Casitas B-lineage lymphoma (Cbl) is an E3 ubiquitin ligase of many tyrosine kinase receptors. The authors previously detected c-Cbl mutation and low protein expression in non-small cell lung cancer (NSCLC). Therefore, it was hypothesized that overexpression of wild-type c-Cbl (c-Cbl WT) exhibits tumor growth inhibition.
Wound healing and transwell assays were conducted to examine cell motility after c-Cbl WT transfection in NSCLC cell lines. The cell cycle was investigated by flow cytometry. A549 and H1299-Luc c-Cbl WT-transfected xenografts and experimental metastasis models were performed to investigate tumor growth and metastasis inhibition in vivo.
Wound healing and transwell assays demonstrated inhibition of migration in the A549 and H226br cells 4 to 24 hours after transfection. Ectopic c-Cbl WT expression was found to reduce cell proliferation at 48 hours in A549 cells. It is important to note that A549 and H1299-Luc cells with ectopic c-Cbl WT expression demonstrated inhibition of tumor growth in vivo. A549 cells overexpressing c-Cbl WT inhibited tumor metastasis in animal models.
Overexpression of tyrosine kinase receptors (RTKs) has been detected in lung cancer. 1, 2 Therefore, promotion of a degradation system of RTKs is a new approach for tumor growth inhibition. 3 Many studies indicate that Casitas B-lineage lymphoma (Cbl) plays an important role in the down-regulation of RTKs through its E3 ubiquitin ligase activity. 4, 5 The Cbl family, especially c-Cbl protein, also is associated with the endocytosis mechanism and thus plays a crucial role in the termination of signaling RTKs such as c-Met and epidermal growth factor receptor (EGFR). 6
c-Cbl mutations were first reported in human acute myeloid leukemia (AML) and other types of leukemia. 7, 8 To the best of our knowledge, our previous study was the first to report that the c-Cbl mutation also occurred in solid tumors. Our data indicated that overexpression of c-Cbl mutations in non-small cell lung cancer (NSCLC) cell lines led to increased cell proliferation and motility. 9 A recent study demonstrated that mutation or knockdown of c-Cbl induced cell migration in breast cancer. 10 Therefore, we hypothesized that the overexpression of wild-type c-Cbl (c-Cbl WT) may exhibit tumor inhibition.
MATERIALS AND METHODS
Cell Culture and c-Cbl Transfection
Human NSCLC cell lines were obtained from the American Type Culture Collection (Manassas, Va). A luciferase-expressing NSCLC cell line, H1299-Luc, was kindly provided by Dr. P.-J. Lu (Institute of Clinical Medicine, National Cheng Kung University, Taiwan). A human NSCLC cell line, AS2, was obtained from Dr. W.-C. Su (Department of Internal Medicine, National Cheng Kung University). A human bronchial cell line, BEAS-2B, was obtained from Dr. P.-C. Yang (Department of Internal Medicine, National Taiwan University).
Vector constructs were previously described by Tan et al. 9 The cells were transfected with c-Cbl WT vector or empty vector control using ExGen500 transfection reagent (Fermentas, Glen Burnie, Md). After 48 hours, cells were collected for Western blot analysis, cell proliferation, migration assays, and animal studies.
Western Blot Analysis, Tissue Western Blot Analysis, and Immunofluorescence Assay
Immunoblotting was performed for various proteins using the conditions described: c-Cbl, 1:200; phosphorylated epidermal growth factor receptor (p-EGFR) (Tyr-1173), 1:200; signal transducer and activator of transcription 3 (STAT3), 1:1000 (Santa Cruz Biotechnology, Santa Cruz, Calif); phosphorylated AKT (p-AKT) (Ser-473), 1:1000; AKT, 1:1000; phosphorylated Met (p-Met) (Tyr-1234/Tyr-1235), 1:500; Met, 1:1000; EGFR, 1:500; phosphorylated STAT3 (p-STAT3) (Tyr-705), 1:2000; phosphorylated ERK (p-ERK) (Thr-202/Tyr-204), 1:1000 (Cell Signaling Technology Inc, Danvers, Mass); ERK, 1:1000; RAS, 1:1000 (Upstate, Billerica, Mass); phosphorylated Src (p-Src) (Tyr-416), 1:2000 (Invitrogen, Carlsbad, Calif); Src, 1:1000 (obtained from Dr. T.-H. Leu, Department of Pharmacology, National Cheng Kung University); phosphorylated FAK (p-FAK) (Tyr-397), 1:200; FAK, 1:1000; and β-actin, 1:5000 (Abcam, Cambridge, UK). For tissue Western blot analysis, xenografts were collected after mice sacrifice, homogenized with CelLytic TMMT lysis buffer (Sigma-Aldrich Corporation, St. Louis, Mo), and used for immunoblotting. Pericellular polyfibronectin assemblies were detected with antifibronectin (1:600; Sigma-Aldrich) 11 using bright field and fluorescent microscopy.
Transient Expression of c-Cbl, Cell Proliferation Analysis, Flow Cytometry, and Wound Healing and Transwell Migration Assays
These assays were performed as previously described. 9
Tumor Growth and Metastasis Analyses In Vivo
Female BALB/c nude mice, ages 5 to 6 weeks, were acquired from the National Laboratory Animal Center (Taipei, Taiwan) after obtaining appropriate Institutional Review Board approval and raised in a pathogen-free environment. Transfected A549 cells (1 × 106) in a volume of 200 μL were injected through the tail vein for in vivo experimental metastasis analysis. The mice were euthanized at the indicated times and lung tumors were photographed with a digital camera. For the tumor growth inhibition assay, transfected A549 or H1299-Luc cells (5 × 106) in a volume of 100 μL were implanted subcutaneously into the mice. The size of the tumor mass was measured and the tumor volume was calculated as 1/2 × length × width 2 in mm3 for the A549 xenograft. The growth of the H1299-Luc xenograft was observed under an IVIS-50 in vivo imaging system (Xenogen Biosciences Corporation, Cranbury, NJ) after injection of an endotoxin-free luciferase substrate (VivoGlo; Promega Corporation, Madison, Wis). The body weight of the mice was measured. Tumors were fixed and stained with hematoxylin and eosin (H & E) for further pathological confirmation.
For continuous variables, group comparisons were performed using analysis of variance (ANOVA) followed by the Sidak adjustment for multiple comparisons. Experiments involving measurements over time were analyzed using repeated measures ANOVA with the Greenhouse-Geisser adjustment.
Ectopic Expression of c-Cbl WT Inhibits Cell Proliferation and Motility
Western blot analysis was performed to examine expression of the c-Cbl protein in various lung cancer cell lines. H2171, H249, A549, H226br, AS2, and H1975 cells demonstrated lower c-Cbl expression than BEAS-2B bronchial epithelial cells (Fig. 1A). Therefore, A549 and H226br cells were selected as the cell models to be used for further investigation. Transient transfection with 8 μg of c-Cbl WT was used for all assays because it demonstrated the highest expression of ectopic c-Cbl protein (Fig. 1B). The cell growth results indicated that c-Cbl WT inhibited cell proliferation (Fig. 1C) and induced a sub-G1 population (Fig. 1D), possibly via reduction of the total c-Met protein level and AKT/ERK survival signaling (Fig. 1E) in A549 cells at 48 hours after transfection.
We performed wound healing and transwell migration assays on A549 and H226br cells that were transiently transfected with c-Cbl WT expression vector. The wound gaps of c-Cbl WT transfection in both the A549 and H226br cells all were significantly larger than noted in control cells transfected with empty vector at 24 hours after transfection (Fig. 2A). A transwell migration assay confirmed that c-Cbl WT expression inhibited the migration ability of both cell lines (Fig. 2B). Ectopic expression of c-Cbl WT in A549 cells inhibited FAK signaling (Fig. 2C) and pericellular polyfibronectin assemblies on the cell surface (Fig. 2D) at 48 hours after transfection compared with the control cells.
c-Cbl WT Transfection Effectively Inhibits Tumor Growth in an Animal Model
To examine whether c-Cbl WT could inhibit tumor growth in vivo, we first transfected c-Cbl WT into A549 or H1299-Luc cells and implanted them subcutaneously into nude mice. Animals with the A549 xenograft expressed c-Cbl WT, resulting in a significant reduction in tumor mass compared with the control group without changes in body weight (Figs. 3A and 3B). Tissue Western blot analyses demonstrated that ectopically expressed c-Cbl WT remained overexpressed at 44 days after xenograft implantation (Fig. 3D). c-Cbl WT transfection-induced antitumor growth was confirmed by a reduction in luciferase intensity in the H1299-Luc xenograft (Fig. 3C).
c-Cbl WT Transfection Effectively Inhibits Tumor Metastasis in an Animal Model
To examine whether c-Cbl WT could inhibit tumor metastasis in vivo, A549 cells with or without ectopic expression of c-Cbl WT were injected into the tail vein of nude mice. After 6 weeks, the animals were sacrificed so that lung tissue could be examined. The c-Cbl WT transfection group demonstrated a significant decrease in metastatic lung tissue weight compared with the control group (Figs. 4A and 4B). H & E staining results demonstrated that the number and size of the metastatic tumor colonies in the lungs of nude mice receiving control A549 cells were significantly higher than in those receiving c-Cbl WT-expressing A549 cells (Fig. 4C).
The c-Cbl E3 ubiquitin ligase induces internalization and ubiquitination of RTKs such as c-Met and EGFR. 7 The signaling experiment using Western blot analyses demonstrated that overexpression of c-Cbl WT decreased the total c-Met protein level but not that of EGFR. This might be because c-Cbl represents only one aspect of EGFR post-translational regulation. 12 Further studies to examine whether transduction of c-Cbl WT is also effective in NSCLC cells with c-Met overexpression or an EGFR mutation are warranted.
c-Cbl has been shown to target other kinases, including platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1), and Src. 13 c-Cbl also functions as a signal transduction molecule affecting pathways such as RAS, phosphoinositide 3-kinase (PI3K)/AKT, and STAT. 13 Although we did not observe changes in RAS and p-STAT status, we cannot rule out other potential effects as contributing factors.
To the best of our knowledge, the current study is the first to demonstrate that ectopic expression of c-Cbl WT inhibits tumor growth and metastasis in lung cancer in vivo. c-Cbl WT-induced inhibition of tumor growth may be mediated by down-regulation of p-AKT (survival) and p-ERK1/2 (proliferation and differentiation) signaling. 13 In addition, an antimetastasis effect may occur through inhibition of p-FAK (motility control) and pericellular polyfibronectin assemblies (tumor colonization). 11 Loss of c-Cbl function in individuals with AML and myelodysplastic syndrome supports the potential clinical therapeutic value of c-Cbl WT gene therapy in patients with hematologic malignancies in addition to those with lung cancer.
Supported in part by grant NSC 99-2628-B-006-004-MY3 and grant DOH98-TD-G-111-024 (to Y.C.W.) and National Cancer Institute grant 5R01CA125541-04 (to R.S.).