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Functional Analysis of c-Met/Hepatocyte Growth Factor Pathway in Malignant Pleural Mesothelioma

¹ Section of Hematology/Oncology, Department of Medicine, University of Chicago Cancer Research Center, University of Chicago Medical Center, Pritzker School of Medicine, Chicago, Illinois; ² Environmental Epidemiology and Biostatistics, National Cancer Research Institute, Largo Rosanna Benzi, Genoa, Italy; ³ Department of Surgery, Division of Cardiovascular and Thoracic Surgery, University of Minnesota Medical School, Minneapolis, Minnesota; and ⁴ Department of Surgery, Division of Thoracic Surgery, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts

Requests for reprints: Ravi Salgia, Section of Hematology/Oncology, Department of Medicine, The University of Chicago Medical Center, Pritzker School of Medicine, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637. Phone: 773-702-4399; Fax: 773-834-1798; E-mail: rsalgia{at}medicine.bsd.uchicago.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
c-Met receptor tyrosine kinase (RTK) has not been extensively studied in malignant pleural mesothelioma (MPM). In this study, c-Met was overexpressed and activated in most of the mesothelioma cell lines tested. Expression in MPM tissues by immunohistochemistry was increased (82%) in MPM in general compared with normal. c-Met was internalized with its ligand hepatocyte growth factor (HGF) in H28 MPM cells, with robust expression of c-Met. Serum circulating HGF was twice as high in mesothelioma patients as in healthy controls. There was a differential growth response and activation of AKT and extracellular signal–regulated kinase 1/2 in response to HGF for the various cell lines. Dose-dependent inhibition (IC₅₀ < 2.5 µmol/L) of cell growth in mesothelioma cell lines, but not in H2052, H2452, and nonmalignant MeT-5A (IC₅₀ >10 µmol/L), was observed with the small-molecule c-Met inhibitor SU11274. Furthermore, migration of H28 cells was blocked with both SU11274 and c-Met small interfering RNA. Abrogation of HGF-induced c-Met and downstream signaling was seen in mesothelioma cells. Of the 43 MPM tissues and 7 cell lines, we have identified mutations within the semaphorin domain (N375S, M431V, and N454I), the juxtamembrane domain (T1010I and G1085X), and an alternative spliced product with deletion of the exon 10 of c-Met in some of the samples. Interestingly, we observed that the cell lines H513 and H2596 harboring the T1010I mutation exhibited the most dramatic reduction of cell growth with SU11274 when compared with wild-type H28 and nonmalignant MeT-5A cells. Ultimately, c-Met would be an important target for therapy against MPM. (Cancer Res 2006; 66(1): 352-61

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Malignant pleural mesothelioma (MPM) is an aggressive malignancy which can be associated with asbestos exposure. Incidence of this disease is expected to increase dramatically over the next few decades. It has been estimated that 250,000 people will die of MPM in Europe in the next three decades (1) and 2,500 to 3,000 new cases are diagnosed each year in the United States with an even higher number worldwide. Recently, the multitargeted antifolate pemetrexed has been approved as the front-line agent in combination with cisplatin for the treatment of mesothelioma (2). Nonetheless, the overall outcome of this disease remains very poor despite current available therapies.

New approaches for the treatment of malignant pleural mesothelioma (MPM) are urgently needed. It was reported that the c-Met receptor tyrosine kinase (RTK) is an important molecule in a variety of malignancies (3–6) and overexpression of the receptor confers a poor prognosis (7, 8). Targeting the HGF/c-Met pathway could become a rational therapeutic approach for the malignant mesothelioma. c-Met is a RTK located on chromosome 7q31, and the gene contains 21 exons, and the protein consists of /ß chains, with the ß-chain MW of 145 kDa. The ligand for c-Met has been identified as hepatocyte growth factor (HGF). Previously, c-Met has been shown to be overexpressed in MPM tumor tissues as compared with normal pleura (9). The c-Met/HGF axis can be involved in cell growth, cell survival, angiogenesis, cell motility/migration, and invasion and metastasis.

There is now a large body of data supporting the inhibition of RTKs as a new paradigm of therapy for various malignancies. For instance, the small-molecule inhibitor imatinib has been used to target BCR/ABL in chronic myelogenous leukemia and c-Kit in gastrointestinal stromal tumors (10). Epidermal growth factor receptor (EGFR) has been targeted in non–small-cell lung cancer with small-molecule inhibitors gefitinib and erlotinib (11). However, novel targeted therapy against c-Met has not come to clinical fruition. Preclinical data suggest that c-Met can be inhibited with antisense/small interfering RNA (siRNA), peptides/antagonists of HGF, small-molecule tyrosine kinase inhibitors, and antibodies directed against c-Met or HGF. We have previously shown that the small-molecule tyrosine kinase inhibitor SU11274 is specific against c-Met and has an IC₅₀ of 1 to 5 µmol/L against a number of lung cancer cell lines (12).

Because the expression and function of c-Met in MPM is not well known, we evaluated the role of c-Met/HGF in MPM. The potential for therapeutic inhibition of c-Met in MPM is attractive, and we determined that SU11274 and c-Met siRNA against c-Met are effective inhibitors of c-Met in MPM cell lines. We have also determined the mutations of c-Met in MPM. Finally, we show that serum HGF levels can be higher in MPM patients as compared with control subjects, thus potentially serving as a biomarker in MPM.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents and antibodies. SU11274 [(3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl) carbonyl]-1H-pyrrol-2-yl} methylene)-N-methyl-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide] was provided by Pfizer, Inc. (San Diego, CA). Human recombinant HGF was purchased from Calbiochem (Cambridge, MA) and fetal bovine serum (FBS) was from Gemini Bioproducts (Woodland, CA). Cell culture media, penicillin, and streptomycin were obtained from Cellgro (Boehringer Ingelheim, Heidelberg, Germany). Polyclonal phosphorylation site–specific c-Met antibodies and phospho-extracellular signal–regulated kinase (ERK)-1/2 (T185/Y187) antibodies were obtained from Biosource International (Camarillo, CA). Antibody against p-AKT (S473) was obtained from Cell Signaling Technology (Beverly, MA), c-Met (c12) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and ß-actin monoclonal antibody and all other chemicals were purchased from Sigma (St. Louis, MO).

Cell lines and cell culture. Malignant pleural mesothelioma (MPM) cell lines H513 (epithelioid) and H2596 (sarcomatoid) were cultured as previously described (13). H28 (epithelioid), H2052 (sarcomatoid), H2452 (biphasic), MSTO-211H (biphasic), and the nonmalignant mesothelial cell line (MeT-5A) were obtained from the American Type Culture Collection (Rockville, MD). MPM cells were cultured routinely in RPMI supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. MeT-5A was cultured in Medium 199 with Earle's balanced salt solution supplemented with 10% FBS, 0.75 mmol/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO₂ at 37°C.

Immunoblot analysis. To examine c-Met expression in mesothelioma and nonmalignant mesothelial cells under basal conditions, subconfluent cells were cultured in medium supplemented with 10% FBS. The cells were then washed with ice-cold PBS and lysed with lysis buffer. To detect the activation of c-Met, AKT, and ERK1/2, cells grown on six-well plates were washed twice with PBS, then replaced with serum-free RPMI for 24 hours. added with HGF (40 ng/mL), and then incubated at 37°C for the specified time durations indicated (0-5 hours). Next, the cells were washed in cold PBS and lysed, followed by SDS-PAGE on a 7.5% gel, and transferred to a nitrocellulose membrane. Immunoblots were then done using standard method as previously described (3, 14). The same membranes were subsequently stripped and reprobed in a similar fashion with different primary antibodies.

Immunohistochemical analysis of c-Met expression by tissue microarray. Expression of c-Met was detected by immunohistochemistry using tissue microarray in tissue cores that were obtained from 21 normal and 66 mesothelioma (47 epithelioid, 3 sarcomatoid, and 16 biphasic) specimens as previously described (15). The tissue samples were obtained and processed in accordance with the Institutional Review Board (IRB)–approved protocols.

Immunofluorescence and confocal microscopy. The expression of c-Met and internalization with HGF was detected in mesothelioma cells by indirect immunofluorescence as previously described (16). Briefly, H28 cells were grown on coated glass coverslips and prestarved in 0.5% bovine serum albumin (BSA)–containing media. After treatment with or without HGF (40 ng/mL, 15 minutes), cells were fixed in ice-cold methanol/acetone (1:1) for 10 minutes and blocked in PBS containing 1% BSA for 30 minutes. Primary antibody against c-Met was incubated at a 1:50 dilution in the blocking solution for 1 hour, followed by three washes with PBS. Cy3-conjugated secondary antibody was incubated for an additional hour in blocking solution at 1:500 dilutions. After three washes with PBS, cells were mounted in fluorescent Vectashield mounting medium with 4',6-diamidino-2-phenylindole (DAPI; H-1300, Vector Laboratories, Burlingame, CA) and images were captured using an Olympus DSU spinning disc confocal microscope and saved as digital images.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The growth effect of HGF and the inhibitory effect of SU11274 for a variety of different mesothelioma cell lines were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (17). The absorbance of each well was measured in a microplate reader at 570 nm. The percentage cell growth was calculated by comparison with the A570 reading obtained from treated versus control cells. Each experiment was done in four replicate wells for each drug concentration. The IC₅₀ value is defined as the concentration needed for a 50% reduction in absorbance calculated from the growth curves.

Mutational analysis. For mutational screening for the coding regions of the c-Met gene, standard PCR and direct DNA sequencing of the c-Met gene were done and analyzed using genomic DNA isolated from 6 mesothelioma cell lines and 1 normal mesothelial cell line as well as cDNA generated from 43 mesothelioma tumor tissue samples as previously described (15).

Gene silencing. siRNA oligonucleotides targeting c-Met mRNA were obtained from Dharmacon, Inc. (Lafayette, CO) and used according to the instructions of the manufacturer for the gene silencing studies. Briefly, a pool of four c-Met-specific 21-nucleotide RNA oligonucleotides forming a 19-bp duplex core with 2-nucleotide 3' overhangs was used in combination. siRNA duplexes were transiently transfected into the H28 and H2596 cells using the DharmaFect transfection reagent according to the instructions of the manufacturer (Dharmacon Research). Mock transfection was done in parallel using control siRNA as negative control. Cells were incubated for 48 to 72 hours before the MTT assay or lysate preparation.

Cell migration assay. In vitro wound assay studies were done using methods as previously described (3). Briefly, cells were plated in six-well tissue culture plates with complete culture media for 24 hours, and then treated with SU11274 for the subsequent 24 hours. Using the p-20 pipette tip, a line was drawn through the middle of the plates to create a "scratch wound." The plates were photographed at 0, 24, and 48 hours using an Olympus IX81 microscope.

Cell motility analysis by time-lapse video microscopy. H28 cells were plated on cell culture dishes and placed into a temperature-controlled chamber at 37°C in an atmosphere of 5% CO_2. The cells were examined by time-lapse video microscopy as previously described (18) using an Olympus IX81 or Axiovert inverted microscopes equipped with a cooled CCD cameras. Control, control siRNA, c-Met siRNA (100 or 200 pmol/mL) transfected, and 2.5 µmol/L SU11274 pretreated (4 hours) H28 cell images were recorded for 2 to 4 hours. Digital video images were saved every minute and cell movement or morphologic changes were analyzed. For movement analysis, the position of cell nuclei was measured every minute with the NIH ImageJ or MetaMorph (Universal Imaging Corp., Downingtown, PA) programs and plotted to show the trace of cell movement. The distance that the cell nucleus transversed for each minute was calculated and the velocity of each cell was determined and compared.

Serum levels of circulating HGF and EGF. The serum HGF and EGF were measured in 23 MPM pretreated patients and 19 healthy controls. The characteristics of all subjects are reported in Table 1. Healthy controls were recruited from a group of blood donors and from nonneoplastic patients with eye diseases, treated in the same hospitals as MPM patients. The diagnosis of MPM was achieved through cytologic or histologic examination of pleural biopsies obtained through thoracoscopy or thoracotomy.

Statistical analysis. All reported HGF and EGF values were the mean of duplicate determinations. Means, medians, and SDs were calculated for cancer and control. A cutoff value was calculated on the basis of the mean ± 2 SD of markers frequency in healthy controls. Differences in the values between the groups under study were analyzed using the Kruskal-Wallis test, and the Mann Whitney U test was used for comparison of two groups. A ² test was used to evaluate the relationship between categorical variables. The Spearman correlation method was used to correlate the HGF level with other variables. The experiments were usually done at least thrice. Statistical significance was tested by two-tailed Student's t test. A two-tailed P value of <0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of c-Met in cell lines and tumor tissues. The expression of c-Met RTK in mesothelioma cell lines (H28, H2052, H2452, MSTO-211H, H513, and H2596) and a nonmalignant mesothelial cell line (MeT-5A) using standard immunoblotting with an antihuman polyclonal c-Met antibody was determined. Robust expression of c-Met was identified in two (H28 and H2452 cells) of the six mesothelioma cell lines and minimal expression was observed in MeT-5A (Fig. 1A). Twenty-one normal and 66 mesothelioma (47 epithelioid, 3 sarcomatoid, and 16 biphasic) specimens on tissue microarray were used for immunohistochemical analysis of c-Met. Normal pleura showed no staining (0) whereas mesothelioma tissues displayed a spectrum of intensities ranging from no expression (0) to strong expression (3+) of c-Met (Fig. 1C and D). There was expression of c-Met in 82% of MPM (1+ to 3+).

View larger version (30K): [in this window] [in a new window]   Figure 1. Expression of c-Met in mesothelioma. A, expression of c-Met in the mesothelioma cell lines. Expression levels of c-Met in seven mesothelioma cell lines (H28, H2052, H2452, MSTO-211H, H513, and H2596) and mesothelial cell line MeT-5A as determined by immunoblotting using anti–total c-Met polyclonal antibody (C-12). The upper 170-kDa protein band represents the precursor form of the glycosylated c-Met and the lower 145-kDa band is the biologically active transmembrane ß-subunit of c-Met (top). Equal loading of lysate is shown by the anti-ß-actin immunoblot (bottom). B, the relative signal intensity for c-Met in each lane was determined by densitometry and expressed as arbitrary unit (fold) after the normalization. C, expression of c-Met in the mesothelioma tissue microarray. Immunohistochemical analysis c-Met RTK expression patterns in normal (n = 21) and mesothelioma (n = 66) tissues using anti-c-Met antibody on tissue microarrays. D, weak immunonegative c-Met staining (0) and immunoreactive strong c-Met staining (1+, 2+, and 3+).

View larger version (44K): [in this window] [in a new window]   Figure 2. A, cell growth of mesothelioma cells with HGF. Equal numbers of mesothelial and mesothelioma cell lines were treated with HGF or no HGF for 48 hours, and then cell growth was studied by MTT assay as described in Materials and Methods. HGF effectively stimulated cell growth in H28, MSTO-211H, and H2596 cell lines whereas there was minimal or no effect in H513, H2052, H2452, and MeT-5A cell lines. Columns, percentage of change in cell growth from control. *, no effect from their respective control. B, activation of signaling kinases in mesothelioma cells with HGF. Immunoblot analysis was done to evaluate the expression and phosphorylation states (as an indicator of activation) of kinases downstream of c-Met/HGF using phosphospecific antibodies. c-Met was differentially phosphorylated on tyrosine autophosphorylation site [pY1230/1234/1235] (top), Cbl-binding site [pY1003] (second), the cell survival proteins AKT [S473] (third), and ERK1/2 [T185/Y187] (fourth) in mesothelial cell line and mesothelioma cell lines. Equal loading of lysate is shown by the anti-ß-actin immunoblot (fifth). C, internalization of c-Met after HGF stimulation. H28 cells on glass coverslips were serum starved and stimulated with HGF (40 ng/mL) at 37°C for 15 minutes. The cells were fixed with methanol and stained for c-Met (red), with DAPI as nuclear counter stain (blue), as described in Materials and Methods. In non-HGF-treated H28 cells, c-Met was primarily located in the plasma membrane, whereas in HGF-stimulated cells, the c-Met receptor was observed mostly in a perinuclear area and cytoplasm. Representative results from more than five fields were examined in three individual experiments.

Internalization of c-Met was also evaluated with immunofluorescence in H28 cells which express high c-Met levels. Treatment of the cells with HGF (40 ng/mL, 15 minutes) led to internalization of c-Met in H28 cells (Fig. 2C). In non-HGF-treated H28 cells, c-Met was primarily distributed in the plasma membrane, whereas in HGF-stimulated cells, the c-Met receptor was readily identifiable in two distinct subcellular locations. An immunofluorescence signal was observed mostly in cytosolic and perinuclear distribution and less in the plasma membrane against c-Met in H28 cells (Fig. 2C).

Effects of SU11274 on mesothelioma cell growth. SU11274 inhibited the cell growth of H28, MSTO-211H, H513, and H2596 cells in a dose-dependent manner with an IC₅₀ between 2 and 3 µmol/L within 48 hours. On the other hand, SU11274 had no inhibitory effect in the H2052 and H2452 mesothelioma cells or in mesothelial cells (MeT-5A) at concentrations even up to 10 µmol/L at 48 hours (Fig. 3). Interestingly, the nonresponding cells did not have a significant alteration in growth with HGF (Fig. 2A).

View larger version (13K): [in this window] [in a new window]   Figure 3. Inhibition of cell growth in mesothelioma cells by SU11274. Cells were plated 24 hours before adding SU11274. Mesothelioma cells and MeT-5A cells were cultured for 48 hours in the absence or presence of increasing concentrations of SU11274 and cell growth was determined by the MTT assay and expressed as percentage of cell growth compared with untreated cells. SU11274 effectively reduces the cell growth of mesothelioma cell lines H28, MSTO-211H, H513, and H2596, but not H2052, H2452, and nonmalignant MeT-5A cells, at 48 hours of incubation. Points, mean of three independent experiments.

View larger version (73K): [in this window] [in a new window]   Figure 4. Inhibition of cell migration and HGF-induced signal transduction of mesothelioma cells by SU11274. A, SU11274 inhibits migration of H28 cells. H28 cells migrate into an in vitro wound created onto a subconfluent plate. Cells were seen to be migrating into the wound over the subsequent 48 hours, and migration was significantly inhibited in the presence of 2.5 µmol/L SU11274. B, SU11274 reduces velocity of the H28 cells. SU11274 (2.5 µmol/L)–treated H28 cells were observed under time-lapse video microscopy and pictures were taken at 0 to 4 hours. The position of cell nucleus was measured and tracked every minute with the NIH ImageJ or MetaMorph (Universal Imaging) programs and plotted to show the trace of nucleus movement. The distance that the cell nucleus transversed and velocity for each minute were calculated to determine the speed of the movement in control and SU11274-treated H28 cells. C, dose-dependent inhibition of HGF-induced c-Met phosphorylation and downstream AKT and ERK1/2 phosphorylation in H28 cells by SU11274. Serum-starved mesothelioma H28 cells were preincubated with vehicle or the indicated concentrations of SU11274 for 12 hours. Cells were treated with HGF (40 ng/mL) for 15 minutes before lysis. Cell lysates were resolved by 7.5% SDS-PAGE and probed with either antibody. Immunoblots were shown for p-c-Met [Y1230/1234/1235] (top), p-c-Met [Y1003] (second), total c-Met (third), the cell survival proteins p-AKT [S473] (fourth), and p-ERK1/2[pT185/pY187] (fifth). Equal loading of lysate was shown by the anti-ß-actin immunoblot (sixth).

We examined the tyrosine phosphorylation of c-Met by immunoblotting in H28 mesothelioma cells in the absence or presence of HGF or SU11274. HGF induced tyrosine phosphorylation of c-Met at both the autophosphorylation site (pY1230/1234/12350) and the regulatory juxtamembrane c-Cbl binding site (pY1003). However, both of the tyrosine phospho-epitopes of c-Met were inhibited by pretreatment of SU11274 in a dose-dependent manner up to near basal level without affecting total steady-state c-Met protein level (Fig. 4C). To examine HGF-mediated signaling events downstream of c-Met activation, ERK1/2 and AKT phosphorylation in the presence of SU11274 was analyzed. As shown in Fig. 4C, SU11274 also inhibited HGF-induced phosphorylation of AKT and ERK1/2 in a dose-dependent manner.

Effects of c-Met siRNA on cell growth and migration in mesothelioma cells. To further investigate the role of c-Met in cell growth and migration of mesothelioma, c-Met expression was down-regulated by transfection of cells with siRNAs. H28 and H2596 cells transfected with c-Met siRNA had substantially reduced (>95%) c-Met expression after 72 hours (Fig. 5A). siRNA-mediated down-regulation of c-Met protein expression in H28 and H2596 cells resulted in inhibition of cell growth and viability as determined by MTT assay. Migration in c-Met siRNA–transfected cells was markedly reduced compared with control siRNA–transfected cells (Fig. 5B).

View larger version (45K): [in this window] [in a new window]   Figure 5. Silencing of c-Met by siRNA in H28 and H2596 cells. A, c-Met expression and cell growth after 72 hours of c-Met siRNA (100 and 200 pmol/mL) transfection. Immunoblot analysis was used to confirm siRNA-directed knockdown of the c-Met expression in H28 and H2596 cells. Columns, mean percentage of cell growth; bars, SE. 0 siRNA represents control siRNA; the expression levels are the same as without siRNA (data not shown). B, control siRNA– (no significant different was observed when compared with no siRNA with the same experimental set up; data not shown) and c-Met siRNA–transfected H28 cells were observed under time-lapse video microscopy and images were taken over 2 hours. The position of cell nucleus was measured, tracked every minute with the NIH ImageJ and MetaMorph (Universal Imaging) programs, and plotted to show the trace of nucleus movement. The distance that the cell nucleus transversed and velocity for each minute were calculated to determine the speed of the movement in control siRNA– and c-Met siRNA–transfected H28 cells.

View larger version (30K): [in this window] [in a new window]   Figure 6. Sequence analysis reveals c-Met mutations in human MPM tumors. DNA sequence analysis was done using genomic DNA extracted from mesothelial, MPM cell lines, and MPM tumor samples. Mutations and sequence variants of c-Met were identified in mesothelioma. A, mutations in the MPM mutational analysis are illustrated schematically in the context of the functional domains of c-Met. B and C, samples from patients (T8, T11, T13, and T42; B) and mesothelioma cell lines (H513 and H2596; C) showed alteration of the c-Met gene. Mutations are indicated in the sequencing chromatogram. No mutations were detected in the normal mesothelial cell line. D, cell lines harboring T1010I mutation exhibited a dramatic reduction of cell growth with SU11274 when compared with wild-type H28 cells and nonmalignant MeT-5A cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Malignant pleural mesothelioma is a difficult illness with current standard therapy involving surgery, chemotherapy, or radiation therapy depending on the extent of the tumor. The link to asbestos in MPM has been established. However, the molecular mechanisms in MPM pathogenesis have not been explored to the fullest extent. Here we analyzed the role of the c-Met RTK in MPM. Table 2 summarizes a majority of our findings within this study. A number of cell lines had robust expression of c-Met whereas the normal mesothelial cells had a much lower amount of c-Met. The overexpression of c-Met in mesothelioma was also confirmed in MPM tumor tissues. Ligand (HGF)-induced c-Met receptor internalization was observed by immunofluorescence microscopy. There were a number of signal transduction cascade mechanisms that were activated with HGF stimulation such as phosphorylation of c-Met and phosphorylation of ERK1/2 and AKT. Because c-Met is expressed and functional in mesothelioma tumor cell lines, we also determined its specific inhibition with SU11274, a small-molecule c-Met inhibitor. In the mesothelioma cell lines, there was a dose-dependent inhibition of cell growth whereas the nonmalignant MeT-5A was not responsive to SU11274. We observed that the cell lines H513 and H2596 harboring the T1010I mutation exhibited a dramatic reduction of cell growth with SU11274 when compared with the nonmalignant MeT-5A cells and wild-type overexpressing H28 cells. Finally, the serum levels of HGF were elevated in mesothelioma subjects, implying that this might be an important biomarker in this disease.

Inhibition of various RTKs offers an exciting opportunity for novel therapeutics, especially in MPM. Unfortunately, clinical trials of imatinib (inhibiting the platelet-derived growth factor receptor and c-Kit pathways) and gefitinib (inhibiting the EGFR pathway) in MPM have not shown any benefit (22). The strategy to target other RTKs, such as c-Met, would be a quite novel approach to inhibit MPM. Previously, c-Met has been shown to be inactivated with c-Met siRNA (23, 24), peptides (25), natural killer cell transcript 4 (26, 27), and antibodies against c-Met (28) or HGF (29). We and others have recently shown that the c-Met tyrosine kinase domain can be inhibited by compounds such as SU11274, PHA665752, and K252a (12, 30, 31). SU11274 treatment in small-cell lung cancer and non–small-cell lung cancer leads to decreased viability, decreased formation of reactive oxygen species, decreased cell motility/migration, and decreased cell signaling to the cytoskeleton (3, 15). SU11274 led to decreased cell growth, motility, and migration and to inhibition of signal transduction by HGF in MPM cell lines. Cell growth of mesothelial cells with HGF was an important determinant to responsiveness to SU11274 (Table 2). As c-Met knockdown with siRNA inhibited cell growth and migration, it indicates that c-Met has an essential role in the cell growth, migration, and progression of mesothelioma. It would now be interesting to pursue the use of inhibitors such as these in vivo. It is also not known if compounds such as SU11274 can synergize with other chemotherapeutic agents such as pemetrexed in MPM.

The results obtained showed that there is a difference in enhancement of growth effects of HGF and the signal transduction effects of HGF in MPM cell lines as shown in Fig. 2 and summarized in Table 2. Interestingly, although the response to HGF in the various cell lines leads to activation of signal transduction, this did not necessarily translate to inhibition of cell growth by the small-molecule inhibitor SU11274. Only cells that were responsive to cell growth by HGF were also responsive to inhibition by SU11274, with the exception of H513 cells. The H513 cell line did not have a growth effect by HGF stimulation. However, by mutational analysis, H513 cells harbored the T1010I c-Met mutation. Despite activation of signaling events by HGF (such as phosphorylation of AKT or ERK1/2), we did not observe increased cell growth in H2052 and H2452 cells. This is of interest because it indicates that in these cells, chronic activation of signaling events involved in a proliferative response eliminates the threshold for ligand-induced proliferation. Nevertheless, it still needs to be determined if there are other biological activities regulated through the c-Met receptor that are regulated by HGF binding.

In two of the cell lines, we also identified a mutation as well as a deletion in the juxtamembrane domain (T1010I). Juxtamembrane domains of RTKs are thought to be key regulators of catalytic functions (32). More recently, the structural basis of the regulatory role (autoinhibition) of the RTK Eph-B2 by the unphosphorylated juxtamembrane domain has been elucidated (32). A germ line mutation of c-Met, P1009S (exon 14), was detected in a patient with gastric carcinoma and is the first such missense mutation to be described affecting the juxtamembrane domain (as opposed to tyrosine kinase domain). The P1009S mutation does not induce ligand-independent activation of c-Met but shows increased persistent response to HGF stimulation when expressed in NIH 3T3 cells (33). Peschand et al. have shown that c-Cbl acts as a negative regulatory protein for c-Met, as well as several other RTKs, by promoting the polyubiquitination of c-Met. The Y1003 juxtamembrane tyrosine, when replaced by phenylalanine, results in the loss of ubiquitination of the Met receptor and has transforming activity in fibroblast and epithelial cells (34). We have also shown that in small-cell lung cancer, R988C and T1010I mutations lead to altered cytoskeletal functions (3). It is unclear at this point what the role of G1085X would be in the pathogenesis of MPM, and this is under evaluation. Besides missense mutations, c-Met-mediated tumorigenesis can be a result of gene amplification, as seen in human gastric carcinoma via the break-fusion-bridge mechanism (35). At this point, it is not known of in some of MPM the gene can be amplified, and this is under evaluation as well.

There were several mutations of the semaphorin domain identified within the MPM tumor tissues. The semaphorin domain is conserved among all semaphorins and is also found present in the plexins and c-Met (36–38). In c-Met, the semaphorin domain is encoded by exon 2. More recently, the three-dimensional conformations of the HGF and heparin-binding sites of c-Met have been established by deletion mutagenesis of the RTK (28). The extracellular ligand–binding domain in the c-Met ectodomain was identified as adopting a seven-blade ß-propeller fold for the semaphorin domain of c-Met, homologous to the ß-propeller fold template seen in the NH₂-terminal domain of -integrin. These three-dimensional models and functional map of the c-Met ectodomain, along with the mutations identified, would undoubtedly facilitate further development of targeted therapeutics against c-Met.

HGF has recently been shown to be an important serum biomarker in small-cell lung cancer (39). In this study, we also show that HGF can be elevated in patients with MPM. Biomarkers are an important tool in clinical diagnosis, prognosis, and therapeutics. Recently, mesothelin has been shown to be important in MPM (40). It is a highly sensitive positive marker for epithelioid mesotheliomas. There was also a strong statistical correlation between histologic type and HGF expression (41). Similar to mesothelin, sarcomatous differentiated tumors stained much weaker with HGF than their epithelial differentiated counterparts did. It would be important in the future to compare mesothelin levels with HGF levels in MPM.

In conclusion, we show that c-Met is expressed and functional in MPM. c-Met can also be inhibited with siRNA and the small-molecule tyrosine kinase inhibitor SU11274. With further preclinical testing to verify c-Met as an important target in mesothelioma, it would be useful to target this receptor in future clinical trials.

	Acknowledgments

 
Grant support: Mesothelioma Applied Research Foundation Grant, American Cancer Society Research Scholar Grant RSG-02-244-02-CCE, Institutional Cancer Research Awards from the University of Chicago Cancer Center with the American Cancer Society and the V-Foundation (R. Salgia), and NIH/National Cancer Institute RO1 grant R01 CA100750-02 (R. Salgia).

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.

Received 12/22/04. Revised 9/15/05. Accepted 10/21/05.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Peto J, Decarli A, La Vecchia C, Levi F, Negri E. The European mesothelioma epidemic. Br J Cancer 1999;79:666–72.[CrossRef][Medline]
2. Vogelzang NJ, Rusthoven JJ, Symanowski J, et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 2003;21:2636–44.[Abstract/Free Full Text]
3. Ma PC, Kijima T, Maulik G, et al. c-MET mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions. Cancer Res 2003;63:6272–81.[Abstract/Free Full Text]
4. Schmidt L, Junker K, Nakaigawa N, et al. Novel mutations of the MET proto-oncogene in papillary renal carcinomas. Oncogene 1999;18:2343–50.[CrossRef][Medline]
5. Otte JM, Schmitz F, Kiehne K, et al. Functional expression of HGF and its receptor in human colorectal cancer. Digestion 2000;61:237–46.[CrossRef][Medline]
6. Knudsen BS, Edlund M. Prostate cancer and the met hepatocyte growth factor receptor. Adv Cancer Res 2004;91:31–67.[Medline]
7. Lee WY, Su WC, Lin PW, Guo HR, Chang TW, Chen HH. Expression of S100A4 and Met: potential predictors for metastasis and survival in early-stage breast cancer. Oncology 2004;66:429–38.[CrossRef][Medline]
8. Lengyel E, Prechtel D, Resau JH, et al. C-Met overexpression in node-positive breast cancer identifies patients with poor clinical outcome independent of Her2/neu. Int J Cancer 2004;113:678–82.
9. Zimmerman RL, Fogt F. Evaluation of the c-Met immunostain to detect malignant cells in body cavity effusions. Oncol Rep 2001;8:1347–50.[Medline]
10. Nadal E, Olavarria E. Imatinib mesylate (Gleevec/Glivec) a molecular-targeted therapy for chronic myeloid leukaemia and other malignancies. Int J Clin Pract 2004;58:511–6.[CrossRef][Medline]
11. Glover KY, Perez-Soler R, Papadimitradopoulou VA. A review of small-molecule epidermal growth factor receptor-specific tyrosine kinase inhibitors in development for non-small cell lung cancer. Semin Oncol 2004;31:83–92.[Medline]
12. Sattler M, Pride YB, Ma P, et al. A novel small molecule met inhibitor induces apoptosis in cells transformed by the oncogenic TPR-MET tyrosine kinase. Cancer Res 2003;63:5462–9.[Abstract/Free Full Text]
13. Hoang CD, Zhang X, Scott PD, et al. Selective activation of insulin receptor substrate-1 and -2 in pleural mesothelioma cells: association with distinct malignant phenotypes. Cancer Res 2004;64:7479–85.[Abstract/Free Full Text]
14. Maulik G, Kijima T, Ma PC, et al. Modulation of the c-Met/hepatocyte growth factor pathway in small cell lung cancer. Clin Cancer Res 2002;8:620–7.[Abstract/Free Full Text]
15. Ma PC, Jagadeeswaran R, Jagadeesh S, et al. Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer. Cancer Res 2005;65:1479–88.[Abstract/Free Full Text]
16. Wang Z, Zhang L, Yeung TK, Chen X. Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation. Mol Biol Cell 1999;10:1621–36.[Abstract/Free Full Text]
17. Dong Y, Ganther HE, Stewart C, Ip C. Identification of molecular targets associated with selenium-induced growth inhibition in human breast cells using cDNA microarrays. Cancer Res 2002;62:708–14.[Abstract/Free Full Text]
18. Kijima T, Maulik G, Ma PC, et al. Regulation of cellular proliferation, cytoskeletal function, and signal transduction through CXCR4 and c-Kit in small cell lung cancer cells. Cancer Res 2002;62:6304–11.[Abstract/Free Full Text]
19. Peschard P, Ishiyama N, Lin T, Lipkowitz S, Park M. A conserved DpYR motif in the juxtamembrane domain of the Met receptor family forms an atypical c-Cbl/Cbl-b tyrosine kinase binding domain binding site required for suppression of oncogenic activation. J Biol Chem 2004;279:29565–71.[Abstract/Free Full Text]
20. Katzmann DJ, Babst M, Emr SD. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 2001;106:145–55.[CrossRef][Medline]
21. Clague MJ. Membrane transport: a coat for ubiquitin. Curr Biol 2002;12:R529–31.[CrossRef][Medline]
22. Kindler HL. Moving beyond chemotherapy: novel cytostatic agents for malignant mesothelioma. Lung Cancer 2004;45 Suppl 1:S125–7.
23. Shinomiya N, Gao CF, Xie Q, et al. RNA interference reveals that ligand-independent met activity is required for tumor cell signaling and survival. Cancer Res 2004;64:7962–70.[Abstract/Free Full Text]
24. Ramos-Nino ME, Scapoli L, Martinelli M, Land S, Mossman BT. Microarray analysis and RNA silencing link fra-1 to cd44 and c-met expression in mesothelioma. Cancer Res 2003;63:3539–45.[Abstract/Free Full Text]
25. Bardelli A, Longati P, Williams TA, Benvenuti S, Comoglio PM. A peptide representing the carboxyl-terminal tail of the met receptor inhibits kinase activity and invasive growth. J Biol Chem 1999;274:29274–81.[Abstract/Free Full Text]
26. Ueda K, Iwahashi M, Matsuura I, et al. Adenoviral-mediated gene transduction of the hepatocyte growth factor (HGF) antagonist, NK4, suppresses peritoneal metastases of gastric cancer in nude mice. Eur J Cancer 2004;40:2135–42.[CrossRef][Medline]
27. Matsumoto K, Nakamura T. NK4 (HGF-antagonist/angiogenesis inhibitor) in cancer biology and therapeutics. Cancer Sci 2003;94:321–7.[CrossRef][Medline]
28. Kong-Beltran M, Stamos J, Wickramasinghe D. The Sema domain of Met is necessary for receptor dimerization and activation. Cancer Cell 2004;6:75–84.[CrossRef][Medline]
29. Cao B, Su Y, Oskarsson M, et al. Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models. Proc Natl Acad Sci U S A 2001;98:7443–8.[Abstract/Free Full Text]
30. Morotti A, Mila S, Accornero P, Tagliabue E, Ponzetto C. K252a inhibits the oncogenic properties of Met, the HGF receptor. Oncogene 2002;21:4885–93.[CrossRef][Medline]
31. Ma PC, Schaefer E, Christensen JG, Salgia R. A selective small molecule c-MET Inhibitor, PHA665752, cooperates with rapamycin. Clin Cancer Res 2005;11:2312–9.[Abstract/Free Full Text]
32. Wybenga-Groot LE, Baskin B, Ong SH, Tong J, Pawson T, Sicheri F. Structural basis for autoinhibition of the Ephb2 receptor tyrosine kinase by the unphosphorylated juxtamembrane region. Cell 2001;106:745–57.[CrossRef][Medline]
33. Lee JH, Han SU, Cho H, et al. A novel germ line juxtamembrane Met mutation in human gastric cancer. Oncogene 2000;19:4947–53.[CrossRef][Medline]
34. Peschard P, Fournier TM, Lamorte L, et al. Mutation of the c-Cbl TKB domain binding site on the Met receptor tyrosine kinase converts it into a transforming protein. Mol Cell 2001;8:995–1004.[CrossRef][Medline]
35. Hellman A, Zlotorynski E, Scherer SW, et al. A role for common fragile site induction in amplification of human oncogenes. Cancer Cell 2002;1:89–97.[CrossRef][Medline]
36. Tessier-Lavigne M, Goodman CS. The molecular biology of axon guidance. Science 1996;274:1123–33.[Abstract/Free Full Text]
37. Tamagnone L, Artigiani S, Chen H, et al. Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 1999;99:71–80.[CrossRef][Medline]
38. Christensen CR, Klingelhofer J, Tarabykina S, Hulgaard EF, Kramerov D, Lukanidin E. Transcription of a novel mouse semaphorin gene, M-semaH, correlates with the metastatic ability of mouse tumor cell lines. Cancer Res 1998;58:1238–44.[Abstract/Free Full Text]
39. Bharti A, Ma PC, Maulik G, et al. Haptoglobin -subunit and hepatocyte growth factor can potentially serve as serum tumor biomarkers in small cell lung cancer. Anticancer Res 2004;24:1031–8.[Abstract/Free Full Text]
40. Li Q, Verschraegen CF, Mendoza J, Hassan R. Cytotoxic activity of the recombinant anti-mesothelin immunotoxin, SS1(dsFv)PE38, towards tumor cell lines established from ascites of patients with peritoneal mesotheliomas. Anticancer Res 2004;24:1327–35.[Medline]
41. Tolnay E, Kuhnen C, Wiethege T, Konig JE, Voss B, Muller KM. Hepatocyte growth factor/scatter factor and its receptor c-Met are overexpressed and associated with an increased microvessel density in malignant pleural mesothelioma. J Cancer Res Clin Oncol 1998;124:291–6.[CrossRef][Medline]

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