ANTICANCER RESEARCH 26: 1029-1038 (2006)
EGF Receptor-related Protein (ERRP) Inhibits Invasion of Colon Cancer Cells and Tubule Formation by Endothelial Cells In Vitro ARUN K. RISHI, RAKESH PARIKH, ANIL WALI, LUKASZ DURKO, LIYUE ZHANG, YINGJIE YU and ADHIP P.N. MAJUMDAR
Veterans Affairs Medical Center, Karmanos Cancer Institute, Departments of Internal Medicine and Pathology, Wayne State University, Detroit, Michigan, U.S.A.
Abstract. Activation of the epidermal growth factor receptor (EGFR) and/or its family member(s) stimulates many processes of carcinogenesis, including cell invasion and the formation of new blood vessels, events that are critically involved in angiogenesis. Interference with the activation of EGFRs, therefore, represents a promising strategy for the development of novel and selective anticancer therapies. Previously, we reported that EGFR-related protein (ERRP), which we have isolated and characterized as a pan-erbB inhibitor, is a potential therapeutic agent for colorectal and other epithelial cancers. The present investigation was undertaken to determine whether ERRP would affect the invasion of colon cancer cells and formation of tubules, and the regulation of these processes. ERRP inhibited tubule formation by aortic endothelial cells and invasion of HCT-116 colon cancer cells through matrigel. These changes were associated with marked reductions in the synthesis and secretion of bFGF, VEGF and TGF-· by HCT-116 cells. Secretion of bFGF and VEGF by aortic endothelial cells was also inhibited by ERRP. Microarray analysis of ERRP-treated HCT-116 cells showed reduced levels of several growth regulatory proteins such as p21Rac1, Stratifin (14-3-3 Sigma), focal adhesion kinase (FAK) and mediators of the Ras-RafERK pathway. ERRP treatments resulted in reduced expression of p21Rac1 and inhibited the constitutive activation of FAK and MEK2 in HCT-116 cells. Transfection of constitutively activate p21Rac1 or MEK2 into HCT-116 cells abrogated ERRP-induced inhibition of growth. In summary, it was
Correspondence to: Adhip P.N. Majumdar, Ph.D., D.Sc., John D. Dingell VA Medical Center, 4646 John R; Room: B-4238, Detroit, MI 48201, U.S.A. Tel: (313) 576-4460, Fax: (313) 576-1112, e-mail:
[email protected] Key Words: EGF receptor, growth factors, EGFR signal transduction, angiogenesis.
0250-7005/2006 $2.00+.40
demonstrated that ERRP not only inhibits cell growth, but also the processes of cell invasion and blood vessel formation that are critical for the development and progression of carcinogenesis. Members of the receptor tyrosine kinase family, including the epidermal growth factor receptor (EGFR), are frequently implicated in experimental models of epithelial cell neoplasia as well as in human cancers (1-4). Overexpression and/or activation of EGFRs, frequently observed in many cancers including colorectal cancer, are associated with resistance to cytotoxic drugs, and are generally considered indicators of poor prognosis (5-8). Inhibition of EGFRs and their signaling processes represents a promising strategy for the development of targeted therapies against a variety of cancers (9, 10). Indeed, several small molecule inhibitors of EGFR such as gefitinib and erlotinib, and monoclonal antibodies directed to either EGFR (cetuximab) or HER-2 (trastuzumab), were developed for the treatment of epithelial cancers (11). Unfortunately, these agents had limited success due, in part, to functional redundancy among EGFRs, and the fact that these agents inhibited a particular member of the EGFR family (6). Moreover, gefitinib was shown to be effective only in a subgroup of non-small cell lung cancer patients with specific mutations in the EGFR (12, 13), while a second mutation in the EGFR kinase domain contributed to acquired resistance of lung adenocarcinomas to gefitinib (14). Since most tumors express multiple members of the EGFR family, identification of inhibitor(s) that target multiple EGFRs is likely to provide a greater therapeutic benefit to a broader range of patients. ERRP (EGF receptor-related peptide), a recently isolated negative regulator of EGFR, that possesses a substantial homology to the extracellular ligand-binding domain of EGFR and its family members, was shown to be a pan-erbB inhibitor that targets multiple members of the
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ANTICANCER RESEARCH 26: 1029-1038 (2006) EGFR family (15,16). We have suggested ERRP could be a potential therapeutic agent for a variety of epithelial cancers, including colon and pancreatic cancers (16-18). With respect to colon cancer, we have demonstrated that intratumoral or subcutaneous injections of purified ERRP inhibit colon cancer HCT-116 cells xenografts in SCID mice with no apparent toxicity (19, 20). Data from in vitro studies have further demonstrated that ERRP inhibits anchoragedependent and -independent proliferation of colon cancer cells (15). These changes have been attributed to attenuation of the basal and ligand-induced activation of the EGFR family members (15, 16). Cell migration and invasion are important processes involved in various physiological and pathological conditions, including tumor development and metastasis (21). Accumulating evidence suggests that EGFRs are key regulators of signal transduction pathways, controlling these processes. In light of the fact that ERRP inhibits the growth of a wide variety of epithelial cancer cells, including colon cancer, we were prompted to examine the effect of ERRP on the processes of invasion of colon cancer cells as well as tubule formation by endothelial cells. Utilizing HCT-116 human colon cancer and rat aortic endothelial cells, we demonstrated that ERRP inhibited cellular growth, tubule formation and migration through matrigel as well as decreasing the production and secretion of a number of proangiogenic factors such as bFGF and VEGF. The expressions as well as activation of Stratifin (14-3-3), Erk 1/2 regulator MEK1, p21Rac1 and FAK proteins, that are associated with processes of proliferation and cell invasion, were also greatly decreased in response to ERRP.
Materials and Methods Cell lines. Colon cancer HCT-116 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and rat aortic endothelial cells were obtained from Dr. Angela Vouyaka at the VA Medical Medical Center, Detroit, Michigan, U.S.A. The cells were maintained in RPMI or DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml streptomycin-penicillin and incubated at 37ÆC in an atmosphere of 95% air and 5% CO2. Assessment of cell growth. Cell growth was assessed by MTT assay as described previously (16, 22). Briefly, aliquots of cells (3x104 cells/ml) in DMEM/10% FBS were plated in a 96-well culture plate with 5 replicates per treatment. After 24 h of plating, the medium was replaced with one containing 2.5% FBS, and the incubation at 37ÆC continued in the absence (control) or presence of recombinant ERRP for various time-periods, as stated in the legends to the figures. All incubations were terminated by adding 20 Ìl of 0.5 g/ml stock of MTT [3-(4,5-dimethylthialzol-2yl)-2,5diphenyltetrazolium bromide] to each well. The reaction was allowed to proceed for 3-4 h at 37ÆC. The culture medium was removed, and formazan crystals were dissolved by adding 0.2 ml of DMSO, and the intensity of color was measured at 570 nm.
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Western blot analysis. Aliquots of approximately 1x105 cells/well were seeded in DMEM/10% FBS in 6-well culture plates. After 24 h of plating, they were serum-starved for 24 h and subsequently incubated for another 24 h or 48h in the absence (controls) or presence of various doses of recombinant ERRP. The incubations were terminated by adding lysis buffer (50 mM Tris pH 7.4, 100 mM NaCl, 2.5 mM EDTA, 1% Triton X-100, 0.5% NP-40, 2.5 mM Na3VO5, 1 mM phenylmethylsulfonyl fluoride, 25 Ìg/ml aprotinin and leupeptin, 50 Ìg/ml Soybean trypsin inhibitor). The lysates were rotated for 30 min at 4ÆC and subsequently centrifuged at 11,000 xg for 15 min at 4ÆC. The supernatant was used for Western blot analysis after determination of the protein concentration by the protein assay kit from Bio-Rad laboratories. Aliquots containing 50 Ìg of protein were separated on a 7.5% sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDSPAGE) and then electroblotted to a nitrocellulose membrane. The membrane was blocked overnight with 5% non-fat dried milk in TBS-T buffer (20 mM Tris, pH 7.6, 100 mM NaCl, 0.1% Tween 20), followed by 3 h of incubation with the primary antibodies (see below) at the manufacturer’s recommended final dilution in TBS-T buffer containing 5% non-fat dried milk at room temperature. After washing 3 times with TBS-T buffer, the membranes were incubated with a horseradish peroxidase-conjugated goat antirabbit IgG (1:5000 dilutions) for 1 h at room temperature. The proteins were visualized using enzyme-linked enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL, USA). The membranes were stripped and probed with ·-tubulin antibodies (Boehringer Mannheim) as an internal control. Signals on the blots were visualized by autoradiography and quantitated by densitometry using the ImageQuant image analysis system (Storm Optical Scanner, Molecular Dynamics, Sunnyvale, CA, USA). Unless otherwise stated, the data are expressed as mean±SD. Where applicable, the results were compared by using the unpaired Student’s t-test, taking p
