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Experimental Therapeutics, Preclinical Pharmacology |
1 Departments of Radiation Oncology and 2 Pathology, University of Michigan, and 3 Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: We selected three human colon carcinoma cell-lines (LoVo, Caco-2, which express activated epidermal growth factor receptor and ErbB-2 family members, and SW620, which does not), and analyzed the effects of CI-1033 both in vitro and in vivo. For in vivo studies LoVo and Caco-2 cells were implanted s.c. in the flank of nude mice. After the tumor reached 
100 mm3, treatment was initiated with 20 mg/kg of CI-1033 (orally once daily x 5 for 3 successive weeks), radiation treatment (a total of 30 Gy given in 2 Gy once daily x 5 for 3 successive weeks), or a combination of both CI-1033 and radiation treatment.
Results: We found that exposure of LoVo and Caco-2, but not SW620 cells, to CI-1033 in the range of 1–3 µM could inhibit constitutive signaling by tyrosine kinases, arrest cell growth, inhibit cells in G1, stimulate expression of p53, and induce apoptosis. The inhibition of cell growth by CI-1033 seemed to produce only minimal radiosensitization in LoVo and Caco-2 cells. In contrast, the combination of CI-1033 and radiation produced significant (P < 0.0005 and P = 0.0002, respectively) and prolonged suppression of tumor growth in both the tumor types when compared with either treatment alone.
Conclusions: These findings suggest that CI-1033 can increase the effectiveness of radiation therapy. The extent of suppression of tyrosine kinase activity by CI-1033, rather than the amount of activity in untreated cells, seemed to be more closely associated with the efficacy of combination treatment.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because of the apparent role of aberrant expression of receptor tyrosine kinases in the uncontrolled growth that occurs in many cancers, there have been intensive efforts to develop agents that inhibit EGFR autophosphorylation and concomitantly epidermal growth factor-stimulated signal transduction (11, 12, 13, 14) . One strategy for inhibition in combination with radiation therapy has involved the use of monoclonal antibodies, which bind to the extracellular domain of the EGFR and of ErbB-2 (15 , 16) . A second approach has been to target the tyrosine kinase itself. The 4-anilinoquinazolines, which bind to the ATP binding domain of the ErbB family of tyrosine kinases can inhibit kinase activity in the nanomolar range (12) . One example of this class of compounds is CI-1033 (14) , a pan ErbB tyrosine kinase inhibitor that irreversibly binds to the receptor tyrosine kinase (17, 18, 19, 20) . Thus, it differs in two potentially important ways from the compounds in this category that have been most intensively investigated in the clinic (ZD1839 and OSI-774), which reversibly bind only the EGFR tyrosine kinase with high affinity (21, 22, 23) .
These observations led us to evaluate the combination of CI-1033 and radiation in cultured human colorectal cancer cells and in tumors. Thousands of patients receive radiation therapy each year for either primary locally advanced rectal cancer, recurrent rectal cancer, or metastatic colorectal cancer, and the potential role of growth factor receptor inhibition in combination therapy has been reviewed (24) . For example in one recent Phase II clinical trail, treatment with a combination of Cetuximab (C225) and Irinotecan produced a 67% response rate in colorectal carcinoma patients (25) . We have shown, using cultured breast cancer and normal breast epithelial cells, that radiation and CI-1033 can interact synergistically both through cytostatic and radiosensitizing effects (26) . In the present report, we have carried out additional studies to determine the mechanism of interaction of CI-1033 and radiation both in cultured colorectal cancer cells and in animals bearing these tumors as xenografts.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Irradiation.
Human colon cancer cell lines Caco-2, LoVo, and SW620, and breast cancer cell lines MCF-7 and MDA-MB-468 (used in control experiments) were obtained from the American Type Culture Collection (Manassas, VA). The LoVo cell line was maintained in Ham’s F-12 medium supplemented with 10% fetal bovine serum. The remaining cell lines were grown in RPMI 1640 supplemented with 10% bovine serum. In this study cells were neither serum starved nor stimulated by epidermal growth factor. Cultures were tested routinely for Mycoplasma infection. For all of the experiments log phase cells were produced by releasing cells from the flasks with PBS containing 0.01% trypsin and 0.20 mM EDTA and plating them into 100-mm culture dishes 2 days before drug or radiation treatments. Irradiations were performed at a dose rate of 1–2 Gy/min at room temperature using an AECL Theratron 80 (60Co) calibrated with an ionization chamber directly traceable to the National Institute of Standards and Technology standard.
Cell Growth and Apoptosis.
Cells were treated with 0, 1, or 3 µM CI-1033 for up to 96 h, and floating (apoptotic) and adherent cells were counted every 24 h using a Coulter Counter (Beckman Coulter, Miami, FL) after the beginning of the treatment. The apoptotic fraction was calculated by taking the ratio of floating cells:total cells (floating plus adherent cells). The values from the control groups were subtracted from the treated group to obtain the final values. To confirm that the floating cells were apoptotic, cells were washed in PBS, fixed in 70% ethanol, stained with 1% propidium iodide, and viewed for nuclear fragmentation using a fluorescent microscope. Greater than 90% of the floating cells and <5% of the adherent cells were apoptotic, as has been confirmed by gel electrophoresis and reported previously (27)
. Approximately 200 cells were counted from random fields.
Clonogenic Cell Survival Assay.
Clonogenic assays were performed using standard techniques as described previously (28)
. Radiation survival data from drug-treated cells were corrected for plating efficiency using an unirradiated plate treated with CI-1033 under the same conditions. Cell survival curves were fitted using the linear-quadratic equation, and the mean inactivation dose was calculated according to the method of Fertil et al. (29)
. The cell survival enhancement ratio was calculated as the ratio of the mean inactivation dose under control conditions divided by the mean inactivation dose after drug exposure. A value significantly greater than one indicates radiosensitization.
Flow Cytometry.
Cells were exposed to bromodeoxyuridine (30 µM) for 15 min just before the termination of the experiment. They were then trypsinized, washed, and resuspended in PBS, fixed by drop-wise addition of 2.5 volumes of ice-cold 70% ethanol, and stored at 4°C until the day of analysis. For two-parameter flow cytometry, cells were processed by treatment with detergent (Triton X-100 in 0.1 N HCl) followed by boiling to permeabilize the cells. They were then incubated with antibromodeoxyuridine antibody (BD PharMingen, San Diego, CA) followed by FITC-goat-antimouse IgG (Sigma Chemical Co.). Cells were then suspended in propidium iodide solution (180 µg/ml) and analyzed in the Flow Cytometry Core of the University of Michigan Cancer Center at Ann Arbor.
Immunoblotting.
Cells were scraped into PBS containing sodium orthovanadate and protease inhibitor mixture (Roche Diagnostic Co., Indianapolis, IN). Detergent-soluble proteins were extracted by incubating cells at 4°C for 15 min in extraction buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 0.1% SDS, 1 mM EDTA, 100 mM NaF, 1 mM Na3Vo4, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin]. After particulate material was removed by centrifugation at 13,000 rpm for 15 min at 4°C, protein concentrations in the supernatant were estimated by Bradford reagent (Bio-Rad Laboratories, Hercules, CA). Aliquots containing 40 µg of protein were diluted with an equal volume of loading buffer [63 mM Tris-HCl, 2% (w/v) SDS, 10% (v/v) glycerol, and 0.005% (w/v) bromphenol blue], heated to 95°C for 5 min, and applied to SDS-polyacrylamide gels. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membrane. Membranes were incubated for 1 h at room temperature in blocking buffer consisting of 3% BSA and 1% normal goat serum in Tris-buffered saline [137 mM NaCl, 20 mM Tris-HCl (pH 7.6), and 0.1% (v/v) Tween 20]. They were subsequently incubated overnight at 4°C with 1 µg/ml primary antibody in blocking buffer, washed, and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (Southern Biotechnology Inc., Birmingham, AL). After three additional washes in Tris-buffered saline, bound antibody was detected by enhanced chemiluminescence plus reagent (Amersham Biosciences, Piscataway, NJ).
Immunoprecipitation.
Cells were scraped as described for immunoblotting. Five x 106 cells were suspended in radioimmunoprecipitation assay buffer [Tris-HCl 50 mM (pH 7.4), NP40 1%, Na-deoxycholate 0.25%, NaCl 150 mM, EDTA 1 mM, phenylmethylsulfonyl fluoride 1 mM, aprotinin, leupeptin, pepstatin 1 µg/ml each, Na3VO4 1 mM, and NaF 1 mM], incubated on ice for 15 min, sonicated (two to four bursts), and incubated again on ice for 15 min. Supernatant was collected by spinning at 13,000 rpm at 4°C. Cell-lysates were cleared by addition of 100 µg normal mouse/rabbit (depending on the host of primary antibody) IgG/5 x 106 cells and addition of protein-G Sepharose beads. Primary antibody (10 µg) was used to immunoprecipitate EGFR or ErbB-2, using protein-G Sepharose. Beads were washed five times in ice-cold radioimmunoprecipitation assay buffer, boiled in 1x SDS loading buffer, and subjected to immunoblot analysis.
Tissue Microarray Construction.
Tissue microarrays were constructed at the University of Michigan Comprehensive Cancer Center Tissue Core by standard methods. For this purpose Caco-2 tumor xenografts and mouse skin tissue were used. Tissue cores from the circled areas of interest were targeted for transfer to the recipient array blocks. The 0.6-mm-diameter tissue microarray cores were spaced at 0.8 mm from core center to core center.
Immunohistochemistry.
After slides were deparaffinized and rehydrated, antigen retrieval was performed using Proteinase K (DAKO Cytomation, Carpinteria CA), before antibody detection. Slides were incubated with primary antibody overnight at 4°C (from 1:75 to 1:200). The Envision+ Peroxidase detection kit (DAKO Cytomation) was used to visualize the signal. Slides were counterstained with Hematoxylin, then dehydrated to Xylene before the coverslip was placed.
RNA Isolation and Reverse Transcription-PCR.
Cells were harvested in TRIzol (Invitrogen Co., Carlsbad, CA) and passed through a 28-gauge needle fitted syringe to shear the DNA. RNA was extracted and purified by phenol-chloroform, precipitated with isopropanol, dissolved in RNase free water, and stored at -20°C until used. RNA (1 µg) was reverse transcribed to cDNA under standard conditions. The cDNA product was then used for amplification of EGFR, ErbB-2, ErbB-3, and ErbB-4 genes by PCR reactions. The primers used in this study were: EGFR: 5'-ACT AGC CAG GAA GTA CTT CC-3' and 5' GGC CTT CTT GGA TCT TTA GT 3' (product size: 398 bp); ErbB-2: 5'-CGG GAG ATC CCT GAC CTG CTG GAA 3' and 5' CTG CTG GGG TAC CAG ATA CTC CTC-3' (product size: 300 bp); ErbB-3: 5'-GAG GCT GAG CTC CAG GAG AA-3' and 5'-CTG GGA CCT GGG AGA GAG AG-3' (product size: 814 bp); and ErbB-4: 5'-CCT CTC CTT CCT GCG GTC TGT-3' and 5'-AAG TCT GGC AAT GAT TTT CTG TGG G-3' (product size: 404 bp).
After PCR amplification, the products were separated on a 2% (w/v) agarose gel in Tris-acetate-EDTA buffer, stained with 0.5 µg/ml ethidium bromide, and photographed under UV light. Glyceraldehyde-3-phospate dehydrogenase transcripts were used as a control for the RNA used to amplify the genes by reverse transcription-PCR.
Tumor Growth Studies.
LoVo or Caco-2 cells (5 x 106) were transplanted in the flank of NU/NU CD-1 nude mice. When tumors had reached an average volume of 100 mm3, mice were randomized into four groups (6 mice/group), and the treatment was initiated. CI-1033 was dissolved in water and was given orally in the morning. The tumors in the combination or radiotherapy only group were irradiated 4 h later based on the observation that receptor tyrosine kinase activity was reduced significantly at that time (see Fig. 2
). Body weight and tumor measurements were taken twice a week. Tumor volume (TV) was calculated according to the equation for a prolate spheroid: TV = (
/6; LxW2), where L and W are the longer and shorter dimensions of the tumor, respectively. Data are expressed as the ratio of tumor volume at various times after treatment compared with the first day of treatment (day 0). Measurements were made until day 15 (Caco-2), day 60, or until the tumor volume increased by approximately a factor of eight (LoVo), at which point the animals were sacrificed to avoid potential discomfort. On day 15 Caco-2 tumor-bearing animals were sacrificed, the tumor and skin tissue were harvested and frozen in Tissue-Tek OCT compound (Electron Microscopy Sciences, Washington, PA), and stored below -70°C until immunostaining was performed. Animals were handled according to the established procedures of the University of Michigan Laboratory Animals Maintenance Manual.
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RESULTS |
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. This confirmed the presence of phospho-EGFR and ErbB-2 in LoVo and Caco2 cells, and the absence of phosphorylated EGFR and ErbB-2 in SW620 cells. We additionally confirmed the presence of constitutively phosphorylated EGFR and ErbB-2 by performing immunoprecipitation with EGFR and ErbB-2 antibodies followed by immunoblotting with an antiphosphotyrosine antibody (data not shown). Finally, the absence of EGFR in SW620 cells was confirmed at the message level by reverse transcription-PCR (Fig. 1B)
. We then assessed the dependence on concentration and duration of exposure of CI-1033 on the phosphorylated epidermal growth factor and ErbB-2 receptors in LoVo, Caco-2, and SW620 cells. These experiments were performed with 1–3 µM CI-1033, as these levels are produced in plasma after a 20 mg/kg oral dose.4
Cells were treated with 1 or 3 µM CI-1033 and subjected to phosphotyrosine immunoblotting from 30 min to 24 h after the beginning of drug exposure (Fig. 2)
. Both 1 and 3 µM CI-1033 inhibited phosphorylation for prolonged time periods. Substantial inhibition of phosphorylation of receptor tyrosine kinases was observed by 30 min after exposure to 3 µM CI-1033 and continued for up to 24 h in the presence of the drug. To provide a positive control as well as a context for the level of expression, MDA-MB-468 cells, which have an EGFR amplification, were also assessed (30)
. Note that the extent of inhibition of phosphorylation by the drug was greatest in MDA-MB-468 and Caco-2 cells, whereas less inhibition was observed in LoVo cells (Fig. 2)
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and was minimally affected by concentrations up to 10 µM for up to 4 days (data not shown). These latter findings suggest that nonspecific effects of CI-1033 are minimal below 10 µM.
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. This CI-1033-induced G1 arrest was not observed in SW620 cells. Similar results were obtained at 48 and 72 h after drug exposure (data not shown). To begin to determine the mechanism of G1 arrest, we assessed p53 levels after drug treatment. We found that CI-1033 produced an elevation in p53 protein in LoVo and Caco-2 that was detectable as early as 4 h after initiation of treatment and was maintained for at least 48 h, whereas SW620 cells (p53 mutant) were not affected (Fig. 5A)
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. Thus, the concentration of CI-1033 required to produce cell death in Caco-2 and LoVo cells was greater than that required to produce cell cycle arrest and was associated with the disappearance of phosphorylated receptor (see Fig. 2
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Effect of CI-1033 on Radiation Sensitivity in Vitro and in Vivo.
We hypothesized that the cell cycle redistribution produced by CI-1033 might also be associated with an increase in cellular radiation sensitivity. To test this hypothesis, Caco-2, LoVo, and SW620 cells were treated with CI-1033 for 24 h (3 µM) and then irradiated (2–8 Gy). Twenty-four h after radiation treatment, cells were assessed for clonogenic survival. We found that CI-1033 produced a modest increase in radiosensitivity of Caco-2 (enhancement ratio = 1.4 ± 0.1) and LoVo cells (1.2 ± 0.04), whereas SW620 cells were not sensitized (Fig. 6)
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. We then assessed in detail the combination of CI-1033 (20 mg/kg/day, which results in a plasma level of approximately 2.5–5 µM at 24 h after oral administration4
) and a clinically applicable course of radiation (15 daily 2 Gy fractions). We observed prolonged growth delay (4.2-fold at day 30 compared with controls) only when CI-1033 and radiation were combined (Fig. 8)
. This increased efficacy was associated with moderate but acceptable toxicity (maximum weight loss during treatment was 12.6 ± 2.2% on day 21; weight recovered completely in all animals by day 35). No increase in skin toxicity was observed (data not shown). Histological evaluation of treated LoVo xenografts revealed islands of viable tumor within specimens resected from tumors receiving radiation or CI-1033 alone, whereas the great majority of the tumor was necrotic after combined treatment (Fig. 9)
. These findings demonstrated that the effect of combining CI-1033 with radiation in vivo appeared to be substantially greater than we would have anticipated based purely on the modest radiosensitizing effects observed in vitro.
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). The initial tumor growth data were similar to those from the LoVo experiments (Fig. 10)
. These animals were sacrificed, the tumors were harvested for tissue microarray construction, and immunostaining was performed. In addition to markers involved in the cellular response to radiation (phospho-EGFR, phospho-ERK 1/2, and p53), we assessed the effect of treatment on the endothelial cells using CD31 antibody. Immunostaining indicated that in tumor from the combination treatment group, phosphorylated EGFR and pERK 1/2 and endothelial cell marker CD31 levels were reduced relative to either CI-1033 or radiation alone. On the other hand, p53 levels appeared to be elevated after combination therapy (Fig. 11)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our finding, that modest in vitro radiosensitization could translate into substantial synergy in vivo, is consistent with those reported from the combination of radiation and cetuximab (15 , 31 , 32) , as well as from investigations of other small molecular tyrosine kinase inhibitors used with chemotherapy (33, 34, 35) . There are several potential explanations for the apparent difference between in vitro and in vivo effects. The simplest explanation, which we proposed earlier (26) , is that potent in vivo synergy might be achieved by the combination of slight radiosensitization, slight cytotoxicity, and inhibition of tumor cell growth resulting from G1 arrest (which is not a radiation-sensitive phase of the cell cycle). Even modest sensitization of each fraction would produce substantial enhancement by the end of a course of treatment. For example, an enhancement ratio of 1.2/fraction for each of 15 fractions would produce a total enhancement ratio of 15.4 (1.215). An additional factor could include the blockage of EGFR stimulation. Schmidt-Ullrich et al. (36) have demonstrated that radiation can increase EGFR tyrosine kinase activity, and it has been hypothesized that this elevation of activity can produce clinical resistance through stimulation of survival pathways and/or tumor growth during treatment. It is possible that additional mechanisms operate, because other studies indicate that tyrosine kinase inhibitors may also block the secretion of angiogenic factors such as vascular endothelial growth factor and interleukin-8.
A better understanding of in vivo mechanisms is crucial in determining the optimal application of this category of drugs. The role of p53 in the response to CI-1033 is not clear. The cell lines that we chose with moderate to high activation of EGFR and ErbB-2 receptor tyrosine kinase contained wild-type p53. Although it is possible that p53 plays a crucial role, G1 arrest and apoptosis have been produced by both cetuximab and small molecule tyrosine kinase inhibitors in cells that do not express p53 (16 , 37) . Likewise, the combination of cetuximab and radiation can increase bax and decrease bcl-2, leading to apoptosis (37) . Preliminary experiments demonstrate that p53 mutant MDA-MB-468 cells undergo growth arrest when exposed to low concentrations of CI-1033,5 suggesting that wild-type p53 function is not crucial to CI-1033-mediated growth arrest under all circumstances. It will be important to elucidate the pathways responsible for G1 arrest and apoptosis, and their relative dependence on p53 in future studies.
The critical role of ERK 1/2 in the EGFR signal transduction pathway (38)
suggests that it could play a role in the mechanism of radiosensitization by CI-1033. Thus, we examined the response of phospho-ERK 1/2 to the combination of CI-1033 and radiation both in vitro and in vivo. In vitro, 3 µM CI-1033 inhibited both proliferation and ERK 1/2 phosphorylation (data not shown), and in vivo (Fig. 11)
a dramatic decrease in phospho-ERK 1/2 was observed with the combination of drug and radiation. These effects were seen at the drug concentrations that inhibit EGFR phosphorylation (see Ref. 4
and Fig. 2
) and that can be achieved in patients. These data suggest that phospho-ERK 1/2 response may be a good candidate for a marker for determining which tumors will respond to therapy with this category of drugs.
It is increasingly becoming clear that blockade of EGFR activation by cetuximab and by small molecule tyrosine kinase inhibitors results in a significant decrease in tumor-cell production of angiogenic growth factors such as basic fibroblast growth factor, vascular endothelial growth factor, and interleukin-8 (39) . The decrease in the angiogenic growth factors, in turn, correlates with a significant decrease in microvessel density and an increase in apoptosis of endothelial cells in human tumor xenografts. Our finding that the combination of CI-1033 and radiation reduced the number of CD31 staining vessels is consistent with other studies and may explain, in part, the observed tumor growth delay when there is minimal in vitro radiosensitization (40) .
In summary, we have found that Caco-2 and LoVo cells, which demonstrated high levels of EGFR and ErbB-2 tyrosine kinase activity, were affected by CI-1033, whereas SW620 cells, which had low levels of receptor tyrosine kinase activity, were unaffected by relatively high concentrations for long time periods. Furthermore, at least in the case of LoVo and Caco-2 cells, the extent of suppression of tyrosine kinase activity by CI-1033, rather than the amount of activity in untreated cells, seemed to be more closely associated with growth inhibition. Thus, the simple finding of receptor overexpression may not guarantee a particular response. Moreover, blocking an overexpressed receptor tyrosine kinase may not always inhibit cell growth, as a hallmark of cancer is aberrant cross-talk between pathways. For instance, initial results using cetuximab combined with chemotherapy for colon cancer (41) have not shown a relationship between receptor content (based on immunohistochemical staining) and response. Although some might interpret this result as indicating that these agents can be given to all patients with hope of success, the overall low clinical response rates (in the range of 20%) would suggest that a more useful direction would be to determine the appropriate target so as to better identify patients who could benefit from treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Theodore S. Lawrence, Department of Radiation Oncology, University of Michigan, 1500 East Medical Center Drive, UH-B2C490, Ann Arbor, MI 48109-0010. Phone: (734) 647-9955; Fax: (734) 763-7371; E-mail: tsl{at}umich.edu
4 Dr. Cho-Ming Loi, Pfizer Global Research and Development (Ann Arbor, MI), personal communication. 
5 M. K. Nyati, unpublished observations.
Received 7/ 1/03; revised 10/ 7/03; accepted 10/16/03.
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), 1 µm (
), or 3 µM (•) CI-1033 once, and adherent cells were counted every day for 4 days by Coulter Counter. Data are presented as the mean for three experiments; bars, ±SE.
; n = 8). The average volumes relative to those at day 0 are shown; bars, ±SE. Horizontal bar, the treatment period.
