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Carcinogenesis Advance Access originally published online on January 12, 2008
Carcinogenesis 2008 29(3):647-655; doi:10.1093/carcin/bgn009
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Gab1 but not Grb2 mediates tumor progression in Met overexpressing colorectal cancer cells

Isolde Seiden-Long1,{dagger}, Roya Navab1,{dagger}, Warren Shih1, Ming Li1, Jane Chow1, Chang Qi Zhu1, Nikolina Radulovich1, Caroline Saucier4 and Ming-Sound Tsao1,2,3,*

1 Ontario Cancer Institute and Princess Margaret Hospital, University Health Network, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9
2 Department of Medical Biophysics, University of Toronto, Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9
3 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1L5 and
4 Depatment of Anatomy and Cell Biology, Faculty of Medicine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4

* To whom correspondence should be addressed. Tel: +416 340 4737; Fax: +416 340 5571; Email: ming.tsao{at}uhn.on.ca


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Hepatocyte growth factor receptor (Met) plays an important role in the progression of multiple cancer types. The overexpression of Met in DLD-1 colon carcinoma cells with kirsten rat sarcoma oncogene homolog (KRAS) oncogene activation resulted in enhanced subcutaneous and orthotopic tumor growth rate and increased metastatic potential. To elucidate the mechanism of this effect, we stably expressed kinase-inactive MetK1110A, Src homology 2 (SH2)-binding domain-inactive MetY1349/1356F, growth factor receptor-bound protein 2 (Grb2) non-binding MetN1358H and mutant receptors with ability to selectively recruit signaling proteins Grb2, src homology domain c-terminal adaptor homolog (Shc), phospholipase c-gamma (PLC{gamma}) and p85 phosphatidyl inositol 3 kinase. As subcutaneous implants, DLD-1 cells that expressed the majority of these receptor constructs failed to recapitulate the tumor growth-enhancing effect of the wild-type Met receptor. The Grb2- and Shc-recruiting Met mutants demonstrated slight but consistent tumor-suppressive activity, whereas the expression of N1358H mutant stimulated tumor growth rate comparable with the wild-type receptor. This suggests that direct Grb2/Shc binding does not contribute to the tumor progression activity of Met receptor. The tumors expressing Grb2- and Shc-recruiting Met receptors demonstrated a marked loss in Grb2-associated adaptor protein 1 (Gab1) protein levels, which was not observed in the cell lines, consistent with a post-translationally regulated process. Moreover, a moderate level of Gab1 overexpression stimulated tumor growth. The findings suggest a delicate balance for intact Y1349/1356 SH2-binding domain to mediate the tumor progression activity of the coactivated Met–rat sarcoma oncogene homolog (RAS) pathways. Selectivity for specific adaptor protein involvement may be the key that determines the tissue- and cell-type specificity of Met-mediated tumorigenicity in human cancers.

Abbreviations: c-Cbl, Casitas B-lineage lymphoma proto-oncogene; Crk, Sarcoma CT10 oncogene homolog; Gab1, Grb2-associated adaptor protein 1; Gab1WT, Gab1 wild type; GFP, green fluorescent protein; HGF, hepatocyte growth factor; KRAS, Kirsten rat sarcoma oncogene homolog; mRNA, messenger RNA; MAPK, mitogen-activated protein kinase; Met, hepatocyte growth factor receptor; MetWT, wild-type Met; PCR, polymerase chain reaction; PI3K, phosphatidyl inositol 3 kinase; PLC, phospholipase c-gamma; RAS, rat sarcoma oncogene homolog; SH2, Src homology 2; Shc, Src homology domain c-terminal adaptor homolog; Shp2, Src homology-containing tyrosine phosphatase 2; ShRNA, short hairpin RNA; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; Tpr, translocated promoter region


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Hepatocyte growth factor (HGF) is a multifunctional cytokine with pleotrophic roles in cell proliferation, migration, morphogenesis and tumor growth (1). While the tumor growth-promoting role of HGF/hepatocyte growth factor receptor (Met) signaling is well established, there are also reports that excessive HGF/Met signaling can lead to tumor growth suppression (2,3). We showed previously that the overexpression of the Met receptor in DLD-1 colon cancer cells harboring oncogenic KRAS mutation results in enhanced tumor growth rate in immune-deficient mice (4). This activity appears as a result of interaction between constitutive activation of Met and KRAS as Met overexpression in an isogeneic cell line with inactivated KRAS oncogene led to suppression of tumorigenicity (4). The tumor-enhancing response appeared dependent on a sustained rather than transient duration of mitogen-activated protein kinase (MAPK) signaling in the DLD-1 cell line. The presence of KRAS mutation represents a selection point in determining whether HGF/Met causes growth promotion or suppression (4). To further delineate the signaling pathways that mediate the tumor progression effect of Met in DLD-1 cells, we have characterized the behavior of signal-specific mutant Met receptors in DLD-1 cells.

Mutant receptors capable of recruiting only specific signal transduction molecules have been used to explore the downstream biochemical pathways of tyrosine kinase receptors. The Met receptor depends on several key C-terminal phosphotyrosine residues for signal transduction. Tyrosine 1356 recruits Grb2 and Shc adaptor proteins for direct binding and activation of the RAS/proto-oncogene serine threonine protein kinase/MAPK (RAS/RAF/MAPK) pathway (5,6). Both the tyrosines 1349 and 1356 are capable of either directly or indirectly recruiting Grb2-associated adaptor protein 1 (Gab1), a large adaptor protein capable of docking with multiple other signaling proteins such as p85, the regulatory subunit of phosphatidyl inositol 3 kinase (PI3K), PLC{gamma}, src homology-containing tyrosine phosphatase 2 (Shp2), sarcoma ct10 oncogene homolog (Crk) and casitas B-lineage lymphoma proto-oncogene (c-Cbl) (7–13). In addition, the Met receptor has the potential to directly recruit p85 PI3K, PLC{gamma} (14) and Crk (15) through tyrosines 1349 and 1356, and c-Cbl (16) via tyrosine 1001. C-Cbl has a role as both an adaptor protein and ubiquitin ligase.

Met was first isolated as translocated promoter region (Tpr)-Met, a constitutively active chimeric receptor (17–19). Many studies that characterized the signaling pathways downstream from the Met receptor have been conducted using the Tpr-Met in cell lines generated from species other than humans. Based on results from these studies, one could putatively infer functional information on key signaling residues of the full-length Met receptor. Similar to Tpr-Met, full-length Met may also be activated in a ligand-independent manner when it is overexpressed (4).

There are many Tpr-Met signaling constructs that have been well characterized. Kinase activity of the receptor can be abrogated by mutating lysine 1110 in the kinase domain to alanine (K1110A) (20,21), whereas binding of downstream signaling molecules to C-terminal Src homology 2 (SH2) domains can be eliminated by mutating the tyrosines 1349 and 1356 to phenylalanine (Y1349/1356F). The binding of Grb2 to the receptor could be specifically abrogated by mutating asparagine 1358 to histidine (N1358H), thus making the SH2-binding motif that specifically recruits Grb2 around tyrosine 1356 (YVNV) to mimic the SH2-binding motif at tyrosine 1349 (YVHV) (22,23). The multifunctional docking site of the Met receptor could also be disabled and replaced with specific binding sites for selective recruitment of one signaling protein at a time (24). These motifs were borrowed from other receptor tyrosine kinases such as epidermal growth factor receptor, platelet-derived growth factor receptor (PDGFR) and tyrosine receptor kinase of nerve growth factor (TrkA).

In this study, we have adapted these constructs into full-length Met receptors to demonstrate that Gab1 and Shp2 and not Grb2/Shc are involved in promoting invasion motility and tumor growth of Met overexpressing colorectal cancer cell line DLD1.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Reagents and cell lines
Phoenix 293T Ampho and DLD-1 cell lines were obtained from the American Type Tissue Culture Collection and were routinely cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY). Antibodies to phospho-p44/p42 MAPK (Thr202/Tyr204), phospho-Met (Tyr1234/Try1235), pSTAT3, pGab1 (Tyr627 and Tyr307), signal transducer and activator of transcription 3 and MAPK were purchased from Cell Signaling Technologies (Beverly, MA). Antibodies for human Met (C28), murine Met (B3) and Grb2 were from Santa Cruz Biotechnology (Santa Cruz, CA), for Shp2 and Shc from BD Biosciences (San Diego, CA), and for Gab1, PLC{gamma} and p85 PI3K from Upstate (Waltham, MA). Human recombinant HGF was purchased from R&D Systems (Minneapolis, MN).

Mutant Met receptor constructs
The pBMN-IRES-GFP plasmid is part of the Pheonix retroviral packaging system provided by Dr Gary Nolan (http://www.stanford.edu/group/nolan/plasmid_maps/pmaps.html). Tpr-Met SH2 domain mutants MetGrb2, MetShc1, MetShc2, Metp85 PI3K and MetPLC{gamma} were constructed as described (24). Tpr-Met shares ~1300 bp 3' sequence with the full-length wild-type Met (MetWT). The MetWT was cloned into the XhoI site of pBluescript II SK+ (Stratagene, La Jolla, CA). To construct full-length Met mutants, a 1200 bp SpeI restriction fragment was excised from pXM-Tpr-Met mutant constructs (the SpeI site is located 3' to the fusion point of Tpr and Met) and cloned into the SpeI site of MetWT, replacing the 3' end with SH2 domain mutants (supplementary Figure 1 is available at Carcinogenesis Online). Nucleotide and amino acid sequences for each expression construct are shown in supplementary Table I (available at Carcinogenesis Online).

Met mutants K1110A, N1358H and Y1349/56F were created by overlap extension polymerase chain reaction (PCR). Briefly, the 3' SpeI fragment of MetWT was amplified in two consecutive cycles of PCR. The first cycle of PCR amplified two fragments for each template. Primers MetSeqE forward (5'-GCC CGA AGT GTA AGC CCA ACT ACA G-3'), K1110A reverse (5'-CAG TGA TTC TGT TCA AGG ATG CCA CAG CAC AGT GAA TTT TC-3'), N1358H reverse (5'-CGG AGC GAC ACA TTT TAC GTG CAC ATA AGT AGC GTT CAC-3') and Y1356F reverse (5'-GAC ACA TTT TAC GTT CAC AAA AGT AGC GTT CAC ATG GAC-3') were employed for the first fragment. Primers Met 3' reverse (5'-GCG AGC TCC TCG AGC TGC AGC GCA TTG GTC CCT G-3'), K1110A forward (5'-GAA AAT TCA CTG TGC TGT GGC ATC CTT GAA CAG AAT CAC TG-3'), N1358H forward (5'-GTG AAC GCT ACT TAT GTG CAC GTA AAA TGT GTC GCT CCG-3') and Y1349F forward (5'-CTA CTT TCA TTG GGG AGC ACT TTG TCC ATG TGA ACG CTA C-3') were used for the second fragment. Following 35 cycles of amplification, both PCR fragments were gel purified and then corresponding fragments were added together in equal concentration for a second 35 cycles of PCR, employing only the flanking MetSeqE and Met 3' reverse primers. The resulting 1300 bp fragments were gel purified and restriction digested with SpeI and XhoI to create 1200 bp Met 3' fragments. Met 1200 bp SpeI/XhoI fragments were subcloned into XhoI cut pBluescript II SK+ together with the MetWT 5' 3400 bp fragment (also restriction digested with XhoI and SpeI) by 3-part ligation. Constructs were then bidirectionally sequence verified to confirm single-base pair mutations (for sequence information, see supplementary Table I, available at Carcinogenesis Online). All the pBMN-Met constructs were created bidirectionally cloning the full-length 4.6 kb Met complementary DNA into the EcoRI and NotI sites of pBMN-IRES-GFP. The EcoRI site was filled and destroyed during cloning.

Establishment of stable cell lines
To generate retroviruses, the Pheonix 293T Amphotropic retroviral packaging line was transfected with pBMN, pBMN-MetWT and pBMN-Met mutants (MetGrb2, MetShc1, MetShc2, Metp85PI3K, MetPLC{gamma}, MetK1110A, MetN1358H or MetY1349/56F) constructs using Lipofectamine PLUS transfection reagent (Invitrogen, Burlington, ON). At 48 h post-transfection, retrovirus-containing media were harvested and cell debris were removed by centrifugation at 1500 r.p.m. for 15 min. DLD1 cells were transduced twice with pBMN, pBMN-Met and pBMN-Met mutants' viral supernatants in the presence of 8 µg/ml polybrene (Sigma Chemical, St Louis, MO), at the time of subculture and again at 48 h after subculture. Approximately 40% of the population expressed green fluorescent protein (GFP) at 72 h post-transduction (data not shown). At 96–120 h after the first transduction, total cell population were trypsinized into single-cell suspension and sorted for high GFP expression by fluorescence-activated cell sorting. Sorting attained a 90–95% pure population of GFP expressing cells. Cell lines were monitored by flow cytometry for GFP expression for 10 passages. No loss of GFP expression occurred in 10 passages (data not shown).

To generate stable cell lines with moderate expression level of Gab1 and DLD-1 Gab1 line that does not recruit Shp2, DLD-1 cell line was transfected with pBabe-puro-Gab1WT and pBabe-puro-Gab1Y627F constructs using Lipofectamine PLUS transfection reagent (Invitrogen). Cells were passaged 48 h post-transduction and clones were selected with 5 µg/ml puromycin.

PCR assay for stable Met constructs
Total genomic DNA was isolated from all Met mutant cell lines using the DNeasy tissue kit (Qiagen, ON, Canada). Intron-spanning PCR primers (Met-E-forward 5'-GCC CGA AGT GTA AGC CCA ACT ACA-3' and Met-D-reverse 5'-CGT GTG TCC ACC TCA TCA TC-3') were designed to selectively amplify inserted Met complementary DNA but not endogenous Met genomic DNA. A 1200 bp fragment at the 3' end of Met was amplified from total cellular genomic DNA by PCR under the following conditions: 5 min at 95°C; 35 cycles (30 s at 95°C, 30 s at 67°C and 1.30 min at 72°C), 7 min at 72°C. The 1200 bp PCR fragment was bidirectionally sequence verified for the appropriate presence of specific Met mutant or wild-type sequence (data not shown).

Small hairpin RNA lentivirus constructs
PLentiLox shShp2 was kindly provided by Dr Ben Neel (Harvard University). The small interfering RNA (siRNA) target sequence (GATTCAGAACACTGGTGAT) for short hairpin (sh)RNA design corresponded to nucleotides 545–560 of the human Shp2 messenger RNA (mRNA) sequence NM_002834 [GenBank] . A control shRNA sequence not complementary to any human gene was from Qiagen's non-silencing control shRNA sequence (TTCTCCGAACGTGTCACGT). Constructs were verified by sequencing.

Lentivirus and transduction
Lentiviruses were prepared by transfecting three plasmids into the 293T cells, as described (25,26). The plasmids were pMDLg/pRRE, the vesicular stomatitis virus envelope plasmid pCMV-VSG, rev-expressing plasmid pRSV-Rev and gene transfer pLentiLox shShp2 or pLentiLox containing the non-specific sequence. The pLentiLox 3.7 vector has a CMV-eGFP cassette. Stocks were stored frozen at –80°C and titered on 293T cells. GFP-positive cells were scored to quantify the titer. For a typical preparation, the titer of an undiluted virus was ~2 x 106 infectious units per milliliter. Cells were transduced as described previously (25,26). To obtain the maximum knockdown with the lowest number of viral particles we used five infectious units per cell in all transductions.

Growth factor stimulation, protein lysates and immunoprecipitations
All cell lines were passaged the day before protein isolation and were ~50% confluent at time of isolation. Growth factor stimulation was conducted by adding HGF (10 ng/ml) to the growth media, and total cellular protein was isolated 5–10 min later. To prepare protein extracts, stimulated and unstimulated cells were washed twice with cold phosphate-buffered saline and lysed for 15 min in ice-cold lysis buffer (50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 8.0, 10% glycerol, 0.5% Triton X-100, 150 mM NaCl, 2 mM ethyleneglycotetraacetic acid (EGTA), 1.5 mM MgCl2, supplemented with 10 µg/ml leupeptin, 10 µg/ml aprotinin, 100 µg/ml phenylmethylsulfonyl fluoride and 1 mM sodium orthovanadate). Protein was extracted from snap-frozen tumor tissue by homogenizing tissue fragments in ice-cold lysis buffer for 30 s. All lysates were centrifuged at 14 000 r.p.m. for 10 min and the supernatant stored at –70°C prior to subsequent analyses.

Cell lysates (1–3 mg) were then immunoprecipitated, respectively, with anti-MAPK, anti-Gab1, anti-Shp2, anti-Akt or anti-PI3K p85 overnight at 4°C. Immunoprecipitates were collected by addition of protein A-sepharose beads, washed three times with lysis buffer and resuspended in Laemmli sample buffer (27). Immunoprecipitates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes followed by immunoblotting with anti-phosphotyrosine antibodies (cell signaling) conjugated to horseradish peroxidase. The blots were revealed by enhanced chemiluminescence followed by radioautography on Kodak X-Omat AR films or using Typhoon 9410 Variable Mode Imager (Amersham, Quebec, Canada).

Samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting. To perform immunoblotting, lysates containing 10–35 µg of protein were resolved on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride or nitrocellulose membrane. Equal protein loading was confirmed by staining the gel with amido black dye or reprobing the blot with antibody against reduced guanosine adenine dinucleotide phosphate. Membranes were blocked with Tris-buffered saline containing 1% Roche blocking reagent (Roche, Dorval, Quebec) or Tris-buffered saline containing 0.1% Tween 20 and 5% non-fat milk. The membrane was incubated with primary antibody, respectively, for 1 h at room temperature or overnight at 4°C in blocking solution. Membranes were washed three times for 5 min each in Tris-buffered saline containing 0.1% Tween 20 and incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse IgG for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence reagents (Roche-Boehringer Mannheim, Dorval, Quebec).

Invasion and migration assays
Tumor cell invasion was assessed in vitro by the reconstituted basement membrane (Matrigel) invasion assay. The chemoinvasion assays were performed using 8 µm polycarbonate filters coated with reconstituted basement membrane (Matrigel; BD Biosciences, San Diego, MA). Prior to coating, Matrigel was diluted with cold distilled water and 14 µg in 60 µl were added to each filter. The coated filters were dried overnight and equilibrated with serum-free RPMI for 2 h. The medium was then removed, the filters placed in 24-well plates and to each filter 5 x 104 cells were added in 100 µl RPMI containing 0.2% bovine serum albumin for a 48 h incubation at 37°C, in a humidified 5% CO2 incubator. Human fibronectin (5 µg/ml in RPMI; Gibco BRL, Burlington, ON) was used as a chemoattractant in the lower chamber. For HGF treatment assays, 50 ng/ml HGF was added to the lower chamber. At the end of the incubation period, the cells on the upper surface of the filters were removed with a cotton swab and the filters fixed in 0.1% glutaraldehyde and stained with 0.2% crystal violet. The number of cells that migrated to the lower side of the filter was counted using a Nikon Upright microscope (Nikon OPTIPHOT) (x100) (Mississauga, Ontario, Canada) using Image pro program. The whole area was counted per filter and two filters were used for each assay condition. Migration assays were carried out in a similar manner using filters, which were precoated with a low concentration of type IV collagen (7.5 µg per filter; Sigma, Oakville, ON) to facilitate cell spreading.

mRNA expression assay
Total RNA (4 µg) was reverse transcribed using Superscript II reverse transcriptase (Invitrogen, Ontario, Canada). A 10 ng equivalent of complementary DNA was used for each quantitative PCR assay performed with the Stratagene Mx3000p Sequence Detection System using SYBR green 2x master mix (Stratagene). Intron-spanning primers for Gab1 PCR amplification were designed using the Primer Express software (Perkin-Elmer Applied Biosystems, Foster City, CA). Predicted PCR product sequences were checked using BLAST for recognition of target and non-target sequences. Dissociation curves were performed as routine verification in order to check the primers for amplification of a single band, and template dilution standard curves (5 log range) were conducted with each primer set to verify a linear relationship between template concentration and Ct values (R2 > 0.9, data not shown). Primer sequences for Gab1 quantification were (forward) 5'-aagactacctgttgctcatcaactgt-3' and (reverse) 5'-ggacgttatcattggagtctgtttc-3'. RNA for standard curve generation was derived from subconfluent DLD-1 Met cell line grown in tissue culture due to its high overall transcriptional activity(4). Absolute transcript concentrations were calculated using a standard curve.

Tumorigenicity assay
In all, 2 x 106 cells were injected subcutaneously in the left shoulder region of 6-week-old male severe combine immunodeficiency mice (n = 4 per cell line). Mice were examined every 2 days and tumor length and width was measured using calipers. Tumor volume was calculated using the following formula: {pi}/6 (length x width2) (28). At 18 days, mice were euthanized by CO2 asphyxiation and tumors were excised. Portions of tumors were snap frozen and stored in liquid nitrogen or fixed in 10% buffered formalin for routine histopathological processing. Difference in tumor growth rates of xenografts was tested using mixed-effects model estimation (29). All statistical analyses were performed using SASv9.0 statistical software (SAS Institute, Cary, NC).

Orthotopic tumorigenicity and metastasis assay
For the tumor fragment implantation procedure, fresh tumor tissue was harvested from the primary xenograft tumor grown in donor animals implanted subcutaneously with the DLD-1 vector control and DLD-1 harboring different mutant Met. Viable tumor tissue was mechanically cut in a sterile petri dishes into ~2 mm diameter pieces by ‘crossed scalpels’ technique. For orthotopic implantation, the peritoneal cavity was surgically exposed, the cecum was isolated and a tumor fragment sutured on the surface of cecum wall using 7.0 Prolene suture. Animals were euthanized by CO2 when they succumbed to the disease or showed morbidity from tumor progression (~9–11 weeks post-implantation). Three-dimensional measurement of the primary tumor, lymph node and diaphragm metastasis were calculated using length x width x height by means of caliper (Manostat Inc., Rennes, Switzerland). Metastasis occurrences were assessed macroscopically and microscopically. Any visible tumor deposit other than the primary tumor was considered as metastasis. The internal organs including lung, liver, kidney, brain, chest wall or bone as well as mediastinal lymph nodes and diaphragm were removed from the euthanized animals. The specimens were weighed, fixed in 10% buffered formalin, serially sectioned and processed for histological examination.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Establishment of mutant Met-expressing DLD-1 cell lines
Stable integration and expression of the full-length mutant Met receptors in DLD-1 cells was monitored for >10 passages and their overexpression was confirmed by western blots (Figure 1A). Expression of MetWT and all but MetK1110A constructs results in the constitutive and HGF-independent activation of the receptor, as detected by western blot with the phospho-Met-specific antibody for tyrosines 1234 and 1235 (Figure 1A). The K1110A construct suppressed the phosphorylation of the Met receptor, even in the presence of the HGF ligand (Figure 1A). All cell lines also demonstrated similar expression levels of relevant downstream signaling adaptor molecules Grb2, Shc, Shp2, p85 PI3K, PLC{gamma} and Gab1 (Figure 1B).


Figure 1
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  		Fig. 1. Establishment of stable mutant Met expressing DLD-1 cell lines. (A) The DLD-1 mutant Met cell lines with or without HGF stimulation (20 ng/ml) were analyzed by western blot for the activated receptor using phospho-Met-specific antibody (pMet, top panel) and the total Met receptor levels using human Met antibody (hMet, bottom panel). (B) The expression of Grb2, Shc, Shp2, p85 PI3K, PLC{gamma} and Gab1, adaptor signaling proteins, were also verified by western blot using their specific antibodies.

 
Tumorigenicity of Met mutant receptors
Total cell population overexpressing the MetWT (positive control) and mutant Met receptors were implanted in the subcutaneous tissue of severe combine immunodeficiency mice and tumor growth was measured over time and compared with that of DLD-1 cells expressing the vector control alone (DLD-1-pBMN). The majority of receptor constructs tested lost the wild-type receptor's capability to increase tumor growth rate of DLD-1 cells in vivo. DLD-1 MetShc2, Metp85 PI3K and MetPLC{gamma} grew subcutaneously at a similar rate to the DLD1-pBMN vector control cells (data not shown). Similarly, the growth of K1110A (Figure 2A) and Y1349/56F (data not shown) cells mimicked the DLD-1-pBMN. Surprisingly, the non-Grb2-binding Met N1358H mutant did not stimulate tumor growth (P < 0.05, Figure 2B), whereas Met-Grb2 (P < 0.05, Figure 2C) and Met-Shc1 (P < 0.05, Figure 2D) mutant's overexpression resulted in slight but consistent reduction in tumor growth rates.


Figure 2
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  		Fig. 2. Grb2- and Shc-recruiting Met mutants suppress tumor growth rate in vivo. Experiment was conducted in duplicate and the data are represented as mean ± SEM (n = 4).

 
To test if the tumor-suppressive effects of the Grb2- and Shc-recruiting mutant receptors were mediated by direct binding of these signal transducers, we also studied the negative control mutants MetGrb2Y/F and MetShc1Y/F. MetGrb2Y/F was able to abrogate the suppressive effect of MetGrb2 (Figure 2C), whereas MetShc1Y/F was not able to do so (Figure 2D).

Met receptor overexpression enhanced metastatic potential of DLD-1 cells
The metastatic potential of colon cancer cell lines is best demonstrated by their orthotopic implantation in the colonic walls of immune-deficient mice. This model mimics the progression of human colon cancer with greater accuracy than subcutaneous implantation alone (30). Similar to the subcutaneous model, both DLD1-Met and DLD1-N1358H Met showed greater orthotopic tumorigenicity than the other mutants (data not shown). More importantly, they also showed an apparent increase in lymph node but not lung metastases (Figure 3A and B). We observed higher lymph node metastasis compared with lung metastases in MetWT (10-fold; P = 0.314) and MetN1358H (11-fold; P = 0.0869) relative to control pBMN, whereas lung metastases were 4-fold higher in MetWT (P = 0.3802) and 6-fold higher in MetN1358H (P = 0.0442) compared with control pBMN.


Figure 3
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  		Fig. 3. Metastatic potential of DLD-1 cells implanted orthotopically is not dependent on Grb2 signaling. (A) Lymph node metastasis total volume per mouse; pBMN-Met compared with vector control or DLD-1. (B) Number of lung metastasis examined microscopically. Data are represented as mean ± SEM.

 
Lack of in vitro surrogate markers
To investigate whether there is a surrogate marker for the in vitro effect of wild-type and N1358H mutant Met receptors observed in vivo, we performed invasion, motility and soft agar growth assays on all mutant Met receptor expressing cell lines and compared them with the negative control DLD-1-pBMN and positive control DLD-1-Met cell lines (supplementary Figure 2, available at Carcinogenesis Online). Overall, there was a lack of consistent correlation between the in vivo and in vitro effects. We believe this is probably due to the predominant effect of KRAS oncogenes over Met receptor activity on these in vitro cellular activities. Interestingly, the K1110A, N1358H and Y1349/56F mutants that putatively had less ability to recruit downstream signaling molecules showed lower anchorage-independent growth capacity in their response to HGF stimulation (supplementary Figure 2B and C, available at Carcinogenesis Online).

Loss of Gab1 protein in Grb2 and Shc1 mutant tumors in vivo
When the levels of signal transduction proteins in the tumor tissues were evaluated by western blot, it was noted that while the expression levels of almost all other adaptor proteins remained similar or slightly increased compared with the control DLD-1-pBMN tumor tissues, Gab1 levels were markedly suppressed in Grb2- and Shc-recruiting tumors (Figure 4A). Importantly, this loss of Gab1 protein expression appeared post-translational, as Gab1 mRNA expression was higher in MetGrb2 and MetShc1 tumors compared with those formed by the pBMN control or MetWT overexpression cells (Figure 4B). The loss of Gab1 protein also appears restricted to the in vivo situation, as it was not seen in cell lines growing in vitro (Figure 1B) or MetGrb2 and MetShc1 tumor cells recultured from tumor tissue (data not shown).


Figure 4
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  		Fig. 4. Gab1 protein level is decreased while mRNA level is increased in DLD-1 MetGrb2 and MetShc tumor tissues. (A) The protein levels of Gab1 in tumors formed by DLD-1 cell lines expressing the control pBMN or mutant Met receptors were evaluated by western blot. (B) The mRNA expression of Gab1 in pBMN, pBMN-Met, Grb2- and Shc-recruiting tumors expressing Met receptor constructs were evaluated using RT-qPCR. Horizontal line indicates median. Data are expressed as relative level of Gab1 mRNA expression as compared with pBMN control.

 
Gab1 and Shp2 are crucial for invasion and motility of DLD-1 cells
To further delineate the role of Gab1, we established stable cell lines with moderately elevated expression level of Gab1 wild type (Gab1WT) and Gab1 mutant incapable of recruiting Shp2 (Gab1Y627F). Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and western blotting confirmed that the Gab1 expression levels was similar in both the Gab1WT and Gab1Y627F cell lines and was 4- to 5-fold higher than in the control DLD1 or DLD-1-pBMN cells (Figure 5A and B). While DLD-1-Gab1WT tumors showed slightly enhanced growth rate compared with the DLD-1-pBMN tumors, Gab1Y627F showed significantly slower growth rate (P < 0.05, Figure 5C). Using the Matrigel invasion assay, Gab1WT resulted in ~6-fold increase in invasiveness compared with controls DLD-1 and DLD-1-pBMN cells, whereas the increase by Gab1Y627F was only 2-fold (Figure 5D). Western blot revealed that the DLD-1-Gab1Y627F mutant cells also showed lower levels of pGab1 (Tyr627), pMAPK and pShp2 levels compared with Gab1WT cells (Figure 5E and F).


Figure 5
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  		Fig. 5. Gab 1 is sufficient to mediate xenograft tumorigenicity and activation of MAPK. (A) Gab1 mRNA expression was analyzed using RT-qPCR in DLD-1 cell lines. *P < 0.05 using Mann–Whitney test compared with empty vector or DLD-1. (B) Western blot analysis of total cell lysates shows the level of Gab1 expression. (C) The tumor growth rates of subcutaneous implanted Gab1WT DLD-1 cell line and Gab1 DLD-1 cell line, which does not recruit Shp2 compared with controls. The experiments were conducted in duplicate with the representative data shown as mean ± SEM (n = 4). (D) Invasiveness of mutant cell lines was expressed relative to the negative control DLD-1-pBMN cell line. *P < 0.05 using Mann–Whitney test. (E) Lysates prepared from 0.8 cm of Gab1WT and Gab1Y627F tumors were analyzed by immunoblot followed by immunoprecipitation for pMAPK, pGab1 (both Tyr 307 and Tyr 627), pShp2, pAkt and PI3K p85. (F) Histogram shows the ratio between the two bands (phosphoprotein and total protein) for the same transducer. The intensity of the bands was measured using the microcomputer imaging device (MCID) program.

 
To confirm the role of Shp2 in invasion and migration of DLD1 cells, Shp2 expression was suppressed by shRNA (Figure 6A and B). There was a significant suppression of the invasiveness and motility of Shp2 down-regulated DLD-1 cells compared with cells transduced by non-specific shRNA. This effect was even more evident in the DLD-1 Met (MetWT) cell line (Figure 6C and D).


Figure 6
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  		Fig. 6. Gab1 regulates invasiveness of DLD-1 cell line through interaction with Shp2. (A) The protein levels of Shp2 protein in DLD-1 cell lines transduced with pLentiLox 3.7 vector carrying shShp2 and/or non-specific (NS) constructs were evaluated by western blot and (B) RT-qPCR. (C) Invasiveness and (D) migration of shShp2-transduced DLD-1 cell lines were expressed relative to their negative control non-specific-transduced cell lines and were verified using in vitro Matrigel invasion assay. *P < 0.05 using Mann–Whitney test, compared with cell lines transduced with control shRNA.

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
The constitutive activation of the HGF/Met signaling in DLD-1 tumor cells expressing KRAS oncogene results in enhancement of tumor growth rate in vivo (4). This work demonstrates further that the DLD-1 cells overexpressing Met also showed greater lung and lymphatic metastasis potential. By using a panel of mutant Met receptor constructs that selectively recruit specific downstream signaling proteins, we demonstrate that the Met-Gab1-Shp2 pathway is the obligate route for tumor progression activity of Met–RAS coactivation, but any effort to increase directly the signaling through Grb2 or Shc adaptors could lead to a diametrically opposed phenotype. The N1358H Met receptor mutant that specifically disallows the binding of Grb2 to the receptor acted like the MetWT and promoted tumor growth. This indicates that Grb2 binding is not necessary for the tumor-enhancing activity of Met in DLD-1 cells. The ability of MetGrb2Y/F to partially reverse the tumor suppression of overactivated MetGrb2 supports an active role for Grb2 signaling in tumor suppression. In contrast, the inability of the MetShc1Y/F construct to restore baseline tumor growth activity suggests that the negative growth effect in cells overexpressing the Shc-recruiting construct could be due to its interference with the binding of a growth-promoting factor such as Gab1 to the receptor.

The negative regulation of tumor growth by Grb2 and Shc recruitment is contradictory to published findings from several sources. It has been reported previously that Grb2 and Shc are responsible for activating the RAS/MAPK signaling cascade downstream from the Met receptor, a process associated with cell growth and proliferation (6,7,24,31). Many downstream pathways have been delineated for Met receptor signaling and most of those studies were conducted using Tpr-Met, which is a constitutively activated chimeric Met receptor with cytoplasmic localization (17–19). However, MetWT receptor can be spontaneously activated if it is present in the cells in sufficient quantity (4). Differences in cellular localization, as well as cell line may influence the results from these studies. Furthermore, many of the studies using human Met receptor are conducted in cell lines that are other than human origin such as MDCK, COS and 3T3 fibroblasts. Many of these lines make no Met receptor to begin with, nor would they be expected to in vivo. For example, 3T3 fibroblasts actually produce HGF in large quantities but do not express Met receptor, which indicates a mainly paracrine role of HGF for this type of cells. Consequently, it is not unexpected that signaling pathways downstream from full-length Met receptor in human colon carcinoma cells can have different biological consequences than those indicated in previous reports. However, it should be noted that while the Grb2-recruiting motif is quite specific, the Shc-binding motif is relatively generic and may possibly recruit additional SH2 domain-containing proteins. It is also possible that there are additional, unidentified adaptors contributing to the phenotype observed in vivo. We have reported previously that Met overexpression in NCI-H1264 non-small cell lung cancer cell line could also exert tumor-suppressive activity (2). Our current finding that Met-induced Grb2 overactivity may lead to tumor suppression may provide an explanation for our previous finding with the H1264 cells. It is possible that Met overexpression in H1264 cells led to preferential signaling through the Grb2/Shc pathway. Furthermore, it is known that oncogenic RAS activation, i.e. downstream of Grb2, may lead to growth arrest or senescence in certain cell lines (32). While the mechanism of this effect remains largely unknown, a cross talk between Met and Fas has been reported to promote apoptosis in the presence of elevated Met receptor levels in hepatic cells (33).

The necessity for preserving both the Y1349 and Y1356 SH2 domains suggest that the coordinated activation of multiple signaling pathways is necessary to affect the tumor progression of Met–RAS interaction. Alternatively, the truly crucial pathway could be the one coordinated by Gab1, a large adaptor protein that can either bind to the Met receptor directly in the multifunctional docking site or can indirectly bind to the receptor via Grb2. Gab1 has numerous sites that may become tyrosine phosphorylated upon association with the Met receptor. Once phosphorylated, Gab1 can associate with p85 PI3K, PLC{gamma}, Grb2, Shc and several other adaptor proteins downstream from the Met receptor such as Crk, c-Cbl and Shp2 (34). The regulation of Gab1 levels by Met may present a mechanism for Met to bypass signaling through its multifunctional docking site and maintain downstream signal propagations. We show that the direct recruitment of Gab1 through Met is sufficient to mediate the contribution of Met to aggressive tumor growth in human colorectal carcinoma. Conversely, we have shown that Met constructs that specifically bind Grb2 or Shc adaptor proteins, and thus could potentially recruit Gab1 indirectly, suppress tumorigenicity. The recruitment of Grb2 and Shc to the receptor may influence Gab1 protein levels and Gab1 levels are putatively key to regulating tumor growth rate, although this mechanism remains to be elucidated. Since the loss of Gab1 protein in Grb2 and Shc mutant receptor tumors were not reflected at mRNA expression level or in vitro, it could be postulated that binding of Grb2 or Shc to the Met receptor could in some way affect the stability, degradation or cycling of the Gab1 protein. Recently, Elferink et al. (35) reported the strict requirement for Grb2 and the ubiquitin ligase activity of Cbl for c-Met endocytosis. Therefore, we assume that in Grb2 Met mutant, the loss of Gab1 is through degradation of Gab1 through ubiquitination by the E3 ubiquitin ligase Cbl. This hypothesis is still under investigation in our laboratory. The adaptor protein Gab1 in this context may represent a crucial determinant of cell fate in Met signaling.

Gab1 is also an essential mediator of Met-regulated epithelial morphogenesis program, a biological process that requires coordinated epithelial cell proliferation, adhesion, migration and invasion (36). Gab1 has been shown to enhance cell growth and transformation of NIH 3T3 cells downstream of the epidermal growth factor receptor (37) and Met (38). Gab1 is also a potential player in the nuclear localization of activated MAPK. Gab1 is capable of binding to MAPK and aiding in nuclear translocation through a chaperone-like function (39). This finding is particularly pertinent as the differential regulation of tumor growth rate by Met is dependent on the Ras pathway via differences in the duration of MAPK activation (4). Transcriptional regulation within colon carcinoma cells was also shown to be most dependent on the presence of a Ras oncogene (4). The dependence of Shp2 binding to Gab1 in establishing the enhanced tumor growth phenotype (9) also indicates that Grb2 binding and signaling through Gab1 potentiates a far different response than direct Grb2 binding to Met. Shp2 binding to Gab1 has been associated with increased MAPK phosphorylation (40). In the context of epidermal growth factor receptor signaling (which has many signaling similarities in common with Met), Shp2 dephosphorylates key residues on the Gab1 protein that allows a RAS-GTPase-activating protein (RAS-GAP) to bind Gab1 and promote RAS activity (41). In this context, in cell lines with activated KRAS oncogene, increased Met levels and subsequent increase in Gab1 levels could either result in an increase in nuclear translocation of activated MAPK or an enhanced activation of the RAS/RAF/Map kinase kinase/MAPK (RAS/RAF/MEK/MAPK) pathway. The nuclear MAPK may then promote cell survival and increased proliferation.

Our results also show that the ability of Gab1 to promote colon cancer tumor progression downstream of Met is dependent on the association of Gab1 with the tyrosine phosphatase Shp2 and activation of MAPK. The association of Gab1 with Shp2 is required for sustained MAPK activation and tumor formation downstream from the Met receptor (40,42). Based on this study, a Gab1 mutant lacking the ability to bind Shp2 (Gab1Y627F) fails to activate MAPK and tumorigenicity downstream of the Met receptor, indicating that activation of MAPK may occur predominantly through the Gab1–Shp2 protein complex during colon cancer progression. Thus, it would appear that although there are several possible signaling routes from Met, all converge on MAPK in tumor growth promotion in DLD-1.

The regulation of Gab1 protein levels by Met receptor may represent a novel mechanism by which downstream signaling specificity may be achieved in receptor tyrosine kinase signaling in vivo. This would stand to reason as Gab1 in many ways has redundant function to Met and can bind almost the same array of downstream signaling molecules. Thus, it would appear that the cell can choose the large multisite adaptor quite specifically and that choice leads to a different phenotype. These findings are of particular interest in the design of targeted therapies for many cancer types overexpressing Met. Gab1 and/or Gab1-dependent signal inhibition has potential as a therapeutic target for tumors with deregulated Met receptor function.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figures 1 and 2 and Table I can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Canadian Institutes of Health Research (MOP-64345) to T.M.-S.; Doctoral Award to I.S.-L.


   Footnotes
 
{dagger} Drs Seiden-Long and Navab contributed equally to this work. Back


   Acknowledgments
 
Dr Morag Park for the Tpr-Met constructs and Dr Gary Nolan (Stanford University) generously allowed the use of his pBMN retroviral transduction system for our studies. We also thank Dr Ti Cai for Gab1WT and Gab1Y627F cDNA, Dr Ben Neel for PLentiLox shShp2 and Ms Trudey Nicklee for helping us with the Microcomputer Imaging Device to measure the intensity of the bands in the western blot analysis.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

  1. Mazzone M, et al. The Met pathway: master switch and drug target in cancer progression. FASEB J. (2006) 20:1611–1621.[Abstract/Free Full Text]
  2. To C, et al. High expression of Met/hepatocyte growth factor receptor suppresses tumorigenicity in NCI-H1264 lung carcinoma cells. Exp. Cell Res. (2002) 273:45–53.[CrossRef][Web of Science][Medline]
  3. Tsao MS, et al. Differential expression of Met/hepatocyte growth factor receptor in subtypes of non-small cell lung cancers. Lung Cancer (1998) 20:1–16.[CrossRef][Web of Science][Medline]
  4. Long IS, et al. Met receptor overexpression and oncogenic Ki-ras mutation cooperate to enhance tumorigenicity of colon cancer cells in vivo. Mol. Cancer Res. (2003) 1:393–401.[Abstract/Free Full Text]
  5. Fixman ED, et al. Efficient cell transformation by the Tpr-Met oncoprotein is dependent upon tyrosine 489 in the carboxy-terminus. Oncogene (1995) 10:237–249.[Web of Science][Medline]
  6. Fixman ED, et al. Pathways downstream of Shc and Grb2 are required for cell transformation by the tpr-Met oncoprotein. J. Biol. Chem. (1996) 271:13116–13122.[Abstract/Free Full Text]
  7. Bardelli A, et al. Gab1 coupling to the HGF/Met receptor multifunctional docking site requires binding of Grb2 and correlates with the transforming potential. Oncogene (1997) 15:3103–3111.[CrossRef][Web of Science][Medline]
  8. Fixman ED, et al. Efficient cellular transformation by the Met oncoprotein requires a functional Grb2 binding site and correlates with phosphorylation of the Grb2-associated proteins, Cbl and Gab1. J. Biol. Chem. (1997) 272:20167–20172.[Abstract/Free Full Text]
  9. Maroun CR, et al. The Gab1 PH domain is required for localization of Gab1 at sites of cell–cell contact and epithelial morphogenesis downstream from the met receptor tyrosine kinase. Mol. Cell. Biol. (1999) 19:1784–1799.[Abstract/Free Full Text]
  10. Nguyen L, et al. Association of the multisubstrate docking protein Gab1 with the hepatocyte growth factor receptor requires a functional Grb2 binding site involving tyrosine 1356. J. Biol. Chem. (1997) 272:20811–20819.[Abstract/Free Full Text]
  11. Weidner KM, et al. Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature (1996) 384:173–176.[CrossRef][Medline]
  12. Garcia-Guzman M, et al. Met-induced JNK activation is mediated by the adapter protein Crk and correlates with the Gab1-Crk signaling complex formation. Oncogene (1999) 18:7775–7786.[CrossRef][Web of Science][Medline]
  13. Lock LS, et al. Identification of an atypical Grb2 carboxyl-terminal SH3 domain binding site in Gab docking proteins reveals Grb2-dependent and -independent recruitment of Gab1 to receptor tyrosine kinases. J. Biol. Chem. (2000) 275:31536–31545.[Abstract/Free Full Text]
  14. Ponzetto C, et al. A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell (1994) 77:261–271.[CrossRef][Web of Science][Medline]
  15. Riordan SM, et al. The beta-subunit of the hepatocyte growth factor/scatter factor (HGF/SF) receptor phosphorylates and associates with CrkII: expression of CrkII enhances HGF/SF-induced mitogenesis. Biochem. J. (2000) 350((Pt 3)):925–932.[CrossRef][Web of Science][Medline]
  16. Peschard P, 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][Web of Science][Medline]
  17. Cooper CS, et al. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature (1984) 311:29–33.[CrossRef][Medline]
  18. Park M, et al. Mechanism of met oncogene activation. Cell (1986) 45:895–904.[CrossRef][Web of Science][Medline]
  19. Rodrigues GA, et al. Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase. Mol. Cell. Biol. (1993) 13:6711–6722.[Abstract/Free Full Text]
  20. Zhu H, et al. Tyrosine 1356 in the carboxyl-terminal tail of the HGF/SF receptor is essential for the transduction of signals for cell motility and morphogenesis. J. Biol. Chem. (1994) 269:29943–29948.[Abstract/Free Full Text]
  21. Zhu H, et al. Receptor chimeras indicate that the met tyrosine kinase mediates the motility and morphogenic responses of hepatocyte growth/scatter factor. Cell Growth Differ. (1994) 5:359–366.[Abstract]
  22. Giordano S, et al. A point mutation in the MET oncogene abrogates metastasis without affecting transformation. Proc. Natl Acad. Sci. USA (1997) 94:13868–13872.[Abstract/Free Full Text]
  23. Fournier TM, et al. Branching tubulogenesis but not scatter of madin-darby canine kidney cells requires a functional Grb2 binding site in the Met receptor tyrosine kinase. J. Biol. Chem. (1996) 271:22211–22217.[Abstract/Free Full Text]
  24. Saucier C, et al. Use of signal specific receptor tyrosine kinase oncoproteins reveals that pathways downstream from Grb2 or Shc are sufficient for cell transformation and metastasis. Oncogene (2002) 21:1800–1811.[CrossRef][Web of Science][Medline]
  25. Dull T, et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. (1998) 72:8463–8471.[Abstract/Free Full Text]
  26. Naldini L, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science (1996) 272:263–267.[Abstract]
  27. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227:680–685.[CrossRef][Medline]
  28. Tomayko MM, et al. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother. Pharmacol. (1989) 24:148–154.[CrossRef][Web of Science][Medline]
  29. Littell RC, et al. Statistical analysis of repeated measures data using SAS procedures. J. Anim. Sci. (1998) 76:1216–1231.[Abstract/Free Full Text]
  30. Pocard M, et al. Efficiency of orthotopic xenograft models for human colon cancers. In Vivo (1996) 10:463–469.[Web of Science][Medline]
  31. Ponzetto C, et al. Specific uncoupling of GRB2 from the Met receptor. Differential effects on transformation and motility. J. Biol. Chem. (1996) 271:14119–14123.[Abstract/Free Full Text]
  32. Bardeesy N, et al. RAS unplugged: negative feedback and oncogene-induced senescence. Cancer Cell (2006) 10:451–453.[CrossRef][Web of Science][Medline]
  33. Wang X, et al. A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Mol. Cell (2002) 9:411–421.[CrossRef][Web of Science][Medline]
  34. Bertotti A, et al. Tyrosine kinase signal specificity: lessons from the HGF receptor. Trends Biochem. Sci. (2003) 28:527–533.[CrossRef][Web of Science][Medline]
  35. Li N, et al. Specific Grb2-mediated interactions regulate clathrin-dependent endocytosis of the cMet-tyrosine kinase. J. Biol. Chem. (2007) 282:16764–16775.[Abstract/Free Full Text]
  36. Pollack AL, et al. Morphogenetic mechanisms of epithelial tubulogenesis: mDCK cell polarity is transiently rearranged without loss of cell–cell contact during scatter factor/hepatocyte growth factor-induced tubulogenesis. Dev. Biol. (1998) 204:64–79.[CrossRef][Web of Science][Medline]
  37. Holgado-Madruga M, et al. A Grb2-associated docking protein in EGF- and insulin-receptor signalling. Nature (1996) 379:560–564.[CrossRef][Medline]
  38. Mood K, et al. Gab1 is required for cell cycle transition, cell proliferation, and transformation induced by an oncogenic met receptor. Mol. Biol. Cell (2006) 17:3717–3728.[Abstract/Free Full Text]
  39. Osawa M, et al. ERK1/2 associates with the c-Met-binding domain of growth factor receptor-bound protein 2 (Grb2)-associated binder-1 (Gab1): role in ERK1/2 and early growth response factor-1 (Egr-1) nuclear accumulation. J. Biol. Chem. (2004) 279:29691–29699.[Abstract/Free Full Text]
  40. Cunnick JM, et al. Regulation of the mitogen-activated protein kinase signaling pathway by SHP2. J. Biol. Chem. (2002) 277:9498–9504.[Abstract/Free Full Text]
  41. Montagner A, et al. A novel role for Gab1 and SHP2 in epidermal growth factor-induced Ras activation. J. Biol. Chem. (2005) 280:5350–5360.[Abstract/Free Full Text]
  42. Maroun CR, et al. The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase. Mol. Cell. Biol. (2000) 20:8513–8525.[Abstract/Free Full Text]
Received June 29, 2007; revised November 15, 2007; accepted December 20, 2007.


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