experiments were performed in triplicate and results represent the averages of two independent experiments.

Analysis of cell viability
Cell death was analyzed by measuring lactate dehydrogenase (LDH) release using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, USA), according to the manufacturer's recommendations. Values of LDH release were normalized to the number of adherent cells per well, measured by crystal violet staining (as described for the thymidine incorporation assay). Experiments were performed in triplicate and results represent the averages of two independent experiments.

Tumor formation in vivo
Cells of the desired cell type were detached from the tissue culture plate, washed with PBS, and resuspended in Ringer's solution. Subsequently, 1 x 106 cells in 100 µl Ringer's solution were subcutaneously injected into immunodeficient SCID/BALB/c recipient mice. Tumor formation was measured periodically by palpation, and the tumor size was determined using a Vernier caliper. Tumor volume was calculated from tumor size using the formula (diameter x diameter x length)/2, and the tumor weight in milligrams was determined as described recently (34). All experiments were performed in triplicate and carried out according to the Austrian guidelines for animal care and protection.

Immunohistochemistry and TUNEL analysis of experimental tumors
SCID mice were killed and the tumors were removed and fixed in 4% phosphate-buffered formaldehyde overnight at 4°C (48). For immunohistochemical analysis, paraffin-embedded sections (4 µM) were treated with citric acid buffer (0.01 M, pH 6.0) before staining with a monoclonal antibody against proliferating cell nuclear antigen (PCNA) (1:100; Dako, Carpinteria, USA). To detect DNA fragmentation in tumor tissues, an in situ cell death detection kit (Roche, Mannheim, Germany) was employed. After incubation of deparaffinized sections with proteinase K (20 µg/ml) for 15 min at 37°C in the presence of fluorescein-labeled dUTP and counter-staining with propidium iodide (1 µg/ml), conventional fluorescence microscopy (Nikon Corporation, Tokyo, Japan) was performed.


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 Abstract
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 Materials and methods
 Results
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 References
 
Immortalized MIM hepatocytes undergo EMT by the cooperation of TGF-ß1 and hyperactive Ha-Ras
We recently demonstrated that the cooperation of TGF-ß1 and hyperactive Ha-Ras causes the conversion of epithelial murine hepatocytes (MMH) to a fibroblastoid and a migratory phenotype (34). This process, referred to as hepatocellular EMT, results in increased malignancy accompanied by invasiveness and metastasis of transformed hepatocytes, and is considered as a crucial event in late stage liver tumorigenesis (36). In this study, we employed immortalized p19ARF null hepatocytes, called MIM-1-4, which express liver-specific markers, show a non-tumorigenic phenotype and are sensitive to Fas-mediated and staurosporine-mediated apoptosis, as recently reported (42). In order to analyze EMT, MIM-1-4 hepatocytes were retrovirally transmitted with a construct bicistronically expressing constitutively active Ha-Ras and GFP, resulting in MIM-R cells (Figure 1A). Comparable to parental MIM-1-4, MIM-R hepatocytes displayed a polarized epithelial morphology and grew in a strictly contact inhibited fashion. Upon TGF-ß1 treatment, MIM-R cells synchronously adopted a fibroblastoid phenotype after 48 h, and generated polylayers without contact inhibition (data not shown). MIM-R hepatocytes treated for >14 days with TGF-ß1, designated MIM-RT, maintained this mesenchymal phenotype (Figure 1A). The phenotypical transition was associated with the disruption of intercellular contacts as detected by confocal immunofluorescence microscopy. Epithelial MIM-R cells revealed localization of the adherence junction components E-cadherin and ß-catenin at cell borders, whereas a cytoplasmic redistribution was found in fibroblastoid MIM-RT cells (Figure 1B). The tight junction component ZO-1 and the desmosome constituent plakoglobin were also found at the cell boundaries of MIM-R, whereas MIM-RT showed a cytoplasmic staining. Apart from ß-catenin, the protein levels of E-cadherin, ZO-1 and plakoglobin were significantly reduced after long-term treatment with TGF-ß1 (Figure 1C). From these data we concluded that the cooperation of oncogenic Ras and TGF-ß is also operative to cause EMT in p19ARF null MIM hepatocytes, which indicates independence of hepatocellular EMT on the genetic background.



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Fig. 1. TGF-ß1 triggers an EMT of oncogenic Ha-Ras expressing MIM-R cells. (A) Phase contrast microscopy of parental MIM-1-4 hepatocytes, MIM-R cells and MIM-R treated for >14 days with TGF-ß1, termed MIM-RT. (B) Confocal immunofluorescence images of MIM-R cells showing localization of E-cadherin, ß-catenin, ZO-1 and plakoglobin at cell-to-cell contacts. Fibroblastoid MIM-RT cells (right panel) exhibit cytoplasmic redistribution of these epithelial markers. (C) Corresponding protein levels in parental MIM-1-4, malignant MIM-R and MIM-RT as indicated by immunoblotting. Tubulin ß is shown as loading control. (D) Transactivation of Smad3 in MIM hepatocytes treated with 2.5 ng/ml TGF-ß1, as analyzed by reporter gene assay: gray bars, MIM-1-4; black bars, MIM-R. The data are representative of at least two independent experiments. The values depicted in (D) represent the mean of triplicate samples of two independent experiments. Error bars denote SD.

 
Oncogenic Ras has been reported to inhibit TGF-ß induced nuclear translocation of Smad2/3 and Smad-dependent transcription due to their aberrant phosphorylation by Erk/MAPK (51). It is noteworthy that no inhibition of Smad-dependent transactivation by oncogenic Ha-Ras was detected in MIM hepatocytes (Figure 1D). Moreover, immunofluorescence microscopy revealed nuclear localization of Smad2/3 as well as Smad4 in MIM-R cells immediately after TGF-ß treatment (20 min) and in long-term TGF-ß treated fibroblastoid MIM-RT (data not shown). Thus, hepatocellular EMT results from the functional collaboration of Ras and TGF-ß signaling rather than from Ras-mediated inhibition of the TGF-ß pathway.

Hepatocellular EMT shows resistance against TGF-ß1 induced growth arrest and apoptosis
In the liver, TGF-ß1 is considered as the critical cytokine which inhibits hepatocyte DNA synthesis and induces apoptosis (23). As expected, parental MIM-1-4 hepatocytes treated for 14 days with 2.5 ng/ml TGF-ß1 in culture showed a significantly reduced cellular turnover after 48 h followed by a continuous loss in cell number due to cell death (Figure 2A). In contrast, MIM-R cells exposed to TGF-ß1 continued proliferation, showing only a slight growth depression between days 5 and 7. Long-term treatment with TGF-ß1 (>7 days) resulted in comparable proliferation kinetics as monitored with untreated MIM-R hepatocytes. Notably, comparable data of proliferation kinetics were obtained after stimulation of cells with TGF-ß2 and TGF-ß3 isoforms (2.5 ng/ml), i.e. loss of MIM-1-4 cells and unperturbed cell-cycle progression of MIM-R hepatocytes (data not shown).



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Fig. 2. MIM-1-4 hepatocytes expressing active Ha-Ras (MIM-R) are protected against TGF-ß induced growth arrest and apoptosis. (A) Proliferation kinetics of untreated parental MIM-1-4 (gray circles) and Ha-Ras expressing MIM-R cells (black circles) compared with those of TGF-ß treated cells (gray and black triangles). A starting population of 1 x 105 cells was plated to monitor cumulative cell numbers over 14 days. The number of cells was determined every 2–3 days using a multichannel cell analyzer (CASY, Schärfe Systems, Germany). (B) [3H]Thymidine (TdR) incorporation (c.p.m.) normalized to crystal violet staining, (C) Caspase 3 activity standardized to protein content and (D) LDH release normalized to crystal violet staining of cells treated with 2.5 ng/ml TGF-ß1 for 24, 48 and 72 h: gray bars, MIM-1-4; black bars, MIM-R. In (B), the TdR incorporation at each time point after TGF-ß treatment is expressed in percent compared to untreated control cells. The values represent mean of triplicate samples of two independent experiments. Error bars denote SD.

 
In order to discriminate between the contribution of TGF-ß mediated cell-cycle defects and cell death events, metabolic thymidine DNA labeling and enzymatic activity assays determining cell death were performed. After 72 h of TGF-ß treatment, thymidine incorporation rates of MIM-R hepatocytes were 14-fold higher than those of parental MIM-1-4 hepatocytes (Figure 2B), indicating that hyperactive Ha-Ras is operative to overcome TGF-ß induced cell-cycle arrest at the early onset of EMT. Interestingly, both parental cells and MIM-R hepatocytes showed caspase 3 activation with similar kinetics and a peak after 48 h of TGF-ß stimulation (Figure 2C). However, MIM-R cells showed lower basal and TGF-ß induced levels of caspase 3 activity. Cell death was significantly increased only in parental MIM-1-4 cells, which displayed a 2-fold higher LDH release after 48 h and 5-fold higher LDH release after 72 h of TGF-ß treatment compared with MIM-R cells (Figure 2D). In contrast, LDH release in MIM-R transiently elevated after 24 h of TGF-ß treatment but decreased to basal levels after 72 h, suggesting that active Ras provides resistance against TGF-ß induced cell death events.

The TGF-ß induced morphological transition from a polarized to a mesenchymal phenotype is completed after 24 h (data not shown), whereas oncogenic Ras overcomes TGF-ß mediated effects on proliferation and survival after 72 h. Thus, hepatocellular EMT is suggested to consist of an induction phase, in which Ras signaling establishes resistance against TGF-ß induced cell-cycle inhibition and apoptosis, whereas in the maintenance phase, Ras promotes growth advantage and survival of malignant hepatocytes. Together, these data show clear evidence that oncogenic Ha-Ras provides both insensitivity to TGF-ß mediated growth arrest and resistance to TGF-ß induced apoptosis upon EMT of MIM hepatocytes.

Analysis of the Ras pathway in hepatocellular EMT: MAPK signaling overcomes the TGF-ß mediated cell-cycle arrest
In order to investigate the collaboration of Ras and TGF-ß with respect to cell-cycle progression and apoptosis, we analyzed the role of two important Ras subeffector pathways, namely the Erk/MAPK pathway and the PI3K-Akt pathway in hepatocellular EMT. We accomplished this task by using two different approaches. On the one hand we employed the commercially available low molecular weight compounds PD184.352 and LY294.002 which have been reported to specifically inhibit Mek1/2 and PI3 kinases, respectively. On the other hand we used a genetic approach by generating cell lines through stable retroviral transmission of MIM-1-4. These cell lines expressed either the Ras mutant S35-V12–Ras or C40-V12–Ras (43) selectively activating MAPK (called MIM-S35) or PI3K signaling (MIM-C40) in response to the cognate signals. Similar to MIM-1-4 and MIM-R cells, MIM-S35 and MIM-C40 cells displayed a polarized epithelial morphology and were able to grow in a strictly contact inhibited manner (data not shown).

Initially, a characterization of Ras mutant hepatocytes revealed high phospho-Akt levels in untreated MIM-C40 cells which transiently decreased after 24 h and again increased to high levels after long-term treatment with TGF-ß (Figure 3A). Interestingly, MIM-R and MIM-S35 hepatocytes also showed strong activation of Akt upon continuous stimulation with TGF-ß, although low levels of active Akt were found in untreated cells. These MIM-R cells exhibited high phospho-Erk levels, which were transiently downregulated after 24 h of TGF-ß stimulation, but increased again upon sustained TGF-ß stimulation. MIM-S35 displayed Erk activation kinetics similar to MIM-R, but with lower activity of Erk in untreated cells. The MIM-C40 cells were devoid of phospho-Erk activation after long-term treatment with TGF-ß. Parental MIM-1-4 hepatocytes, however, showed no significant Erk or Akt phosphorylation. It is important to note that V12-C40 and V12-S35 mutants expressed by the corresponding cell lines are not constitutively active but become activated in response to the cognate signals and subsequently activate either PI3K or MAPK. Moreover, these cell lines still harbor endogenous Ras which can be stimulated independent of exogenous Ras. Thus, it cannot be excluded that the employed cell lines show activation of both subeffector pathways at various time points.



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Fig. 3. MAPK but not PI3 kinase signaling abrogates TGF-ß mediated cell-cycle inhibition upon hepatocellular EMT. (A) MIM-R, MIM-S35, MIM-C40 and MIM-1-4 hepatocytes harvested after treatment with TGF-ß for the indicated time periods (hours) were processed for western blotting. Total Erk and Akt levels are shown as controls for phospho-Erk and phospho-Akt, respectively. LT, long-term treatment with TGF-ß1 for 14 days; MIM-1-4 LT cells were not viable and therefore not shown (see also Figure 2A). The data are representative of three independent experiments. (B) Proliferation kinetics of untreated MIM-C40 (white circles) and MIM-S35 cells (gray circles) compared with those of TGF-ß treated cells (white and gray triangles). (C) [3H]Thymidine (TdR) incorporation (c.p.m.) normalized to crystal violet staining of cells treated with 2.5 ng/ml TGF-ß. Light gray bars, MIM-1-4; white bars, MIM-C40; dark gray bars, MIM-S35; black bars, MIM-R. In (C), the TdR incorporation at each time point after TGF-ß treatment is expressed in percent compared with untreated control cells. The values represent the mean of triplicate samples of two independent experiments. Error bars denote SD.

 
Proliferation kinetics of Ras subeffector mutants treated with TGF-ß over a period of 14 days revealed that MIM-S35 continued proliferation comparable to MIM-R hepatocytes (Figures 3B and 2A), whereas MIM-C40 cells showed a reduction in cell number (Figure 3B). In order to determine the contribution of cell-cycle inhibition to the observed cumulative cell numbers, a thymidine incorporation assay was performed, showing that the activation of the MAPK pathway confers insensitivity towards TGF-ß induced growth arrest of MIM-R and MIM-S35 cells. In contrast, MIM-C40 cells failed to display a significant growth advantage as compared with parental MIM-1-4 cells and showed a constitutive cell-cycle arrest after treatment with TGF-ß (Figure 3C). These data suggest an essential role of MAPK signaling in abolishing the TGF-ß mediated cell-cycle inhibition, indicated to be necessary for progression in hepatocellular EMT.

MAPK confers resistance against TGF-ß induced apoptosis in MIM-1-4 hepatocytes
Since the PI3K–Akt pathway was reported to protect cells from TGF-ß induced apoptosis in a mammary EMT model (52), we analyzed the contributions of Ras subeffectors to survival in hepatocellular EMT. After 48 h of TGF-ß stimulation, all cell lines displayed ~4- to 6-fold caspase 3 activation, whereby parental MIM-1-4 cells showed highest basal and TGF-ß induced values, followed by MIM-C40, MIM-S35 and MIM-R cells (Figure 4A). Although all cell lines showed caspase 3 activation after 48 h of TGF-ß stimulation compared with the untreated samples, release of LDH and thus execution of cell death after 72 h of TGF-ß was observed in MIM-1-4 and MIM-C40 cells, but not in MIM-S35 and MIM-R cells (Figure 4B). These results could be confirmed by inhibitor studies in MIM-R cells, demonstrating that inhibition of the Erk/MAPK pathway employing PD184.352 leads to massive caspase 3 activation and release of LDH into the medium, whereas the PI3 kinase inhibitor LY294.002 caused only weak effects (Figure 4C and D). Taken together, these data indicate the crucial role of MAPK in rescuing hepatocytes from TGF-ß mediated apoptosis.



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Fig. 4. MAPK confers resistance against TGF-ß mediated apoptosis during hepatocellular EMT. (A, C) Caspase 3 activity standardized to protein content, and (B, D) LDH release normalized to crystal violet staining. Light gray bars, MIM-1-4; white bars, MIM-C40; dark gray bars, MIM-S35; black bars, MIM-R. In (C and D), MIM-R hepatocytes were employed for pharmacological inhibition. C, untreated control cells; T, stimulation with 2.5 ng/ml TGF-ß for 48 h; PD, 24 h treatment with the Mek1/2 inhibitor PD184.352; LY, treatment for 24 h with the PI3 kinase inhibitor LY294.002. The values represent the mean of triplicate samples of two independent experiments. Error bars denote SD.

 
Next we addressed the question whether the mechanisms active in the induction of EMT are also operative during the maintenance phase. Therefore, we investigated the fibroblastoid cell lines MIM-RT, MIM-ST and MIM-CT which were generated from MIM-Ras, MIM-S35 and MIM-C40 cells, respectively, by long-term treatment with TGF-ß (over 14 days) (Figure 5A). It is to be noted that MIM-CT cells required growth factor supplementation such as insulin, IGF-II and TGF-{alpha} in order to overcome TGF-ß mediated cell-cycle defects and apoptosis. Nevertheless, the proliferation rates of MIM-CT cells in vitro were reduced as compared with those of MIM-RT and MIM-ST cells (Figure 5B). Western blot analysis revealed that in spite of growth factor supplementation of MIM-CT hepatocytes, Erk activation was significantly lower than in MIM-ST and MIM-RT cells. On the other hand, activation of Akt could be observed in all fibroblastoid cell lines (Figure 5C).



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Fig. 5. Molecular features of fibroblastoid MIM-RT, MIM-ST and MIM-CT cells. (A) Phase contrast images showing the fibroblastoid morphology. (B) Proliferation rates of TGF-ß treated fibroblastoid cell lines. A starting population of 1 x 105 cells was each plated to monitor cumulative cell numbers over several days. Black diamonds, MIM-RT; gray squares, MIM-ST; white triangles, MIM-CT. (C) Analysis of Erk/MAPK and Akt activation in MIM-RT, MIM-ST and MIM-CT cells by western blotting. (D) Caspase 3 activity standardized to protein content and (E) LDH release normalized to crystal violet staining. C, control cells; PD, 24 h treatment with PD184.352; LY, 24 h treatment with LY294.002. The data are representative of at least two independent experiments. The values depicted in (B), (D) and (E) represent the mean of triplicate samples of two independent experiments. Error bars denote SD.

 
To detect the contribution of Ras subeffectors to the survival of TGF-ß mediated apoptosis, we investigated the effects of pharmacological inhibitors in MIM-RT cells. The caspase 3 assay showed a dramatic increase of caspase 3 activity in cells treated with the MAPK inhibitor PD184.352 and almost no effect in cells treated with the PI3K inhibitor LY294.002 (Figure 5D). Treatment with PD184.352 and TGF-ß resulted in highest LDH release, whereas LY294.002 caused only moderate cell death (Figure 5E). In conclusion, these data provide strong evidence that MAPK signaling is indispensable for cell survival during the induction as well as the maintenance of hepatocellular EMT.

MAPK signaling confers tumorigenic potential to MIM-1-4 hepatocytes in vivo and drastically accelerates malignancy in cooperation with TGF-ß
To confirm the results obtained in vitro, we next analyzed the tumorigenic potential of the various cell lines in vivo. Therefore, we subcutaneously injected MIM-1-4, MIM-C40, MIM-S35 and MIM-R hepatocytes, as well as the fibroblastoid cell lines MIM-RT, MIM-ST and MIM-CT into immunocompromised recipient SCID mice. As expected, parental MIM-1-4 cells were not able to form tumors (data not shown). PI3K activating MIM-C40 and MIM-CT cells also failed to form tumors in vivo (data not shown), although MIM-C40 cells in culture were able to survive the TGF-ß mediated effects and establish fibroblastoid MIM-CT cells when supplemented with growth factors. In contrast, both MIM-S35 and MIM-R formed tumors, indicating that MAPK signaling is sufficient to confer tumorigenic potential. Interestingly, MIM-S35 hepatocytes produced even faster growing tumors than MIM-R cells. Moreover, the cooperation of MAPK and TGF-ß accelerated the tumor growth tremendously in vivo, since MIM-ST and MIM-RT cells formed larger tumors in comparison with the untreated samples (Figure 6A and B). The increased tumor size was due to an enhanced proliferation in cooperation with TGF-ß since the immunohistochemistry of tumor sections revealed strongly increased nuclear PCNA staining in TGF-ß treated MIM-RT tumor cells compared with untreated MIM-R (Figure 6C, upper panel). Quantitatively, in MIM-RT tumor sections ~64.6% (±3.9) of cells showed nuclear PCNA staining whereas in MIM-R tumors only ~40.4% (±5.3) stained positively. Similar results were found in MIM-ST and MIM-S35 tumors, which showed 74.7% (±6.9) and 44.8% (±5.3) of cells with nuclear PCNA, respectively (data not shown). This observation is of particular interest since proliferation kinetics obtained in vitro (Figure 2A) showed no growth advantage of MIM-R and MIM-S35 hepatocytes in cooperation with TGF-ß.



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Fig. 6. TGF-ß in cooperation with MAPK accelerates tumor formation in vivo. (A) Tumor size and (B) tumor weight after subcutaneous injection of cells into immunodeficient SCID mice. Untreated MIM-S35 (gray bars) and MIM-R cells (black bars) as well as those pre-treated with TGF-ß cells were capable of forming tumors in vivo, whereas MIM-C40 and MIM-1-4 cells failed to show a tumorigenic phenotype. Plain bars, untreated cells; hatched bars, TGF-ß pretreatment for 14 days. The values represent the mean of three independent experiments. Error bars denote SD. (C) Sections from MIM-R derived tumors were immunohistochemically analyzed for PCNA expression. DNA fragmentation in tumor sections was detected by TUNEL assay. The data are representative of at least three independent experiments.

 
Studies on cell death in vivo by TUNEL analysis of histological sections revealed a moderate increase of DNA fragmentation in tumors generated by MIM-RT hepatocytes compared with tumors of MIM-R cells (Figure 6C, lower panel). However, cell death events were rare in both, i.e. 0.73% (±0.12) and 0.37% (±0.10), respectively.

In summary, the growth advantage of TGF-ß pre-treated samples is by far exceeding the moderately increased cell death, thus displacing the net balance between cell-cycle progression and cell death towards the accumulation of tumorigenic hepatocytes.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the current study we present a novel hepatocellular EMT model which is based on the use of immortalized p19ARF null MIM-1-4 hepatocytes. In contrast to a multitude of transformed hepatic cell lines described in the literature, MIM hepatocytes have been recently characterized to express a broad panel of liver-specific markers and to show a highly differentiated and non-tumorigenic phenotype as well as competence to liver-intrinsic Fas-mediated apoptosis (42). Intriguingly, these cultured MIM hepatocytes harbor liver reconstituting activity after intrasplenic transplantation into Fas-injured livers of SCID mice, thereby acting as hepatic liver progenitors in vivo. Through the expression of constitutively active Ha-Ras and the concomitant stimulation with TGF-ß, epithelial Ras-transformed hepatocytes underwent EMT which was characterized by the acquisition of a spindle-shaped morphology and a dramatic increase in malignancy. Here we show first evidence that the MAPK signal transduction cascade is critical for the cooperation with TGF-ß in the late stage of liver tumorigenesis.

EMT tumor models are suggested to represent in vitro correlates to local tumor invasion and metastasis in vivo, providing versatile cellular tools to study molecular mechanisms involved in carcinoma progression (3537). MIM-1-4 hepatocytes undergoing EMT by the cooperation of oncogenic Ha-Ras and TGF-ß show a variety of characteristics described also for human HCCs, thus underlining the in vivo relevance of the employed cellular tumor model. These features include loss or dislocation of the tumor suppressor E-cadherin (5355), nuclear accumulation of ß-catenin (9,56) and autocrine secretion of TGF-ß1 (1,2). Moreover, our hepatocellular EMT model provides an important advantage which is the combination of investigating cells in culture and the opportunity to study the growth behavior and characteristics of different cell lines in vivo. In particular, the study of hepatocytes after orthotope transplantation into the livers of recipient SCID mice is of an exceptional relevance, since the exogenous hepatocytes are relocated in their natural environment (M. Mikula, E. Fuchs and W. Mikulits, manuscript in preparation).

It is noteworthy that autocrine TGF-ß production could be observed in MIM-1-4 hepatocytes which underwent EMT. After orthotopic transplantation of MIM-RT cells into the liver of immunocompromised SCID mice and tumor formation, recovered cells displayed a fibroblastoid phenotype and secreted 5-fold higher amounts of TGF-ß as compared with epithelial recultivated cells from MIM-R tumors (data not shown). This is of particular relevance, since the transplanted cells did not receive exogenous TGF-ß stimulation after recultivation, and the fibroblastoid morphology persisted throughout long-term passaging, confirming that the EMT phenotype is stable under physiological conditions. As well, fibroblastoid MIM-RT cells cultured in vitro by semi-conditioned medium without in vivo passage maintained their phenotype longer than 2 weeks, most probably due to autocrine TGF-ß secretion (data not shown). From the data in vitro and in vivo, we concluded that hepatocellular EMT consists of (1) an induction phase, in which Ras signaling establishes the resistance against TGF-ß induced cell-cycle inhibition and apoptosis, and (2) a maintenance phase, in which TGF-ß autocrine production allows persistence of EMT. In our experimental approach, the induction phase occurred within 72 h after TGF-ß stimulation, whereas the maintenance phase has been observed to be well established 14 days post-TGF-ß treatment. However, the precise timing of these suggested phases remains to be elucidated by studies on (ir)reversibility of the EMT characteristics.

The implication of Ras signaling pathways in TGF-ß mediated EMT are controversially discussed, since different reports describe the PI3K-Akt (26) and MEK-Erk/MAPK signaling (38,39) as indispensable in TGF-ß induced EMT. Furthermore, activation of Raf-MAPK pathway has been shown to protect MDCK cells from apoptotic stimuli provided by TGF-ß (41), whereas PI3K activity was required for protection from TGF-ß induced apoptosis in mammary epithelial cells (52). Moreover, both Ras subeffectors have been found to be implicated in proliferation (57,58). These findings indicate that the different Ras effector functions might depend on the cell type and the cellular context. By employing genetic as well as pharmacological approaches, we demonstrate that Erk/MAPK activity is necessary and sufficient to protect MIM hepatocytes against TGF-ß mediated cell-cycle arrest and apoptosis. Thus, our data provide first evidence that the Erk/MAPK signaling on its own is crucial in conferring tumor-promoting effects of TGF-ß in late stage liver tumorigenesis.

In agreement with these data, Erk/MAPK plays an important role also in human hepatocarcinogenesis, since Erk/MAPK has been found to be activated in 58% of examined human HCC and its activity was positively correlated with tumor size (59). In addition, activated MEK–MAPK pathway has been described to be associated with survival of liver cancer cells in human HCC, whereby overexpression and phosphorylation of MAPK have been detected in 91% (42 of 46) and 69% (32 of 46) of the HCCs examined (60). In line with these data, treatment of human HepG2 and Hep3B hepatic cancer cells with MEK1/2 specific inhibitors U0126 and PD98059 led to growth inhibition and apoptosis, whereas inhibition of PI3K, JNK and p38 kinase activities caused only a mild apoptosis (60). Interestingly, investigation of primary hepatocytes revealed that epidermal growth factor generated survival signals were dependent on MEK-Erk/MAPK activation, whereas both PI3K and MEK-Erk/MAPK cascades have been found to be necessary for the activation of DNA synthesis (61). In our model of hepatocarcinogenesis, PI3K activation could be observed upon long-term treatment with TGF-ß in MIM-R hepatocytes and, most surprisingly, also in MAPK activated MIM-S35 cells, indicating that endogenous Ras in MIM-S35 hepatocytes is stimulated to activate this pathway. This observation leaves open questions of how PI3K signaling is activated and what the consequences of PI3K activation are. Although we could not assign a specific role to the PI3K–Akt pathway during hepatocellular EMT, it must be noted that MIM-C40 well differ from the parental MIM-1-4 hepatocytes, since the MIM-C40 cells could survive the TGF-ß mediated effects and establish fibroblastoid MIM-CT cells when supplemented with growth factors.

Unexpectedly, all cell lines showed caspase 3 activation after 48 h TGF-ß stimulation compared with the untreated samples, whereas release of LDH and thus execution of cell death after 72 h TGF-ß was observed only in MIM-1-4 and in MIM-C40, but not in MIM-S35 and MIM-R cells (Figure 4A and B). This phenomenon of caspase 3 activation without subsequent cell death, as observed in MIM-R and MIM-S35 hepatocytes, has already been described for several cell types including 423 embryonic rat cells (62), T lymphocytes (6365), Ras-overexpressing Ba/F3 cells (66) and non-small cell lung cancer (67). The underlying mechanism still remains to be elucidated by employing the highly synchronous induction of TGF-ß mediated hepatocellular EMT.

Subcutaneous injection of cells into SCID mice allowed the identification of tumorigenic potential and provided additional information about the growth behavior in a physiological, albeit not in the natural context of hepatocytes. The proliferation of tumor cells in vivo (Figure 6) showed discrepancy to the observed proliferation rates of the corresponding cell lines in vitro (Figures 2A, 3B and 5B), since the fibroblastoid cell lines MIM-RT and MIM-ST did not display any growth advantage compared with the epithelial MIM-R and MIM-S35 hepatocytes in culture, respectively. Interestingly, preliminary data showed nuclear ß-catenin staining in sections of tumors generated by fibroblastoid MIM-RT cells, whereas immunohistochemistry of differentiated MIM-R tumors revealed localization of ß-catenin at intercellular contacts (M. Mikula, E. Fuchs and W. Mikulits, manuscript in preparation). In comparison, cultured MIM-RT cells did not show any nuclear ß-catenin accumulation (Figure 1B). Wnt signaling leads to phosphorylation and inhibition of glycogen synthase kinase (GSK)-3ß thereby increasing the cytoplasmic levels of free ß-catenin, allowing ß-catenin to translocate to the nucleus and to stimulate Lef/Tcf-dependent transcription of Wnt target genes such as c-myc and cyclin D1 (68,69). Activation of Wnt signaling upon cooperation with TGF-ß might be one of the reasons for the differences between observations in vitro and the tumor growth in vivo. Accordingly, nuclear ß-catenin accumulation has been found to be closely associated with areas of high cell proliferation in liver tumors developed by c-myc/TGF-ß1 transgenic mice, indicating that constitutive deregulation of the Wnt/TGF-ß signaling may play an important role in hepatocarcinogenesis (70).

Somatic mutations of ß-catenin were observed in 19–26% of HCCs (56,71), and furthermore two regulators of ß-catenin in Wnt signaling, Axin and APC, have also been reported to be mutated in HCCs, although with relatively low frequencies (72,73). Nevertheless, ~50% of human HCC revealed nuclear accumulation of ß-catenin (56). The relationship between ß-catenin activation, tumor grade and clinical outcome in human HCC is controversially discussed. Some studies demonstrated a significant correlation between nuclear accumulation of ß-catenin, high grade of differentiation and better prognosis (74,75) or described no correlation with clinical outcome (76). In contrast, other investigations revealed nuclear accumulation of ß-catenin associated with poor survival (77) and enhanced nuclear staining in invasive compartments of tumors (78). In HCCs of transgenic mouse models, activation of ß-catenin was found to be associated with a more benign phenotype and a relatively stable genome (79). Thus, further analysis of the role of ß-catenin signaling in our hepatocellular tumor model might be very promising. In this context it is worth mentioning that the activation of PI3K–Akt pathway is known to cause phosphorylation and inhibition of GSK-3ß thereby increasing the cytoplasmic levels of free ß-catenin, allowing ß-catenin to translocate to the nucleus and to stimulate Lef/Tcf-dependent transcription (80). This could provide a possible function of PI3 kinase activation. Even though differences in tumor growth between TGF-ß treated and untreated malignant hepatocytes might be due to ß-catenin, the discrepancy between growth rates of tumors generated by MIM-R and MIM-S35 still remains to be elucidated.

In summary, we present a valuable cellular model of hepatocarcinogenesis based on p19ARF null hepatocytes which shows a variety of characteristics observed in human HCC. In this model, a significant increase in malignancy is observed by EMT caused through the cooperation of Ha-Ras and TGF-ß. The analysis of the Ras pathway by genetic and pharmacological strategies identified MAPK to be functional in protecting hepatocytes from TGF-ß induced cell-cycle arrest and apoptosis. This experimental finding should be taken into consideration for the understanding of liver tumor progression and the development of novel concepts in liver cancer therapy.


    Acknowledgments
 
This work was supported by grants from the ‘Fonds zur Förderung der wissenschaftlichen Forschung’, FWF P15435, and by the ‘Herzfeldersche Family Foundation’, Austria.


    References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 24, 2004; revised January 27, 2005; accepted February 1, 2005.





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