Hereditary papillary renal carcinoma (HPRC) is characterized by multiple, bilateral papillary renal carcinomas. Previously, we demonstrated missense mutations in the tyrosine kinase domain of the MET proto-oncogene in HPRC and a subset of sporadic papillary renal carcinomas. In this study, we screened a large panel of sporadic papillary renal carcinomas and various solid tumors for mutations in the MET proto-oncogene. Summarizing these and previous results, mutations of the MET proto-oncogene were detected in 17/129 sporadic papillary renal carcinomas but not in other solid tumors. We detected five novel missense mutations; three of five mutations were located in the ATP-binding region of the tyrosine kinase domain of MET. One novel mutation in MET, V1110I, was located at a codon homologous to an activating mutation in the c-erbB proto-oncogene. These mutations caused constitutive phosphorylation of MET when transfected into NIH3T3 cells. Molecular modeling studies suggest that these activating mutations interfere with the intrasteric mechanism of tyrosine kinase autoinhibition and facilitate transition to the active form of the MET kinase. The low frequency of MET mutations in noninherited papillary renal carcinomas (PRC) suggests that noninherited PRC may develop by a different mechanism than hereditary papillary renal carcinoma.
The MET proto-oncogene encodes a receptor tyrosine kinase that is widely expressed in epithelial cells; the ligand for the MET receptor is hepatocyte growth factor/scatter factor (HGF/SF). Increased expression of the MET proto-oncogene has been found in a number of human cancers implicating MET in their pathogenesis (Jeffers et al., 1996; Di Renzo et al., 1995). These studies have not determined whether the MET proto-oncogene was involved in the origin or progression of these neoplasms. The recent demonstration of germline and somatic mutations in the MET proto-oncogene in papillary renal carcinomas directly implicated the MET gene in the pathogenesis of papillary renal cancer. Schmidt et al. (1997) found missense mutations in the tyrosine kinase domain of the MET proto-oncogene in families with papillary renal carcinomas and in a subset of sporadic papillary renal carcinomas. Studies by Jeffers et al. (1997) demonstrated that the observed mutations in MET produced constitutive phosphorylation of the MET protein, and malignant transformation of NIH3T3 cells.
To determine the frequency of MET proto-oncogene mutations in sporadic papillary renal carcinomas, to determine whether mutations in the MET proto-oncogene play a role in the pathogenesis of other solid tumors, and to identify additional mutations, we performed mutation analysis on a large panel of sporadic papillary renal carcinomas and various solid tumors. The results indicate that mutations in the MET proto-oncogene are uncommon in sporadic papillary renal carcinomas and not detected in the panel of solid tumors. Five novel missense mutations in MET were detected. Molecular modeling studies were performed to predict the effect these mutations would have on the regulation of the kinase activity of the MET protein.
Frequency of detection of MET mutations in sporadic papillary renal carcinomas and metanephric adenomas
We tested 129 sporadic papillary renal carcinomas, and two metanephric adenomas for mutations in the MET gene. The sporadic papillary renal carcinoma panel included 79 matched normal and tumor DNAs, seven papillary renal carcinoma cell lines, and 43 paraffin-embedded or frozen tumor samples from patients with multiple or single papillary renal carcinomas and no family history of renal neoplasia. Summarizing these and previously reported data (Schmidt et al., 1997, 1998), MET mutations were detected in 17/129 (13%) sporadic papillary renal carcinoma samples (Table 1). Eight of the 17 apparent sporadic MET mutations were germline, even though there was no known family history of renal cancer in these patients.
Novel mutations in the MET proto-oncogene
In this study we identified five novel missense mutations in the MET proto-oncogene in the sporadic papillary renal carcinoma panel. Mutations and clinical details describing these patients are summarized in Table 2.
We identified three apparently unrelated individuals with papillary renal carcinoma (patients 5946, 5274 and 6088) and the identical germline MET proto-oncogene mutation changing a valine to an isoleucine at codon 1110. The patients with the V1110I mutation were from the United States, Sweden and Germany. The V1110I change was not detected in 300 normal chromosomes making it unlikely that this change was a rare polymorphism. It was not possible to test whether the mutation segregated with the disease because there was only one affected individual in each family. The V1110I mutation was located in the highly conserved, glycine-rich, ATP-binding region of the tyrosine kinase domain. Previously, an activating mutation was detected at an homologous codon in the chicken proto-oncogene, c-erbB (V157I) (Figure 1) (Shu et al., 1990, 1991, 1994). The amino acid changes, valine→isoleucine, were identical in the MET and c-erbB mutations.
MET H1112L and H1112Y
Previously, a mutation in MET codon H1112 (H1112R) was found to predispose to HPRC in two large North American families (Schmidt et al., 1998). In this study, we identified two additional missense mutations in codon 1112, H1112L and H1112Y. MET mutation H1112Y was detected in the germline of patient 6285 and as a somatic change in the papillary renal carcinoma of patient 5458. MET mutation H1112L was detected as a somatic change in the papillary renal carcinoma of patient 6134. The H1112L and H1112Y mutations were not detected in 300 normal chromosomes making it unlikely that these changes were rare polymorphisms. The H1112 mutations were immediately adjacent to the ATP-binding domain (GXGXXGXV) and the V110I mutation (Figure 1).
A new mutation in codon 1124, H1124D, was identified in a sporadic papillary renal tumor from archival tissue. (Patient 6052). No accompanying normal was available for comparison. This change was not observed in 300 normal chromosomes. The location of this mutation is in proximity to the conserved, ATP binding region and adjacent to two highly conserved amino acids, alanine 1126 and lysine 1128 (Figure 1).
Missense mutations were detected previously in MET codon 1248. (Y1248C and Y1248H) (Schmidt et al., 1997). In this study, we detected another germline mutation in codon 1248, Y1248D, in patient 6059, which was not found in 300 normal chromosomes.
A MET mutation in a renal papillary neoplasm
Metanephric adenoma is a recently recognized, benign, papillary renal tumor characterized by trisomy of chromosomes 7 and 17 (Brown et al., 1997; Grignon and Eble, 1998). No MET mutations were detected in two metanephric adenomas analysed in this study. A third sample, 6046, a renal papillary neoplasm, was examined histologically by three pathologists who disagreed on the diagnosis of metanephric adenoma. MET mutation Y1248C was detected in sample 6046. Normal DNA was not available for comparison.
A French patient, 6067, with papillary renal carcinoma and no known family history of kidney cancer, was found to have a germline MET mutation, V1238I, previously identified in a Spanish HPRC family (Schmidt et al., 1997).
A MET polymorphism, T1010I
We detected a C to T sequence change at nt 3223 (T1010I) in exon 14 in the papillary renal carcinoma cell line ACHN (Borden et al., 1982), and in the germline of a patient with a family history of papillary renal carcinoma. This change was located in the juxtamembrane region of the intracellular portion of MET. The T1010I change did not segregate with the disease (inherited from the unaffected parent) and may represent a rare polymorphism. The amino acid change T1010I was not found in 150 normal chromosomes.
Evaluation of various human tumors for mutations in the MET proto-oncogene
Previously, we observed that some patients with a germline MET mutation (H1112R) had other nonrenal malignancies (pancreatic carcinoma, malignant melanoma, carcinoma of the common bile duct) (Zbar et al., 1994; Zbar and Lerman, 1998, in press; Schmidt et al., 1997, 1998). Other investigators had found that breast, prostate and ovarian carcinomas were characterized by a loss of heterozygosity that mapped to chromosome 7q31, the region containing the MET proto-oncogene (Lin et al., 1996; Zenklusen et al., 1994, 1995; Huang et al., 1998). These studies raised the possibility that mutations in the MET proto-oncogene might play a role in the pathogenesis of certain solid tumors.
We screened 199 sporadic human tumors for mutations in the tyrosine kinase domain of the MET proto-oncogene (Table 1) by SSCP or DHPLC. Gliomas, prostate and Wilms' tumors were selected for study because they have been found to have trisomy of chromosome 7, a genetic change consistently found in papillary renal carcinomas. Malignant melanoma, pancreatic cancer and colon cancer were tested because they have been found as secondary malignancies in some HPRC family members. In addition, we tested tumors from bladder and thyroid, neoplasms which overexpress MET. No mutations in the MET proto-oncogene were detected in these samples.
Functional studies of novel mutations in the MET proto-oncogene
Previously, we demonstrated that missense mutations in the tyrosine kinase domain of the MET proto-oncogene, when introduced into mouse MET cDNA, caused constitutive phosphorylation of the MET protein and malignant transformation of NIH3T3 cells (Jeffers et al., 1997). In this study we evaluated the ability of the MET mutations described in this report to cause constitutive phosphorylation of MET and malignant transformation of NIH3T3 cells.
Western analysis of the mutant MET transfected NIH3T3 cell lines is shown in Figure 2. Constitutive phosphorylation of MET protein was stimulated by the V1110I, H1112L and H1124D mutations at levels comparable to the weakly activating MET mutant, M1149T, described by Jeffers et al. (1997) and included for comparison (Figure 2a). The Y1248D and H1112Y mutations resulted in intermediate levels of constitutive phosphorylation, comparable to Y1248H described by Jeffers et al. (1997) (Figure 2a). No phosphorylation of MET was seen in the cell lines transfected with wild type MET or empty vector. Western analysis of the mutant MET transfected NIH3T3 cell lines revealed MET protein expression at levels comparable to wild type for all five novel mutations (Figure 2b).
These new MET mutations, when introduced into mouse MET cDNA and transfected into NIH3T3 cells, stimulated focus formation. Mutants Y1248D, V1110I, H1112Y, H1112L and H1124D produced an average of 2, 12, 30, 36 and 36 foci per μg DNA, respectively, from three separate experiments (data not shown). No foci were produced in transfections with the wild type MET construct or empty vector. It should be noted that when MET T1010I, thought to be a rare polymorphism, was introduced into NIH3T3 cells, neither focus formation nor constitutive MET phosphorylation was observed (data not shown).
Predicted effect of MET mutation on the activation of the MET protein based on molecular modeling studies
To study the mechanism by which missense mutations in the MET proto-oncogene produce constitutive activation, we constructed a three dimensional model (3-D) of the catalytic core of the MET tyrosine kinase domain (amino acid residues 1096 – 1355) based on the crystal structure of the unphosphorylated form of the insulin receptor (IRK) (Hubbard et al., 1994). Sequence identity of 40% in this region and the presence of three critical tyrosine residues in the activation loop of these two receptors suggests that the IRK structure should be a good approximation of MET. The model can be superimposed on the structure of IRK with the root mean square difference (rmsd) of 0.66 Å between 249 equivalent C-α pairs. The tyrosine kinase domain is divided into amino terminal and carboxy terminal lobes separated by a large cleft (Figure 3a). In the catalytically inactive, unphosphorylated form, access to the ATP and substrate binding sites is blocked by the activation loop. Y1252 is hydrogen bonded to the catalytic aspartic acid, D1222, while the side chain of Y1253 resides in a substrate binding pocket formed mainly by residues from the P+1 loop. F1241 from the kinase conserved triplet DFG occupies the adenine binding pocket, and together with G1242 create a steric hindrance which forces the two lobes apart. This conformation of the activation loop is further stabilized by hydrophobic interactions of the phenolic ring of Y1248 with the alkyl portions of arginines 1226 and 1188 (Figure 3b) and hydrogen bonds through its hydroxyl group to A1182 and N1185 (data not shown).
The proposed mechanism of autoregulation of kinase activity assumes that the activation loop can adopt an alternative conformation which allows for the transition to the active form of the enzyme (Hubbard et al., 1994). Trans-phosphorylation of tyrosines in the activation loop strongly shifts the equilibrium toward the active conformation.
Mutations may activate the MET kinase by destabilizing the inhibitory conformation of the activation loop. For example, as shown in Figure 3b, the replacement of valine 1110 with isoleucine results in a steric clash with phenylalanine 1241 forcing it out of the adenine binding pocket, and releasing the steric hindrance which prevents rearrangement of the two lobes. Substitution of the phenolic ring of the buried Y1248 by polar side chains of aspartic acid, histidine or cysteine would also strongly favor the activated form (Figure 3b). As revealed by the crystal structure of the activated IRK, the phosphotyrosine at this codon is fully exposed to the cytosol (Hubbard, 1997).
We screened a panel of sporadic papillary renal carcinomas and solid tumors to determine the frequency of mutation of the MET proto-oncogene in sporadic papillary renal carcinoma and in various nonrenal tumors, and to identify novel mutations in the MET proto-oncogene. Summarizing these and our previous results, we detected a total of 17 mutations in 129 (13%) sporadic papillary renal carcinomas and 0 mutations in two metanephric adenomas and 199 solid tumors (Tables 1 and 3).
In our previous report, mutation analysis was concentrated in exons 16 – 19 of the tyrosine kinase domain (Schmidt et al., 1997). However it was possible that we failed to detect MET mutations located outside the tyrosine kinase domain. Therefore, the cysteine-rich extracellular domain (exons 5 and 7), the transmembrane region (exon 13) and intracellular portion of the protein (exons 14 – 20), were sequenced in their entirety in 79 sporadic papillary renal carcinomas and exons 14 – 19 were sequenced in 43 paraffin block DNAs and seven papillary renal cell lines. The results of this study suggest that MET mutations occur in only a small proportion of sporadic papillary renal carcinomas. Independent studies suggest that sporadic papillary renal carcinoma is genetically heterogeneous, and may also be caused by mutations in the TFE3 and PPRC genes (Sidhar et al., 1996; Weterman et al., 1996).
Trisomy 7 is a characteristic of papillary renal carcinomas, other neoplasms and some normal tissues (Kovacs, 1993; Ermis et al., 1995). Recently, studies of tumors in patients with hereditary papillary renal carcinoma showed that the chromosome 7 carrying the inherited mutant MET allele was duplicated in these tumors (Fischer et al., 1998; Zhuang et al., 1998). This observation raised the possibility that the MET gene might be mutated and duplicated in tumors characterized by trisomy 7. Our data which show MET mutations in 13% of sporadic papillary renal carcinomas and data of Kovacs (1993), showing trisomy 7 in 95% of papillary renal carcinomas indicate that most papillary renal carcinomas are characterized by trisomy 7 without MET mutations. Since polysomy of chromosome 7 has been associated with a proliferative advantage in normal cells (Ermis et al., 1995), the increased dosage of MET and its ligand (both located on chromosome 7) may be responsible for a growth advantage of papillary renal carcinoma cells with trisomy 7, but without MET mutation. The low frequency of MET mutations in sporadic PRC is underscored by the fact that eight of 17 mutations identified in our studies were germline (with no known family history of disease) and may represent examples of de novo mutational events.
We did not detect mutations in the MET gene in 199 nonrenal solid tumors. MET mutations may be present in other tumor types not included in our panel, or may be present in some of these tumors at a low percentage, undetectable in our relatively small sample set. It also may be possible that mutations exist in regions of the MET gene not screened in this study.
Metanephric adenoma is a recently recognized benign tumor of the kidney. Brown et al. (1997) identified trisomy of chromosomes 7 and 17 in these tumors. We tested two metanephric adenomas and found no mutations in the MET proto-oncogene.
In this study we report five novel mutations identified in a large panel of sporadic papillary renal carcinomas, which were shown to be transforming and cause constitutive phosphorylation of MET. These new mutations may best be analysed in the context of previously identified mutations in the MET proto-oncogene. The 15 different mutations detected to date were missense and were located in exons 16 – 19 of the MET proto-oncogene in the tyrosine kinase domain. These 15 different mutations involved ten codons: V1110, H1112, H1124, M1149, V1206, L1213, V1238, D1246, Y1248 and M1268. Codons V1110, H1112, V1238, D1246, Y1248 and M1268 were mutated in more than one sample. Codons H1112 and Y1248 were altered by three different missense mutations. These codons appear to be particularly critical in regulating tyrosine kinase activity because each of these codons was mutated to three different amino acids and still produced malignant disease. Mutations were clustered in the activation loop and the glycine rich-ATP binding region.
We previously reported the presence of striking homologies between MET proto-oncogene mutations and mutations in other receptor tyrosine kinases (Schmidt et al., 1997; Zbar et al., 1998). MET mutation M1268T is homologous to RET mutation M918T (the RET M918T mutation is responsible for most cases of multiple endocrine neoplasia type 2B); MET proto-oncogene mutations D1246H and D1246N are homologous to c-KIT mutation D816V (the c-Kit mutation D816V is responsible for systemic mastocytosis).
Here, we report another striking homology between an activating mutation in MET, V1110I, and a corresponding activating mutation in c-erbB, V157I (Shu et al., 1990). Both mutations change a valine to an isoleucine in the highly conserved glycine rich ATP-binding pocket. Detailed studies of the c-erbB mutation, V157I, have been performed (Shu et al., 1990, 1991, 1994). These observations demonstrate that there are several conserved codons in the tyrosine kinase domain which, when mutated, lead to uncontrolled cellular proliferation. The identification of such codons may be important in analysing the role of other tyrosine kinases in malignancy (Santoro et al., 1998).
Regulation of kinase activity by autophosphorylation of tyrosines in the activation loop is a common regulatory mechanism used by several receptor tyrosine kinases (RTK). Autophosphorylation occurs in trans and requires ligand-induced receptor dimerization (Ullrich and Schlessinger, 1990). Ligand-independent activating mutations within the catalytic core of RTKs appear to interfere with the intrasteric mechanism of autoinhibition. This can occur either by a direct alteration of contacts between the residues of the activation loop and those from the main body of the catalytic domain, or by increasing flexibility at critical points of the structure to facilitate subdomain movement. Molecular modeling studies described here suggest this type of activating mechanism for MET mutations in papillary renal carcinoma. Further biochemical studies will provide the experimental evidence to support this model of mutation-induced MET RTK activation.
Materials and methods
The panel of papillary renal carcinomas included 79 tumor DNAs with matching normal DNA, 21 paraffin-embedded tumor samples from patients with multiple or single papillary renal tumors, 22 paraffin-embedded or frozen tumor samples from patients with bilateral papillary renal carcinoma without a family history of renal neoplasia, and seven papillary renal carcinoma cell lines. The two metanephric adenomas and 199 nonrenal solid tumor DNAs were obtained from collaborators listed in acknowledgements.
Mutation analysis of the MET gene
We sequenced the cyteine-rich extracellular region (exons 5 and 7) and the intracellular portion (exons 13 – 20) of the MET proto-oncogene in 79 sporadic papillary renal carcinomas. (Exons 16 – 19 were sequenced in 60 of 79 samples for a previous study.) DNA from an additional 43 papillary renal carcinomas and seven PRC cell lines were sequenced in exons 14 – 19. DNAs from two metanephric adenomas and 199 solid nonrenal tumors were screened for mutations in exons 14 – 19 in the MET proto-oncogene by PCR-based single strand conformation polymorphism (PCR – SSCP) (Schmidt et al., 1993, 1997), or by denaturing high performance liquid chromatography (DHPLC).
Nucleotides were numbered according to the scheme in GenBank Accession number J02958 with nucleotide −194 in the cDNA (Park et al., 1987) given the number 1. Amino acids were numbered as in the reference by Park et al. (1987). The primers used for amplification of MET exons for PCR were described previously (Duh et al., 1997). PCR products from samples that showed an aberrant SSCP or DHPLC shift were sequenced on both strands from two independent PCR reactions as described previously (Schmidt et al., 1995).
DHPLC was performed as described (Underhill et al., 1996). Products were run on a Varian/Rainin machine using a DNASep column (Transgenomics, San Jose, CA, USA) and the DNA was detected by UV fluorescence at 254 nm. Samples were run in 0.1 M TEAA pH 7 buffer containing acetonitrile. Samples were injected at 17.5% acetonitrile, raised to 30.5% at 1 min, to 34% over the next 4 min and to 50% for the last 2 min. Melting temperatures for heteroduplexes formed between mutant and wild type strands of the PCR products were calculated using a program developed by Peter Oefner available at http:.//lotka.standford.edu.
NIH3T3 transformation assay
NIH3T3 cells (CRL 1658) were obtained from the American Type Culture Collection, (Rockville, MD, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% calf serum (Life Technologies Gaithersburg, MD, USA). The pMB1 expression vector containing the wild-type mouse MET cDNA (pMB11) was used for mutation construction (Jeffers et al., 1997). To construct the MET mutants, the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used with pMB11 as the template. Mutations were verified by sequencing both strands of DNA in the region of interest.
Transfections were performed as previously described with a plasmid containing the mutant or wild-type MET construct, a plasmid conferring resistance to G418 (pSV2neo) and Lipofectamine (Life Technologies, Gaithersburg, MD, USA). Focus formation assays were performed as described (Jeffers et al., 1997).
MET phosphorylation and expression
The NIH3T3 cell lines transfected with mouse MET mutant constructs were grown in DMEM/10% calf serum (Life Technologies, Gaithersburg, MD, USA) supplemented with Geneticin (G418; Life Technologies). Pools of 100 or more cells were used for expression and phosphorylation studies. Western analysis was performed as described (Jeffers et al., 1997). SDS – PAGE (8%) was performed under reducing conditions and protein was electrotransferred to Immobilon-P (Millipore, Bedford, MA, USA). Western blotting was performed using the anti-MET antibody SP260 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or the anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, Lake Placid, NY, USA) and the ECL detection system (Amersham, Arlington Heights, III, USA).
To study the mechanism of ligand-independent MET activation by mutations found in patients with sporadic papillary renal carcinoma, homology modeling using crystal structure coordinates of the insulin receptor (1IRK) (40% sequence identity with MET) from the Brookhaven Protein Data Bank was performed with the program package LOOK version 2.0 (Molecular Applications Group, Palo Alto, CA, USA). INSIGHT II version 95.0 (Biosym/MSI, San Diego, CA, USA) was used for the analysis of the model, side chain manipulations and preparation of illustrations.
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We thank Drs Scott Kern, Bert Vogelstein, Marcel Mannens, Marius Skudlinski, Gloria Niehans, Holger Moch, Webster Cavanee, Sandra Bigner, Burt Feuerstein, McClellan Walther, Kimberly Newsom Johnson/Vanderbilt Tissue Procurement Lab, Sharon Howard/Fox Chase Cancer Center, Paolo Radice, Michael Hughson, Masahiro Yao, Suzanne Topalian, Virginia LiVolsi, and Bruce Weintraub for providing tumor samples or DNA for mutation analysis. In addition we thank Dr Gerd Riedasch, Department of Urology, Rupert-Karls-University, Heidelberg, Germany for clinical evaluation of PRC patients and Dr JA Brown, Mayo Clinic (Rochester, MN, USA) for karyotypic and histologic analyses of metanephric adenomas. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000 and under contract with ABL (MM). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. The publisher or recipient acknowledges right of the US Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.
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