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© The Rockefeller University Press,
0021-9525/2001//1023 $5.00
The Journal of Cell Biology, Volume 153, Number 5,
, 2001 1023-1034
Original Article |
Activation of the Met Receptor by Cell Attachment Induces and Sustains Hepatocellular Carcinomas in Transgenic Mice
rwang{at}cgl.ucsf.edu
Overexpression is the most common abnormality of receptor tyrosine kinases (RTKs) in human tumors. It is presumed that overexpression leads to constitutive activation of RTKs, but the mechanism of that activation has been uncertain. Here we show that overexpression of the Met RTK allows activation of the receptor by cell attachment and that this form of activation can be tumorigenic. Transgenic mice that overexpressed Met in hepatocytes developed hepatocellular carcinoma (HCC), one of the human tumors in which Met has been implicated previously. The tumorigenic Met was activated by cell attachment rather than by ligand. Inactivation of the transgene led to regression of even highly advanced tumors, apparently mediated by apoptosis and cessation of cellular proliferation. These results reveal a previously unappreciated mechanism by which the tumorigenic action of RTKs can be mediated, provide evidence that Met may play a role in both the genesis and maintenance of HCC, and suggest that Met may be a beneficial therapeutic target in tumors that overexpress the receptor.
Key Words: Met • receptor tyrosine kinase signaling • tumorigenesis • transgenic mouse • cell adhesion
© 2001 The Rockefeller University Press
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Introduction |
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The MET protooncogene was first encountered in a chromosomal translocation that had generated the TRP-MET oncogene in cultured tumor cells treated with a chemical carcinogen (Cooper et al. 1984; Park et al. 1987). The gene encodes a 170-kD protein (p170met), Met, that is processed to generate the cell surface receptor Met (p190met), composed of a glycosylated extracellular 
subunit (p50met) and a transmembrane β subunit (p140met) (Gonzatti-Haces et al. 1988; Giordano et al. 1989). The β subunit possesses protein-tyrosine kinase activity and a "docking site" for cell-signaling molecules (Naldini et al. 1991a; Ponzetto et al. 1994). The ligand for Met is a growth factor known as both scatter factor and hepatocyte growth factor (HGF) (Bottaro et al. 1991; Naldini et al. 1991c), which elicits multiple biological responses, including proliferation, migration, invasion, and morphogenesis (Bardelli and Comoglio 1997; Vande Woude et al. 1997; Birchmeier and Gherardi 1998).
MET has been implicated in various human cancers. Cells transformed by MET are tumorigenic in experimental animals (Bardelli and Comoglio 1997; Vande Woude et al. 1997). Mutant alleles of MET have been found in renal, hepatocellular, and squamous cell carcinomas of humans (Schmidt et al. 1997; Olivero et al. 1999; Park et al. 1999; Di Renzo et al. 2000). Transgenic mice expressing activated mutant forms of Met develop tumors (Liang et al. 1996; Jeffers et al. 1998). Moreover, the gene is overexpressed in a variety of epithelial tumors of humans, including those of liver, prostate, colon, breast, and skin (Stuart et al. 2000). Despite this evidence, the role of MET in the genesis of human tumors remains uncertain. In particular, Met is overexpressed in some epithelial tumors while the ligand is absent (Rusciano et al. 1995, Rusciano et al. 1996; Hiscox et al. 1997; Ueki et al. 1997). This discrepancy diminishes the possibility that autocrine or paracrine activation of the Met receptor contributed to the genesis of the epithelial tumors.
Here we provide the first evidence that wild-type MET can be tumorigenic in vivo. Since HGF is not expressed or activated in normal liver and often absent from the tissue of hepatocellular carcinoma (HCC) (Kinoshita et al. 1989; D'Errico et al. 1996; Noguchi et al. 1996; Ueki et al. 1997), HGF-independent activation of Met might play a role in the genesis of liver cancer. We created transgenic mice that expressed human MET in hepatocytes under the control of tetracycline. Although human Met (hMet) cannot respond to murine HGF (Bhargava et al. 1992; Rong et al. 1992), it was enzymatically active in the hepatocytes, and the activity was dependent on cell adherence, but not HGF. The transgenic mice developed HCC that regressed when the transgene was inactivated. These results indicate that overexpression of Met may play a role in the genesis and maintenance of human HCC, provide an explanation for why HGF need not be present in tumors that overexpress Met, and suggest that cell adherence can be an alternative activation mechanism for RTKs relevant to human cancers.
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Materials and Methods |
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Transfection
HeLa cells expressing tTA (CLONTECH Laboratories, Inc.) were cotransfected with a plasmid containing the murine Met driven by the tetracycline-responsive control element and a plasmid containing the hygromycin resistance gene as a selectable marker. Stable cells expressing murine Met were selected with Hygromycin B at 200 µg/ml. HeLa cells transiently expressing Trk/Met were obtained by transfecting HeLa cells expressing tTA with a plasmid containing trk/met driven by a tetracycline-responsive element (TRE) and cells were harvested 48 h after transfection. The murine Met cDNA was a gift from Dr. G. Vande Woude (Van Andel Research Institute, Grand Rapids, MI). The Trk/Met cDNA was a gift from Dr. Walter Birchmeier (Max Delbruck Center for Molecular Medicine, Berlin, Germany).
Cell Attachment and HGF Stimulation
Detached cells were released from plates by treating with PBS containing 0.04% EDTA, washed in PBS, and cell pellets were loosened by tapping the tubes before lysis. Attached cells were plated on dishes and incubated in PBS for 15 min at 37°C before lysis on the plates. For HGF stimulation, 40 ng/ml human recombinant HGF (R&D Systems) was added to the cells for 15 min at 37°C in serum-free medium. For attached cells, HGF was added onto the plates. For detached cells, HGF was added into the tube and the tube was inverted every 2 min to ensure the cells were kept in suspension.
Cell Surface Protein Labeling with Biotin
Cells were labeled with biotin (Pierce Chemical Co.) 0.5 mg/ml in PBS for 15 min on ice, and the reactions were stopped with 50 mM ice-cold NH4Cl/PBS for 10 min on ice. Cells then were lysed, and Met was immunoprecipitated with anti-Met antibodies. The immunocomplexes were resolved in SDS-PAGE and visualized with horseradish peroxidase–conjugated Streptavidin.
Immunoprecipitation and Immunoblotting
Antiphosphotyrosine monoclonal antibody 4G10 was produced from the 4G10 hybridoma provided by D. Morrison (National Cancer Institute, Frederick, MD). Anti-hMet and anti–murine Met (mMet) antibodies, both rabbit polyclonal antipeptide antibodies, were purchased from Santa Cruz Biotechnology, Inc.
Cell lines were starved in serum-free media supplemented with 1% bovine serum albumin for 18–24 h, and primary cells were starved in media containing 0.5% serum overnight before experiments. Isolated hepatocytes were analyzed without serum starvation. Lysis of cells and immunoblotting were performed as described (Wang et al. 1996). Where required, cells were first detached from dishes with PBS containing 0.04% EDTA before lysis.
Cell lysates were cleared by centrifugation at 14,000 g for 10 min, incubated with antibodies for 2 h, and then with protein G–Sepharose beads for 1 h at 4°C. The beads were washed three times with lysis buffer. Immune complexes were boiled in Laemmli's sample buffer for electrophoresis.
Transgenic Mice
A 4.5-kb XhoI and HindIII fragment of human MET cDNA, a gift of Dr. G. Vande Woude,was blunt-ended and inserted into pUHG 10-3 plasmid, a gift from H. Bujard (European Molecular Biology Laboratory, Heidelberg, Germany), at the XbaI site. The 6.5-kb XhoI and AseI DNA fragments containing the TRE, hCMV minimal promoter with heptamerized upstream tet-operators, human MET coding sequence, and the rabbit beta-globin intron and Poly (A) sequence were used for microinjection (see Fig. 4 A). Mice were derived from the FVB/N strain according to standard methods (Hogan et al. 1994). TRE-MET mice were screened by PCR analysis using primers derived from the TRE sequence (5'-GTCGAGTAGGCGTGTACG-3') and the human MET sequence (5'-GAATGACATTCTGGATGGGTG-3'). LAP-tTA mice were a gift from Dr. Herman Bujard (European Molecular Biology Laboratory, Heidelberg, Germany) (Kistner et al. 1996), and the line used in this study was bred to FVB/N predominant background in Bruce Counklin's lab at the University of California at San Francisco. The PCR primers used for genotyping these mice were described by Redfern et al. 1999. Doxycycline was administered either in water at 0.2 mg/ml (Sigma-Aldrich) or in chow diet (Bio-Serv).
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Histology
Tissues were fixed in 10% buffered formalin, and 5 µm paraffin sections were stained with hematoxylin and eosin.
Immunohistochemistry Detection of hMet
Formalin-fixed paraffin sections were deparaffinized before staining. Sections were treated with citrate buffer for 10 min at 95°C for antigen retrieval. The endogenous peroxidase activity was blocked by a 5-min treatment with 5% H202/PBS. Sections were incubated with 10% goat serum for 4 h, then with rabbit anti-hMet antibodies at 4°C overnight. The primary antibody was detected by using the Elite ABC rabbit kit (Vector Laboratories). The enzymatic activity of horseradish peroxidase was detected by DAB staining (Vector Laboratories).
Detection of Cell Proliferation and Apoptosis
Mice were injected intraperitoneally with 10 mg/ml BrdU (Boehringer) at doses of 0.01 ml/g 2 h before time of killing. Tissues were fixed and processed for paraffin sections. Slides were immunostained with an antibody to BrdU using a BrdU staining kit (Zymed Laboratories). In situ apoptotic cells were detected using the ApopTag Fluorescein apoptosis detection kit (Intergen) according to the manufacturer's instructions.
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Results |
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We also asked whether the overexpressed Met might be abnormally processed or localized. Cell surface proteins were labeled with biotin and examined by electrophoresis in both native and reducing conditions. The samples of Met obtained from NCI-H441 and A549 cells were labeled equally well. Under reducing conditions, the and β subunits from both sources separated as expected during electrophoresis, whereas under native conditions they remained associated with one another (Fig. 2 a). We conclude that the abundant Met produced in NCI-H441 cells is processed normally and transported to the cell surface.
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The signal initiated by activation of Met is transduced by a downstream cascade of biochemical events (Naldini et al. 1991b; Ponzetto et al. 1994; Royal and Park 1995; Weidner et al. 1996; Boccaccio et al. 1998). Among the earliest of these events is the recruitment of Grb2 protein to a binding domain on Met (Ponzetto et al. 1994). We found that activation of Met by cell attachment increased the amount of Grb2 that could be coprecipitated with Met (Fig. 2 b). Thus, it is likely that activation of Met by attachment initiates a cascade of intracellular signaling.
Conditional Expression of hMet in Hepatocytes of Transgenic Mice
The preceding results raised the possibility that overexpression of Met might lead to activation of the receptor by cell attachment and subsequent tumorigenesis. Although Met is overexpressed in human HCC, HGF is frequently absent, suggesting that Met can be activated by a ligand-independent mechanism in this disease (Ueki et al. 1997). To test this hypothesis, we expressed human MET in hepatocytes and examined tumor development in mice. By using human MET in the transgene, we precluded response to intrinsic mouse HGF (Bhargava et al. 1992; Rong et al. 1992). Thus, any activation of the transgenic receptor was likely to arise from cell attachment within liver tissue.
We first generated mice in which transgenic human MET is under the control of the TRE (Kistner et al. 1996; Fig. 4 a).
MET is controlled by a TRE (Kistner et al. 1996), which allows the inducible expression of the transgene (Fig. 4 a). The TRE-MET mice were then crossed with LAP-tTA mice, in which expression of the tetracycline transactivator protein is under the control of the promoter for the liver-specific liver-activating protein gene (Kistner et al. 1996). The cross produced offspring that were transgenic for both TRE-MET and LAP-tTA. The doubly transgenic mice should express hMet in the liver, and the expression should be suppressed by the administration of doxycycline.
Examination by immunohistochemistry revealed abundant expression of hMet in hepatocytes of the doubly transgenic mice, but not in controls (Fig. 4 b). Human Met was expressed in hepatocytes (Fig. 4 b, middle panel) of the normal liver parenchyma (Fig. 4 b, middle, black arrow) as well as hyperplastic nodules (Fig. 4 b, middle, arrowheads; Fig. 5, c–e). Staining for the protein was especially pronounced at the margins of cells (Fig. 4 b, inset), as expected for a cell surface receptor. Expression was not apparent in other types of cells in the hepatic tissue (Fig. 4 b, middle, purple arrow), nor in other tissues examined, including lung, spleen, kidney, and pancreas (data not shown). Expression of hMet could be repressed by feeding the transgenic mice the tetracycline analogue doxycycline (Fig. 4 b, right).
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In contrast, mice of lines 3 and 4 were healthy at birth, but began to die at 4 mo postpartum (Fig. 5 a). Like in lines 1 and 2, 
85% of the deaths could be attributed to HCC, which appeared as an abdominal mass at 6 mo of age or later. The incidence of HCC was again 60% by 1 yr of age (Fig. 5 b).
About 15% of deaths occurred without evidence of tumor at autopsy in all MET transgenic lines when Met expression was turned on in adult animals. These deaths were often associated with abnormal gross and histological pathology of the liver. The animals were usually cachectic and jaundiced before deaths. These observations suggest that the mice died of a liver disease, the nature of which will require further analysis. We have not detected abnormalities in other organs except occasional pale kidneys in moribund tumor-bearing mice.
We monitored the development of tumors by histological analysis (Fig. 5, c–f). By 60 wk of age, small foci of hyperplasia appeared, often surrounding the central vein of the hepatic lobule (Fig. 5 c). Typically, a couple of these foci were detected per section, suggesting that dozens of these foci were present throughout the liver. These foci of hyperplasia had become bigger, increased in number by a factor of five, and uniformly distributed throughout the liver by 4 mo of age (Fig. 5 d). The cells in these foci were still recognizable as hepatocytes, but had developed foamy cytoplasm containing fat deposits that could be identified by staining with oil red-O (data not shown). These pathological changes are characteristic of the hyperplasia that typically precedes the development of HCC in humans (Ferrell et al. 1993).
Zones of progression to malignancy became apparent within the fields of hyperplasia by 6 mo of age (Fig. 5e and Fig. f). Within these zones, cells were poorly organized and less well differentiated than normal, resembling the dysplastic precursors of HCC in the human liver (Fig. 5 e; Ferrell et al. 1993). Individual livers contained multiple dysplastic foci, suggesting that tumor progression was occurring independently in multiple clonal lineages. In addition, the surrounding liver also showed atypical cytologic changes affecting hepatocytes. These included cellular and nuclear enlargement, as well as disorganization of cell plate and lobular structures. With further progression over time, the malignant foci enlarged and developed the trabeculae typical of advanced HCC (Fig. 5 f). The trabeculae were frequently separated from one another by blood-filled spaces, lined by endothelial cells, and reminiscent of widened hepatic sinusoids. Areas of necrosis were also common in the fully developed tumors. We conclude that the transgenic mice had developed HCC. The penetrance of tumorigenesis was >60% (Fig. 5 b). The area of this typical HCC was usually found in only one or two lobes of the liver, suggesting that a subset of hyperplastic foci proceed to HCC.
Activation of Transgenic Met by Cell Attachment
Our in vitro experiments show that Met can be activated when tumor cells are adherent. Since hepatocytes are adherent in liver tissues, it seemed likely that hMet from the tumor could be activated. We confirmed this expectation by using Western blotting to analyze lysates of the transgenic liver tumor (Fig. 6 a). The analysis revealed that hMet was phosphorylated on tyrosine and, thus, presumably enzymatically active. In contrast, the endogenous mMet in control liver contained no detectable phosphotyrosine (Fig. 6, lane 2').
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Sustained Expression of Met Is Required for the Maintenance of HCC
Transgenic Met activated by cell attachment apparently triggered the genesis of HCC. To determine whether Met might also be required for maintenance of the tumor, we treated 11 moribund mice of line 3 and 13 of line 4 with doxycycline to repress expression of the transgene (see above). Within 1 mo, 21 of these mice had regained their health and lost the abdominal swelling demonstrative of tumor mass (Fig. 7, a and b). One mouse from line 3 and two from line 4 died within 1 wk of doxycycline treatment. Six mice treated with doxycycline for 4 mo have remained healthy throughout.
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Histological analysis was used to examine livers from mice that had been treated for 4 mo with doxycycline (Fig. 7, g–j). The bulk of the liver appeared microscopically normal (Fig. 7 g). The residual nodule of abnormal tissue could be identified by a transition from normal tissue to an area of necrosis, scarring, calcification, and inflammatory reaction (Fig. 7h and Fig. i), the last mostly composed of neutrophils and macrophages (Fig. 7 j). We conclude that inactivation of the human MET transgene led to full regression of the hepatic tumors with reconstitution of normal tissue architecture.
Regression of HCC Was Accompanied by Apoptosis and Diminished Cellular Proliferation
We explored two possible explanations for regression of the tumors: arrest of the cell cycle and apoptosis. The proliferative activity of tissue was examined by labeling with BrdU. Numerous cells were proliferating in the liver tumors elicited by Met (Fig. 8 a). But within 3 d of instituting treatment with doxycycline, proliferation was virtually undetectable (Fig. 8 b), much as is found in a normal liver (data not shown). The absence of proliferation persisted in the remaining scar tissue when doxycycline treatment had been extended to either 20 d or 4 mo (data not shown). We conclude that removal of the stimulus from the transgenic hMet led to prompt cessation of cellular proliferation, even in the cells of advanced malignancies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Little is known about the molecular pathogenesis of HCC. One clue has been the observation that the protooncogene MET is frequently overexpressed in HCC (Prat et al. 1991; D'Errico et al. 1996; Noguchi et al. 1996; Ueki et al. 1997). Other evidence for the tumorigenicity of overactive MET has come from studies with transformation of cells in vitro (Iyer et al. 1990; Rong et al. 1992; Bardelli and Comoglio 1997; Giordano et al. 1997; Vande Woude et al. 1997), the finding of mutant alleles of MET in human cancers (Schmidt et al. 1997; Olivero et al. 1999; Park et al. 1999; Di Renzo et al. 2000), and the occurrence of mammary tumors in transgenic mice that express mutant versions of Met with constitutive activity using the metallothionein 1 promoter (Liang et al. 1996; Jeffers et al. 1998). Here we demonstrate a novel activation mechanism that does not rely on mutation of Met and demonstrate for the first time that overexpression of wild-type Met in hepatocytes of mice enables ligand-independent activation of the receptor that leads to HCC with a high penetrance.
Activation of the Met Receptor in the Absence of Ligand
The ligand for the Met receptor is not produced in normal liver and is often absent from the tissue of HCC (Kinoshita et al. 1989; D'Errico et al. 1996; Noguchi et al. 1996; Ueki et al. 1997). These observations diminish the prospect for autocrine or paracrine activation of Met, thus calling into doubt any role for the receptor in the genesis of HCC. We can now offer an alternative possibility. Overexpression of Met can lead to activation of the receptor by cell adherence, eliminating the requirement for ligand stimulation. In accord with this view, we were able to elicit HCC by overexpressing hMet in the hepatocytes of mice. The transgenic receptor was constitutively active and this activity receded when hepatocytes were dissociated into suspensions of single cells. The activity of Met could not be attributed to the endogenous ligand, because the transgene encodes the human version of Met, which cannot respond to murine HGF (Bhargava et al. 1992; Rong et al. 1992). Moreover, hMet in hepatocytes that were derived from HCC-bearing transgenic mice did not respond to recombinant human HGF. Therefore, we conclude that the transgenic Met was activated by cell adherence in a ligand-independent fashion.
The mechanism by which cell attachment activates Met remains obscure, but appears to be dependent on overexpression. Overexpression of Met potentiates the receptor activation by cell adherence and eliminates the requirement for ligand stimulation (Fig. 3 and Fig. 6; Wang, R., and J.M. Bishop, unpublished results). Therefore, we were able to elicit HCC by overexpressing hMet in the hepatocytes of mouse liver. Previous work failed to produce HCC in mice expressing a transgene of wild-type Met (Jeffers et al. 1998). Our findings suggest an explanation for that result. The level of Met expression may have been insufficient to permit activation of the receptor by cell adherence, and this form of activation may be the only means by which Met can be activated in HCC pathogenesis. However, genetic backgrounds of the mice differed in these two studies, and this might also explain the different outcomes.
We could not explain the activation of Met either by mutations or by abnormal processing or localization of the protein. In addition, we doubt that the extracellular domain of Met plays a role in the activation, because replacement of this domain with its unrelated counterpart from the Trk receptor had no effect on activation by cell attachment. The cytoplasmic domain of Met is obviously essential to activation, because it carries the enzymatic activity of the receptor. But membrane anchorage may also be required for activation by cell attachment, and such anchorage would be provided by the Trk component of the hybrid protein. The receptors for PDGF and macrophage-stimulating protein can also be activated by cell adhesion independent of the ligand (Sundberg and Rubin 1996; Danilkovitch-Miagkova et al. 2000). This activation is apparently mediated by signaling arising from cell surface integrins, but we and others have shown previously that such signaling may not explain the ligand-independent activation of Met (Rusciano et al. 1996; Wang et al. 1996).
We have attempted to address this problem in several ways, and the results support our hypothesis. But it is still possible that a mechanism other than adhesion might account for the results. One such possibility would be cross-talk between Met and another receptor. We have sought evidence for this without success.
The in vitro studies performed to date have detected activation of Met by cell attachment only in certain tumor cells. We have also shown that the activation of Met by cell attachment occurred in the hepatocytes isolated from tumor-bearing transgenic mice. It is possible that secondary events during tumor development may also contribute to adhesion-mediated activation of Met. A further puzzle is represented by the observation that Met, which can be activated by cell attachment in some tumor cell lines and in the hepatocytes from HCC-containing transgenic livers, is accompanied by refractoriness to HGF. We can presently offer no explanation for this observation.
The Role of Met in the Maintenance of HCC
It has not been clear whether genetic lesions responsible for initiation of tumorigenesis would eventually cease to play a role as the tumor progressed to malignancy (Hanahan and Weinberg 2000). Recent work has shown that sustained activity of an oncogene is required for maintenance of tumors elicited by myc, ras, and bcr/abl (Chin and DePinho 2000). We now show that tumors elicited by transgenic MET will regress if the transgene is inactivated, even at advanced stages of tumor progression. This finding raises the possibility that the Met receptor may be a beneficial target for the treatment of HCC and other malignancies, particularly when the receptor is overexpressed in tumor tissue.
There are at least three possible explanations for the tumor regression described here. First, malignant cells might differentiate into normal hepatocytes when the MET transgene is inactivated. This mechanism may explain the regression of hematopoietic malignancies when the expression of transgenic MYC is repressed (Felsher and Bishop 1999), but we presently have no evidence that it contributes to the regression of HCC elicited by transgenic Met.
Second, Met is among the numerous cell surface receptors whose activity can elicit and sustain the cell division cycle (Stuart et al. 2000). Thus, the repression the transgenic Met expression might lead to proliferative arrest on the part of the malignant hepatocytes. We present evidence that this is indeed the case.
Third, apoptosis of tumor cells could contribute to the reduction in tumor mass. Our data demonstrated the rapid onset of apoptosis in tumor tissue soon after the MET transgene had been inactivated, but apoptosis diminished once tumor regression was well advanced. The apoptotic response may be held in check in tumors by Met itself (Amicone et al. 1997), but it would be unleashed when the activity of Met is withdrawn, and diminishes as tumor tissue is replaced by the regrowth of normal hepatocytes.
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Acknowledgments |
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We also thank help from the Cell and Tissue Biology Core facility at the University of California at San Francisco Liver Center, supported by funds from the National Institutes of Health (5P30DK26743). This work was supported by postdoctoral fellowships from the Leukemia Society of America and American Heart Association to R. Wang, and funds from the National Institutes of Health (CA44338) and the G.W. Hooper Research Foundation.
Submitted: 16 January 2001
Revised: 23 March 2001
Accepted: 23 April 2001
Abbreviations used in this paper: HCC, hepatocellular carcinoma; HGF, hepatocyte growth factor; NHEM, normal human epidermal melanocyte; RTK, receptor tyrosine kinase; TRE, tetracycline-responsive element.
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