Certain tumor cell responses to the growth factor–inducible early response gene product CCN1/Cyr61 overlap with those induced by the hepatocyte growth factor (HGF)/c-Met signaling pathway. In this study, we investigate if Cyr61 is a downstream effector of HGF/c-Met pathway activation in human glioma cells. A semiquantitative immunohistochemical analysis of 112 human glioma and normal brain specimens showed that levels of tumor-associated Cyr61 protein correlate with tumor grade (P < 0.001) and with c-Met protein expression (r2 = 0.4791, P < 0.0001). Purified HGF rapidly upregulated Cyr61 mRNA (peak at 30 minutes) and protein expression (peak at 2 hours) in HGF−/c-Met+ human glioma cell lines via a transcription- and translation-dependent mechanism. Conversely, HGF/c-Met pathway inhibitors reduced Cyr61 expression in HGF+/c-Met+ human glioma cell lines in vitro and in HGF+/c-Met+ glioma xenografts. Targeting Cyr61 expression with small interfering RNA (siRNA) inhibited HGF-induced cell migration (P < 0.01) and cell growth (P < 0.001) in vitro. The effect of Cyr61 on HGF-induced Akt pathway activation was also examined. Cyr61 siRNA had no effect on the early phase of HGF-induced Akt phosphorylation (Ser473) 30 minutes after stimulation with HGF. Cyr61 siRNA inhibited a second phase of Akt phosphorylation measured 12 hours after cell stimulation with HGF and also inhibited HGF-induced phosphorylation of the Akt target glycogen synthase kinase 3α. We treated preestablished subcutaneous glioma xenografts with Cyr61 siRNA or control siRNA by direct intratumoral delivery. Cyr61 siRNA inhibited Cyr61 expression and glioma xenograft growth by up to 40% in a dose-dependent manner (P < 0.05). These results identify a Cyr61-dependent pathway by which c-Met activation mediates cell growth, cell migration, and long-lasting signaling events in glioma cell lines and possibly astroglial malignancies. Cancer Res; 70(7); 2932–41
- Hepatocyte growth factor
- glioma siRNA
Aberrations in receptor tyrosine kinase (RTK) systems activate a multitude of oncogenic mechanisms that increase overall malignancy. The RTK c-Met and its multifunctional ligand hepatocyte growth factor (HGF) are overexpressed or hyperactivated in many solid neoplasms and correlate with poor patient survival in select malignancies. HGF/c-Met pathway activation can induce a variety of cell type–dependent and context-dependent biological responses, such as migration/invasion, proliferation, angiogenesis, and cytoprotection (1–5). Inhibiting HGF and/or c-Met expression or function potently inhibits tumor xenograft formation, growth rates, and angiogenesis in many cancer model systems (6–9). The link between c-Met and malignant progression has stimulated the search for downstream mediators of these responses, in hope of understanding c-Met signaling and identifying new potential therapeutic targets.
c-Met is a potent activator of multiple signaling pathways, such as phosphatidylinositol 3-kinase (PI3K)/Akt, Ras/mitogen-activated protein kinase, signal transducer and activator of transcription (STAT), and NFκB, which activate a wide variety of oncogenic targets (10, 11). Expression microarray analysis of human glioblastoma cells stimulated with HGF resulted in the identification of several genes that are upregulated by c-Met activation. One HGF-responsive gene identified is the cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed (CCN) family member Cyr61 (12). Cyr61 is a secreted heparin-binding protein that associates with the cell surface and extracellular matrix (13–15). Cyr61, which is normally involved in wound healing, chondrogenesis, and neovascularization in vivo, is a growth factor–inducible and hypoxia-inducible early response gene that interacts with various cellular integrins to induce the expression of a diverse array of genes (14–18). Purified Cyr61 protein has been shown to regulate cell adhesion, stimulate chemotaxis, augment growth factor–induced DNA synthesis, foster cell survival, and enhance angiogenesis in vivo (19, 20). Cyr61 expression has been linked to poor outcomes in a multitude of solid tumors (21, 22), is implicated in increased tumorigenicity, and is overexpressed in invasive breast cancer and astrocytoma cell lines (23–28). Moreover, forced expression of Cyr61 in a low-grade U343 glioma model markedly enhanced tumorigenicity and vascularization in vivo. Xie and colleagues (20) showed that multiple integrins, especially αvβ3, were upregulated by Cyr61 protein along with an increase in integrin-linked kinase (ILK) activity and Akt activation.
The overlapping phenotypic effects of c-Met activation and Cyr61 suggest a novel mechanism by which the HGF/c-Met pathway influences cell behavior. This study investigates whether the HGF/c-Met pathway and Cyr61 are correlated in human gliomas and whether HGF induces Cyr61 in glioma models. Furthermore, we investigate if inhibiting Cyr61 in HGF-dependent glioma models alters HGF-induced cell survival, migration, and Akt activation. Our findings show that Cyr61 induction contributes to HGF-dependent glioma cell Akt activation, migration, cell proliferation, and tumor xenograft growth.
Materials and Methods
Cell culture and reagents
U373MG, U87MG, and SNB-19 cell lines were originally obtained from the American Type Culture Collection. Serial routine testing in our laboratory shows that these cells have maintained their characteristic phenotypic and gene expression patterns. U87MG cells were grown in MEM with Earle salts and l-glutamine (MEM 1X; Mediatech, Inc.) supplemented with 10% fetal bovine serum (FBS; Gemini Bioproducts, Inc.), 2 mmol/L sodium pyruvate (Mediatech), 0.1 mmol/L MEM nonessential amino acids (Mediatech), and penicillin-streptomycin (Mediatech). U373MG and SNB-19 cells were grown in DMEM low glucose with l-glutamine and sodium pyruvate supplemented with 10% FBS and penicillin-streptomycin as previously described (29). All cells were grown at 37°C in a humidified incubator with 5% CO2.
Antibodies were obtained from the following sources and used at the indicated dilutions: CCN1/Cyr61 (rabbit, 1:1,000; Abcam), phospho–glycogen synthase kinase 3α/β (GSK3α/β; Ser9/21; rabbit, 1:1,000) and phospho-Akt (Ser473; rabbit, 1:1,000; Cell Signaling Technology), Cyr61 (rabbit, 1:1,000) and actin (rabbit, C-11; 1:1,000; Santa Cruz Biotechnology), total Akt (mouse, 1:500; BD Biosciences), and secondary antibodies labeled with spectrally distinct near-IR dyes IRDye 800 CW (goat anti-mouse, 1:15,000) and IRDye 680 CW (goat anti-rabbit, 1:20,000; LI-COR Biosciences). Anti-HGF L2G7 murine monoclonal antibodies (mAb) and the isotype-matched control 5G8 were obtained from Galaxy Biotech, LLC and used as indicated (30).
Glioma xenografts were generated as previously described (29). Six- to 8-wk-old female mice (National Cancer Institute, Frederick, MD) were anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (5 mg/kg). For subcutaneous xenografts, nu/nu mice received 4 × 106 cells in 0.05 mL of plain medium s.c. in the dorsal flank. When tumors reached 200 mm3, the mice were randomly divided into groups (n = 5 per group) and received the indicated doses of either L2G7 or isotype-matched control mAb (5G8) in 0.1 mL PBS i.p. as previously described (30). Tumor volumes were estimated by measuring two dimensions [length (a) and width (b)] and calculating volume as V = ab2/2 (30, 31). At the end of each experiment, tumors were excised and frozen in liquid nitrogen, and protein was extracted for immunoblot analysis.
For intracranial xenografts, Scid/beige mice received 1 × 105 cells/2 μL by stereotaxic injection into the right caudate/putamen (29). L2G7 or 5G8 mAb was administered as above. Groups of mice (n = 5) were sacrificed by perfusion fixation at the indicated times, and the brains were removed for histologic studies. Vibratome/microtome perfusion-fixed tumor xenograft sections were subjected to quantitative IR immunofluorescence by simultaneously staining with primary antibodies specific for Cyr61 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using methods described by Kearns and colleagues (ref. 32; http://www.licor.com). Secondary antibodies labeled with two spectrally distinct near-IR dyes (IRDye 800 CW goat anti-mouse, 1:10,000; IRDye 680 CW goat anti-rabbit, 1:10,000) were used to simultaneously detect and quantify Cyr61 relative to GAPDH. Computer-assisted signal quantification was performed using the Odyssey Infrared Imager from LI-COR Biosciences.
The Johns Hopkins University Institutional Animal Care and Use Committee approved all animal protocols used in this study.
Immunohistochemistry and immunofluorescence
Cryostat or paraffin-embedded sections were stained with anti-Cyr61, anti-total Met, or anti-Ki67 using previously described methods (29). Biotinylated-conjugated secondary antibodies followed by incubation with 3,3′-diaminobenzidine peroxidase substrate were used to detect primary antibodies. Sections were counterstained with Gill's hematoxylin solution. We analyzed three to four random fields per histologic section and two sections per tumor to generate an average value per individual tumor. The percent area of antibody staining or proliferation indices was determined by computer-assisted quantification using ImageJ software (http://rsb.info.nih.gov/ij/). The rabbit IgG control values were determined in adjacent serial sections for each field and subtracted from the raw Cyr61 or Met expression value (as determined by computer-assisted image analysis software) to generate the final expression levels. Values >2 SDs above the normal human brain clinical specimens were used as the cutoff point for overexpression of Cyr61 or Met in gliomas (20, 28).
Northern blot analysis
Total RNA was harvested from cells using Qiagen RNeasy kits according to the manufacturer's recommendations. Ten micrograms of RNA per sample were denatured with deionized glyoxal, combined with RNA loading buffer, and subjected to electrophoresis in either duplicate or triplicate on a 1.0% agarose gel containing ethidium bromide for 2.5 h at 60 V. RNA was transferred to a nylon membrane (Nytran, Schleicher & Schuell Bioscience) overnight in 10× SSC (1.5 mol/L NaCl, 0.15 mol/L sodium citrate). The cDNA probe for Cyr61 was synthesized using oligonucleotide primers (sense, 5′-GGUUUACUUACGCUGGAUGtt-3′; antisense, 5′-CAUCCAGCGUAAGUAAACCtg-3′) designed from accession NM_001554. Reverse transcription-PCR was performed using U373MG RNA as a template, and PCR products were subcloned in TOPO PCR cloning vectors (Invitrogen, Inc.) and sequenced before use. Northern blot probes were generated for Cyr61 and 28S rRNA with [32P]dCTP (Amersham-Pharmacia) using a random priming labeling kit (Roche Diagnostics) according to the manufacturer's specifications. Membranes were prehybridized for 4 h at 42°C and then hybridized overnight at 42°C in a rotating oven. Membranes were washed thrice in 1× SSC–0.1% SDS at 50°C. Radioactivity was quantified using the Bio-Imaging Analyzer BAS 2500 (Fuji Medical Systems). All blots were stripped and then rehybridized with cDNA probe specific for 28S rRNA. Data from Western blots consist of control and experimental lanes quantified from the same membrane. Results are expressed relative to 28S rRNA. Each figure derived from these methods represents data from a single blot and single exposure condition. For a subset of experiments, representative lanes (shown below quantitative data) that most closely approximate the mean quantitative values were selected and repositioned from the original single blots to eliminate replicate lanes.
Cyr61 small interfering RNAs
Predesigned small interfering RNAs (siRNA) for Cyr61 were obtained from Ambion, Inc. and Dharmacon. Three siRNAs (ID 144771, ID 144772, and ID10013; NM_001554) were screened for knockdown efficiency by Northern and Western blotting as described above. siRNA ID 144771 and Cyr61 siRNA ON-TARGETplus Human SMARTpool (Dharmacon) were used for experimental studies. U373 cells were plated in 10-cm-diameter tissue culture dishes at 5 × 105 per plate in high serum medium (10% FBS). The next day, the medium was replaced with low serum medium (0.1% FBS). siRNAs were prepared using siPort Lipid (Ambion) for in vitro studies as instructed by the manufacturer to obtain a final working siRNA concentration of 50 nmol/L. One hour after replacing the medium, siRNA complexes were added to cells and allowed to incubate for 48 h. For in vivo studies, siRNAs were prepared using in vivo JET-PEI (Polyplus Transfection) as per the manufacturer's instructions to obtain a final treatment concentration of 50 to 500 pmol of Cyr61 siRNA in a total volume of 50 μL.
Total protein was extracted from glioma xenografts and cells using radioimmunoprecipitation assay buffer (1% Igepal, 0.5% sodium deoxycholate, and 0.1% SDS in PBS) containing fresh 1× protease and 1× phosphatase inhibitors (Calbiochem) at 4°C. Tissue extracts were sonicated on ice and centrifuged at 5,000 rpm at 4°C for 5 min. Supernatants were assayed for protein concentrations by Coomassie protein assay (Pierce) according the manufacturer's recommendations. Aliquots of 40 or 60 μg of total protein were combined with Laemmli loading buffer containing β-mercaptoethanol and subjected to SDS-PAGE according to the method of Towbin and colleagues (33, 34) with some modifications. For immunoblot analyses, proteins were electrophoretically transferred to nitrocellulose membrane with a semidry transfer apparatus (GE Healthcare) at 50 mA for 60 min. Membranes were incubated for 1 h in Odyssey Licor Blocking Buffer at room temperature and then overnight with primary antibodies at 4°C in 5% bovine serum albumin in TBS containing 0.1% Tween 20 (TBS/T). Membranes were then washed thrice with TBS/T, incubated with secondary antibody at 1:10,000 for 1 h in TBS/T, and washed thrice with TBS/T, followed by washing twice with TBS. Proteins were detected and quantified using the Odyssey Infrared Imager (LI-COR Biosciences).
U373MG cells were plated in 60-mm gridded plates and treated with designated siRNAs. After 24 h, cells were treated with mitomycin C (50 μg/mL) for 2 h, and cells on each side of the grid lines were removed by scraping. Cells were placed in low serum (0.1% FBS) medium and stimulated with 50 ng/mL HGF for 6 d. Cells were fixed and stained with cresyl violet. Computer-assisted image software was used to quantify cell migration relative to the grid line border.
Statistical analysis consisted of one-way ANOVA followed by the Tukey or Dunnet's multiple comparison test using Prism (GraphPad Software, Inc.). The χ2 test and t test were used to analyze the association of Cyr61 (or Met) with each clinical characteristic (age, gender, pathology, and grade). P < 0.05 was considered significant. All experiments reported here represent at least three independent replications. Data are represented as means ± SEM.
Cyr61 expression correlates with Met expression in human gliomas
Previous reports have shown that Cyr61 and Met expression both independently correlate with progression and malignancy in human gliomas (4, 28, 35). A correlation between Cyr61 and Met expression would support a potential mechanistic link between these proteins in glioma pathogenesis. We quantified the levels of Cyr61 and Met expression in 100 gliomas and 12 normal brain specimens by performing semiquantitative immunohistochemistry on a paraffin-embedded human glioma tissue microarray (Biomax, Inc.; Fig. 1). Met and Cyr61 were found to be overexpressed >2 SDs higher (>3-fold expression) than normal brain using criteria set forth by Xie and colleagues (28) in 35% and 41% of glioma samples, respectively. Overexpression of either Cyr61 or Met correlated with tumor grade (P < 0.0001; Fig. 1A and B). We found that 27 of 33 (82%) and 29 of 33 (88%) WHO grade IV samples overexpressed Met and Cyr61, respectively. In comparison, 3 of 25 (12%) and 3 of 23 (13%) WHO grade 3 and 2 samples, respectively, overexpressed Met and 8 of 25 (32%) and 3 of 23 (13%) WHO grade 3 and 2 samples, respectively, overexpressed Cyr61. Using a 95% confidence interval and regression analysis, we found that Cyr61 positively correlated with Met expression in human gliomas (P < 0.0001; Fig. 1C).
HGF/scatter factor–mediated expression of Cyr61 in glioma cell lines
Previous gene expression microarray analyses of U373 glioma cells treated with HGF identified Cyr61 as a HGF-inducible gene product (25). We hypothesized that HGF-induced glioma cell responses are mediated, in part, by Cyr61. To evaluate this hypothesis, we first defined the time course and magnitude of Cyr61 mRNA production and protein expression in response to HGF. Human U373 and SNB19 glioma cell lines were treated with HGF (50 ng/mL) for various time points, after which total cellular RNA, total cellular protein, and conditioned medium were isolated for Northern blot analysis or immunoblot analysis. Cyr61 mRNA expression was induced maximally 30 to 60 minutes after stimulation with HGF (Fig. 2A). Protein levels increased maximally 2 to 4 hours after HGF stimulation (Fig. 2B). Cyr61 protein accumulated in conditioned medium for up to 36 hours after HGF stimulation (Fig. 2C). HGF stimulation also induced Cyr61 in SNB19 (∼50% increase) and U87 (∼350% increase) cells (Supplementary Fig. S1A). We also examined U373 glioma cells engineered to stably express HGF (3) to determine if autocrine c-Met activation correlated with basal expression of Cyr61. The HGF-expressing clonal cell line had a ∼2-fold higher expression of Cyr61, in comparison with wild-type cells and HGF-negative transfected controls (Supplementary Fig. S1B). Both cycloheximide and actinomycin D potently inhibited HGF-induced Cyr61 expression, consistent with a transcriptional and translational mechanism of expression induction (Supplementary Fig. S1C).
Anti-HGF therapy inhibits Cyr61 protein expression concurrent with tumor growth inhibition in glioma xenograft models
We asked if Cyr61expression is altered by c-Met pathway inhibition in HGF/c-Met–dependent glioma xenografts. Mice bearing preestablished c-Met+/HGF+ subcutaneous glioma xenografts were treated with neutralizing anti-HGF mAbs (L2G7) or with isotype-matched control mAb (5G8) as previously reported (30). Anti-HGF therapy significantly inhibited the growth of both U87 tumor xenografts (Fig. 3A) and U373-SF55 xenografts (Fig. 3B). Anti-HGF therapy concurrently reduced xenograft Cyr61 expression by ∼50% (P < 0.05) in U373-SF55 tumors and by ∼25% in U87 tumors compared with controls. We also assessed the effect of anti-HGF mAbs on U87 intracranial tumor xenografts using quantitative dual wavelength near-IR integrative immunohistofluorescence imaging, which allows the simultaneous assessment of Cyr61 and GAPDH on tumor-specific regions of interest in histologic sections (Fig. 3C). L2G7 significantly inhibited Cyr61 expression by ∼20% (P < 0.05) in comparison with control mAb.
Cyr61 knockdown inhibits HGF-induced Akt signaling
HGF is a potent activator of the PI3K/Akt pathway and typically upregulates phospho-Akt maximally within 30 minutes of c-Met activation in c-Met–expressing cells. Akt activation remains above nonstimulated levels as long as 24 hours after HGF stimulation in glioma cell lines. We hypothesized that Cyr61 induction contributes to HGF-induced Akt activity at these later time points. Cyr61 siRNA was used under conditions that achieved near-complete inhibition of HGF-induced Cyr61 expression as assessed by Western blot analysis (Fig. 4A; Supplementary Fig. S2A). We measured the time course of HGF-induced Akt phosphorylation (Ser473) in U373MG glioma cells treated with either control siRNA or Cyr61 siRNA. HGF induced a ∼3-fold increase in Akt phosphorylation at 30 minutes in all treatment groups, indicating that Cyr61 expression inhibition has no effect on the early phase of HGF-induced Akt phosphorylation. In contrast to that observed 30 minutes after HGF stimulation, the induction of Akt phosphorylation at both 2 and 12 hours after HGF stimulation was completely inhibited by Cyr61 siRNA (Fig. 4B). We examined the effect of Cyr61 expression knockdown on HGF-induced phosphorylation of the Akt target GSK3α. At 24 hours after HGF stimulation, phospho-GSK3α (Ser21) was increased ∼1.8-fold (P < 0.05) in control cells and in cells treated with scramble siRNA. Cyr61 siRNA inhibited HGF-induced phosphorylation of GSK3α (Ser21; P < 0.05) in comparison with scramble siRNA–treated cells 24 hours after HGF stimulation (Fig. 4C; Supplementary Fig. S2A).
Cyr61 knockdown inhibits HGF-induced glioma cell migration and proliferation
Because HGF induces Cyr61 expression, we hypothesized that Cyr61 expression contributes to the migration response of HGF-stimulated glioma cells. Cyr61 siRNAs significantly inhibited HGF-induced migration of growth-arrested cells using a monolayer wound assay. Glioma cell migration was increased ∼50% (P < 0.01) by HGF in control and scramble siRNA–treated cells (Fig. 5A). In contrast, HGF-induced migration was almost completely inhibited in Cyr61 siRNA–treated cells (Fig. 5A). U373 and U87 cells were treated with Cyr61 siRNAs ± HGF; cell growth was quantified using MTT cell viability assays, and cell death was quantified by lactate dehydrogenase (LDH) release assay. Cyr61 siRNAs blunted HGF-induced cell proliferation by ∼40% in U373 glioma cells (Fig. 5B). U87 glioma cells express both HGF and c-Met, creating an autocrine loop for Met pathway activation. Cyr61 siRNAs also blunted cell proliferation by approximately 40% to 55% in U87 glioma cells, consistent with the inhibition of a HGF-dependent autocrine pathway (Fig. 5C). Cyr61 siRNA had no effect on LDH release under both basal and HGF-stimulated conditions (Supplementary Fig. S2B and C). Thus, Cyr61 contributes to HGF-dependent migration and cell proliferation without influencing cell viability.
Targeting Cyr61 inhibits glioma growth in vivo
The in vitro findings led us to investigate whether Cyr61 expression knockdown decreases tumor xenograft growth in vivo. We examined the effect of transient Cyr61 siRNA treatment on the growth of HGF-dependent subcutaneous U87 tumor xenografts. U87 glioma cells were implanted in the flanks of nude mice, and xenografts were allowed to grow to ∼200 mm3. Control and Cyr61 siRNAs complexed to the JET-PEI in vivo transfection reagent were injected intratumorally once per day for 5 consecutive days. All mice were sacrificed 24 hours after the last intratumoral injection. Cyr61 siRNAs at a concentration of 500 pmol statistically significantly inhibited both U87 glioma xenograft growth (Fig. 6A) and Cyr61 expression (Fig. 6B) by ∼40% compared with animals treated with control siRNA (P < 0.05). The antitumor effects of Cyr61 siRNAs on U87 glioma xenograft growth could be explained, in part, by decreases in the cell proliferation response. Cyr61 siRNAs significantly inhibited tumor cell proliferation (i.e., Ki67 labeling) by ∼57% compared with animals treated with control siRNA (P < 0.001; Fig. 6C).
RTKs have emerged as potent regulators of tumor malignancy. Understanding the multifaceted mechanisms of oncogenic RTK signaling will influence the application of currently approved therapies and potentially elucidate novel targets for future drug development. Here, we show that the downstream effects of HGF/c-Met pathway activation are partly mediated through the Cyr61 oncogene. Specifically, we show that intratumoral Cyr61 levels correlate with the RTK Met in human tumors and that inhibiting this immediate early gene abrogates Akt-dependent cell signaling and biological responses to HGF/c-Met activation. Specifically, Cyr61 siRNAs inhibited HGF-dependent glioma cell growth and migration in vitro and glioma xenograft growth in vivo. Our findings show that Cyr61 induction prolongs HGF-induced Akt-dependent signaling and its downstream biological effects. These results also suggest that glioma-associated Cyr61 protein levels can serve as a marker of HGF/c-Met pathway inhibition in HGF-dependent tumors.
Solid malignancies are commonly associated with multiple coactivated receptor pathways, and the heterogeneous genetic backgrounds of these tumor subtypes warrant investigation into common RTK-regulated signaling molecules that may serve as therapeutic targets for drug development. Cyr61 has been implicated in tumor progression, exerting its effects by increasing cell proliferation, migration, and angiogenesis (23–28). Furthermore, Cyr61 is induced by a wide array of growth factors and signaling molecules involved in tumor malignancy, such as epidermal growth factor and platelet-derived growth factor (24, 36). Recent evidence also suggests that Cyr61 transcription is mediated by activation of STAT3, a known RTK-initiated signaling cascade, suggesting a potential pathway by which RTK-mediated malignancies induce their effects (37). Induction of Cyr61 in these tumor models points to a potentially broader role for this oncogene in RTK-regulated malignancies and suggests that Cyr61 could be a potential node of convergence for multiple RTK signaling pathways.
Hyperactivation of the PI3K/Akt pathway is strongly associated with tumor malignancy. Here, we show that Cyr61 contributes to HGF-induced activation of Akt and the phosphorylation of the Akt target GSK3α. Although not investigated in this study, this Akt response is likely to be mediated, at least in part, by focal adhesion kinase (FAK) and ILK, intracellular tyrosine kinases involved in integrin-mediated signaling, cell migration, cell proliferation, and cell adhesion. Xie and colleagues (20) have previously shown that forced expression of Cyr61 in low tumorigenic cell lines leads to increased ILK activity, Akt activation, and the malignant phenotype of glioma cells. Furthermore, RGD peptide integrin inhibitors such as cilengitide and neutralizing antibody blockade of the main binding partner of Cyr61, the integrin αvβ3, are shown to decrease ILK activity, Akt activation, and cell proliferation in glioma and ovarian cancer cells (38, 39). Previous studies found that siRNA directed to FAK and ILK inhibits Akt activation; however, a direct link between Cyr61 and either FAK or ILK remains to be determined.
Our results reveal a novel pathway for HGF-induced Akt activation and suggest that the Cyr61-dependent prolongation of Akt activity is important in HGF-induced cell responses. These studies help to elucidate the regulatory mechanisms underlying HGF/c-Met pathway activation while also providing evidence supporting a mechanism of Akt activation that is RTK induced via a nonclassic pathway.
Disclosure of Potential Conflicts of Interest
B. Lal: licensing agreement, Galaxy Biotech. J. Laterra: licensing agreement, Galaxy Biotech; consultant/advisory board, Merck. The other authors disclosed no potential conflicts of interest.
Grant Support: NIH grants NS32148 (J. Laterra) and CA129192 (J. Laterra), United Negro College Fund/Merck Science Initiative (C.R. Goodwin), and American Federation for Aging Research (C.R. Goodwin).
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.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received September 25, 2009.
- Revision received January 3, 2010.
- Accepted January 21, 2010.
- ©2010 American Association for Cancer Research.