Abstract
Mutations in Met have been identified in human papillary renal carcinomas. We have shown previously that these mutations deregulate the enzymatic activity of Met and that NIH 3T3 cells expressing mutationally activated Met are transformed in vitro and are tumorigenic in vivo. In the present investigation, we find that mutant Met induces the motility of Madin-Darby canine kidney cells in vitro and experimental metastasis of NIH 3T3 cells in vivo, and that the Ras-Raf-MEK-ERK signaling pathway, which has been implicated previously in cellular motility and metastasis, is constitutively activated by the Met mutants. We also report that transgenic mice harboring mutationally activated Met develop metastatic mammary carcinoma. These data confirm the tumorigenic activity of mutant Met molecules and demonstrate their ability to induce the metastatic phenotype.
The Met tyrosine kinase is a high-affinity receptor for hepatocyte growth factor/scatter factor (HGF/SF) (1, 2). Both Met and HGF/SF are expressed in numerous tissues in which their expression is confined predominantly to cells of epithelial and mesenchymal origin, respectively (3, 4). Signaling via this receptor–ligand pair is essential for normal murine embryological development (5–7) and has been shown to affect a wide range of biological activities including angiogenesis, cellular motility, growth, invasion, and morphogenic differentiation (reviewed in ref. 8).
In addition to mediating a variety of normal cellular processes, Met-HGF/SF signaling also has been implicated in the generation and spread of human tumors (reviewed in ref. 9). For example, Met was isolated originally as the product of a human oncogene, tpr-met, which encodes a constitutively dimerized/activated chimeric Met protein possessing transforming ability (10, 11). The generation of an autocrine loop as a result of the coexpression of wild-type Met and HGF/SF molecules in the same cell also is oncogenic (9). The tumorigenicity of both Tpr-Met and autocrine Met-HGF/SF signaling has been verified in transgenic mouse models (12, 13). Met activation also has been shown to promote the metastatic spread of cancer, a finding that likely is due to its stimulatory effects on a variety of processes such as angiogenesis, cellular motility, and protease secretion (9).
Recently, missense mutations in Met were found to be associated with human papillary renal carcinomas (14), and these mutations subsequently were shown to deregulate the enzymatic activity of this receptor, thereby unleashing its oncogenic potential (15). In the present investigation, we examined the effect of mutationally activated Met in various cell types and as a transgene in mice. We show that mutant Met induces the motility of Madin-Darby canine kidney (MDCK) cells and metastasis of NIH 3T3 cells and that transgenic mice expressing this oncogenic form of Met may develop metastatic mammary carcinoma.
MATERIALS AND METHODS
Cell Lines.
NIH 3T3 cells and culture conditions have been described (15). MDCK cells were obtained from Michael Stoker (Imperial Cancer Research Fund Cell Interactions Laboratory, Cambridge, U.K.) and maintained in DMEM (Life Technologies, Gaithersburg, MD) containing 10% fetal bovine serum (FBS; Life Technologies).
Constructs.
For expression in cultured cells, murine Met (38) or Trk-Met (ref. 18, a gift from Walter Birchmeier, Max-Delbruck-Center for Molecular Medicine, Berlin) cDNAs were subcloned into the pMex expression vector, which utilizes the Moloney murine sarcoma virus long terminal repeat promoter (39). For expression in transgenic mice, the murine Met cDNA was subcloned into expression vector 2999 (a gift from Richard Palmiter, Howard Hughes Medical Institute, University of Washington, Seattle), which contains the murine metallothionein-1 promoter flanked by 5′ and 3′ control regions (24). Site-directed mutagenesis was performed by using the QuikChange kit (Stratagene), and mutations were verified by sequencing both strands of DNA in the area of interest.
Transgenic Mouse Generation.
Metallothionein-Met constructs expressing mutationally activated Met were digested with KspI/ClaI, and the resulting ≈22-kb fragment was microinjected into fertilized eggs derived from C57BL/6NCR × C3H/HeNCR F2 mice (40). Founders were identified by dot blotting of tail DNA by using a probe derived from the polyadenylation region of the metallothionein construct.
Motility Assay.
Transfections were performed by using the calcium phosphate coprecipitation method (41). MDCK cells in 90-mm plastic dishes were cotransfected with 19 μg of the plasmid of interest plus 1 μg of a vector [pSV2Neo (42)] conferring resistance to G418 (Life Technologies). After 3 days the cells were split into 150-mm dishes containing DMEM/10% FBS supplemented with 800 μg/ml G418. The cultures were fed every 3–4 days, stained after 2 weeks, and examined microscopically for evidence of scattering.
Focus Formation Assay and Generation of Stable Cell Lines.
These procedures were performed as described (15). In some cases, culture media was supplemented with nerve growth factor (NGF; 2.5S, Boehringer Mannheim).
In Vivo Tumorigenicity and Experimental Metastasis Assay and Generation of Tumor Explants.
These procedures were performed as described (43).
Western Blotting.
Western analysis for Met, phosphotyrosine, and actin was performed as described (15, 44), as was that for ERK1/2 (21).
Northern Blotting.
RNA was prepared by using TRIzol according to the manufacturer’s protocol (Life Technologies) and resolved on an agarose-formaldehyde gel that was treated with 0.05 M NaOH before being transferred to supported nitrocellulose (Life Technologies) (45). The probe, which consisted of the entire murine Met cDNA (38), was labeled by random priming according to the manufacturer’s protocol (Boehringer Mannheim) and hybridized to the filter for 18 hr at 42°C. Filters then were washed to 0.2× SSC at 55°C and exposed to film.
RESULTS
Mutationally Activated Met Induces Metastasis of NIH 3T3 Cells.
We demonstrated previously that mutationally activated Met mediates the transformation and tumorigenicity of NIH 3T3 cells (15), and we wanted to determine whether this oncogenic form of Met also induced their metastasis. For this purpose, we utilized the experimental metastasis assay in which pools of G418-resistant NIH 3T3 cells expressing equal levels of wild-type or mutationally activated Met (15) are injected i.v. into the tails of nude mice, which subsequently are examined for evidence of lung metastasis. We found that cells expressing mutationally activated Met (M1268T) are highly metastatic in this assay, creating a severe lung metastasis burden in five of five animals sacrificed 3–4 weeks after injection; in contrast, cells expressing wild-type Met are much less metastatic, inducing a low metastatic burden in three of nine animals sacrificed 7–9 weeks after injection (Table 1).
Table 1.
Metastatic activity of mutationally activated Met in NIH 3T3 cells
| Met construct* | Metastasis†
|
|---|---|
| No. mice with lung metastasis/no. mice injected | |
| Control | 0/5 |
| Wild type | 3/9 |
| M1268T | 5/5 |
The control construct is the empty pMex expression vector; the wild-type construct encodes murine Met: M1268T encodes mutationally activated murine Met. Each Met molecule is in the pMex vector, which utilizes the Moloney murine sarcoma virus long terminal repeat promoter.
NIH 3T3 cells were cotransfected with the indicated construct plus pSV2 Neo, selected as pools of G418-resistant cells, and injected i.v. into the tail vein of nude mice at 5 × 105 cells per animal. Animals were sacrificed after 7–9 weeks (control and wild type) or 3–4 weeks (M1268T) and examined for lung metastasis. Animals positive for metastasis presented with low (cells expressing wild-type Met) or severe (cells expressing mutant Met) metastatic burden.
Mutationally Activated Trk-Met Induces in Vitro Transformation and in Vivo Tumorigenicity/Metastasis of NIH 3T3 Cells.
Although the activity of mutant Met is clearly distinguishable from that of wild-type Met in the experimental metastasis assay, NIH 3T3 cells expressing wild-type Met nonetheless are capable of a low degree of metastasis (Table 1; ref. 16), consistent with our finding that NIH 3T3 cells expressing wild-type Met are weakly tumorigenic (15, 17). Since the basal level of metastasis induced by wild-type Met potentially could interfere with an analysis of weakly activating Met mutations, we wanted to utilize a system in which the background level of metastasis induced by wild-type Met was zero. It is likely that the metastasis mediated by wild-type Met is due to the generation of an autocrine Met-HGF/SF loop, since NIH 3T3 cells make HGF/SF (ref. 17 and data not shown), and autocrine signaling by this receptor–ligand pair has been shown to induce the metastasis of a number of cell types (9). To circumvent this problem, we introduced the activating mutations into the Met portion of a chimeric molecule composed of the extracellular domain of the NGF receptor (Trk) fused to the intracellular domain of Met (18). The wild-type Trk-Met chimera has been shown to faithfully mediate Met-associated biological activities in response to NGF (18), and since NIH 3T3 cells do not express NGF (19), receptor activation due to autocrine ligand stimulation should not occur.
Before assessing the metastatic ability of cells expressing mutationally activated Trk-Met, we analyzed their transforming and tumorigenic properties (Table 2). In the focus formation assay, we found that the wild-type Trk-Met construct generated approximately the same basal number of foci as did the empty vector control (13 vs. 8 foci/μg DNA, respectively), while Trk-Met harboring each of four independent, activating mutations generated significantly more foci (from 68 to >300 foci/μg DNA, depending on the mutation). When performed in the presence of exogenous NGF, all of the Trk-Met constructs (including wild type) generated a large number of foci (>300 foci/μg DNA), whereas the number of foci produced by empty vector control did not increase over background levels (Table 2). This result confirms that autocrine Met stimulation is transforming for NIH 3T3 cells and serves to control for the integrity of the wild-type Trk-Met construct, and it indicates that mutationally activated Met molecules reside in a partially activated state that can be enhanced further through ligand stimulation.
Table 2.
Activity of mutationally activated Trk-Met in NIH 3T3 cells
| TRK-MET construct* | Focus formation† (no. foci/μg DNA)
|
Tumorigenicity‡
|
Metastasis§
|
||
|---|---|---|---|---|---|
| NGF (−) | NGF (+) | No. mice with tumors/ no. mice injected | Mean tumor size in mm2 | No. mice with lung metastasis/ no. mice injected | |
| Control | 8 | 5 | 0/5 | 0 | 0/5 |
| Wild type | 13 | >300 | 0/5 | 0 | 0/5 |
| M1268T | >300 | >300 | 5/5 | 245 | 4/4 |
| L1213V | 74 | >300 | 5/5 | 133 | 4/4 |
| Y1248H | 105 | >300 | 5/5 | 257 | 5/5 |
| D1246H | 68 | >300 | 5/5 | 99 | 4/4 |
The control construct is the empty pMex expression vector; the wild-type construct encodes murine Met; M1268T, L1213V, Y1248H, and D1246H encode mutationally activated Met. Each Met molecule is in the pMex vector, which utilizes the Moloney murine sarcoma virus long terminal repeat promoter.
NIH 3T3 cells transfected with the indicated constructs were scored for focus formation after approximately 2 weeks. Samples receiving NGF were treated with this ligand at 100 ng/ml throughout the duration of the assay. Similar results were obtained in two independent experiments.
NIH 3T3 cells were cotransfected with the indicated construct plus pSV2 Neo, selected as pools of G418-resistant cells, and injected s.c. into nude mice at 8 × 105 cells per animal. Tumors were measured after 17 days. Mice injected with cells expressing wild-type Trk-Met were tumor-free even after 2 months.
NIH 3T3 cells were cotransfected with the indicated construct plus pSV2 Neo, selected as pools of G418-resistant cells, and injected i.v. into the tail vein of nude mice at 8 × 105 cells per animal. Animals were sacrificed after 4 weeks (or earlier for animals in distress) and examined for lung metastasis. All animals positive for metastasis presented with severe metastatic burden. Mice injected with cells expressing wild-type Trk-Met were metastasis-free even after 2 months.
NIH 3T3 cells stably expressing wild-type or mutationally activated Trk-Met were generated and characterized (Fig. 1). When lysates from these cells were blotted with anti-Met antibody, a doublet specific for cells transfected with the Trk-Met constructs was evident (Fig. 1A, top blot). The high- and low-molecular-mass species of the doublet represent mature and precursor Trk-Met protein, respectively (data not shown). Reblotting of the filter with antiphosphotyrosine antibody (second blot from top) demonstrates that mutationally activated Trk-Met proteins (lanes 3–6) exhibit enhanced autophosphorylation relative to wild-type Trk-Met (lane 2), consistent with previous results using nonchimeric Met constructs (15). Reprobing with an anti-actin antibody (third blot from top) shows that an equal quantity of protein was analyzed for each sample.
Figure 1.
Western analysis of NIH 3T3 cells expressing mutationally activated Trk-Met. (A) Samples labeled control and wild type are from cells stably transfected with empty vector or vector expressing wild-type Trk-Met, respectively. All other samples are from cells stably transfected with vectors expressing Trk-Met harboring the indicated activating Met mutation. Cells were cultured in DMEM/10% CS supplemented with 800 μg/ml G418, and 50 μg of cell lysate/sample was resolved on a 4–15% gel and examined under nonreducing conditions (top three blots). Cells were cultured in DMEM/1% CS for 24 hr, and 10 μg of cell lysate/sample was resolved on a 10% gel and examined under reducing conditions (bottom two blots). The following primary antibodies were used (top to bottom): anti-Met, antiphosphotyrosine, anti-actin, antiactive (i.e., phosphorylated) ERK1/2, and anti-ERK1/2. (B) Samples labeled with a “P” are from a pool of cells stably transfected with Trk-Met harboring the indicated activating Met mutation, which were used as the inoculum for tumor induction. Samples labeled with a “T” are explanted tumors derived from cells expressing the indicated Trk-Met mutant. Cells were cultured in DMEM/10% CS supplemented with 800 μg/ml G418, and 50 μg cell lysate/sample was resolved on a 4–15% gel and examined by Western analysis under nonreducing conditions using anti-Met (Upper) or anti-actin antibody (Lower). Molecular mass markers are indicated on the left.
Since the Ras-Raf-MEK-ERK pathway has been implicated in motility, transformation, invasion, and metastasis (20–23), we investigated whether mutationally activated Trk-Met affected this signaling pathway. When lysates from NIH 3T3 cells expressing the various Trk-Met molecules were blotted with antiactive (i.e., phosphorylated) ERK1/2 antibody, we found that cells expressing mutationally activated Trk-Met exhibit elevated levels of active ERK1/2 relative to cells expressing wild-type Trk-Met or control cells transfected with empty vector (Fig. 1A, fourth blot). Reprobing of the filter with anti-ERK1/2 antibody shows that all cells express approximately equal levels of total ERK1/2 protein (Fig. 1A, bottom blot).
After s.c. injection into nude mice, NIH 3T3 cells stably expressing wild-type Trk-Met are nontumorigenic throughout a 2-month observation period (Table 2), thereby eliminating the background tumorigenicity exhibited by NIH 3T3 cells expressing nonchimeric wild-type Met (15, 17). In contrast, cells expressing mutationally activated Trk-Met are highly tumorigenic, giving rise to tumors in all animals 1–2 weeks after injection. Tumor measurements taken at 17 days postinjection show that there is a good correlation between focus-forming ability in vitro and tumor-forming ability in vivo (Table 2). Cultured explants from these tumors continue to express activated Trk-Met and do so at a level greater than that of parental cells comprising the respective tumor inoculums (Fig. 1B).
Having established that the Met mutations are activating in the context of the Trk-Met chimera, we next examined the metastatic potential of the various Trk-Met-expressing NIH 3T3 cells by using the experimental metastasis assay (Table 2). We found that cells expressing wild-type Trk-Met are incapable of inducing metastasis, while cells expressing mutationally activated Trk-Met are highly metastatic. Differences among the various Met mutants with regard to metastatic potential were not evident since cells expressing each of the mutants induced severe metastatic burden by 4 weeks after injection. These results unambiguously demonstrate that mutationally activated Trk-Met molecules are capable of inducing the metastatic phenotype.
Mutationally Activated Met Induces “Scattering” (Motility) in MDCK Cells.
To test whether mutationally activated Met mediates cellular motility, an activity closely associated with the metastatic phenotype, MDCK cells were utilized. These cells grow as compact, multicellular colonies (illustrated in Fig. 2B) that disperse (i.e., become scattered/motile) in response to Met-HGF/SF signaling (3). To assess the ability of mutationally activated Met to induce their spontaneous motility, MDCK cells were cotransfected with a vector conferring resistance to G418, together with a vector expressing wild-type or mutationally activated nonchimeric Met. After a 2-week selection in G418, the percentage of colonies exhibiting a “scattered” (motile) phenotype (illustrated in Fig. 2C) was recorded (Fig. 2A). Our findings indicate that each of the three Met mutants examined generates a significantly higher percentage of scattered colonies than does wild-type Met, with the most activating Met mutation (M1268T) exhibiting a 3-fold increase. These data also demonstrate that the Met mutants are active in cells other than NIH 3T3. We suspect that the relatively low percentage of colonies exhibiting the scattered phenotype after transfection with even the most strongly activated Met molecule (21% with Met mutant M1268T) may be due to the low percentage of G418-resistant colonies expressing exogenous Met after cotransfection.
Figure 2.
“Scattering” (motility) of MDCK cells expressing mutationally activated Met. Cells were cotransfected with a vector conferring resistance to G418, together with a vector encoding wild-type or mutationally activated Met, and selected in G418. (A) The percentage of colonies exhibiting a motile/scattered phenotype was recorded. The results represent the mean of two independent experiments in which a minimum of 100 colonies/sample were examined. (B) A representative nonscattered parental colony. (C) A representative “scattered” (motile) colony induced by mutationally activated Met (L1213V).
Mutationally Activated Met Induces Metastatic Mammary Adenocarcinoma in Transgenic Mice.
We next tested the mutant Met molecules for activity in transgenic animals. To this end, two independent, strongly activating mutations (M1268T and Y1248H) were expressed in transgenic mice under the control of the metallothionein promoter (24). Before being used for the generation of transgenic mice, the constructs were expressed in NIH 3T3 cells, which subsequently were assessed for tumorigenicity in nude mice (Table 3). We found that cells expressing either Met mutant under the control of the metallothionein promoter were highly tumorigenic (1- to 2-week latency), while cells expressing wild-type Met from this promoter were only weakly tumorigenic (4- to 5-week latency). The metallothionein-Met constructs give rise to an mRNA transcript of the predicted size of ≈5 kb in NIH 3T3 cells, which is easily distinguished from the endogenous murine Met transcript of ≈7.5 kb (Fig. 3A).
Table 3.
Tumorigenic activity of mutationally activated Met in NIH 3T3 cells under the control of the metallothionein promoter
| MET construct* | Tumorigenicity†
|
|
|---|---|---|
| No. mice with tumors/no. mice injected | Mean tumor size in mm2 | |
| Wild type | 0/5 | 0 |
| M1268T | 5/5 | 234 |
| Y1248H | 5/5 | 238 |
The wild-type construct encodes murine Met; M1268T and Y1248H encode mutationally activated murine Met. Each Met molecule is in vector 2999, which utilizes the metallothionein promoter.
NIH 3T3 cells were cotransfected with the indicated construct plus pSV2 Neo, selected as pools of G418-resistant cells, and injected s.c. into nude mice at 5 × 105 cells per animal. Tumors were measured after 19 days. Mice injected with cells expressing wild-type Met developed tumors at 4–5 weeks.
Figure 3.
Transgene expression in mammary carcinomas induced by mutationally activated Met. Fifteen micrograms of total RNA/sample was resolved on a 1.2% agarose-formaldehyde gel and examined by Northern analysis by using an anti-Met probe. (A) RNA was obtained from parental NIH 3T3 cells (lane 1) or NIH 3T3 cells stably transfected with wild-type Met under the control of the metallothionein promoter (lane 2). Note the appearance of an ≈5-kb Met-reactive transcript [designated Met transgene (TG)] only in transfected cells. The ≈7.5-kb transcript represents endogenous Met (38). (B) RNA was obtained from normal murine kidney (lane 1) or mammary tumors (MT) from founder transgenic animals harboring the indicated activating Met mutation (lanes 2 and 3). Note the strong expression of the Met transgene (TG) in tumor tissue relative to endogenous Met in these samples and to normal kidney [a tissue that expresses high levels of Met (38)]. The migration of 28S and 18S ribosomal RNA is indicated on the left.
Founder animals harboring each metallothionein–mutant Met construct were selected and mated with nontransgenic partners (two males and two females for construct Y1248H; two males and four females for construct M1268T). Most founders exhibited severe breeding difficulties and either had no offspring over many months of continual breeding or did not transmit the transgene. However, two 10-month-old female founder animals, one expressing Met mutant M1268T and the other expressing mutant Y1248H, developed overt tumors that were diagnosed as type B mammary adenocarcinomas (Fig. 4 and data not shown). Northern analysis showed strong Met transgene expression in both mammary tumors (Fig. 3B). Moreover, both of these tumors exhibited metastasis, with the tumor derived from the Y1248H founder metastasizing to the lung, lymph node, kidney, heart (Fig. 4 C– F, respectively), and cecum (not shown), and the tumor from the M1268T founder metastasizing to the lung (not shown). Immunostaining confirmed that the primary tumor and metastasis depicted in Fig. 4 strongly express the Met receptor (not shown).
Figure 4.
Histopathology of mammary carcinomas and metastasis induced by mutationally activated Met in transgenic mice. Tissues were paraffin-embedded, sectioned, stained with hematoxylin and eosin, and photographed at ×80, 33, 66, 33, 50, and 25 (A–F, respectively). (A and B) Primary mammary carcinoma. (C) Metastasis (METS) to the lung (LG). Note the alveoli at the bottom of the photograph. (D) Metastasis to the lymph node (LN). (E) Metastasis to the kidney (KD). Note the constriction of the renal artery (RA) by the metastasis, leading to kidney infarct. (F) Metastasis to the heart (HT). Note the presence of metastatic cells in a thrombus (TH) lodged in the left ventricle. All samples shown are from a female founder expressing Met mutant Y1248H.
We also pathologically examined some of the remaining founders at approximately 1 year of age. One male from each construct and one female from construct M1268T exhibited no significant pathology, while one female from construct M1268T exhibited mammary hyperplasia (not shown). Some founders were not examined because they were found dead and their state of decomposition precluded pathological analysis.
We also generated founder animals harboring the metallothionein–wild-type Met construct and mated them to nontransgenic partners. In contrast to the results obtained with mutationally activated Met, these founders exhibited normal breeding behavior and transmitted the Met transgene to roughly one-half of their offspring. In addition, no mammary tumors or hyperplasia were found after the pathological examination of the original founders at 10–12 months of age, nor have any mammary tumors been observed or diagnosed in transgenic female offspring (n = 21 between the ages of 8 and 12 months), some of which (n = 7) have been in continual mating for many months.
DISCUSSION
We have demonstrated that mutationally activated Met or Trk-Met induces the experimental metastasis of NIH 3T3 cells in nude mice. During the metastatic process, cells detach from a primary tumor, enter and exit the circulatory system, and begin growing in a secondary location (reviewed in ref. 25). The experimental metastasis assay we used is thought to simulate the later stages of this pathway. In addition to activities such as angiogenesis and protease secretion, the metastatic process is believed to require cell motility. Thus, our finding that mutationally activated Met stimulates the motility of MDCK cells is consistent with its metastasis-promoting capability. Moreover, this demonstrates that the Met mutants are active in cells other than NIH 3T3.
Not all oncogenes enhance metastatic potential. For example, NIH 3T3 cells transformed by Myc or p53 are tumorigenic but nonmetastatic in the experimental metastasis assay (26), while NIH 3T3 cells transformed by tyrosine kinases, including Tpr-Met or autocrine Met-HGF/SF (9, 26), are both tumorigenic and metastatic. Recently, work from our laboratory using Ras effector-domain mutants has shown that experimental metastasis in NIH 3T3 cells appears to require the constitutive activation of ERK1/2 (21). ERK1/2 also has been implicated in the processes of cell motility and invasion (20, 22). Thus, our finding that ERK1/2 is stimulated in cells expressing mutationally activated Met is consistent with the Ras-Raf-MEK-ERK signaling pathway, playing an important role in metastasis mediated by mutant Met molecules. This pathway also has been shown to be activated by Tpr-Met and ligand-activated wild-type Met (27–29).
Our finding that the Met mutations are activating in the context of the Trk-Met chimera, as evidenced by the ability of mutationally activated Trk-Met to induce the transformation, tumorigenicity, and metastasis of NIH 3T3 cells, demonstrates that the extracellular portion of Met is dispensable for the biological activity of the Met mutants. However, it should be noted that the efficiency of NIH 3T3 focus formation by mutationally activated Trk-Met is somewhat reduced relative to nonchimeric Met (15), and the foci are generally smaller (data not shown). The reason for this phenomena is unknown, but could be because of the production of endogenous HGF/SF by NIH 3T3 cells. Nevertheless, the relative biological activity of the individual mutations remains unchanged in the context of Trk-Met (Table 2) or nonchimeric Met (15) (i.e., M1268T > Y1248H > D1246H). We have demonstrated previously that the biological potency of the individual Met mutations in the context of nonchimeric Met correlates with enzymatic activity (15), and the same is likely to be true for the Met mutations in the context of Trk-Met.
The finding that mutationally activated Trk-Met is biologically active in NIH 3T3 cells, which apparently do not make NGF (Table 2; ref. 19), supports the hypothesis that this molecule and, by analogy, nonchimeric Met do not require ligand for activity. Consistent with this hypothesis, we found that mutationally activated nonchimeric Met is biologically active in MDCK cells, which do not produce HGF/SF (3). However, since the transforming activity of mutationally activated Trk-Met is increased by ligand (Table 2), activated Met molecules apparently reside in a partially activated state that can be enhanced further through ligand stimulation.
A role for mutationally activated Met in tumorigenicity and metastasis is supported by our finding that transgenic founder animals expressing mutant Met develop metastatic mammary carcinoma. The tumors analyzed in the present report are very likely to be a consequence of the Met transgene for the following reasons. First, our transgenic mice are derived from crosses between C57BL/6 and C3H mice, which exhibit a very low incidence of spontaneous mammary carcinoma (30), and we have not found any mammary carcinomas in founders or transgenic offspring from control mice harboring the wild-type Met transgene. Second, the tumors from both founders strongly express the mutant Met transgene. Third, mammary carcinoma is the predominant form of malignancy found in female transgenic mice expressing the Tpr-Met oncogene (13), or HGF/SF (12) under the control of the same promoter (i.e., metallothionein) utilized in the present investigation. While neither of those reports mentions the presence of metastasis, the metastatic spread of murine mammary carcinoma has been observed previously (31, 32). Although the incidence of metastasis from murine mammary carcinoma often is quite low, systems have been described in which mammary tumors are metastatic (31, 32).
How do our findings relate to human malignancy? Thus far, activating Met mutations have been found only in papillary renal carcinoma (14). Although not highly vascularized, these tumors do metastasize (33, 34), a finding that is consistent with our results that implicate Met in the metastatic process. In addition, a couple of patients with Met-mediated hereditary papillary renal carcinoma developed carcinoma of the breast (35). While the role of activated Met in the development of these secondary tumors is unknown currently, HGF/SF-Met signaling has been implicated previously in human breast carcinoma (I. Tsarfaty, W. G. Alvord, J. Resau, R. T. Altstock, R. Lidereau, I. Bieche, F. Bertrand, J. Horev, R. L. Klabansky, I. Keydar, and G.F.V.W., unpublished results; refs. 36 and 37), and our transgenic model suggests that mammary tissue may be particularly susceptible to the oncogenic effects of mutationally activated Met. Thus, the mutant Met transgenic mice described in this report may be a relevant model system for studying Met-induced human breast carcinoma and may prove useful for evaluating therapeutics aimed at inhibiting Met signaling.
Acknowledgments
We thank Richard Palmiter and Walter Birchmeier for the metallothionein and Trk-Met expression vectors, respectively; Linda Miller, Jim Resau, Eric Hudson, Marilyn Powers, Leo Lee, Terry Sweeney, Oscar Smith, Deborah Swing, Bryn Eagleson, and Lisa Secrest for technical support; Richard Frederickson for performing the artwork and photography; and Ave Cline for typing the manuscript. This research was sponsored in part by the National Cancer Institute, Department of Health and Human Services, under contract with Advanced BioScience Laboratories, and in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract N01-CO-56000.
ABBREVIATIONS
- MDCK
Madin-Darby canine kidney
- NGF
nerve growth factor
- HGF/SF
hepatocyte growth factor/scatter factor
Footnotes
This paper was submitted directly (Track II) to the Proceedings Office.
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