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Cell Growth & Differentiation Vol. 11, 185-190, April 2000
© 2000 American Association for Cancer Research


Articles

Lung-targeted Expression of the c-Raf-1 Kinase in Transgenic Mice Exposes a Novel Oncogenic Character of the Wild-Type Protein1

Eugen Kerkhoff, Lev M. Fedorov, Renate Siefken, Annette O. Walter, Thomas Papadopoulos and Ulf R. Rapp2

Institut für medizinische Strahlenkunde und Zellforschung, Universität Würzburg, D-97078 Würzburg, Germany [E. K., L. M. F., R. S., A. O. W., U. R. R.], and Pathologisches Institut, Histologisches Labor, Friedrich-Alexander-Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany [T. P.]

Abstract

The c-Raf-1 kinase is a downstream effector of Ras signaling. Both proteins are highly oncogenic when they are mutationally activated, but only the Ras GTPase is frequently mutated in naturally occurring tumors. Although the c-Raf-1 protein was found to be amplified in different lung cancer cell lines, overexpression of the wild-type c-Raf-1 protein was shown to be insufficient to transform cultured cells. Here we have addressed the question of whether overexpression of the wild-type c-Raf-1 kinase can induce lung cancer in mice. We show that lung-targeted expression of oncogenically activated or wild-type c-Raf-1 proteins induces morphologically indistinguishable lung adenomas in transgenic mice. Compared with mice transgenic for the activated c-Raf-1-BxB, tumor development is delayed and occurs at a lower incidence in wild-type c-Raf-1 transgenic mice. Our studies show that the c-Raf-1 expression level is a critical parameter in tumor development and should be analyzed in more detail to evaluate its potential in the induction of cancer.

Introduction

Receptor tyrosine kinase signal transduction pathways are major regulators of cell proliferation and are frequently found to be mutated and activated during tumor development (1 , 2) . After ligand binding to receptor tyrosine kinases, the Ras GTPase is shifted from the GDP to the GTP loaded form (3) . Ras-GTP recruits the Raf protein to the cellular membrane, where the kinase becomes activated and transmits its signal via a cascade of successive phosphorylation events (4 , 5) . Mutations that lead to constitutive activation of the tyrosine kinase receptors are found frequently in naturally occurring tumors and have been shown to be highly transforming in cultured cells (1 , 2) . The Ras GTPase becomes oncogenically activated by single amino acid substitutions, which inhibit the GTPase activity and cause the Ras protein to persist in its activated GTP-bound status (1 , 6) . Of all oncogenes, the ras gene has the highest mutation rate in human tumors (6) . The Raf kinase is a downstream effector of Ras signaling (4 , 5) . The Raf protein consists of a NH2-terminal regulatory domain mediating the interaction with Ras and a COOH-terminal kinase domain (4) . Cell culture experiments have shown that deletion of the NH2-terminal regulatory domain leads to a constitutively active kinase that is highly oncogenic in animals as well as in cultured cells (7 , 8) . Single amino acid substitutions that activate Raf kinase for Mek3 phosphorylation have been found to be transforming in cell culture experiments (9 , 10) . Despite the high transforming potential of its upstream activators, mutationally activated Raf kinases could not be identified in naturally occurring tumors. Analysis of the expression levels of the wild-type c-Raf-1 protein in human lung tumor cell lines and biopsy material from malignant tissues revealed increased expression levels of the c-Raf-1 kinase in the majority of the transformed cells, raising the possibility that overexpression of the wild-type protein may contribute to oncogenic transformation (11) . In contrast to NH2-terminal deletion mutants or activated Ras proteins, overexpression of the wild-type c-Raf-1 kinase was shown to be insufficient to transform cultured cells (7 , 8) . Evidence for an oncogenic potential of the wild-type Raf kinase came from the observation that overexpression of the c-Raf-1 kinase sensitizes cells for Ras transformation (12) . Activated Ras expressed at low levels and wild-type c-Raf-1 overexpression were shown to cooperate in cell transformation in tissue culture experiments. Here we have addressed the question of whether lung-targeted overexpression of c-Raf-1 protein in transgenic mice is tumorigenic. We show that high expression levels of the c-Raf-1 protein lead to the development of lung adenomas within 1 year of age.

Results and Discussion

To evaluate a possible function of the c-Raf-1 protein in tumorigenesis, we have generated transgenic mice [(C57BL/6xDBA-2)F1] expressing the wild-type human c-Raf-1 protein or the oncogenically activated NH2-terminal deletion mutant c-Raf-1-BxB under the control of human SP-C promoter (Fig. 1Citation ). c-Raf-1-BxB is a constitutively active c-Raf-1 kinase and is highly transforming in cell culture experiments (13) .4 In mice, the SP-C gene is expressed in type II epithelial cells lining the lung alveoli (14, 15, 16) . The promoter region of the human SP-C gene has been shown to direct the expression of chimeric genes in transgenic animals in a lung epithelial cell-specific manner (15) . The SP-C promoter has been used frequently for lung-targeted expression of transgenes (14, 15, 16, 17, 18, 19, 20, 21) . Tumor formation could only be observed in those transgenic mice in which the expression of an oncogene was directed by the SP-C promoter (16 , 17) . We have established 11 independent mouse strains carrying the SP-C-c-raf-1 transgene and 2 mouse strains carrying the SP-C-c-raf-1-BxB transgene (Table 1)Citation . Gene integration has been verified by Southern hybridization (data not shown). In addition, we have generated mice transgenic for sequences encoding the isolated NH2-terminal regulatory domain of the c-Raf-1 protein (c-Raf-1-C4). The c-Raf-1-C4 protein acts as an inhibitor of Ras function by interacting with the Ras protein without being able to transmit a signal (5 , 22) . Although two SP-C-c-Raf-1-C4 founders were germ-line transmitting, no transgene expression could be detected by Northern hybridization (data not shown). This is consistent with the absence of lung pathology in these mice (data not shown).



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Fig. 1. Structure of the c-Raf-1 and c-Raf-1-BxB proteins and recombinant DNA constructs directing the expression of the raf sequences under the control of the SP-C promoter. For the generation of transgenic mice, expression cassettes of the SP-C-c-raf-1 or SP-C-c-raf-1-BxB vectors were released by digestion with the NdeI and NotI restriction endonucleases. The purified DNA fragments were injected into fertilized eggs of C75BL/6xDBA-2 F1 mice. The c-Raf-1-BxB protein lacksthe regulatory NH2-terminal sequences of the c-Raf-1 protein, including the Ras interaction domain. In contrast to the wild-type c-Raf-1 kinase, which requires activating upstream regulators, the c-Raf-1-BxB kinase is constitutively active.

 

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Table 1 Tumor development in SP-C-c-Raf-1 and SP-C-c-Raf-1-BxB transgenic mice

 
Both c-Raf-1-BxB-expressing strains develop lung tumors within 1–2 months of age (Table 1)Citation . Confluent tumor development was observed within 5–7 months, accompanied by an 8–10-fold increase in lung tissue mass compared with wild-type mice. Histologically, tumors consisted of cuboid epithelia comparable to alveolar type II cells (Fig. 2Citation ). The tumor cells showed a glandular proliferation, lining the alveolar walls in a lepidic fashion. Sometimes an intra-alveolar micropapillary growth pattern was observed. The grade of nuclear atypia and nuclear polymorphism of the tumor cells was minimal. Single tumor cell mitoses were detectable. Tumor invasion in intrapulmonary vessels or infiltration of bronchial and pleural tissue has never been observed. Extensive examination of extrapulmonary tissues provided no evidence of metastasis. Thus far, the lesions were classified as multicentric alveolar adenomas developed from alveolar type II cells. Analysis of the transgene expression in SP-C-c-Raf-1-BxB mice shows a lung-specific expression of the c-Raf-1-BxB protein (Fig. 3Citation A). Low expression of the oncogenic kinase could also be detected in kidney (Fig. 3A)Citation . This is consistent with several cases of hydronephrosis, which we have observed in SP-C-c-Raf-1 transgenic mice (Table 1)Citation . Comparison of transgene expression in SP-C-c-Raf-1-BxB mice in the early stages of tumor development (3 months) and in the late stages of tumor development, when the tumor cells have reached confluence (8 months), shows a strong amplification of c-Raf-1-BxB protein expression at later stages, indicating that cells expressing the transgene are amplified during tumor development (Fig. 3B)Citation . This result has been confirmed by immunohistochemical staining of the tumor tissue (Fig. 3C)Citation . Backcrossing of SP-C-c-Raf-1-BxB-23 mice to C57BL6 mice (five generations) or the transfer of the SP-C-c-raf-1-BxB allele to 129/Sv mice by crossing (three generations) results in 100% tumor penetrance, indicating that the tumorigenesis is not dependent on the genetic background of C57BL6xDBA-2 mice.



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Fig. 2. Histological analyses of lung tissues from wild-type mice and mice transgenic for lung-targeted expression of the c-Raf-1 or c-Raf-1-BxB proteins. Lung tissues from a 5-month-old wild-type mouse (wt), a 12-month-old SP-C-c-Raf-1-74 mouse (c-Raf-1-74), and a 5-month-old SP-C-c-Raf-1-BxB-23 mouse (BxB-23) were fixed in paraformaldehyde, embedded in paraffin, sectioned at 4 µm, and stained with H&E. The lung of the wild-type mouse exhibited no tumor tissue. In mice transgenic for c-Raf-1, we detected single tumor foci, whereas in mice transgenic for c-Raf-1-BxB expression, the tumor incidence was much higher, resulting in multiple foci.

 


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Fig. 3. Expression of the c-raf-1 and c-raf-1-BxB transgenes. A, the expression of the c-Raf-1-BxB protein of SP-C-c-Raf-1-BxB-11 mice in different tissues has been analyzed by immunoblot experiments. Strong expression of the c-Raf-1-BxB protein was detected in the lung. In addition, we detected a weak expression of c-Raf-1-BxB kinase in kidney. B, the expression of the c-Raf-1 or c-Raf-1-BxB proteins was highly increased during tumor development in SP-C-c-Raf-1 and SP-C-c-Raf-1-BxB mice. The expression of the c-Raf-1 and c-Raf-1-BxB proteins has been analyzed by immunoblot experiments in lung tissues of the following: (a) a 5-month-old wild-type mouse with no tumor (wt, Lane 5); (b) a 3-month-old SP-C-c-Raf-1-BxB-23 mouse with no tumor (c-Raf-1-BxB-23, Lane 3); (c) an 8-month-old SP-C-c-Raf-1-BxB-23 mouse with tumors (c-Raf-1-BxB-23, Lane 8); (d) a 1-month-old SP-C-c-Raf-1-87 mouse with no tumor (c-Raf-1-87, Lane 1); (e) a 10-month-old SP-C-c-Raf-1-87 mouse with tumors (c-Raf-1-87, Lane 10); and (f) SP-C-c-Raf-1–87 isolated tumor tissue (c-Raf-1–87, Lane T). Equal protein loading was verified by Ponceau S staining of the blotted proteins. C, immunohistochemical analyses of c-Raf-1/c-Raf-1-BxB protein expression in lung tissue of wild-type mice and lung tumor tissue of SP-C-c-Raf-1-BxB-23 mice. Paraffin-embedded sections of lung tissues of wild-type mice (wt) and lung tumor tissue of SP-C-c-Raf-1-BxB-23 (BxB-23) mice were deparaffinized, and the expression of the c-Raf-1/c-Raf-1-BxB proteins was analyzed using c-Raf-1-specific antibodies (brown stain). The samples were counterstained with hematoxylin (blue stain).

 
To our surprise, mice transgenic for c-Raf-1 expression under the control of the SP-C promoter also develop lung adenomas. Compared with SP-C-c-Raf-1-BxB mice, the onset of tumor development is delayed. The earliest foci of transformed cells can be detected in SP-C-c-Raf-1 mice at about 7 months of age. The tumors are also lung adenomas and are histologically indistinguishable from those found in SP-C-c-Raf-1-BxB mice (Fig. 2)Citation . The incidence of transformation in the case of SP-C-c-Raf-1 mice is much lower. Lungs of those mice exhibit several isolated foci per lung, whereas the lungs of SP-C-c-Raf-1-BxB mice are covered with numerous foci (Fig. 2)Citation . Six of 11 SP-C-c-Raf-1 transgenic mouse strains did not develop lung cancer within 12–16 months of age (Table 1)Citation . Within the strains that develop lung cancer, the percentage of mice with lung adenomas ranged from 30–100% (Table 1)Citation . The delayed onset as well as the variable incidence of the lung adenomas in the different transgenic mouse strains strongly indicates that secondary mutations are necessary for c-Raf-1 to induce cell transformation. As in SP-C-c-Raf-1-BxB mice, the tumor tissue of SP-C-c-Raf-1 mice shows a strong amplification of c-Raf-1 protein expression (Fig. 3B)Citation , underlining the oncogenic character of c-Raf-1 overexpression. The nature of the high transgene expression in the tumor cells remains unclear. Future experiments analyzing the genomic structure of the tumor cells may reveal whether the increases in expression levels are due to gene or transgene amplification.

c-Raf-1 phosphorylates and activates the Mek protein, which subsequently phosphorylates and activates the Erk kinases. To analyze the activity of the Raf/Mek/Erk signal transduction pathway, we have compared the degree of Erk phosphorylation in wild-type, SP-C-c-Raf-1-87, and SP-C-c-Raf-1-BxB-23 mice (Fig. 4Citation ). Mice expressing the activated c-Raf-1-BxB show an increased level of Erk phosphorylation. Although the analyzed SP-C-c-Raf-1-87 transgenic mouse (age, 10 months) expressed elevated levels of the c-Raf-1 protein and developed a lung tumor, the level of the phosphorylated Erk was not increased compared with the phosphorylation found in the wild-type mouse (Fig. 4)Citation . The degree of Erk phosphorylation has been shown to correlate with tumor development. Tumor progression is highly reduced in the presence of agents inhibiting the Erk activator Mek (23) . In this respect, the lower Erk phosphorylation in the SP-C-c-Raf-1-87 mouse may reflect the longer latency found in the c-Raf-1 transgenic mice. The activation status of the c-Raf-1 kinase in SP-C-c-Raf-1 mice remains to be analyzed in more detail. In particular, the question of whether the c-Raf-1 transgenic mice exhibit a slightly increased Raf activity compared with wild-type mice and whether such a small difference can be sufficient for tumor development has to be analyzed.



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Fig. 4. Lung tumor tissue isolated from a SP-C-c-Raf-1-BxB-23 mouse exhibits increased Erk phosphorylation. Lung tissue of a wild-type mouse (wt) and lung tumor tissues of SP-C-c-Raf-1-BxB-23 (8 months old; c-Raf-1-BxB-23) and SP-C-c-Raf-1-87 (10 months old; c-Raf-1-87) mice were lysed, and the proteins were separated by SDS-PAGE. The amount of phosphorylated Erk proteins was determined by immunoblot using phospho-Erk-specific antibodies. The expression of the c-Raf-1 and c-Raf-1-BxB proteins was detected with c-Raf-1-specific antibodies. As a loading control, the amount of Cdk4 protein was determined with the help of Cdk4-specific antibodies.

 
In contrast to tissue culture experiments, our data show that the amplification of wild-type c-Raf-1 kinase is oncogenic and causes lung adenomas in mice transgenic for lung-targeted expression of the kinase. This emphasizes the possible role of the wild-type c-Raf-1 kinase in cancer development in man. Screening of a wide spectrum of different tumor types will be necessary to evaluate the importance of c-Raf-1 overexpression in naturally occurring tumors. Mice transgenic for the lung-specific expression of oncogenically activated c-Raf-1-BxB and for the wild-type c-Raf-1 kinase do not show deregulated cell proliferation in all epithelial cells expressing the transgenes. In both cases, we detected isolated foci of transformed cells instead of hyperproliferation of the entire tissue. This implies that the cells need further changes to transform. The constitutively activated c-Raf-1-BxB kinase generates more foci in a shorter time interval than the wild-type c-Raf-1 kinase, indicating that for the wild-type kinase to become oncogenic, additional cellular mutations are necessary. Oncogenic Ras cooperates with c-Raf-1 in transformation of tissue culture cells (12) . The Ki-ras gene is frequently mutated in lung tumors (24) and can therefore be regarded as a potential secondary cooperating mutation. Sequence analyses of genomic DNA isolated from tumor tissue of SP-C-c-Raf-1 (14 tumor samples analyzed) and SP-C-c-Raf-1-BxB (10 tumor samples analyzed) mice did not reveal any Ki-ras mutations in the frequently altered codons 12, 13, and 61 (data not shown). Future experiments will elucidate which additional mutations cooperate with wild-type c-Raf-1 compared with the mutations needed for activated c-Raf-1-BxB. Those results will be important to understand the mechanism by which the overexpression of wild-type c-Raf-1 protein acts as an oncogene.

Because of the long delay phase in the onset of c-Raf-1-induced tumorigenesis, the transgenic SP-C-c-Raf-1 mice provide a unique system to study environmental influences in lung cancer development and can now be used for the preclinical testing of novel cancer drugs.

Materials and Methods

Plasmids.
The SP-C-c-raf-1 vector was generated by releasing a 3-kb DNA human c-raf-1 cDNA fragment from pUC13-c-raf-1 (25) by digestion with the EcoRI restriction endonuclease and insertion of the c-raf-1 cDNA into the SP-C-3.7-SV40 plasmid (16) , which was linearized by EcoRI digestion. SP-C-3.7-SV40 contains the 3.7-kb promoter region of the human SP-C and a SV40 small T intron and polyadenylation signal. The SP-C-c-raf-1-BxB vector was generated by releasing a 1.4-kb c-raf-1-BxB cDNA fragment lacking sequences encoding the NH2-terminal regulatory domain of the c-Raf-1 kinase from the pKRSPA-c-raf-1-BxB vector (22) by ClaI/XbaI digestion. The c-raf-1-BxB fragment was inserted into the SP-C-3.7-SV40 plasmid, which was linearized by digestion with the EcoRI restriction endonucleases. The sticky ends of the vector and insert DNAs were blunt-ended before ligation.

Generation of Raf Transgenic Mice.
The expression cassette of the SP-C-raf-1 and SP-C-c-raf-1-BxB vectors was released by digestion with the NdeI and NotI restriction endonucleases. The resulting 7.1-kb (SP-C-raf-1) and 5.5-kb (SP-C-raf-1-BxB) fragments were purified by preparative agarose gel electrophoresis. Transgenic mouse lines were generated by injection of recombinant DNA fragments into fertilized eggs of C57BL/6xDBA-2 F1 mice from Harlan Winkelmann (Borchen, Germany). Live-born animals were analyzed for transgene integration by standard Southern blot and PCR techniques.

Histopathological Evaluation.
Animals were sacrificed and subjected to complete autopsy. Dissected tissue samples were fixed in 3.7% formaldehyde in PBS. Specimens were embedded in paraffin, sectioned at 4 µm, and stained with H&E.

Immunohistochemistry.
Paraffin-embedded sections were deparaffinized, rehydrated, and microwaved in 10 mM sodium citrate buffer (pH 5.5). After antigen retrieval, the samples were washed with water, incubated in 20% sucrose in PBS at 4°C for 30 min, washed with PBS, placed in blocking buffer (2.5% goat serum in PBS) for 40 min, and subsequently incubated in the presence of an anti-c-Raf-1 rabbit polyclonal antiserum (30K; Rapp Laboratory) at room temperature overnight. Antigen-antibody complexes were detected with an immunoperoxidase staining system (Vectastain ABC Kit; Vector Laboratories). The sections were counterstained with hematoxylin.

Immunoblot.
Frozen organs were homogenized in protein sample buffer [60 mM Tris-HCl (pH 6.8), 10% glycerol, 3% SDS, 5% 2-mercaptoethanol, and 0.005% bromphenol blue) with the help of an Ultra-Turrax blender. The protein lysates were separated by SDS-PAGE and blotted onto nitrocellulose membranes. The immunodetection was performed as described previously (26) . The following primary antibodies were used: (a) c-Raf-1, SP-63 (rabbit polyclonal antibody, 1:1000, Rapp Laboratory); (b) phospho-Erk, phospho-p44/p42 mitogen-activated protein kinase antibodies (rabbit polyclonal antibody, affinity purified, 1:400, New England Biolabs); and (c) Cdk4, C-22 (rabbit polyclonal antibody, affinity purified, 1 µg/ml, Santa Cruz Biotechnology).

Acknowledgments

We thank J. Whitsett for providing the SP-C-3.7/SV40 vector. We also thank A. Ganscher, O. Sakk, and E. V. Chernigovskaya for technical assistance and advice and S. Pfränger for excellent photographic reproduction.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Bundesministerium für Bilding, Wissenschaft, Forschung, und Technologie, Bonn, Germany (Interdisziplinäres Zentrum für Klinische Forschung, Erlangen, Teilprojekt B20; Vorhaben 01KV9504/7) and the Wilhelm Sander-Stiftung. E. K., L. M. F., and R. S. contributed equally to this work. Back

2 To whom requests for reprints should be addressed, at Institut für medizinische Strahlenkunde und Zellforschung, Universität Würzburg, Versbacher Strasse 5, D-97078 Würzburg, Germany. Phone: 49-931-201-5141; Fax: 49-931-201-3835. Back

3 The abbreviations used are: Mek, mitogen-activated protein/extracellular signal-regulated kinase kinase; SP-C, surfactant protein C; Erk, extracellular signal-regulated kinase; Cdk, cyclin-dependent kinase. Back

4 J. Troppmair and U. R. Rapp, unpublished observations. Back

Received for publication 11/ 8/99. Revision received 2/ 8/00. Accepted for publication 2/12/00.

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