Cancer Research 67, 6012, July 1, 2007. doi: 10.1158/0008-5472.CAN-07-0022
© 2007 American Association for Cancer Research
Inhibition of Tumor Metastasis by a Growth Factor Receptor Bound Protein 2 Src Homology 2 Domain–Binding Antagonist
Alessio Giubellino1,
Yang Gao1,
Sunmin Lee2,
Min-Jung Lee2,
James R. Vasselli1,
Sampath Medepalli1,
Jane B. Trepel2,
Terrence R. Burke, Jr.3 and
Donald P. Bottaro1
1 Urologic Oncology Branch, 2 Medical Oncology Branch, National Cancer Institute, Bethesda, Maryland; and 3 Laboratory of Medicinal Chemistry, National Cancer Institute, Frederick, Maryland
Requests for reprints: Donald P. Bottaro, Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, Building 10, CRC 1 West, Room 3961, 10 Center Drive, MSC 1107, Bethesda, MD 20892-1107. Phone: 301-402-6499; Fax: 301-402-0922; E-mail: dbottaro{at}helix.nih.gov.
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Abstract
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Metastasis, the primary cause of death in most forms of cancer, is a multistep process whereby cells from the primary tumor spread systemically and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates; however, our understanding of metastasis at the molecular level is still rudimentary. Growth factor receptor binding protein 2 (Grb2) is a widely expressed adapter protein with roles in epithelial cell growth and morphogenesis, as well as angiogenesis, making it a logical target for anticancer drug development. We have previously shown that a potent antagonist of Grb2 Src homology-2 domain–binding, C90, blocks growth factor–driven cell motility in vitro and angiogenesis in vivo. We now report that C90 inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. These results support the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establish a critical role for Grb2 Src homology-2 domain–mediated interactions in this process. [Cancer Res 2007;67(13):6012–6]
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Introduction
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Metastasis, clinically the most important process in the progression of most types of cancer, remains poorly understood at the molecular level (1, 2). Patients with metastatic disease have far less favorable survival rates when compared with tumors discovered and treated while still confined to the primary site of occurrence. Furthermore, metastasis can insidiously appear many years after the primary tumor is removed or treated, due to the presence and growth of undetectable micrometastasis. These facts underscore the desperate need for therapies specifically targeting micrometastatic disease. Such agents have the potential to dramatically improve survival rates for most cancers.
Metastasis is a multistep process (3, 4). Primary tumor cells must first invade the surrounding tissue through a coordinated process of matrix remodeling and motility, requiring, among other things, focal adhesion complex recycling at the leading edge of the lamellipodia (5). Cells breaching the vasculature reach distant sites via the systemic circulation, then extravasate, invade new tissue, and proliferate again. Rational drug development strategies targeting these cellular events have focused on a variety of molecules, including critical regulators of extracellular matrix, cell motility, adhesion, and survival (2). The ability of several growth factors widely implicated in cancer, such as hepatocyte growth factor (HGF), to stimulate cell motility, morphogenesis, and cell cycle progression makes their signaling pathway components particularly attractive drug targets (6–8).
Growth factor receptor binding protein 2 (Grb2) is a ubiquitously expressed adapter protein that links activated tyrosine kinases (TK) to the Ras/mitogen-activated protein kinase (MAPK) pathway via selective recognition of the phosphotyrosyl (pY) peptide motif pYXNX (where N is asparagine and X is any residue) by its Src homology-2 (SH2) domain and constitutive binding of Sos1 by its two SH3 domains (9). Many growth factor receptor TKs, nonreceptor TKs (e.g., focal adhesion kinase or FAK), intracellular effectors of these pathways and phosphotyrosine phosphatases, possess the pYXNX motif recognized by the Grb2 SH2 domain (9). The unique structural features and phosphopeptide selectivity of the Grb2 SH2 domain have facilitated the development of small synthetic binding antagonists that potently disrupt functions mediated by its interaction with cognate proteins (10). We previously showed potent blockade of growth factor–stimulated cell motility, matrix invasion, and morphogenesis in various cell models, as well as angiogenesis in vivo, by this class of compounds (11, 12). We now show that a prototype of this compound class, C90, can inhibit tumor metastasis in two different murine models. These results support the potential efficacy of these compounds in reducing the spread of primary solid tumors, and establish a critical role for Grb2 SH2 domain–mediated interactions in this process.
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Materials and Methods
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Reagents and cell culture. The Grb2-SH2 domain–binding antagonist C90 was prepared as described (11). PC3M-luc-C6 cells (Xenogen Corp.) were derived from PC3M cells by stable transfection with firefly luciferase and maintained according to the instructions of the manufacturer. B16-F1 murine melanoma cells stably transfected with luciferase were maintained in DMEM + 10% fetal bovine serum. Recombinant HGF was obtained from R&D Systems.
Cell migration assays. Cell migration was measured using modified Boyden chambers as described (11). PC3M and B16 cells were seeded at 200,000 and 150,000 cells per chamber, respectively. B16 cell migration was stimulated with serum (10%) and membrane bottoms were coated with fibronectin (10 µg/mL). Mean values from four fields (1 x 1.4 mm) were calculated for each of triplicate wells per condition. IC50 values were determined using GraphPad Prism software.
Cell proliferation assays. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PC3M or B16 cells (25 x 103) were incubated in 96-well plates for 24 h before serum deprivation and treatment with the indicated concentrations of C90, in the presence and absence of HGF (PC3M) or serum (B16) for 36 h. An MTT solution (1 mg/mL; Sigma) was added and plates were incubated at 37°C for 3 h before measuring absorbance at 562 nm (Bio-TEK instruments).
Immunoprecipitation and immunoblot analysis. c-Met/Grb2 interaction was analyzed by immunoprecipitation and immunoblotting as described (11). FAK-Grb2 interaction was analyzed similarly, using different anti-FAK antibodies for immunoprecipitation (Upstate) and immunoblotting (BD Biosciences), and anti-Grb2 for immunoblotting (Santa Cruz Biotech). Antibodies for phospho- and total Akt and MAPK immunoblotting were from Cell Signaling and were used as described (13).
Immunofluorescence photomicroscopy. Cells plated in chamber slides were serum-deprived in the presence or absence of C90 (1 µmol/L) for 24 h, stimulated with HGF (50 ng/mL) for 1 h, then fixed and permeabilized. Slides were blocked with 5% bovine serum albumin in PBS for 1 h, incubated with anti-FAK antibodies (BD Biosciences) overnight at 4°C, washed, then incubated with Texas red–labeled secondary antibody (Molecular Probes) and Alexa488-conjugated phalloidin (Molecular Probes) for 1 h. Slides were washed with PBS, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes), and epifluorescence images were digitally acquired using an Olympus photomicroscope and IPLab software (Scanalytics).
Tumor xenograft models. Cells were treated ex vivo for 16 h with C90 (1 µmol/L), harvested, washed in PBS, and resuspended at the appropriate cell density for injection in the presence of C90 (10 µmol/L). Cells were delivered hypodermically into the dorsal tail vein (B16-luc, 2 x 106 cells) or s.c. into the flank (PC3M-luc, 3 x 106 cells; B16-luc, 1 x 106 cells) of 12-week-old male SCID/Beige mice (Taconic, Inc.). Primary tumor volume was calculated by caliper measurements based on the formula (length x length x width) / 6 and tumor mass was measured on a microbalance. On the indicated days, mice were injected with D-luciferin (150 mg/kg; Xenogen) and imaged (IVIS Imaging System 100 Series; Xenogen) for 1 or 5 min. Total body bioluminescence was quantified by integrating the photonic flux (photons per second) through a uniform region of interest. All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Results, expressed as mean ± SD, were compared by Student's t test.
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Results and Discussion
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C90 blocks Grb2/c-Met and Grb2/FAK interactions in intact cells. Serum-deprived PC3M cells were treated with C90 for 16 h prior to brief stimulation with HGF and coimmunoprecipitation analysis of Grb2/cMet interaction (Fig. 1A
). Consistent with prior studies, Grb2/c-Met binding was strictly ligand-dependent (11). Treatment with C90 completely disrupted c-Met/Grb2 interaction in PC3M cells at concentrations as low as 30 nmol/L (Fig. 1A). The selectivity of C90 for the SH2 domain of Grb2 was investigated in the same cells by analyzing HGF-stimulated activation of Akt and MAPK downstream of c-Met (Fig. 1B). Disruption of ligand-stimulated interaction between c-Met and the phosphatidylinositol 3-kinase (PI3K) SH2 domain, the latter being structurally distinct from the SH2 domain of Grb2, would be expected to block PI3K-dependent activation of Akt (14). Yet, Akt activation was completely unaffected by C90 treatment, even at a dose 10-fold higher than that sufficient to disrupt c-Met/Grb2 binding (Fig. 1B, top). Similarly, pathways to MAPK activation other than via Grb2-mediated recruitment of Sos1/Ras persist in the presence of C90 (Fig. 1B), further supporting its strict selectivity for Grb2 SH2 domain–mediated interactions. Oncogenically relevant Grb2 SH2 domain–mediated interactions with proteins other than c-Met were also blocked by C90 treatment (Fig. 1C). Immunoprecipitation of FAK from HGF-stimulated cells revealed that Grb2/FAK interaction peaked within 30 min; C90 treatment reduced this interaction to control levels (Fig. 1C) and dramatically reduced the number of focal adhesions observed via immunofluorescent staining with anti-FAK antibodies (Fig. 1D). These results indicate a critical role for Grb2 in focal adhesion formation, in addition to its other known roles in mediating HGF-stimulated cell motility.
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Figure 1. The Grb2 SH2 domain antagonist C90 inhibits HGF-stimulated c-Met/Grb2 and FAK/Grb2 interaction. A, PC3M cells were serum-deprived and treated with the indicated concentrations of C90 (in nmol/L) for 16 h, then left without further treatment (left) or stimulated briefly with HGF (50 ng/mL; right). Cell lysates were immunoprecipitated with anti-Grb2, resolved by SDS-PAGE, then immunoblotted with anti–c-Met (top) or anti-Grb2 (bottom). B, PC3M cells were serum-deprived and treated with the indicated concentrations of C90 (in nmol/L) for 16 h, then left without further treatment (left) or stimulated briefly with HGF (50 ng/mL; right). Cell lysates were resolved by SDS-PAGE and subjected to immunoblot analysis for phospho-Akt and total Akt (top), or phospho-MAPK and total MAPK (bottom). C, PC3M cells were serum-deprived and treated with C90 (1 µmol/L) as indicated for 16 h, then left without further treatment (left) or stimulated briefly with HGF (50 ng/mL; right) for the times indicated. Cell lysates were immunoprecipitated with anti-FAK antibody, resolved by SDS-PAGE and subjected to immunoblot analysis with anti-Grb2 (top), or anti-FAK (bottom). D, PC3M cells were serum-deprived and left untreated (Control, left) or treated with C90 (1 µmol/L; C90, right) for 16 h, then both groups were stimulated with HGF (50 ng/mL) for 60 min. Cells were formalin-fixed and stained for immunofluorescence photomicroscopy with anti-FAK Texas red (red), phalloidin-Alexa488 (green), and DAPI (blue). White arrows, representative focal adhesions (objective magnification, x60).
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C90 inhibits metastatic tumor cell migration in vitro. The Grb2 SH2 domain–binding antagonism was associated with diminished HGF-stimulated migration in both metastatic cell lines in vitro (Fig. 2
). C90 significantly inhibited PC3M cell migration over a range of concentrations with an IC50 of 150 nmol/L (Fig. 2A; R2 = 0.98, P < 0.0003). Consistent with prior studies (11) and with persistent PI3K and MAPK signaling, C90 had no significant effect on PC3M cell proliferation over the same dose range (Fig. 2B). C90 treatment also resulted in significant, dose-dependent inhibition of serum-stimulated B16 cell migration (Fig. 2C; IC50 = 650 nmol/L, R2 = 0.89, P < 0.01). As anticipated, no significant effect on B16 cell proliferation was associated with C90 treatment over this dose range (Fig. 2D).
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Figure 2. The Grb2 SH2 domain antagonist C90 inhibits HGF-stimulated migration of PC3M cells and B16 cells in vitro. A, dose-response curve of migration by C90-treated PC3M cells across a semipermeable barrier in a modified Boyden chamber in the presence of HGF (50 ng/mL;  ). Values on the Y-axis are mean cell number per 10x microscopic field. Bars, SD; where no bars are seen, the errors were smaller than the symbol sizes. B, dose-response curve of C90-treated PC3M cell proliferation after 48 h as measured by MTT assay and expressed as a percentage of maximum, in the absence ( ) or presence () of HGF (50 ng/mL). Bars, SD. C, dose-response curve of C90-treated B16 cell migration across a semipermeable barrier in a modified Boyden chamber in the presence of 10% serum ( ). Values on the Y-axis are mean cell number per 10x microscopic field. Bars, SD. D, dose-response curve of C90-treated B16 cell proliferation after 48 h as measured by MTT assay and expressed as a percentage of maximum, in the absence ( ) or presence () of 10% serum. Bars, SD.
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C90 inhibits tumor metastasis in mice. To minimize the amount of drug needed for these studies, cells were treated ex vivo before being introduced into the animals. PC3M human prostate adenocarcinoma cells were stably transfected with luciferase (PC3M-luc-C6) to enable bioluminescence imaging (BLI) and were treated for 16 h with 1 µmol/L of C90 prior to s.c. injection into the flanks of SCID/beige mice (six per group). With primary PC3M tumor growth over 3 weeks, cells spontaneously metastasize to the lungs (15). Within 1 week, the intense BLI signal of the primary tumors obscured the signal generated by lung metastases (data not shown); for this reason, at day 20 postinjection, animals were injected with luciferin immediately before sacrifice and lungs were removed for imaging ex vivo. As shown in Fig. 3A
, the mean bioluminescent intensity from the lungs of mice receiving C90-treated cells was significantly (
50%) lower than control levels (P < 0.05). As anticipated, analysis of the PC3M primary tumors revealed no differences in mass or volume between C90-treated and control groups (Fig. 3B), confirming the absence of any cytoreductive or cytotoxic effects of this compound.
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Figure 3. C90 inhibits lung metastasis in vivo. A, PC3M-luc cells were treated for 24 h with C90 (1 µmol/L, open column) or vehicle (control, filled column) and injected s.c. into the flanks of SCID/Beige mice. Animals (six per group) were sacrificed 20 d postinjection and lung metastatic burden was assessed by BLI ex vivo. All animals injected with cells developed tumors. Results were averaged from the peak light-emitting exposure from each group and plotted as photons per second (total flux) for a uniformly defined region encompassing the lungs. Bars, SD. B, primary tumors from animals described in (A) were removed from the flank, weighed and measured with calipers. Columns, mean tumor mass (left) or volume (right) for each group; bars, SD. C, B16-luc cells were treated for 24 h with C90 (1 µmol/L, open column) or vehicle (control, filled column) and injected i.v. into the dorsal tail vein of SCID/Beige mice. Animals (six per group) were sacrificed 7 d postinjection and lung metastatic burden was assessed by BLI ex vivo. All animals injected with cells developed lung metastases. Results were averaged from the peak light-emitting exposure from each group and plotted as photons per second (total flux) for a uniformly defined region encompassing the lungs. Bars, SD. D, to measure the effect of C90 treatment on B16-luc cell growth as a primary tumor, cells were treated for 24 h with C90 (1 µmol/L, open column) or vehicle (control, filled column) as in (C) and injected s.c. into the flanks of SCID/Beige mice (seven per group). After 14 d, primary tumors were removed from the flank, weighed, and measured with calipers. All animals injected with cells developed tumors. Columns, mean tumor mass (left) or volume (right) for each group; bars, SD.
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B16 cell cultures stably transfected with luciferase (B16-luc) for in vivo BLI were similarly treated for 16 h with 1 µmol/L of C90 prior to harvest and resuspension for tail vein injection (six mice per group). As shown in Supplementary Fig. S1A, SCID/Beige mice injected with untreated cells displayed an intense bioluminescence signal corresponding to a consistent and remarkable number of lesions in the lungs after 1 week, whereas the bioluminescence signal from lungs of animals receiving C90-treated B16-luc cells was significantly lower (P 
0.05). Animals were then sacrificed and lung bioluminescence was determined ex vivo as for PC3M with similar (50%) signal inhibition associated with C90 pretreatment (Fig. 3C). To exclude the possibility that C90 treatment affected tumor cell viability, identically prepared cells were injected s.c. in the flanks of SCID/Beige mice and tumor growth rate (Supplementary Fig. S1B) as well as final tumor mass and volume were measured (Fig. 3D). Consistent with cell proliferation studies, no differences were noted in the growth of primary B16 tumors derived from control or C90-treated cells.
Consistent with BLI analyses, lungs from the B16 melanoma control animal group, as observed macroscopically, displayed a dramatic and devastating number of pigmented lesions relative to lungs removed from the C90-treated animal group or lungs from normal animals not receiving tumor cells (Fig. 4A
). Histopathologic analysis confirmed the presence of numerous large metastatic lesions in the lungs of animals receiving untreated B16 cells and far fewer, significantly smaller, lesions in the lungs of animals receiving C90-treated cells (Fig. 4B and C). Histopathologic analysis of lungs from animals receiving PC3M cells confirmed the presence of spontaneous metastatic lesions, with striking differences in both size and number between control and C90-treated groups (Fig. 4D).
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Figure 4. Macroscopic and histopathologic analysis of tumor metastases. A, representative photographs of lungs from animals receiving untreated B16-luc metastatic melanoma cells (control) or C90-treated cells (C90) via tail vein injection; normal lungs (normal) were included for comparison. B, representative H&E-stained whole lung sections from animals receiving untreated B16-luc metastatic melanoma cells (control) or C90-treated cells (C90) via tail vein injection; normal lungs (normal) were included for comparison (magnification, x1.5). Black arrows, individual metastases produced by melanoma cells. C, representative H&E-stained sections of lungs removed from animals receiving untreated B16-luc metastatic melanoma cells (control, left) or C90-treated cells (C90, right) via tail vein injection (objective magnification, x10; inset, x20). D, representative H&E-stained sections of lungs removed from animals receiving untreated PC3M metastatic prostate adenocarcinoma cells (control, left) or C90-treated cells (C90, right) via s.c. injection into the flank (objective magnification, x10; inset, x20).
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The significant inhibition of metastasis observed in these models is consistent with the potent effects of this class of compounds on cell motility, invasion, and associated cellular processes. Seeking a proxy for Grb2 SH2 domain–binding antagonism in vivo, expression microarray studies on control and drug-treated cell cultures were done to identify drug-responsive genes. Among several genes, the expressions of which were significantly modulated by drug treatment (data not shown), N-cadherin was selected for further study because of its well-documented importance in tumor invasiveness (16, 17). Array results were first confirmed by real-time PCR (Supplementary Fig. S2A). N-cadherin mRNA transcripts levels in PC3M cell cultures that had been treated with C90 for 3 days were consistently suppressed by 50% relative to untreated cells (P < 0.0001). Analysis of primary PC3M tumor samples obtained 10 days postimplantation, after measurable solid tumors had formed in all animals, revealed that N-cadherin expression was suppressed by >50% in tumors derived from C90-treated cells relative to untreated controls (Supplementary Fig. S2B; P < 0.0001). These results provide evidence of a biologically relevant drug effect in tumor tissue in vivo and reveal that Grb2 SH2 domain–mediated signaling promotes increased N-cadherin expression in aggressively malignant cells.
Conclusions. We report that treatment of tumor cells with the Grb2 SH2 domain antagonist C90 substantially reduced the metastatic burden to lungs detected in mice 1 week (B16) or 3 weeks (PC3M) after injection. Several lines of evidence suggest that the primary mode of drug action is the disruption of cell motility and invasiveness; no significant effects on tumor cell viability or proliferation rate were observed in vitro or in vivo. The ability of C90 administered to cells prior to their implantation in animals to significantly inhibit tumor spread suggests that it acts early in the metastatic process, consistent with a model in which a small, metastatically competent fraction of the tumor cell population invade and colonize distant sites soon after their introduction into the host (18).
Further work will be needed to optimize drug delivery and dosing; nonetheless, the observed activity of this class of compounds in vivo in two aggressive tumor models strongly supports their potential efficacy as antimetastatic drugs. Our studies also reveal that whereas Grb2 signaling is suspected of contributing to tumor progression and malignancy by promoting cell proliferation as well as adhesion and motility, only the latter are susceptible to blockade through SH2 domain antagonism alone. Combining Grb2 SH2 domain antagonists with agents that target cell cycle progression or survival may lead to the development of effective and comprehensive anticancer therapies.
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Acknowledgments
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Grant support: Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
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.
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Footnotes
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Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Received 1/ 3/07.
Revised 3/30/07.
Accepted 5/ 2/07.
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Abstract
Introduction
