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[Cancer Research 60, 4678-4681, September 1, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Inhibition of Epidermal Growth Factor Receptor Tyrosine Kinase Fails to Suppress Adenoma Formation in ApcMin Mice but Induces Duodenal Injury1

Steve R. Ritland, Sandra J. Gendler, Lawrence J. Burgart, David W. Fry, James M. Nelson, Alexander J. Bridges, Larry Andress and William E. Karnes, Jr.2

Tumor Biology Program, Mayo Clinic, Scottsdale, Arizona 85259 [S. R. R., S. J. G.]; Department of Cancer Research, Pfizer Global Research and Development, Ann Arbor, Michigan 48105 [D. W. F., J. M. N., A. J. B., L. A.]; and Department of Pathology [L. J. B.] and Gastrointestinal Research Unit [W. E. K.], Mayo Clinic, Rochester, Minnesota 55905


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
A highly selective, p.o. bioavailable irreversible inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, N-[4-(3-chloro-4-fluoro-phenylamino)-quinazolin-6-yl]-acrylamide (CFPQA), was evaluated for its ability to prevent intestinal adenoma formation in ApcMin mice. Ten-week continuous dietary exposure to CFPQA at doses sufficient to abolish intestinal EGFR tyrosine phosphorylation failed to affect intestinal tumor multiplicity or distribution but induced flat mucosal lesions in the duodenum characteristic of chronic injury. Intestinal trefoil factor, an intestinal peptide that mediates antiapoptotic effects through an EGFR-dependent mechanism, was notably absent in adenomas but was highly expressed in flat duodenal lesions. We conclude that chronic inhibition of EGFR tyrosine kinase by CFPQA does not prevent adenomas in ApcMin mice but may induce duodenal injury.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Because of its role in malignant transformation and growth, EGFR3 is an attractive target for treatment and prevention of cancer (1, 2, 3) . Preclinical studies show that inhibition of EGFR activation induces cytostasis and apoptosis in a variety of carcinoma cell lines in vitro and has antitumor activity against epidermoid, breast, ovarian, lung, and nasopharyngeal carcinoma in vivo (4, 5, 6) , and Phase I studies in humans with advanced malignancies are under way .

Selective inhibitors of EGFR tyrosine kinase induce caspase-dependent apoptosis in colon cancer and adenoma cell lines (7, 8, 9) , perhaps by preventing EGFR-mediated antiapoptotic signals through the phosphatidylinositol 3'-kinase-Akt pathway (10, 11, 12) . ITF/TFF3 also activates this antiapoptotic pathway and confers resistance of colon cancer cells to apoptosis induced by DNA-damaging agents, serum deprivation, or ceramide through an EGFR-dependent mechanism (13) . Of particular interest in colorectal cancer, EGFR and its family members interact with and phosphorylate ß-catenin after activation by EGFR ligands or ITF/TFF3 (14, 15, 16) . ß-Catenin accumulates early during colorectal carcinogenesis, following functional loss of the adenomatous polyposis coli (APC) gene product (17) , and is considered to mediate the transforming and antiapoptotic effects of Apc loss through its actions as a transcriptional activator of antiapoptotic genes when complexed with the Lef/Tcf family of transcription factors (18 , 19) . The impact of EGFR signaling on the oncogenic function of ß-catenin remains unknown. However, together, these observations suggest that EGFR may play a central role in antiapoptotic signaling during colorectal carcinogenesis generated by EGFR ligands, ITF/TTF3, and perhaps ß-catenin. To determine whether EGFR-mediated signals are critical for adenoma formation induced by Apc loss, we examined the effects of a 10-week continuous exposure to a highly selective, p.o. bioavailable inhibitor of EGFR tyrosine kinase activity, CFPQA (PD179651; Ref. 20 ), at three different oral doses on tumor multiplicity and distribution in the C57BL/6J-ApcMin mouse model of intestinal tumorigenesis.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Mice.
The ApcMin mouse is a genetic model of intestinal tumorigenesis driven by a germ-line mutation at codon 850 of the APC gene analogous to FAP in human (21) . Inbred C57BL/6J-ApcMin mice were produced by breeding from stock animals purchased from The Jackson Laboratory (Bar Harbor, ME) and screened for the Min/+ genotype using a previously described PCR assay (22) . Study mice were housed in an AAALAC-approved specific pathogen-free facility using forced air Thoren microisolator cages (three mice/cage), monitored for health every 2–3 days, and weighed weekly. Mice were weaned onto the AIN-93G powdered diet at 25–30 days of age and assigned systematically to drug treatment and control groups to normalize the distribution of males and females and to avoid clustering of individual mice from single litters. At the end of the study, an intestinal tissue sample was taken from each animal and subjected to Min/+ screening PCR to verify the Min/+ genotype as described previously (22) .

Diet and Drug Treatment.
The test drug, CFPQA, is a member of the quinazolin family of irreversible selective inhibitors of EGFR tyrosine kinase (1) , and its structure, biochemical properties, and selectivity are comparable to those of the well-characterized analogue PD168393 (4 , 20) . CFPQA has an IC50 of 0.75 nM against the isolated enzyme and an IC50 of 3.1 nM against epidermal growth factor-stimulated receptor phosphorylation in A431 epidermoid cancer cells (20) . Similar IC50 values have been determined against murine EGFR.4 CFPQA has minimal activity against other receptor tyrosine kinases. Like another closely related quinazolin, PD153035, CFPQA induces cytostasis and apoptosis of colorectal cancer cell lines at concentrations near its IC50 for inhibition of EGFR tyrosine kinase activity (7) . However, unlike PD153035, CFPQA is well suited for in vivo chemopreventative studies because it has greater aqueous solubility and is p.o. bioavailable.

Food and water were freely available at all times for the duration of the study. Study mice were fed either powdered AIN-93G rodent diet alone (Dyets, Inc., Bethlehem, PA) or AIN-93G plus drug prepared immediately before use by extensive manual mixing of milled drug with powdered diet. Fresh diet was provided every 2–3 days, and all unused AIN-93G diet was stored at 4°C for a period not exceeding 3 months from the time of purchase. Both piroxicam and CFPQA are stable in dry chow at 22°C for over a week (22) .4 All experimental groups were treated continuously from the time of weaning (25–30 days of age) until the time of sacrifice (99–111 days of age). Experimental groups included an AIN-93G vehicle control (n = 13 mice), a piroxicam positive control (200 ppm piroxicam; n = 6 mice; Ref. 23 ), and three CFPQA treatment groups (50, 200, and 500 ppm CFPQA; n = 10 mice/group). Based on the average food consumption per day and the average weight of mice in this study, approximate dosage conversions were as follows: (a) 500 ppm {cong} 100 mg/kg/day; (b) 200 ppm {cong} 40 mg/kg/day; and (c) 50 ppm {cong}10 mg/kg/day. Three additional supplementary studies were performed: (a) four ApcMin mice were treated with 500 ppm CFPQA for 80 days and then observed off treatment for 4 weeks before sacrifice to assess the reversibility of CFPQA-induced duodenal lesions (described in "Results"); (b) four ApcMin mice were treated with 500 ppm CFPQA for 80 days and then treated with 200 ppm piroxicam for 10 days before sacrifice to assess the effects of piroxicam on CFPQA-induced duodenal lesions; and (c) four wild-type C57BL/6J mice were treated with 500 ppm CFPQA for 80 days before sacrifice to assess whether duodenal lesions were dependent on the ApcMin genotype.

Tumor Enumeration.
Methods used for determining intestinal tumor multiplicity and distribution in ApcMin mice have been described in detail (23) . Briefly, the entire gastrointestinal tract was dissected, washed extensively with PBS, and fixed with methacarn (60% methanol, 30% chloroform, and 10% acetic acid), and tumors were evaluated by dark-field microscopy. The smallest tumors scored by this method were 0.2 mm in diameter, and the site of each tumor was recorded using a calibrated stage micrometer (Klarmann Rulings) to facilitate distribution analysis. For tumor distribution analysis, duodenum, jejunum, ileum, and colon were each divided into three equal segments.

Immunoprecipitation and Western Blot.
Frozen tissue was homogenized in 10 volumes of ice-cold lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM sodium EDTA, 1 mM sodium EGTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride], and supernatant representing 1 mg of total protein was rotated at 4°C for 2 h with 2 µg of EGFR antibody (clone 1005; Santa Cruz Biotechnology, Santa Cruz, CA). Fifty µl of protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) were added and rotated overnight at 4°C. Immune complexes were washed five times with precipitate wash buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, and 0.02% sodium azide], resuspended with 30 µl of loading buffer (2% SDS and 30 mM ß-mercaptoethanol), heated to 100°C for 5 min, and centrifuged. Thirty µl of the lysate were loaded and separated on a 4–20% polyacrylamide gel, and the proteins were transferred electrophoretically to nitrocellulose membrane. The membrane was washed once in TNA and blocked overnight in TNA containing 5% BSA and 1% ovalbumin. The membrane was blotted for 2 h with antiphosphotyrosine antibody (1 µg/ml) in blocking buffer (Upstate Biotechnology, Inc., Lake Placid, NY) and then washed twice in TNA, washed once in TNA containing 0.05% Tween-20 and 0.05% NP40, and washed twice in TNA. The membranes were then incubated for 2 h in blocking buffer containing 0.1 µCi/ml 125I-protein A and then washed again as described above. After the blots were dry, they were loaded into a film cassette and exposed to XAR X-ray film for 1–7 days. Band intensities were determined with a Molecular Dynamics laser densitometer.

Immunohistochemistry.
Expression of trefoil peptides was determined by immunohistochemistry using rabbit polyclonal antibodies kindly provided by Dr. Daniel Podolsky (Massachusetts General Hospital, Boston, MA) raised against rat ITF/TFF3 and human SP (SP/TFF2), both recognizing the murine forms. Sections (5 µm) of methacarn-fixed, paraffin-embedded tissues were dewaxed and rehydrated, treated with 2% hydrogen peroxide in 100% methanol for 10 min, and then rinsed in PBS (pH 7.4). Slides were then blocked with 50% FCS/PBS for 30–45 min, followed by a 1-h incubation with 100–200 µl of primary antibody diluted in 15% FCS/PBS at a 1:100 ratio for ITF and a 1:500 ratio for SP in a closed humidified chamber. Slides were then rinsed three times in PBS and incubated with 100–200 µl of antirabbit conjugated to horseradish peroxidase secondary antibody (DAKO, Carpinteria, CA) diluted 1:1000 in 15% FCS/PBS for 1 h in the chamber. After three to four additional rinses in PBS, 200 µl of 3,3'-diaminobenzidine solution prepared according to the instructions supplied with the 3,3'-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Burlingame, CA) were added for 7 min. Slides were then washed in water and stained with 200 µl of hematoxylin for 15–30 s, followed by several washes with PBS. Slides were then permount coverslipped and analyzed using a Zeiss Axiovert S100TV microscope and a Spot digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Images were batch-processed for consistent color balance and contrast using Adobe Photoshop version 4.0.

Statistical Analysis.
End points for efficacy included intestinal tumor multiplicity and distribution, body weight, serum drug levels, and EGFR phosphorylation level in intestinal mucosa. Statistical comparisons of tumor multiplicity between chemoprevention study groups were performed using Wilcoxon’s rank-sum or two-sample t tests. All Ps <0.05 were considered significant.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
CFPQA Steady-State Serum Levels and Effects on Intestinal EGFR Tyrosine Phosphorylation.
Chronic oral administration of CFPQA to ApcMin mice through a food/drug mixture produced serum levels of 11.4 ± 2.5 nM (for 50 ppm), 105 ± 26 (for 200 ppm), and 282 ± 69 nM (for 500 ppm). Serum exposure levels attained for 200 and 500 ppm CFPQA were sufficient to abolish detectable EGFR tyrosine phosphorylation in the intestinal mucosa of ApcMin mice by immunoprecipitation and Western blot (Fig. 1)Citation . Therefore, the 200 and 500 ppm doses of CFPQA produced exposure levels sufficient to modulate the pharmacodynamic marker (EGFR autophosphorylation) in the target tissue (intestinal mucosa).



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  		Fig. 1. Effects of 5-day treatment with 500 ppm CFPQA on EGFR tyrosine phosphorylation in jejunal mucosa of ApcMin mice. EGFR was immunoprecipitated from tissue lysates, separated by SDS-PAGE, and analyzed by Western analysis with antibodies specific for phosphotyrosine (top) or EGFR (bottom) as described in "Materials and Methods." Lane 1, animals fed the AIN-93G diet alone; Lane 2, animals treated with 500 ppm CFPQA; Lane 3, positive control lysate from EGFR-overexpressing A431 epidermoid cancer cells. Molecular weight markers are shown on the right. Bands representing the Mr 180,000 EGFR are indicated by the arrowheads.

 
CFPQA Does Not Affect Adenoma Multiplicity or Distribution in ApcMin Mice.
Despite adequate systemic exposure and confirmed modulation of EGFR tyrosine phosphorylation, none of the tested doses of CFPQA caused significant changes in intestinal tumor multiplicity or distribution in ApcMin mice (Fig. 2, A and B)Citation . An apparent increase in tumor multiplicity in the first portion of the duodenum (Fig. 2Citation B, D1) was judged to be an artifact after histological analysis revealed nonadenomatous mucosal lesions confined to the duodenum in the CFPQA-treated animals (described below). In contrast, piroxicam (positive control) dramatically reduced adenoma multiplicity in the duodenum, jejunum, and ileum (>95% reduction; P < 0.01 versus the AIN-93G control group). These findings suggest that adenoma multiplicity in the ApcMin mouse is not dependent on EGFR tyrosine kinase activity.



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  		Fig. 2. Effects of CFQPA on tumor multiplicity and distribution in ApcMin mice. Tumors were counted in three equal segments each of duodenum (D1, D2, and D3), jejunum (J1, J2, and J3), ileum (I1, I2, and I3), and colon (C1, C2, and C3) as described in "Materials and Methods." A shows the average total number of tumors in each treatment group (error bars, SD). B shows the average number of tumors in each segment in each treatment group (shown in the legend). The apparent increase in D1 tumors in CFPQA-treated animals is believed to be an artifact due to the duodenal lesions described in "Results" and shown in Figs. 3Citation 4Citation . Numbers of animals in each group are shown in parentheses in the legend. Treatment groups labeled CQ/50, CQ/200, and CQ/500 represent CFPQA at 50, 200, and 500 ppm, respectively.

 
CFPQA Induces Duodenal Lesions.
No significant differences in body weights or overall health between any of the treatment groups were noted. However, all ApcMin and wild-type mice treated with 200 or 500 ppm CFPQA for 10 weeks exhibited flat mucosal lesions restricted to the duodenum. Histologically, the duodenal lesions exhibited absent villi, expanded hyperchromatic proliferative zones with an increased frequency of mitotic figures and glandular crowding, and underlying Brunner’s gland hypertrophy without mucosal ulceration or erosion (Fig. 3)Citation . The duodenal changes induced by CFPQA were reversible on cessation of treatment (gross observation 4 weeks after the end of treatment) but were not affected by simultaneous treatment with piroxicam (200 ppm; data not shown). Taken together, these observations suggest that the CFPQA-induced duodenal lesions develop independently of Apc status and represent reactive rather than neoplastic changes.



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  		Fig. 3. Histology of duodenal lesions induced by CFQPA using H&E staining. The left panel shows duodenal mucosa (segment D1) from a representative ApcMin mouse after 10 weeks of treatment with AIN-93G diet alone. The right panel shows flat lesion (F) and underlying Brunner’s gland (B) in segment D1 from a representative ApcMin mouse after 10 weeks of treatment with 500 ppm CFPQA. x160.

 
ITF/TFF3 Expression in Adenomas and Duodenal Lesions.
We then performed immunohistochemical analysis of adenomas, normal intestinal mucosa, and the duodenal lesions of CFPQA-treated and control mice for protein expression of ITF/TFF3, as well as SP (SP/TFF2), a trefoil peptide expressed primarily in the neck cells of gastric glands (24) . Specific ITF/TFF3 (Fig. 4)Citation but not SP/TFF2 (data not shown) immunoreactivity was present in cells of the crypts, rare villus goblet cells, and the overlying mucous layer of normal intestinal mucosa of treated and untreated animals. Adenomas exhibited no detectable immunoreactivity for either peptide (Fig. 4)Citation . However, CFPQA-induced duodenal lesions showed strong ITF/TFF3 immunoreactivity of isolated cells throughout the entire thickness of the lesions, consistent with the histological impression that these lesions represent expanded regenerative crypts (Fig. 4)Citation . The absence of ITF/TFF3 immunoreactivity in adenomas suggests that this peptide does not play a role in ApcMin adenoma development or in the resistance of adenomas to chemoprevention by CFPQA.



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  		Fig. 4. ITF/TFF3 expression. Positive ITF/TFF3 immunostaining shows as a brown color. Blue staining is hematoxylin. x160. Images include duodenal mucosa (left) and adenomas (A; right) from representative ApcMin mice after 10 weeks of treatment with AIN-93G diet alone (top) or 500 ppm CFPQA (bottom).

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In the present study, chronic dietary exposure to 200 and 500 ppm of the selective EGFR tyrosine kinase inhibitor CFPQA achieved steady-state serum concentrations well above the in vitro IC50 for inhibition of EGFR tyrosine kinase activity and abolished detectable EGFR tyrosine phosphorylation in intestinal mucosa but had no effect on tumor multiplicity or distribution of adenomas in ApcMin mice. Similar concentrations of CFPQA or its closely related analogue, PD153035, are sufficient to induce cytostasis and apoptosis in EGFR-dependent colorectal cancer cell lines within 24 h (7) .4 Together, these results suggest that EGFR-mediated proliferative and survival signals are not critical for early stages of colorectal carcinogenesis. We speculate that other signaling systems may be activated that compensate for or protect against the antitumor effects of EGFR inhibition or that EGFR is not involved in adenoma formation induced by Apc loss. Neither ITF/TTF3 nor SP/TTF2 was expressed in adenomas, suggesting that these antiapoptotic gut peptides (13) are not involved in adenoma formation and do not confer resistance of adenomas to chemoprevention by CFPQA.

Although the ApcMin mouse is considered to be a genetically relevant model of human colorectal carcinogenesis triggered by APC loss, there are important phenotypic differences between ApcMin mice and humans with FAP. Compared with adenomas in FAP, ApcMin adenomas infrequently progress to frank carcinomas, they are distributed more densely in the small intestine than in the colon, and they lack the genetic complexity and instability of human colorectal neoplasms (25) . Furthermore, in contrast to reports that ITF/TFF3 is expressed in human adenomas and colorectal cancers (26) , we found no detectable ITF/TFF3 expression in the adenomas of ApcMin mice. Thus, the lack of antitumor activity for CFPQA in the ApcMin model cannot be generalized to human colorectal adenomas and carcinomas.

Although no gross toxicity was detected, doses of CFPQA sufficient to abolish mucosal EGFR tyrosine phosphorylation induced hyperproliferative duodenal lesions in both ApcMin and wild-type C57Bl6/J mice. Unlike adenomas, these lesions expressed ITF/TFF3, developed in wild-type mice as well as ApcMin mice, and were reversible in mice held from CFPQA treatment for 4 weeks before sacrifice, suggesting that they are reactive and not neoplastic. The lesions showed characteristics of a regenerative response to injury with absent villi, glandular crowding, and expanded proliferative zone. Interestingly, piroxicam, which depletes mucosal cytoprotective prostaglandins through inhibition of cyclooxygenases I and II, did not exacerbate these lesions. Future studies using any of the several classes of EGFR tyrosine kinase inhibitors that are chemically distinct from the quinazolones will help determine whether these lesions are a response to a secondary (off-target) activity or to EGFR inhibition per se.

In summary, we have shown that the EGFR-selective tyrosine kinase inhibitor CFPQA does not inhibit intestinal tumor formation in the ApcMin mouse despite inhibition of the target enzyme EGFR in intestinal mucosa. Although no gross toxicities were detected, doses of CFPQA sufficient to abolish mucosal EGFR tyrosine phosphorylation induced reversible duodenal lesions suggestive of chronic injury in both ApcMin and wild-type C57BL/6J mice. These findings may have important implications regarding the role of EGFR-mediated signals in intestinal carcinogenesis and for the future development of EGFR-targeted therapies for chemoprevention of adenomas.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the valuable contributions of Dr. Daniel Podolsky (Massachusetts General Hospital, Boston, MA), Anita Jennings (Mayo Clinic, Scottsdale, Histology Core), Dr. Thomas Lidner (Mayo Clinic, Scottsdale, Pathology), and members of the Mayo Clinic, Scottsdale, Transgenic Animal Core.


   FOOTNOTES
 
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.

1 Supported by NIH Grants R29 CA7194 (to W. E. K.), RO1 DK 56373-01 (to W. E. K.), and RO1 CA64389 (to S. J. G.) and by the Mayo Foundation and the Mayo Comprehensive Cancer Center. Back

2 To whom requests for reprints should be addressed, at 2-435 Alfred Gastrointestinal Research Unit, Saint Mary’s Hospital, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Phone: (507) 284-6635; E-mail: karnes.william{at}mayo.edu Back

3 The abbreviations used are: EGFR, epidermal growth factor receptor; CFPQA, N-[4-(3-chloro-4-fluoro-phenylamino)-quinazolin-6-yl]-acrylamide; ITF, intestinal trefoil factor; TFF, ; SP, spasmolytic peptide; FAP, familial adenomatous polyposis; TNA, 10 mM Tris (pH 7.2), 150 mM NaCl, and 0.01% sodium azide. Back

4 William E. Karnes, Jr., unpublished observations. Back

Received 4/27/00. Accepted 7/19/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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K. J. Hare, B. Hartmann, H. Kissow, J. J. Holst, and S. S. Poulsen
The Intestinotrophic Peptide, GLP-2, Counteracts Intestinal Atrophy in Mice Induced by the Epidermal Growth Factor Receptor Inhibitor, Gefitinib
Clin. Cancer Res., September 1, 2007; 13(17): 5170 - 5175.
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Q.-W. Fan, K. M. Specht, C. Zhang, D. D. Goldenberg, K. M. Shokat, and W. A. Weiss
Combinatorial Efficacy Achieved Through Two-Point Blockade within a Signaling Pathway--A Chemical Genetic Approach
Cancer Res., December 15, 2003; 63(24): 8930 - 8938.
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R. B. Roberts, L. Min, M. K. Washington, S. J. Olsen, S. H. Settle, R. J. Coffey, and D. W. Threadgill
Importance of epidermal growth factor receptor signaling in establishment of adenomas and maintenance of carcinomas during intestinal tumorigenesis
PNAS, January 24, 2002; (2002) 32678499.
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 Proc. Natl. Acad. Sci. USAHome page
R. B. Roberts, L. Min, M. K. Washington, S. J. Olsen, S. H. Settle, R. J. Coffey, and D. W. Threadgill
Importance of epidermal growth factor receptor signaling in establishment of adenomas and maintenance of carcinomas during intestinal tumorigenesis
PNAS, February 5, 2002; 99(3): 1521 - 1526.
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