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Epidermal growth factor receptor-related protein: a potential therapeutic agent for colorectal cancer
GASTROENTEROLOGY 2003;124:1337–1347
Epidermal Growth Factor Receptor–Related Protein: A Potential Therapeutic Agent for Colorectal Cancer DOROTA J. MARCINIAK,* LATHIKA MORAGODA,* RAMZI M. MOHAMMAD,*,‡ YINGJIE YU,* KIRAN K. NAGOTHU,* AMRO ABOUKAMEEL,* FAZLUL H. SARKAR,‡,§ VOLKAN N. ADSAY,§ ARUN K. RISHI,*,‡,㛳 and ADHIP P. N. MAJUMDAR*,㛳 *Department of Internal Medicine, Wayne State University School of Medicine, Detroit; ‡Karmanos Cancer Institute, Detroit; §Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan; 㛳Veterans Affairs Medical Center, Detroit, Michigan
Background & Aims: Epidermal growth factor receptor is frequently implicated in epithelial cancers and is, therefore, being considered as a potential target for therapy. Recently, we reported the isolation and characterization of epidermal growth factor receptor–related protein, a negative regulator of epidermal growth factor receptor. To discern whether epidermal growth factor receptor– related protein could be an effective therapeutic agent for colorectal cancer, we generated epidermal growth factor receptor–related protein fusion protein and studied its effect on the growth of colon cancer cells in vivo and in vitro. We also studied whether epidermal growth factor receptor–related protein expression is altered in colorectal cancer. Methods: A 55-kilodalton epidermal growth factor receptor–related protein fusion protein with V5 and His tags was generated in a drosophila expression system and subsequently purified by a His antibody affinity column. Rabbit polyclonal antibodies against epidermal growth factor receptor–related protein were used to examine the expression of epidermal growth factor receptor–related protein. Results: Epidermal growth factor receptor–related protein expression was found to be high in benign human colonic epithelium but low in adenocarcinoma. Exposure of the colon cancer cell lines HCT-116 and Caco-2 to purified recombinant epidermal growth factor receptor–related protein caused a marked inhibition of proliferation, as well as attenuation of basal and ligand-induced stimulation of epidermal growth factor receptor phosphorylation. Epidermal growth factor receptor–related protein-induced inhibition of proliferation of colon cancer cells was prevented by epidermal growth factor receptor–related protein antibodies. Reduced epidermal growth factor receptor phosphorylation was partly due to sequestration of epidermal growth factor receptor ligands by epidermal growth factor receptor–related protein, resulting in the formation of inactive heterodimers with epidermal growth factor receptor. Intratumoral or subcutaneous (away from the tumor site) injections of purified epidermal growth factor receptor–related protein caused regression of palpable colon cancer xenograft tumors in some severely compromised immunodeficient mice and
arrested tumor growth in others. Conclusions: We propose that epidermal growth factor receptor–related protein inhibits cellular growth by attenuating epidermal growth factor receptor signaling processes and is an effective therapeutic agent for colorectal cancer.
embers of the receptor tyrosine kinase family are frequently implicated in experimental models of epithelial cell neoplasia, as well as in human cancers.1–3 There is increasing evidence to support the concept that the malignant behavior of some tumors is sustained by deregulated activation of certain growth factor receptors. Such deregulation could be due to structural alterations of the receptor itself4; to the establishment of an autocrine loop, whereby the cells produce growth factors that stimulate their own growth5–7; or to the loss of a suppressor of receptor function. One of the best studied receptor signaling systems from this family is that controlled by the epidermal growth factor (EGF) receptor (EGFR), whose expression and enzyme activity have been linked to a number of malignancies, including cancer of the colon.8 –11 EGFR and its ligand transforming growth factor (TGF)-␣, a structural and functional analogue of EGF, are overexpressed in preneoplastic and neoplastic colonic mucosa.12–14 Moreover, cell lines derived from adenocarcinomas of various gastrointestinal tissues, including the colon, overexpress TGF-␣ and its receptor EGFR.15–17 Because of EGFR’s role in the development and progression of many epithelial cancers, efforts have
M
Abbreviations used in this paper: DMEM, Dulbecco’s modified Eagle medium; DMSO, dimethyl sulfoxide; DSS, disuccinimidyl suberate; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERRP, epidermal growth factor receptor–related protein; FBS, fetal bovine serum; MoAb, monoclonal antibody; ORF, open reading frame; SCID, severely compromised immunodeficient; TGF, transforming growth factor. © 2003 by the American Gastroenterological Association 0016-5085/03/$30.00 doi:10.1016/S0016-5085(03)00264-6
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been made to use EGFR as a potential target for epithelial cancer therapy. Several approaches, including monoclonal antibodies to EGFR and pharmacological inhibitors of EGFR tyrosine kinase, have been used, but with limited success, primarily because of toxicity or lack of specificity. Therefore, identification of endogenous factors that may inhibit EGFR activation and its signaling pathways is of paramount therapeutic importance. To this end, we recently reported the isolation and characterization of a negative regulator of EGFR, referred to as ERRP (EGFR-related protein; US patent 6,399,743; GenBank accession no. AF187818), which possesses a significant homology to the extracellular ligand binding domain of EGFR.18 Transfection of ERRP complementary DNA (cDNA) into the colon cancer cell lines HCT116 and Caco-2 not only inhibits proliferation in matrixdependent and independent systems, but also decreases EGFR activation, as evidenced by reduced tyrosine phosphorylation and tyrosine kinase activity of the receptor.18 Taken together, the results suggest that ERRP inhibits proliferation of colon cancer cells by attenuating EGFR function. These results raise the possibility that ERRP could be an effective therapeutic agent for colorectal cancer. To test this possibility, we generated ERRP protein by using the drosophila expression system and studied the effect of purified recombinant ERRP on the growth of colon cancer cells in vitro and in vivo. We show that recombinant ERRP inhibits proliferation and EGFR activation in colon cancer cell lines in vitro and suppresses the growth of tumors in severely compromised immunodeficient (SCID) mice xenografts of colon cancer cells.
Materials and Methods Cell Lines Colon cancer cell lines HCT-116 and Caco-2 and monkey kidney COS-7 cells, obtained from the American Type Culture Collection (Rockville, MD), were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (10,000 U/mL), streptomycin (10,000 U/mL), and amphotericin (25 L/mL) at 37°C in an atmosphere of 95% air and 5% CO2.
Cell Proliferation This was assessed by 3-(4,5-dimethylthialzol-2yl)-2,5diphenyltetrazolium bromide assay according to the procedure described by Sigma Chemical Co. (St. Louis, MO). Briefly, aliquots of cells (3 ⫻ 104/mL) in DMEM/10% FBS were plated in a 96-well culture plate with 5 replicates per treatment. After 24 hours of plating, the medium was replaced with that containing 2.5% FBS, and the incubation at 37°C continued in the absence (control) or presence of recombinant ERRP, as
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stated in the figure legends. In some experiments, incubations were performed in the absence (control) or presence of recombinant ERRP or recombinant ERRP together with either anti-ERRP antibodies (1:500 final dilution) or normal rabbit serum (1:500 final dilution). All incubations were terminated by adding 20 L of 0.5 g/mL stock of 3-(4,5-dimethylthialzol2yl)-2,5-diphenyltetrazolium bromide to each well. The reaction was allowed to proceed for 3– 4 hours at 37°C. The culture medium was removed, formazan crystals were dissolved by adding 0.2 mL of dimethyl sulfoxide (DMSO), and the intensity of color was measured at 570 nm.
Establishment of Tumors in Severely Compromised Immunodeficient Mice SCID mice were obtained from Taconic Laboratories (German Town, NY). After a period of adaptation, 2 to 3 mice were subcutaneously (sc) injected on each flank with approximately 106 HCT-116 cells. When tumors developed, mice were killed; tumors were dissected, cut into small fragments, and subsequently transplanted sc into similarly conditioned animals (n ⫽ 6 for each group) by using a 12-gauge trocar. Mice were checked 3 times a week for tumor development. Once palpable tumors developed, groups of 6 mice were removed randomly for the ERRP efficacy trial.
Generation of Polyclonal Antibodies to ERRP Polyclonal antibodies to ERRP were raised in rabbits as described previously.19 Briefly, this was achieved by scanning protein databases and identifying a region of ERRP comprising 27 amino acids (nucleotide 1580 –1661), referred to as a U region, that showed no homology with any known protein sequence in the current database.18 Further analysis of the U region showed that the mid portion of the peptide with 15 amino acid residues (amino acid residues of a 15-mer peptide) possessed the most antigenic property. The above 15-mer peptide was synthesized and subsequently used by Sigma–Genosys (Woodlands, TX) to raise antibodies in rabbits. In Western blot analysis, antibodies reacted strongly with a 55-kilodalton protein19 that corresponded well with the calculated molecular mass of ERRP, the open reading of which is composed of 479 amino acids. No cross-reactivity with EGFR or any of its family members was noted.19
Immunohistochemical Staining This was performed as described previously.19 Briefly, 5-m sections of paraffin-embedded tissues were deparaffinized and microwaved for 15 minutes in 1 L of citrate buffer. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide and subsequently incubated with 5% horse serum to block nonspecific binding. The slides were then incubated at room temperature for 2 hours with polyclonal antibodies to ERRP. Negative controls were performed with antigen-neutralized ERRP antibodies. Antigen neutralization was accomplished by preincubating ERRP antibodies at 4°C
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for 16 hours with the antigen (1 g/mL of phosphate-buffered saline; 1 part ERRP antibody and 5 parts antigen). ERRP antibodies, used as positive controls, were also similarly treated after dilution with phosphate-buffered saline. All primary antibodies were used at a final dilution of 1:1000. After incubation with primary antibodies, the avidin-biotin technique was performed with matched components (secondary biotinylated antibody and avidin-peroxidase complex) from the DAKO labeled streptavidin-biotin system (Carpentia, CA) according to the manufacturer’s suggested protocol. The slides were reacted with amino ethyl carbazole, counterstained with Harris’ hematoxylin, and examined by a pathologist blinded to sample coding. At least 10 well-oriented crypts on each slide and 5 slides from each sample were examined under high power.
Generation of ERRP Fusion Protein The drosophila expression system was used to generate ERRP fusion protein. To create stable cell lines expressing ERRP fusion protein, the expression vector pMT/ V5-HisA (Invitrogen, Carlsbad, CA) containing the entire open reading frame (ORF) of ERRP cDNA was co-transfected into drosophila S2 cells with a pCoHygro plasmid (Invitrogen), which confers hygromycin resistance. The transfectants were selected with hygromycin at a concentration of 300 g/mL, and individual sublines were propagated in media containing 150 g/mL of hygromycin. The stable cell lines were induced with 0.5 mmol/L CuSO4 to express the ERRP fusion protein. Western blot analysis with polyhistidine antibodies (Invitrogen) of cell lysates and the conditioned media from CuSO4-treated cells showed a single protein band with a molecular weight ratio (Mr) of approximately 55 kilodaltons (Figure 1) that corresponded well with the calculated molecular mass of ERRP. Detection of ERRP in the conditioned medium together with the fact that the ORF of ERRP contains a signal peptide composed of 24 amino acids, which are similar but not identical to those found in human or rat EGFR,18 suggests that ERRP is a secretory protein. The reason for detecting a substantially higher amount of ERRP in cell lysate, compared with the conditioned medium, was that a total of approximately 30 ⫻ 106 cells were cultured in 10 mL of growth medium. After 24 hours of induction with CuSO4 , cells were obtained by centrifugation and lysed in 1 mL of lysis buffer, and 10 L (one hundredth of the volume) of the lysate and 30 L (three thousandths of the volume) of the conditioned medium were subjected to Western blot analysis. ERRP was purified from the crude cell lysate by using polyhistidine antibodies conjugated to Sepharose 4B (Pharmacia, Piscataway, NJ), as described previously.20The bound protein was eluated with 3 mol/L of sodium thiocyanate,20 and the fractions containing ERRP were pooled and dialyzed against 20 mmol/L of HEPES, pH 7.5, and subsequently concentrated by using Amicon (Beverly, MA) filter units.
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Immunoprecipitation and Western Blot Analysis of EGFR This was performed according to our standard protocol.18,21 Briefly, aliquots of cell lysate containing 1- or 1.5-mg proteins were incubated with polyclonal anti-EGFR antibodies (Upstate Biotechnology, Lake Placid, NY) and Sepharose G at 4°C for 3 hours. Immunoprecipitates were resolved on a 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The electrophoresed proteins were transferred to nitrocellulose membranes and subsequently probed with anti-phosphotyrosine antibodies, as described below. After visualization of protein bands by an enhanced chemiluminescence detection system, the membranes were stripped and reprobed with antiEGFR antibodies as described previously.18
Expression and Secretion of ERRP COS-7 cells were transiently transfected with expression plasmid encoding ERRP fused with polyhistine and cMyc tags to examine ERRP secretion. First, ERRP cDNA (GenBank accession no. AF187818) containing entire proteinencoding ORFs but lacking the translation termination codon and the 3⬘-untranslated region was subcloned into the vector plasmid pcDNA-3/Myc-His-A (Invitrogen) to generate ERRP-Myc-His–tagged clone 1. Next, the vector plasmid (control) or the ERRP-Myc-His–tagged clone 1 was independently and transiently transfected into COS-7 cells, essentially as described previously.18 Approximately 24 hours after transfection, the media were collected and the cells harvested and lysed. The media (1 mL) or the lysates (0.5 mg) were separately used for immunoprecipitation with anti-EGFR (raised against the cytoplasmic domain of EGFR; Upstate Biotechnology), anti– c-Myc, or anti-polyhistidine antibodies (Cell Signaling, Beverley, MA). The immunoprecipitates were subjected to Western blot analysis with anti– c-Myc or anti-polyhistidine antibodies.
Cross-linking of ERRP and EGFR This was performed with crude membrane fractions of serum-starved HCT-116 cells, prepared according to Walker and Burgess.22 Cross-linking was conducted by using 2 mmol/L of disuccinimidyl suberate (DSS) according to the manufacturer’s instructions (Pierce, Rockford, IL). Briefly, aliquots of membranes containing the same amount of protein (0.5 mg) were incubated in the absence (control) or presence of 10 nmol/L of TGF-␣, ERRP (5 g/mL), or both for 1 hour at 4°C. The reaction was terminated by adding DSS in DMSO or an equivalent volume of DMSO alone and incubating further at room temperature for 30 minutes, as described by Walker and Burgess.22 After quenching with 20 mmol/L of Tris (pH 7.5), the reaction was mixed with an equal volume of lysis buffer (10 mmol/L of HEPES, pH 7.2; 150 mmol/L of NaCl; 2.5 mmol/L of Na3VO4; 1 mmol/L of phenylmethylsulfonyl fluoride; 2.5 mmol/L of EDTA; 25 g/mL of aprotinin, leupeptin, and pepstatin A; 0.5 % Triton X-100; and 0.5% Nonidet P-40). After clarification at 11,000 ⫻ g for 15 min-
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Figure 1. Western blot with polyhistidine antibodies showing ERRP fusion protein in S2 cells incubated in the absence or presence of CuSO4 and in the conditioned media (Med) from CuSO4-treated S2 cells.
utes at 4°C, the supernatants were used to detect dimers and monomers of EGFR by immunoprecipitation followed by Western blot analysis, as described previously.18 Solubilized membranes were incubated overnight at 4°C with sheep polyclonal antibodies to EGFR and 30 L of protein A–sepharose beads. The beads, after washing with TT buffer (50 mmol/L of Tris-HCl, pH 7.6, 0.15 mol/L of NaCl, and 0.5% Tween 20), were subjected to electrophoresis on a 5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes, which were subsequently probed with anti-EGFR antibodies to detect homoand heterodimers of EGFR. Protein bands were visualized by a enhanced chemiluminescence detection system.
Statistical Analysis Unless otherwise stated, data are expressed as mean ⫾ SEM. Where applicable, the results were compared by using the unpaired Student t test and analysis of variance for nonparametric data. P ⬍ 0.05 was designated as the level of significance.
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the media of COS-7 cells transfected with ERRP-MycHis–tagged clone 1 plasmid, but not in the media derived from the vector (control) transfected cells (Figure 2). In an analogous experiment, ERRP-fusion protein was immunoprecipitated with c-Myc antibodies followed by Western blots with polyhistidine antibodies. Again, ERRP was detected in the media (data not shown). Taken together, the results suggest that ERRP is a secretory protein. Because ERRP is a negative regulator of EGFR, we hypothesize that increased activation of EGFR associated with colorectal cancer could partly be the result of loss of ERRP, a suppressor of EGFR function. To test this possibility, we examined the expression of ERRP by immunohistochemical analyses in surgical specimens from 10 subjects. A representative photomicrograph is shown in Figure 3; Figure 3A represents hematoxylin and eosin staining of the specimen, showing benign and adenocarcinoma regions in the tumor. Indeed, data from immunohistochemical analyses showed that expression of ERRP was high in benign human colonic epithelium but low in invasive adenocarcinoma in all the specimens tested (Figure 3B). Higher magnification of benign mucosa and invasive adenocarcinoma shows that ERRP localizes primarily to the basolateral membranes in benign mucosa (similar to EGFR) but loses that polarized distribution in invasive adenocarcinoma (Figure 3C and D). The observed increase in ERRP immunoreactivity in benign colonic mucosa could not be attributed to nonspecific staining, because little or no visible staining occurred in normal colonic epithelium when ERRP an-
Results The fact that the ORF of ERRP contains a signal peptide composed of 24 amino acids, together with our observation that ERRP is present in the conditioned media derived from drosophila S2 cells (Figure 1), suggests that ERRP is a secretory protein. To further test this possibility that ERRP is a secretary protein, we generated a construct expressing polyhistidine and Myctagged ERRP protein. This construct or the vector plasmid (control) was subsequently transfected into COS-7 cells, and the presence of ERRP in the conditioned media was determined by immunoprecipitation and subsequent Western blot analysis. ERRP present in the conditioned media was immunoprecipitated with polyhistidine antibodies followed by Western blot with c-Myc antibodies. We found Myc-polyhistidine–tagged ERRP protein in
Figure 2. Western blot showing ERRP levels in the conditioned media of COS-7 cells 24 hours after transfection with either the ERRP-MycHis–tagged clone 1 (lane 2) or only the vector pcDNA-3 (control; lane 1). One milliliter of conditioned medium was subjected to immunoprecipitation with polyhistidine antibodies, and the immunoprecipitates were subsequently analyzed by a 12% sodium dodecyl sulfate– polyacrylamide gel electrophoresis followed by Western blot with anti– c-Myc antibodies. IP, immunoprecipitation.
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Figure 3. A representative photomicrograph showing changes in ERRP immunoreactivity in the benign human colonic epithelium and the adjacent invasive adenocarcinoma (denoted by arrows in A and B).
tibodies were neutralized with the antigen before use (Figure 4B). As expected, a marked ERRP staining was observed when ERRP antibodies were used (Figure 4A). Because epithelial malignancies, including colorectal cancer, show increased EGFR activation,10 –14,23 it is plausible that the observed loss of ERRP is partly responsible for increased EGFR activation in colorectal tumors. Previously, we showed that expression of ERRP in the colon cancer cell lines HCT-116 and Caco-2 inhibits proliferation and EGFR activation,18 raising the possibility that ERRP could be an effective inhibitor of colorectal tumors. To test this hypothesis, we studied the effect of recombinant ERRP on proliferation and EGFR phosphorylation of colon cancer cell lines. Indeed, we observed that the recombinant ERRP inhibited proliferation of the colon cancer cell lines HCT-116 and Caco-2 in a dose-dependent manner, showing a 60%–70% reduction with the highest dose of ERRP (8 –10 g/mL) when compared with the corresponding control (Figure 5A and B). A 72-hour time-course study with HCT-116 cells was performed to determine the growth-inhibitory properties of ERRP. As shown in Figure 5C, recombinant ERRP (5 g/mL) remained effective throughout the experimental period, causing a 50%– 60% inhibition of proliferation. The ERRP-induced inhibition of proliferation of HCT-116 cells was prevented by adding antiERRP antibodies, whereas preimmune rabbit serum was ineffective in preventing this inhibition (Figure 5D). Our earlier observation that overexpression of ERRP in Caco-2 cells, resulting in inhibition of proliferation, was accompanied by a concomitant reduction in tyrosine phosphorylation and tyrosine kinase activity of EGFR18 suggests that ERRP inhibits proliferation by attenuating EGFR activation.18 To determine whether recombinant ERRP will affect EGFR function, changes in the levels of
tyrosine-phosphorylated EGFR in Caco-2 cells were examined after exposure to increasing concentrations of recombinant ERRP for 48 hours. Indeed, we found that a high dose of ERRP greatly inhibited the basal tyrosine phosphorylation of EGFR in Caco-2 cells (Figure 6A). Because ligand binding is the primary cause of activation of EGFR, we examined the effect of recombinant ERRP on ligand-induced stimulation of EGFR phosphorylation in HCT-116 and Caco-2 cells. We observed that exposure of 48-hour serum-starved HCT-116 or Caco-2 cells to either 5% serum or 10 nmol/L of TGF-␣ caused a marked stimulation of tyrosine phosphorylation of EGFR when compared with the control (Figure 6B and C). However, the serum-induced or TGF-␣–induced stimulation of EGFR tyrosine phosphorylation was greatly attenuated by purified recombinant ERRP (Figure 6B and C). Levels of EGFR in either HCT-116 or Caco-2 cells, however, were unaffected by these treatments (Figure 6B and C). Taken together, the results
Figure 4. A representative photomicrograph showing changes in ERRP immunoreactivity in the normal human colon when incubated with (A) ERRP antibodies and (B) antigen-neutralized ERRP antibodies.
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Figure 5. Effect of purified recombinant ERRP on proliferation of the colon cancer cell lines HCT-116 and Caco-2. Dose-dependent changes in the proliferation of HCT-116 and Caco-2 cells are depicted in (A) and (B). Cell lines were exposed to increasing concentrations of purified recombinant ERRP for 48 hours. Time-course changes in proliferation in response to 5 g/mL of purified recombinant ERRP are shown in (C). (D) The effect of recombinant ERRP (5 g/mL) on proliferation of HCT-116 cells in the absence or presence of ERRP antibodies or preimmune rabbit serum. Each value represents mean ⫾ SEM of 5– 6 observations. *P ⬍ 0.01 compared with control.
suggest that purified recombinant ERRP is biologically active and inhibits proliferation by attenuating EGFR function. The mechanism by which ERRP results in attenuation of EGFR activation is poorly understood. However, because ERRP possesses a substantial homology to the ligand binding domain of the EGFR, we hypothesize that ERRP binds the EGFR ligands and subsequently forms inactive heterodimers with EGFR, leading to inhibition of EGFR phosphorylation. To test this hypothesis, we studied the effects of TGF-␣ on the formation of ERRP/EGFR heterodimers in HCT-116 cells in the absence or presence of purified ERRP. Aliquots of crude membranes from serum-starved HCT-116 cells, containing the same amount of protein, were incubated in the absence or presence of TGF-␣ (10 nmol/L), ERRP (5 g/mL), or both. To detect dimers of EGFR and those formed between EGFR and ERRP, the reaction mixtures were treated with and without DSS, a cross-linking agent, followed by immunoprecipitation with antiEGFR antibodies. The immunoprecipitates were then subjected to Western blot analysis with anti-EGFR an-
tibodies to detect homo- and heterodimers of EGFR. We anticipated that incubation of HCT-116 cells with TGF-␣ and ERRP would result in the formation of ERRP/EGFR heterodimers with an Mr of approximately 225 kilodaltons (170 kilodaltons for EGFR ⫹ 55 kilodaltons for ERRP) and EGFR homodimers of 340 kilodaltons. Indeed, this was found to be the case. In the presence of TGF-␣ and ERRP, we detected 3 distinct protein bands with Mr of 340, 220, and 170 kilodaltons, representing homodimers of EGFR, heterodimers of ERRP/EGFR, and EGFR monomers, respectively (Figure 7A). A faint ERRP/EGFR heterodimer band of 220 kilodaltons was also observed when incubations were performed in the absence of ERRP but containing TGF-␣ (Figure 7A). Formation of a 220-kilodalton heterodimer in the absence of exogenous ERRP is probably due to the presence of endogenous ERRP. Neither a 340nor a 220-kilodalton protein band was detected when incubations were performed in the absence of TGF-␣ and ERRP (Figure 7A). Lack of DSS also failed to produce any dimers (Figure 7A). No heterodimers were noted when the cells were incubated with purified ERRP but
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Figure 6. Western blots showing changes in the levels of tyrosine phosphorylated (p-EGFR) and total EGFR in (A) Caco-2 cells after 48 hours of incubation at 37°C in the absence or presence of increasing concentrations of purified recombinant ERRP and (B) in HCT-116 and (C) Caco-2 cells after exposure to either 5% serum or 10 nmol/L of TGF-␣ in the absence or presence of purified recombinant ERRP (5 g/mL) for 7 minutes at 37°C. At the end of the incubation period, cell lysates from these experiments containing 1 or 1.5 mg of protein were subjected to immunoprecipitation with anti-EGFR antibodies, and the immunoprecipitates were subjected to Western blot analysis with anti-phosphotyrosine antibodies. After visualization of protein bands by enhanced chemiluminescence detection, the membranes were stripped and reprobed with anti-EGFR antibodies for determination of total EGFR levels.
without TGF-␣ (data not shown). This could be due to the sensitivity of the methodology applied. However, to determine whether EGFR and ERRP will form a com-
Figure 7. (A) Detection of dimers of EGFR and ERRP in HCT-116 membranes after incubation in the presence or absence of TGF-␣, ERRP, or both and subsequently incubating in the reaction mixture with the cross-linking agent, DSS. Negative control was devoid of DSS. (B) Western blot showing the formation of an ERRP–EGFR complex in COS-7 cells transfected with ERRP-Myc-His–tagged clone 1 or the vector only (control). Twenty-four hours after transfection, cell lysates containing 1 mg of protein were incubated overnight at 4°C with anti-EGFR antibodies, and the immunoprecipitates were subsequently subjected to Western blot analysis with c-Myc antibodies. IP, immunoprecipitation.
plex in the absence of exogenous TGF-␣, we immunoprecipitated EGFR from COS-7 cell lysates derived from transfection of Myc-His–tagged ERRP and subjected the immunoprecipitates to Western blot analysis with c-Myc antibodies. We detected ERRP in the immunoprecipitates of EGFR (Figure 7B), indicating formation of an EGFR–ERRP complex in COS-7 cells. To avoid crosscontamination between EGFR and ERRP, we immunoprecipitated EGFR with EGFR antibodies that were raised against the cytoplasmic domain of EGFR (Upstate Biotechnology). Taken together, the results corroborate our hypothesis that ERRP heterodimerizes with EGFR and that formation of this heterodimer is facilitated in the presence of TGF-␣, one of the primary ligands of EGFR. To study the therapeutic potential of ERRP, an efficacy trial using SCID mice with palpable tumors derived from HCT-116 cells was conducted, in which purified recombinant ERRP or vehicle (control) was administered either intratumorally every other day for 8 days or sc on the back of each mouse (away from the tumor site) for 5 consecutive days. Regression of tumors in some animals and arrested growth of tumors in others was observed at the end of intratumoral or sc injections of ERRP treat-
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ment (Figure 8A and B). In most SCID mice, tumors were not visible at 10 –12 days after discontinuation of ERRP treatment. A representative photograph in Figure 8C shows tumor growth after treatments with ERRP or vehicle (control). None of the animals treated with ERRP lost weight or showed any visible signs of toxicity. However, when ERRP treatment was discontinued, tumors began to grow, but at much slower rate when compared with the controls, and the delay in tumor growth between the ERRP-treated and control groups was statistically significant at the level of P ⬍ 0.01 (Figure 8D and E). Taken together, the data in Figure 8 suggest that ERRP is effective in suppressing tumor growth. The activity score of ERRP, as shown in Table 1, was calculated to be 3⫹ on a scale of 1⫹ to 4⫹, with 4⫹ being considered as highly active in inducing a complete regression of tumors.24 Furthermore, when tumor responses are determined by T/C, ERRP is considered to be active against this type of human cancer (T/C is an indicator of antitumor effectiveness, where a value of ⱕ42% is indicative of a significant antitumor activity24). T/C values for ERRP were calculated to be 18.5% and 22.3% after intratumoral and sc injections of ERRP, respectively (Table 1), indicating a high antitumor activity for ERRP. It should also be noted that 1 in 5 mice injected sc with ERRP remained free of tumors after 60 days of discontinuation of ERRP treatment (Figure 8E).
Discussion Accumulating evidence suggests that downstream blocking of the EGFR signaling pathway is an effective therapeutic approach for the treatment and prevention of many epithelial malignancies, particularly those whose growth is regulated by the EGF family of peptides. This may be the most relevant area for use of ERRP. There are several strategies under investigation to block binding of growth factor to receptor. Previously, Wagner et al.25showed that transfection into pancreatic cancer cells (PANC-I) of a human EGFR cDNA fragment, generated by inserting a synthetic linker to express only the extracellular domain of the receptor, resulted in a marked inhibition of EGF/TGF-␣–induced EGFR tyrosine phosphorylation, anchorage-independent growth, and increased sensitivity of cells to cis-platinum. Recently, in a similar study, Matsuda et al.26 showed that infection of 4 pancreatic cell cancer cell lines (ASPC-1, COLO-357, PANC-1, and T3M4) with an adenoviral vector encoding a truncated EGFR markedly attenuated EGF and heparin-binding EGF-dependent cell growth, EGFR family tyrosine phosphorylation, and phosphorylation of mitogen-activated protein kinase, c-Jun NH2-
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terminal kinase, p38 mitogen-activated protein kinase, and activating transcription factor 2. Taken together, the data show that high levels of truncated EGFR, generated by molecular biology manipulations, modulate the EGFR signaling process and play a role in regulating cellular growth. ERRP, a naturally occurring molecule, also seems to regulate cellular growth by modulating EGFR function. Our observation that recombinant ERRP inhibits basal, as well as serum- and TGF-␣–induced, stimulation of EGFR phosphorylation in colon cancer cell lines suggests that ERRP inhibits growth by attenuating EGFR signaling processes. Our current data suggest that sequestration of the EGFR ligands by ERRP results in the formation of inactive heterodimers between ERRP and EGFR. This may partly be responsible for the attenuation of EGFR function. In view of this, we postulate that the loss of ERRP, as we have observed in colonic adenocarcinoma, may allow for increased activation of EGFR in colon cancer. Although the precise mechanism is unknown, one plausible explanation could be that the loss of ERRP with a concomitant reduction in sequestration of EGFR ligands will result in increased availability of free ligands for binding to and activation of EGFR. Therapeutic approaches to targeting the EGFR protein have usually taken the form of antibodies against the receptor or pharmacological inhibitors of the associated tyrosine kinase activity. A number of antibodies that block EGFR activation have been developed. The monoclonal antibody (MoAb) 225 against EGFR has been shown to induce growth inhibition of human cancer cell lines and xenograft tumor models.27–29 MoAb targeted to the c-erbB-2/HER-2 receptor, Herceptin (Genetech, Inc., San Francisco, CA), has been approved for breast cancer treatment in clinical trials. A phase II study using recombinant humanized anti c-erbB-2/HER-2 MoAb as a sensitizer for chemotherapy with cis-diaminedichloroplatinum in patients with tumors refractory to cis-diaminedichloroplatinum showed improved clinical outcome over those previously reported with either therapeutic alone, and without increased toxicity.30 Additionally, small molecule inhibitors of EGFR—OSI774 (CP 358,774) and Iressa (AstraZeneca, Wilmington, DE)— have progressed to clinical trials in EGFR-expressing cancers, with mixed results. Given the complexity at the molecular and cellular level, no single therapeutic agent is likely to emerge as the “magic bullet” in the treatment of cancer. Nonetheless, evidence suggests that targeting the EGF receptor is an effective therapeu-
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Figure 8. Changes in tumor weight after (A) intratumoral (7.5 g per tumor) and (B) subcutaneous (15 g per mouse) injections of purified recombinant ERRP over 8 and 5 days, respectively. (C) Representative photograph showing the growth of tumors in SCID mice xenografts of HCT-116 cells 12 days after discontinuation of intratumoral injections of ERRP (right) or vehicle (left), and survival curves of SCID mice after intratumoral (D) and subcutaneous (E) injections of purified ERRP or vehicle.
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Table 1. Antitumor Activity of ERRP in SCID Mice Xenografts Bearing Tumors of a Colon Cancer Cell Line, HCT-116 Log10 Kill Agent Control (vehicle) ERRP Control (vehicle) ERRP
Route
No. Animals
T/C (%)
T/C (days)
Net
Gross
Activity score
Tumor weight range (mg)
sc sc IT IT
5 5 5 5
100 22.3 100 8.5
0 14 0 16
0 2.10 0 1.30
0 1.35 0 2.40
(⫺⫺) (⫹⫹⫹) (⫺) (⫹⫹⫹)
288–1268 0–344 40–847 14–642
NOTE. Subcutaneous (sc) injections of 15 g of ERRP per mouse per day for 5 days were given on the back of mice. Intratumoral (IT) injection of 7.5 g per palpable tumor was given directly into each palpable tumor every other day for 8 days. T/C is an indicator of antitumor effectiveness, where a value of ⱕ42% is considered to possess a significant antitumor activity. If log10 kill values (net and gross) are added as a criterion, ERRP showed a clinically meaningful activity. Activity rating scores of 3⫹ and 4⫹ are considered “active” and “highly active,” respectively, in inducing a partial or complete regression of tumors.
tic strategy in cancers in which there is overexpression or activation of this protein. On the basis of results obtained thus far, we believe that ERRP may be a suitable candidate for inhibiting EGFR signaling pathways and, in turn, colorectal tumor growth. The novelty of ERRP is that it is an endogenous secretory protein,18 which could be administered systemically to inhibit EGFR signaling pathways leading to diminution of cellular growth. Our observation that sc injections of ERRP cause regression or arrest the growth of tumors in SCID mice xenografts of HCT-116 cells supports our contention that ERRP could be administered systemically and remains biologically active. We conclude that ERRP is a potential therapeutic agent for colorectal cancer.
References 1. Yarden Y, Ullrich A. Growth factor receptor tyrosine kinases. Ann Rev Biochem 1987;57:443– 487. 2. Candena DL, Gill GN. Receptor tyrosine kinases. FASEB J 1992; 6:2332–2337. 3. Joensuu HJ, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D, Silberman SL, Capdeville R, Dimitrijevic S, Druker B, Demetri GD. Effect of the tyrosine kinase inhibitor ST1571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2000;344:1052–1056. 4. Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A, Schlessinger J. Close similarity of epidermal growth factor receptor and v-erbB oncogene protein sequences. Nature 1984;307:521–527. 5. Cuttitta F, Carney N, Mulshine J, Moody TW, Fedorko J, Fischler A, Minna JD. Bombesin-like peptides can function as autocrine growth factors in human small cell lung cancer. Nature 1985; 316:823– 826. 6. Betsholtz C, Heldin CH, Nister M, Ek B, Heldin CH. Co-expression of a PDGF-like growth factor and PDGF receptors in human osteosarcoma cell line: implications for autocrine receptor activation. Cell 1984;39:447– 457. 7. Sporn MB, Roberts AB. Autocrine growth factor and cancer. Nature 1985;313:745–747. 8. Culig Z, Hobisch A, Cronauer MV, Radmayr C, Zhang J, Thurnher M, Bartsch G. Regulation of prostatic growth and function by peptide growth factors. Prostate 1996;28:392– 405. 9. Barnard JA, Beauchamp J, Russell WE, DuBois RN, Coffey RJ. Epidermal growth factor-related peptides and their relevance to
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gastrointestinal pathophysiology. Gastroenterology 1995;108: 564 –580. Khasharyarsha K, Schirrmacher V, Lichtner RB. EGF-receptor in neoplasia. Cancer Metastasis Rev 1993;12:255–274. Gullick WJ. Prevalence of aberrant expression of the epidermal growth factor receptor in human cancers. Br Med Bull 1991;47: 87–98. Malecka-Panas E, Kordek R, Biernat W, Tureaud J, Majumdar APN. Differential activation of total and EGF receptor tyrosine kinase in rectal mucosa in patients with adenomatous polyps, ulcerative colitis and colon cancer. Hepatogastroenterology 1997;44:435– 440. Relan NK, Saeed A, Ponduri K, Fligiel SEG, Dutta S, Majumdar APN. Identification and evaluation of the role of endogenous tyrosine kinases in azoxymethane induction of proliferative processes in the colonic mucosa of rats. Biochim Biophys Acta 1995;1244:368 –376. Coffey R Jr, Shipley GD, Moses HL. Production of transforming growth factors by human colon cancer lines. Cancer Res 1986; 46:1164 –1169. Ohmura E, Okada M, Onoda N, Kamiya Y, Murakami H, Tsushima T, Shizume K. Insulin-like growth factor and transforming growth factor alpha as autocrine growth factors in human pancreatic cancer cell growth. Cancer Res 1990;50:103–107. Yoshida K, Kyo E, Tsuda T, Tsujino T, Ito M, Niimoto M, Tahara E. EGF and TGF-alpha, the ligands of hyperproduced EGFR in human esophageal carcinoma cells, act as autocrine growth factor. Int J Cancer 1990;45:131–135. Anzano MA, Rieman D, Pritchett W, Bowen-Pope DF, Greig R. Growth factor production by human colon carcinoma cell lines. Cancer Res 1989;49:2898 –2904. Yu Y, Rishi AK, Turner JJ, Liu D, Black E, Moshier JA, Majumdar APN. Cloning of a novel EGFR related peptide: a putative negative regulator of EGFR. Am J Physiol Cell Physiol 2001;280:C1083– C1089. Feng J, Adsay NV, Kruger M, Majumdar APN, Sarkar FH. Expression of ERRP in normal and neoplastic pancreata and its relationship to clinicopathological parameters in pancreatic adenocarcinoma. Pancreas 2002;25:342–349. Majumdar APN, Tureaud J, Relan NK, Kessel A, Dutta S, Hatfield J, Fligiel SEG. Increased expression of pp60c-src in gastric mucosa of aged rats. J Gerontol 1994;49:B110 –B116. Xiao Z-Q, Majumdar APN. Increased in vitro activation of EGFR by membrane-bound EGF from gastric and colonic mucosa of aged rats. Am J Physiol Gastrointest Liver Physiol 2001;281:G111– G116. Walker F, Burgess AW. Reconstitution of the high affinity epidermal growth factor receptor on cell-free membranes after transmodulation by platelet-derived growth factor. J Biol Chem 1991;266:2746 –2752.
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23. Roberts RB, Min L, Washington MK, Olsen SJ, Settle SH, Coffey RJ, Threadgill DW. Importance of epidermal growth factor receptor signaling in establishment of adenomas and maintenance of carcinomas during intestinal tumorigenesis. Proc Natl Acad Sci U S A 2002;99:1521–1525. 24. Mohammad RM, Varterasian ML, Almactchy VP, Hannoudi GN, Pettit GR, Al-Khatib A. Successful treatment of human chronic lymphocytic leukemia xenografts with combination biological agents auristatin PE and bryostatin 1. Clin Cancer Res 1998;4: 1337–1343. 25. Wagner M, Cao T, Lopez ME, Hope C, Van Nostrand K, Kobrin MS, Fan HU, Buchler MW, Korc M. Expression of a truncated EGF receptor is associated with inhibition of pancreatic cancer cell growth and enhanced sensitivity to cisplatinum. Int J Cancer 1996;68:782–787. 26. Matsuda K, Idezawa T, You XJ, Kothari NH, Fan H, Korc M. Multiple mitogenic pathways in pancreatic cancer cells are blocked by a truncated epidermal growth factor receptor. Cancer Res 2002;62:5611–5617. 27. Scott GK, Robles R, Park JW. A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. Mol Cell Biol 1993;13:2247–2257.
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28. Wu X, Fan Z, Masui H, Rosen N, Mendelsohn J. Apoptosis induced by an anti-epidermal growth factor receptor monoclonal antibody in human colorectal carcinoma cell line and its delay by insulin. J Clin Invest 1995;95:1897–1905. 29. Karnes WE, Weller SG, Adjei PN, Kottke TJ, Glenn KS, Gores GJ, Kaufmann SH. Inhibition of epidermal growth factor receptor kinase induces protease-dependent apoptosis in human colon cancer cells. Gastroenterology 1998;114:930 –939. 30. Armstrong DK, Kaufman SC, Ottaviano YL, Ottaviano YL, Furuya Y, Buckley JA, Isaacs JT, Davidson NE. Epidermal growth factormediated apoptosis of MDA-468 human breast cancer cells. Cancer Res 1994;54:5280 –5283.
Received August 14, 2002; Accepted January 27, 2003. Address requests for reprints to: Adhip P. N. Majumdar, Ph.D., D.Sc., Research Service, 151 VA Medical Center, 4646 John R., Detroit, Michigan 48201. e-mail: [email protected]; fax: (313) 5761112. Supported by grants to Dr. Majumdar from the Department of Veterans Affairs and the National Institute on Aging (AG 14343).
Epidermal Growth Factor Receptor–Related Protein: A Potential Therapeutic Agent for Colorectal Cancer DOROTA J. MARCINIAK,* LATHIKA MORAGODA,* RAMZI M. MOHAMMAD,*,‡ YINGJIE YU,* KIRAN K. NAGOTHU,* AMRO ABOUKAMEEL,* FAZLUL H. SARKAR,‡,§ VOLKAN N. ADSAY,§ ARUN K. RISHI,*,‡,㛳 and ADHIP P. N. MAJUMDAR*,㛳 *Department of Internal Medicine, Wayne State University School of Medicine, Detroit; ‡Karmanos Cancer Institute, Detroit; §Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan; 㛳Veterans Affairs Medical Center, Detroit, Michigan
Background & Aims: Epidermal growth factor receptor is frequently implicated in epithelial cancers and is, therefore, being considered as a potential target for therapy. Recently, we reported the isolation and characterization of epidermal growth factor receptor–related protein, a negative regulator of epidermal growth factor receptor. To discern whether epidermal growth factor receptor– related protein could be an effective therapeutic agent for colorectal cancer, we generated epidermal growth factor receptor–related protein fusion protein and studied its effect on the growth of colon cancer cells in vivo and in vitro. We also studied whether epidermal growth factor receptor–related protein expression is altered in colorectal cancer. Methods: A 55-kilodalton epidermal growth factor receptor–related protein fusion protein with V5 and His tags was generated in a drosophila expression system and subsequently purified by a His antibody affinity column. Rabbit polyclonal antibodies against epidermal growth factor receptor–related protein were used to examine the expression of epidermal growth factor receptor–related protein. Results: Epidermal growth factor receptor–related protein expression was found to be high in benign human colonic epithelium but low in adenocarcinoma. Exposure of the colon cancer cell lines HCT-116 and Caco-2 to purified recombinant epidermal growth factor receptor–related protein caused a marked inhibition of proliferation, as well as attenuation of basal and ligand-induced stimulation of epidermal growth factor receptor phosphorylation. Epidermal growth factor receptor–related protein-induced inhibition of proliferation of colon cancer cells was prevented by epidermal growth factor receptor–related protein antibodies. Reduced epidermal growth factor receptor phosphorylation was partly due to sequestration of epidermal growth factor receptor ligands by epidermal growth factor receptor–related protein, resulting in the formation of inactive heterodimers with epidermal growth factor receptor. Intratumoral or subcutaneous (away from the tumor site) injections of purified epidermal growth factor receptor–related protein caused regression of palpable colon cancer xenograft tumors in some severely compromised immunodeficient mice and
arrested tumor growth in others. Conclusions: We propose that epidermal growth factor receptor–related protein inhibits cellular growth by attenuating epidermal growth factor receptor signaling processes and is an effective therapeutic agent for colorectal cancer.
embers of the receptor tyrosine kinase family are frequently implicated in experimental models of epithelial cell neoplasia, as well as in human cancers.1–3 There is increasing evidence to support the concept that the malignant behavior of some tumors is sustained by deregulated activation of certain growth factor receptors. Such deregulation could be due to structural alterations of the receptor itself4; to the establishment of an autocrine loop, whereby the cells produce growth factors that stimulate their own growth5–7; or to the loss of a suppressor of receptor function. One of the best studied receptor signaling systems from this family is that controlled by the epidermal growth factor (EGF) receptor (EGFR), whose expression and enzyme activity have been linked to a number of malignancies, including cancer of the colon.8 –11 EGFR and its ligand transforming growth factor (TGF)-␣, a structural and functional analogue of EGF, are overexpressed in preneoplastic and neoplastic colonic mucosa.12–14 Moreover, cell lines derived from adenocarcinomas of various gastrointestinal tissues, including the colon, overexpress TGF-␣ and its receptor EGFR.15–17 Because of EGFR’s role in the development and progression of many epithelial cancers, efforts have
M
Abbreviations used in this paper: DMEM, Dulbecco’s modified Eagle medium; DMSO, dimethyl sulfoxide; DSS, disuccinimidyl suberate; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERRP, epidermal growth factor receptor–related protein; FBS, fetal bovine serum; MoAb, monoclonal antibody; ORF, open reading frame; SCID, severely compromised immunodeficient; TGF, transforming growth factor. © 2003 by the American Gastroenterological Association 0016-5085/03/$30.00 doi:10.1016/S0016-5085(03)00264-6
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been made to use EGFR as a potential target for epithelial cancer therapy. Several approaches, including monoclonal antibodies to EGFR and pharmacological inhibitors of EGFR tyrosine kinase, have been used, but with limited success, primarily because of toxicity or lack of specificity. Therefore, identification of endogenous factors that may inhibit EGFR activation and its signaling pathways is of paramount therapeutic importance. To this end, we recently reported the isolation and characterization of a negative regulator of EGFR, referred to as ERRP (EGFR-related protein; US patent 6,399,743; GenBank accession no. AF187818), which possesses a significant homology to the extracellular ligand binding domain of EGFR.18 Transfection of ERRP complementary DNA (cDNA) into the colon cancer cell lines HCT116 and Caco-2 not only inhibits proliferation in matrixdependent and independent systems, but also decreases EGFR activation, as evidenced by reduced tyrosine phosphorylation and tyrosine kinase activity of the receptor.18 Taken together, the results suggest that ERRP inhibits proliferation of colon cancer cells by attenuating EGFR function. These results raise the possibility that ERRP could be an effective therapeutic agent for colorectal cancer. To test this possibility, we generated ERRP protein by using the drosophila expression system and studied the effect of purified recombinant ERRP on the growth of colon cancer cells in vitro and in vivo. We show that recombinant ERRP inhibits proliferation and EGFR activation in colon cancer cell lines in vitro and suppresses the growth of tumors in severely compromised immunodeficient (SCID) mice xenografts of colon cancer cells.
Materials and Methods Cell Lines Colon cancer cell lines HCT-116 and Caco-2 and monkey kidney COS-7 cells, obtained from the American Type Culture Collection (Rockville, MD), were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (10,000 U/mL), streptomycin (10,000 U/mL), and amphotericin (25 L/mL) at 37°C in an atmosphere of 95% air and 5% CO2.
Cell Proliferation This was assessed by 3-(4,5-dimethylthialzol-2yl)-2,5diphenyltetrazolium bromide assay according to the procedure described by Sigma Chemical Co. (St. Louis, MO). Briefly, aliquots of cells (3 ⫻ 104/mL) in DMEM/10% FBS were plated in a 96-well culture plate with 5 replicates per treatment. After 24 hours of plating, the medium was replaced with that containing 2.5% FBS, and the incubation at 37°C continued in the absence (control) or presence of recombinant ERRP, as
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stated in the figure legends. In some experiments, incubations were performed in the absence (control) or presence of recombinant ERRP or recombinant ERRP together with either anti-ERRP antibodies (1:500 final dilution) or normal rabbit serum (1:500 final dilution). All incubations were terminated by adding 20 L of 0.5 g/mL stock of 3-(4,5-dimethylthialzol2yl)-2,5-diphenyltetrazolium bromide to each well. The reaction was allowed to proceed for 3– 4 hours at 37°C. The culture medium was removed, formazan crystals were dissolved by adding 0.2 mL of dimethyl sulfoxide (DMSO), and the intensity of color was measured at 570 nm.
Establishment of Tumors in Severely Compromised Immunodeficient Mice SCID mice were obtained from Taconic Laboratories (German Town, NY). After a period of adaptation, 2 to 3 mice were subcutaneously (sc) injected on each flank with approximately 106 HCT-116 cells. When tumors developed, mice were killed; tumors were dissected, cut into small fragments, and subsequently transplanted sc into similarly conditioned animals (n ⫽ 6 for each group) by using a 12-gauge trocar. Mice were checked 3 times a week for tumor development. Once palpable tumors developed, groups of 6 mice were removed randomly for the ERRP efficacy trial.
Generation of Polyclonal Antibodies to ERRP Polyclonal antibodies to ERRP were raised in rabbits as described previously.19 Briefly, this was achieved by scanning protein databases and identifying a region of ERRP comprising 27 amino acids (nucleotide 1580 –1661), referred to as a U region, that showed no homology with any known protein sequence in the current database.18 Further analysis of the U region showed that the mid portion of the peptide with 15 amino acid residues (amino acid residues of a 15-mer peptide) possessed the most antigenic property. The above 15-mer peptide was synthesized and subsequently used by Sigma–Genosys (Woodlands, TX) to raise antibodies in rabbits. In Western blot analysis, antibodies reacted strongly with a 55-kilodalton protein19 that corresponded well with the calculated molecular mass of ERRP, the open reading of which is composed of 479 amino acids. No cross-reactivity with EGFR or any of its family members was noted.19
Immunohistochemical Staining This was performed as described previously.19 Briefly, 5-m sections of paraffin-embedded tissues were deparaffinized and microwaved for 15 minutes in 1 L of citrate buffer. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide and subsequently incubated with 5% horse serum to block nonspecific binding. The slides were then incubated at room temperature for 2 hours with polyclonal antibodies to ERRP. Negative controls were performed with antigen-neutralized ERRP antibodies. Antigen neutralization was accomplished by preincubating ERRP antibodies at 4°C
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for 16 hours with the antigen (1 g/mL of phosphate-buffered saline; 1 part ERRP antibody and 5 parts antigen). ERRP antibodies, used as positive controls, were also similarly treated after dilution with phosphate-buffered saline. All primary antibodies were used at a final dilution of 1:1000. After incubation with primary antibodies, the avidin-biotin technique was performed with matched components (secondary biotinylated antibody and avidin-peroxidase complex) from the DAKO labeled streptavidin-biotin system (Carpentia, CA) according to the manufacturer’s suggested protocol. The slides were reacted with amino ethyl carbazole, counterstained with Harris’ hematoxylin, and examined by a pathologist blinded to sample coding. At least 10 well-oriented crypts on each slide and 5 slides from each sample were examined under high power.
Generation of ERRP Fusion Protein The drosophila expression system was used to generate ERRP fusion protein. To create stable cell lines expressing ERRP fusion protein, the expression vector pMT/ V5-HisA (Invitrogen, Carlsbad, CA) containing the entire open reading frame (ORF) of ERRP cDNA was co-transfected into drosophila S2 cells with a pCoHygro plasmid (Invitrogen), which confers hygromycin resistance. The transfectants were selected with hygromycin at a concentration of 300 g/mL, and individual sublines were propagated in media containing 150 g/mL of hygromycin. The stable cell lines were induced with 0.5 mmol/L CuSO4 to express the ERRP fusion protein. Western blot analysis with polyhistidine antibodies (Invitrogen) of cell lysates and the conditioned media from CuSO4-treated cells showed a single protein band with a molecular weight ratio (Mr) of approximately 55 kilodaltons (Figure 1) that corresponded well with the calculated molecular mass of ERRP. Detection of ERRP in the conditioned medium together with the fact that the ORF of ERRP contains a signal peptide composed of 24 amino acids, which are similar but not identical to those found in human or rat EGFR,18 suggests that ERRP is a secretory protein. The reason for detecting a substantially higher amount of ERRP in cell lysate, compared with the conditioned medium, was that a total of approximately 30 ⫻ 106 cells were cultured in 10 mL of growth medium. After 24 hours of induction with CuSO4 , cells were obtained by centrifugation and lysed in 1 mL of lysis buffer, and 10 L (one hundredth of the volume) of the lysate and 30 L (three thousandths of the volume) of the conditioned medium were subjected to Western blot analysis. ERRP was purified from the crude cell lysate by using polyhistidine antibodies conjugated to Sepharose 4B (Pharmacia, Piscataway, NJ), as described previously.20The bound protein was eluated with 3 mol/L of sodium thiocyanate,20 and the fractions containing ERRP were pooled and dialyzed against 20 mmol/L of HEPES, pH 7.5, and subsequently concentrated by using Amicon (Beverly, MA) filter units.
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Immunoprecipitation and Western Blot Analysis of EGFR This was performed according to our standard protocol.18,21 Briefly, aliquots of cell lysate containing 1- or 1.5-mg proteins were incubated with polyclonal anti-EGFR antibodies (Upstate Biotechnology, Lake Placid, NY) and Sepharose G at 4°C for 3 hours. Immunoprecipitates were resolved on a 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The electrophoresed proteins were transferred to nitrocellulose membranes and subsequently probed with anti-phosphotyrosine antibodies, as described below. After visualization of protein bands by an enhanced chemiluminescence detection system, the membranes were stripped and reprobed with antiEGFR antibodies as described previously.18
Expression and Secretion of ERRP COS-7 cells were transiently transfected with expression plasmid encoding ERRP fused with polyhistine and cMyc tags to examine ERRP secretion. First, ERRP cDNA (GenBank accession no. AF187818) containing entire proteinencoding ORFs but lacking the translation termination codon and the 3⬘-untranslated region was subcloned into the vector plasmid pcDNA-3/Myc-His-A (Invitrogen) to generate ERRP-Myc-His–tagged clone 1. Next, the vector plasmid (control) or the ERRP-Myc-His–tagged clone 1 was independently and transiently transfected into COS-7 cells, essentially as described previously.18 Approximately 24 hours after transfection, the media were collected and the cells harvested and lysed. The media (1 mL) or the lysates (0.5 mg) were separately used for immunoprecipitation with anti-EGFR (raised against the cytoplasmic domain of EGFR; Upstate Biotechnology), anti– c-Myc, or anti-polyhistidine antibodies (Cell Signaling, Beverley, MA). The immunoprecipitates were subjected to Western blot analysis with anti– c-Myc or anti-polyhistidine antibodies.
Cross-linking of ERRP and EGFR This was performed with crude membrane fractions of serum-starved HCT-116 cells, prepared according to Walker and Burgess.22 Cross-linking was conducted by using 2 mmol/L of disuccinimidyl suberate (DSS) according to the manufacturer’s instructions (Pierce, Rockford, IL). Briefly, aliquots of membranes containing the same amount of protein (0.5 mg) were incubated in the absence (control) or presence of 10 nmol/L of TGF-␣, ERRP (5 g/mL), or both for 1 hour at 4°C. The reaction was terminated by adding DSS in DMSO or an equivalent volume of DMSO alone and incubating further at room temperature for 30 minutes, as described by Walker and Burgess.22 After quenching with 20 mmol/L of Tris (pH 7.5), the reaction was mixed with an equal volume of lysis buffer (10 mmol/L of HEPES, pH 7.2; 150 mmol/L of NaCl; 2.5 mmol/L of Na3VO4; 1 mmol/L of phenylmethylsulfonyl fluoride; 2.5 mmol/L of EDTA; 25 g/mL of aprotinin, leupeptin, and pepstatin A; 0.5 % Triton X-100; and 0.5% Nonidet P-40). After clarification at 11,000 ⫻ g for 15 min-
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Figure 1. Western blot with polyhistidine antibodies showing ERRP fusion protein in S2 cells incubated in the absence or presence of CuSO4 and in the conditioned media (Med) from CuSO4-treated S2 cells.
utes at 4°C, the supernatants were used to detect dimers and monomers of EGFR by immunoprecipitation followed by Western blot analysis, as described previously.18 Solubilized membranes were incubated overnight at 4°C with sheep polyclonal antibodies to EGFR and 30 L of protein A–sepharose beads. The beads, after washing with TT buffer (50 mmol/L of Tris-HCl, pH 7.6, 0.15 mol/L of NaCl, and 0.5% Tween 20), were subjected to electrophoresis on a 5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes, which were subsequently probed with anti-EGFR antibodies to detect homoand heterodimers of EGFR. Protein bands were visualized by a enhanced chemiluminescence detection system.
Statistical Analysis Unless otherwise stated, data are expressed as mean ⫾ SEM. Where applicable, the results were compared by using the unpaired Student t test and analysis of variance for nonparametric data. P ⬍ 0.05 was designated as the level of significance.
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the media of COS-7 cells transfected with ERRP-MycHis–tagged clone 1 plasmid, but not in the media derived from the vector (control) transfected cells (Figure 2). In an analogous experiment, ERRP-fusion protein was immunoprecipitated with c-Myc antibodies followed by Western blots with polyhistidine antibodies. Again, ERRP was detected in the media (data not shown). Taken together, the results suggest that ERRP is a secretory protein. Because ERRP is a negative regulator of EGFR, we hypothesize that increased activation of EGFR associated with colorectal cancer could partly be the result of loss of ERRP, a suppressor of EGFR function. To test this possibility, we examined the expression of ERRP by immunohistochemical analyses in surgical specimens from 10 subjects. A representative photomicrograph is shown in Figure 3; Figure 3A represents hematoxylin and eosin staining of the specimen, showing benign and adenocarcinoma regions in the tumor. Indeed, data from immunohistochemical analyses showed that expression of ERRP was high in benign human colonic epithelium but low in invasive adenocarcinoma in all the specimens tested (Figure 3B). Higher magnification of benign mucosa and invasive adenocarcinoma shows that ERRP localizes primarily to the basolateral membranes in benign mucosa (similar to EGFR) but loses that polarized distribution in invasive adenocarcinoma (Figure 3C and D). The observed increase in ERRP immunoreactivity in benign colonic mucosa could not be attributed to nonspecific staining, because little or no visible staining occurred in normal colonic epithelium when ERRP an-
Results The fact that the ORF of ERRP contains a signal peptide composed of 24 amino acids, together with our observation that ERRP is present in the conditioned media derived from drosophila S2 cells (Figure 1), suggests that ERRP is a secretory protein. To further test this possibility that ERRP is a secretary protein, we generated a construct expressing polyhistidine and Myctagged ERRP protein. This construct or the vector plasmid (control) was subsequently transfected into COS-7 cells, and the presence of ERRP in the conditioned media was determined by immunoprecipitation and subsequent Western blot analysis. ERRP present in the conditioned media was immunoprecipitated with polyhistidine antibodies followed by Western blot with c-Myc antibodies. We found Myc-polyhistidine–tagged ERRP protein in
Figure 2. Western blot showing ERRP levels in the conditioned media of COS-7 cells 24 hours after transfection with either the ERRP-MycHis–tagged clone 1 (lane 2) or only the vector pcDNA-3 (control; lane 1). One milliliter of conditioned medium was subjected to immunoprecipitation with polyhistidine antibodies, and the immunoprecipitates were subsequently analyzed by a 12% sodium dodecyl sulfate– polyacrylamide gel electrophoresis followed by Western blot with anti– c-Myc antibodies. IP, immunoprecipitation.
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Figure 3. A representative photomicrograph showing changes in ERRP immunoreactivity in the benign human colonic epithelium and the adjacent invasive adenocarcinoma (denoted by arrows in A and B).
tibodies were neutralized with the antigen before use (Figure 4B). As expected, a marked ERRP staining was observed when ERRP antibodies were used (Figure 4A). Because epithelial malignancies, including colorectal cancer, show increased EGFR activation,10 –14,23 it is plausible that the observed loss of ERRP is partly responsible for increased EGFR activation in colorectal tumors. Previously, we showed that expression of ERRP in the colon cancer cell lines HCT-116 and Caco-2 inhibits proliferation and EGFR activation,18 raising the possibility that ERRP could be an effective inhibitor of colorectal tumors. To test this hypothesis, we studied the effect of recombinant ERRP on proliferation and EGFR phosphorylation of colon cancer cell lines. Indeed, we observed that the recombinant ERRP inhibited proliferation of the colon cancer cell lines HCT-116 and Caco-2 in a dose-dependent manner, showing a 60%–70% reduction with the highest dose of ERRP (8 –10 g/mL) when compared with the corresponding control (Figure 5A and B). A 72-hour time-course study with HCT-116 cells was performed to determine the growth-inhibitory properties of ERRP. As shown in Figure 5C, recombinant ERRP (5 g/mL) remained effective throughout the experimental period, causing a 50%– 60% inhibition of proliferation. The ERRP-induced inhibition of proliferation of HCT-116 cells was prevented by adding antiERRP antibodies, whereas preimmune rabbit serum was ineffective in preventing this inhibition (Figure 5D). Our earlier observation that overexpression of ERRP in Caco-2 cells, resulting in inhibition of proliferation, was accompanied by a concomitant reduction in tyrosine phosphorylation and tyrosine kinase activity of EGFR18 suggests that ERRP inhibits proliferation by attenuating EGFR activation.18 To determine whether recombinant ERRP will affect EGFR function, changes in the levels of
tyrosine-phosphorylated EGFR in Caco-2 cells were examined after exposure to increasing concentrations of recombinant ERRP for 48 hours. Indeed, we found that a high dose of ERRP greatly inhibited the basal tyrosine phosphorylation of EGFR in Caco-2 cells (Figure 6A). Because ligand binding is the primary cause of activation of EGFR, we examined the effect of recombinant ERRP on ligand-induced stimulation of EGFR phosphorylation in HCT-116 and Caco-2 cells. We observed that exposure of 48-hour serum-starved HCT-116 or Caco-2 cells to either 5% serum or 10 nmol/L of TGF-␣ caused a marked stimulation of tyrosine phosphorylation of EGFR when compared with the control (Figure 6B and C). However, the serum-induced or TGF-␣–induced stimulation of EGFR tyrosine phosphorylation was greatly attenuated by purified recombinant ERRP (Figure 6B and C). Levels of EGFR in either HCT-116 or Caco-2 cells, however, were unaffected by these treatments (Figure 6B and C). Taken together, the results
Figure 4. A representative photomicrograph showing changes in ERRP immunoreactivity in the normal human colon when incubated with (A) ERRP antibodies and (B) antigen-neutralized ERRP antibodies.
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Figure 5. Effect of purified recombinant ERRP on proliferation of the colon cancer cell lines HCT-116 and Caco-2. Dose-dependent changes in the proliferation of HCT-116 and Caco-2 cells are depicted in (A) and (B). Cell lines were exposed to increasing concentrations of purified recombinant ERRP for 48 hours. Time-course changes in proliferation in response to 5 g/mL of purified recombinant ERRP are shown in (C). (D) The effect of recombinant ERRP (5 g/mL) on proliferation of HCT-116 cells in the absence or presence of ERRP antibodies or preimmune rabbit serum. Each value represents mean ⫾ SEM of 5– 6 observations. *P ⬍ 0.01 compared with control.
suggest that purified recombinant ERRP is biologically active and inhibits proliferation by attenuating EGFR function. The mechanism by which ERRP results in attenuation of EGFR activation is poorly understood. However, because ERRP possesses a substantial homology to the ligand binding domain of the EGFR, we hypothesize that ERRP binds the EGFR ligands and subsequently forms inactive heterodimers with EGFR, leading to inhibition of EGFR phosphorylation. To test this hypothesis, we studied the effects of TGF-␣ on the formation of ERRP/EGFR heterodimers in HCT-116 cells in the absence or presence of purified ERRP. Aliquots of crude membranes from serum-starved HCT-116 cells, containing the same amount of protein, were incubated in the absence or presence of TGF-␣ (10 nmol/L), ERRP (5 g/mL), or both. To detect dimers of EGFR and those formed between EGFR and ERRP, the reaction mixtures were treated with and without DSS, a cross-linking agent, followed by immunoprecipitation with antiEGFR antibodies. The immunoprecipitates were then subjected to Western blot analysis with anti-EGFR an-
tibodies to detect homo- and heterodimers of EGFR. We anticipated that incubation of HCT-116 cells with TGF-␣ and ERRP would result in the formation of ERRP/EGFR heterodimers with an Mr of approximately 225 kilodaltons (170 kilodaltons for EGFR ⫹ 55 kilodaltons for ERRP) and EGFR homodimers of 340 kilodaltons. Indeed, this was found to be the case. In the presence of TGF-␣ and ERRP, we detected 3 distinct protein bands with Mr of 340, 220, and 170 kilodaltons, representing homodimers of EGFR, heterodimers of ERRP/EGFR, and EGFR monomers, respectively (Figure 7A). A faint ERRP/EGFR heterodimer band of 220 kilodaltons was also observed when incubations were performed in the absence of ERRP but containing TGF-␣ (Figure 7A). Formation of a 220-kilodalton heterodimer in the absence of exogenous ERRP is probably due to the presence of endogenous ERRP. Neither a 340nor a 220-kilodalton protein band was detected when incubations were performed in the absence of TGF-␣ and ERRP (Figure 7A). Lack of DSS also failed to produce any dimers (Figure 7A). No heterodimers were noted when the cells were incubated with purified ERRP but
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Figure 6. Western blots showing changes in the levels of tyrosine phosphorylated (p-EGFR) and total EGFR in (A) Caco-2 cells after 48 hours of incubation at 37°C in the absence or presence of increasing concentrations of purified recombinant ERRP and (B) in HCT-116 and (C) Caco-2 cells after exposure to either 5% serum or 10 nmol/L of TGF-␣ in the absence or presence of purified recombinant ERRP (5 g/mL) for 7 minutes at 37°C. At the end of the incubation period, cell lysates from these experiments containing 1 or 1.5 mg of protein were subjected to immunoprecipitation with anti-EGFR antibodies, and the immunoprecipitates were subjected to Western blot analysis with anti-phosphotyrosine antibodies. After visualization of protein bands by enhanced chemiluminescence detection, the membranes were stripped and reprobed with anti-EGFR antibodies for determination of total EGFR levels.
without TGF-␣ (data not shown). This could be due to the sensitivity of the methodology applied. However, to determine whether EGFR and ERRP will form a com-
Figure 7. (A) Detection of dimers of EGFR and ERRP in HCT-116 membranes after incubation in the presence or absence of TGF-␣, ERRP, or both and subsequently incubating in the reaction mixture with the cross-linking agent, DSS. Negative control was devoid of DSS. (B) Western blot showing the formation of an ERRP–EGFR complex in COS-7 cells transfected with ERRP-Myc-His–tagged clone 1 or the vector only (control). Twenty-four hours after transfection, cell lysates containing 1 mg of protein were incubated overnight at 4°C with anti-EGFR antibodies, and the immunoprecipitates were subsequently subjected to Western blot analysis with c-Myc antibodies. IP, immunoprecipitation.
plex in the absence of exogenous TGF-␣, we immunoprecipitated EGFR from COS-7 cell lysates derived from transfection of Myc-His–tagged ERRP and subjected the immunoprecipitates to Western blot analysis with c-Myc antibodies. We detected ERRP in the immunoprecipitates of EGFR (Figure 7B), indicating formation of an EGFR–ERRP complex in COS-7 cells. To avoid crosscontamination between EGFR and ERRP, we immunoprecipitated EGFR with EGFR antibodies that were raised against the cytoplasmic domain of EGFR (Upstate Biotechnology). Taken together, the results corroborate our hypothesis that ERRP heterodimerizes with EGFR and that formation of this heterodimer is facilitated in the presence of TGF-␣, one of the primary ligands of EGFR. To study the therapeutic potential of ERRP, an efficacy trial using SCID mice with palpable tumors derived from HCT-116 cells was conducted, in which purified recombinant ERRP or vehicle (control) was administered either intratumorally every other day for 8 days or sc on the back of each mouse (away from the tumor site) for 5 consecutive days. Regression of tumors in some animals and arrested growth of tumors in others was observed at the end of intratumoral or sc injections of ERRP treat-
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ment (Figure 8A and B). In most SCID mice, tumors were not visible at 10 –12 days after discontinuation of ERRP treatment. A representative photograph in Figure 8C shows tumor growth after treatments with ERRP or vehicle (control). None of the animals treated with ERRP lost weight or showed any visible signs of toxicity. However, when ERRP treatment was discontinued, tumors began to grow, but at much slower rate when compared with the controls, and the delay in tumor growth between the ERRP-treated and control groups was statistically significant at the level of P ⬍ 0.01 (Figure 8D and E). Taken together, the data in Figure 8 suggest that ERRP is effective in suppressing tumor growth. The activity score of ERRP, as shown in Table 1, was calculated to be 3⫹ on a scale of 1⫹ to 4⫹, with 4⫹ being considered as highly active in inducing a complete regression of tumors.24 Furthermore, when tumor responses are determined by T/C, ERRP is considered to be active against this type of human cancer (T/C is an indicator of antitumor effectiveness, where a value of ⱕ42% is indicative of a significant antitumor activity24). T/C values for ERRP were calculated to be 18.5% and 22.3% after intratumoral and sc injections of ERRP, respectively (Table 1), indicating a high antitumor activity for ERRP. It should also be noted that 1 in 5 mice injected sc with ERRP remained free of tumors after 60 days of discontinuation of ERRP treatment (Figure 8E).
Discussion Accumulating evidence suggests that downstream blocking of the EGFR signaling pathway is an effective therapeutic approach for the treatment and prevention of many epithelial malignancies, particularly those whose growth is regulated by the EGF family of peptides. This may be the most relevant area for use of ERRP. There are several strategies under investigation to block binding of growth factor to receptor. Previously, Wagner et al.25showed that transfection into pancreatic cancer cells (PANC-I) of a human EGFR cDNA fragment, generated by inserting a synthetic linker to express only the extracellular domain of the receptor, resulted in a marked inhibition of EGF/TGF-␣–induced EGFR tyrosine phosphorylation, anchorage-independent growth, and increased sensitivity of cells to cis-platinum. Recently, in a similar study, Matsuda et al.26 showed that infection of 4 pancreatic cell cancer cell lines (ASPC-1, COLO-357, PANC-1, and T3M4) with an adenoviral vector encoding a truncated EGFR markedly attenuated EGF and heparin-binding EGF-dependent cell growth, EGFR family tyrosine phosphorylation, and phosphorylation of mitogen-activated protein kinase, c-Jun NH2-
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terminal kinase, p38 mitogen-activated protein kinase, and activating transcription factor 2. Taken together, the data show that high levels of truncated EGFR, generated by molecular biology manipulations, modulate the EGFR signaling process and play a role in regulating cellular growth. ERRP, a naturally occurring molecule, also seems to regulate cellular growth by modulating EGFR function. Our observation that recombinant ERRP inhibits basal, as well as serum- and TGF-␣–induced, stimulation of EGFR phosphorylation in colon cancer cell lines suggests that ERRP inhibits growth by attenuating EGFR signaling processes. Our current data suggest that sequestration of the EGFR ligands by ERRP results in the formation of inactive heterodimers between ERRP and EGFR. This may partly be responsible for the attenuation of EGFR function. In view of this, we postulate that the loss of ERRP, as we have observed in colonic adenocarcinoma, may allow for increased activation of EGFR in colon cancer. Although the precise mechanism is unknown, one plausible explanation could be that the loss of ERRP with a concomitant reduction in sequestration of EGFR ligands will result in increased availability of free ligands for binding to and activation of EGFR. Therapeutic approaches to targeting the EGFR protein have usually taken the form of antibodies against the receptor or pharmacological inhibitors of the associated tyrosine kinase activity. A number of antibodies that block EGFR activation have been developed. The monoclonal antibody (MoAb) 225 against EGFR has been shown to induce growth inhibition of human cancer cell lines and xenograft tumor models.27–29 MoAb targeted to the c-erbB-2/HER-2 receptor, Herceptin (Genetech, Inc., San Francisco, CA), has been approved for breast cancer treatment in clinical trials. A phase II study using recombinant humanized anti c-erbB-2/HER-2 MoAb as a sensitizer for chemotherapy with cis-diaminedichloroplatinum in patients with tumors refractory to cis-diaminedichloroplatinum showed improved clinical outcome over those previously reported with either therapeutic alone, and without increased toxicity.30 Additionally, small molecule inhibitors of EGFR—OSI774 (CP 358,774) and Iressa (AstraZeneca, Wilmington, DE)— have progressed to clinical trials in EGFR-expressing cancers, with mixed results. Given the complexity at the molecular and cellular level, no single therapeutic agent is likely to emerge as the “magic bullet” in the treatment of cancer. Nonetheless, evidence suggests that targeting the EGF receptor is an effective therapeu-
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Figure 8. Changes in tumor weight after (A) intratumoral (7.5 g per tumor) and (B) subcutaneous (15 g per mouse) injections of purified recombinant ERRP over 8 and 5 days, respectively. (C) Representative photograph showing the growth of tumors in SCID mice xenografts of HCT-116 cells 12 days after discontinuation of intratumoral injections of ERRP (right) or vehicle (left), and survival curves of SCID mice after intratumoral (D) and subcutaneous (E) injections of purified ERRP or vehicle.
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Table 1. Antitumor Activity of ERRP in SCID Mice Xenografts Bearing Tumors of a Colon Cancer Cell Line, HCT-116 Log10 Kill Agent Control (vehicle) ERRP Control (vehicle) ERRP
Route
No. Animals
T/C (%)
T/C (days)
Net
Gross
Activity score
Tumor weight range (mg)
sc sc IT IT
5 5 5 5
100 22.3 100 8.5
0 14 0 16
0 2.10 0 1.30
0 1.35 0 2.40
(⫺⫺) (⫹⫹⫹) (⫺) (⫹⫹⫹)
288–1268 0–344 40–847 14–642
NOTE. Subcutaneous (sc) injections of 15 g of ERRP per mouse per day for 5 days were given on the back of mice. Intratumoral (IT) injection of 7.5 g per palpable tumor was given directly into each palpable tumor every other day for 8 days. T/C is an indicator of antitumor effectiveness, where a value of ⱕ42% is considered to possess a significant antitumor activity. If log10 kill values (net and gross) are added as a criterion, ERRP showed a clinically meaningful activity. Activity rating scores of 3⫹ and 4⫹ are considered “active” and “highly active,” respectively, in inducing a partial or complete regression of tumors.
tic strategy in cancers in which there is overexpression or activation of this protein. On the basis of results obtained thus far, we believe that ERRP may be a suitable candidate for inhibiting EGFR signaling pathways and, in turn, colorectal tumor growth. The novelty of ERRP is that it is an endogenous secretory protein,18 which could be administered systemically to inhibit EGFR signaling pathways leading to diminution of cellular growth. Our observation that sc injections of ERRP cause regression or arrest the growth of tumors in SCID mice xenografts of HCT-116 cells supports our contention that ERRP could be administered systemically and remains biologically active. We conclude that ERRP is a potential therapeutic agent for colorectal cancer.
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Received August 14, 2002; Accepted January 27, 2003. Address requests for reprints to: Adhip P. N. Majumdar, Ph.D., D.Sc., Research Service, 151 VA Medical Center, 4646 John R., Detroit, Michigan 48201. e-mail: [email protected]; fax: (313) 5761112. Supported by grants to Dr. Majumdar from the Department of Veterans Affairs and the National Institute on Aging (AG 14343).