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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Regression of Early and Intermediate Stages of Colon Cancer by Targeting Multiple Members of the EGFR Family with EGFR-Related Protein

Eva M. Schmelz, Hu Xu, Radha Sengupta, Jianhua Du, Sanjeev Banerjee, Fazlul H. Sarkar, Arun K. Rishi and Adhip P.N. Majumdar
DOI: 10.1158/0008-5472.CAN-07-0536 Published June 2007

Abstract

A role of the epidermal growth factor receptor (EGFR) family has been suggested in colon cancer etiology, progression, and/or severity. Our recently identified pan-erbB inhibitor EGFR-related protein (ERRP) targets EGFRs by attenuating their activation and subsequent signaling leading to cellular growth inhibition. In the present study, we evaluated the therapeutic effectiveness of ERRP on early and intermediate stages of colon cancer by examining regression of chemically induced aberrant crypt foci (ACF) in the colon of CF1 mice and intestinal adenomas in APCMin+/− (Min) mice. After formation of ACF or adenomas, the mice were injected (i.p.) with ERRP (50 μg/mouse) for 10 consecutive days. This treatment significantly reduced the number of ACF from 25.0 ± 3.0 (controls) to 14.9 ± 1.6 (ERRP-treated; P = 0.011) and also reduced their size (P < 0.01). In Min mice, ERRP caused the regression of adenomas throughout the small intestine (P < 0.05) and reduced their size (P < 0.001). This could partly be attributed to inhibition of proliferation and stimulation of apoptosis in the intestinal mucosa and was associated with decreased activation of several EGFR family members, suppression of downstream effector nuclear factor κB and down-regulation of cyclooxygenase-2. ERRP-induced attenuation of EGFR activation could be due to increased sequestration of the ligand(s) by ERRP, rendering them unavailable for binding to and activation of the receptor. In conclusion, our data show that ERRP is effective in regressing both early and intermediate intestinal lesions and could be an effective therapeutic agent for colon cancer. [Cancer Res 2007;67(11):5389–96]

  • Colon cancer
  • EGFR
  • ErbB-2
  • ErbB-3
  • proliferation
  • apoptosis
  • NF-κB
  • COX-2

Introduction

Recent advances in molecular and cellular biology have aided in the understanding of the molecular pathogenesis of cancer. A number of genes have now been identified as abnormally expressed in cancer and, thus, serve as targets for therapeutic intervention. The epidermal growth factor (EGF) receptor (EGFR) family, which consist of EGFR itself and three related receptors called ErbB-2/HER-2, ErbB-3/HER-3, and ErbB-4/HER-4 (collectively called EGFRs), is one of these genes. Each receptor, with the exception of ErbB-2, is activated by one or more EGF-related peptides, such as EGF, transforming growth factor (TGF)-α, heparin-binding (HB)-EGF, and amphiregulin. Binding of the ligand induces homodimerization or heterodimerization of the receptors and phosphorylation at specific tyrosine residues at the intracellular domain. These phosphorylation sites provide docking sites for adapter proteins, which, in turn, activate downstream signaling pathways. Except for ErbB-3, all EGFR family members possess tyrosine kinase activity and are not only involved in the regulation of cell proliferation and survival but also in differentiation, migration, and angiogenesis ( 1).

Aberrant expression or activity of EGFR and/or its family member(s) has been associated with the development and progression of many malignancies ( 2). In particular, the overexpression of ErbB-2 in breast, ovarian, and colorectal cancers is associated with an extremely poor clinical prognosis ( 3– 6) and early relapses ( 7, 8). In head and neck, bladder, and lung cancers, the overexpression of EGFR receptors and their ligands (EGF/TGF-α) are predictive of poor outcome and disease recurrence ( 9, 10). Overexpression of EGFRs has also been reported in colon cancer ( 11).

It is becoming increasingly apparent that interference with EGFR activation and/or its signal transduction pathways represent a promising strategy for the development of novel and selective anticancer therapies ( 12). Indeed, several drugs, such as cetuximab, trastuzumab, gefitinib, and erlotinib, that target either EGFR or HER-2 have been developed ( 13). However, their limited success may be the result of the overexpression of more than one member of the EGFR family in solid tumors. Furthermore, coexpression of multiple EGFR family members leads to an enhanced transforming potential and worsened prognosis ( 3, 6). Therefore, identification of inhibitors targeting multiple members of the EGFR family may provide a therapeutic benefit to a broad range of patient population.

EGFR-related protein (ERRP), a 53 to 55 kDa protein which we isolated and characterized as a pan-erbB inhibitor, targets multiple members of the EGFR family ( 14– 17). Our studies suggest that ERRP could be a potential therapeutic agent for colorectal cancer ( 18, 19) and perhaps other epithelial cancers as well ( 20– 22). We have reported that ERRP inhibits growth and attenuates basal and ligand-induced activation of EGFR and/or its family members in several colon and other epithelial cancer cell lines that express varying levels of EGFRs with no apparent change in growth of fibroblast cell lines ( 15). Furthermore, comparison with cetuximab (MoAb to EGFR) or trastuzumab (MoAb to HER-2) has revealed that whereas cetuximab or trastuzumab is effective in inhibiting growth of epithelial cancer cells that express high levels of EGFR or HER-2, respectively, ERRP is effective against cancer cells that express varying levels of EGFR and/or its family members ( 15, 20– 22).

ERRP has been shown to prevent the growth of SCID mice xenografts of colon and pancreatic cancer cells without any signs of toxicity ( 18, 19, 22). However, it remains to be determined whether ERRP will be effective in the treatment of existing colonic lesions in established immunocompetent rodent models. In the present study, we investigated the therapeutic effectiveness of ERRP in early and intermediate stages of colon cancer by examining the disappearance of confirmed aberrant crypt foci (ACF) in a carcinogen-induced colon cancer model and existing adenomas in a genetically defined mouse model. The treatment of rodents with carcinogens induces the same multistep progression described in humans and is such a widely accepted model to test chemopreventive and chemotherapeutic potency of various agents. ACF, which were initially described by Bird ( 23), have also been detected in humans with colon cancer ( 24, 25) and are thought to be precursors of adenomas and carcinomas ( 4) and, thus, represent the earliest stages of colon cancer. The multiple intestinal neoplasia (APCMin+/−, Min) mice spontaneously develop tumors throughout the intestinal tract due to mutations in the adenomatous polyposis coli gene, which are also found in patients with familial adenomatous polyposis and in 40% to 80% of patients with sporadic colon cancer ( 26, 27). Min mice provide an excellent model for intermediate stages of colon cancer. Both models show aberrant expression of EGFRs. In carcinogen-treated animals, ACF and colon tumors frequently overexpress not only EGFR ( 28– 31) but also ErbB-2 and ErbB-3 ( 32). Overexpression of ErbB-4 is not as frequent and seems to be more relevant in later stages of colon cancer ( 5). Whereas overexpression of EGFR has been reported in Min tumors and in colonocytes of Min mice ( 33), no information is available on other EGFR family members. Thus, these rodent models provide ideal molecular targets for therapeutic approaches and were used in the present studies to investigate the regression of early and intermediate stages of colon cancer.

Materials and Methods

Animals. Female CF1 mice (5 weeks) were purchased from Charles Rivers. Min mice (8 weeks; male C57BL/6J-APCMin+/−) and C57BL/6J littermates (the genetic background of Min mice) were obtained from The Jackson Laboratory. The mice were housed at 10 per cage. They had free access to regular rodent chow and tab water and were maintained in a relative humidity of 50% to 60%, a temperature of 23°C, and a 12:12 h light-dark cycle. They were monitored for any signs of illness. All procedures involving animals were approved by the Animal Investigation Committee at Wayne State University School of Medicine.

Generation of recombinant ERRP. ERRP fusion protein was generated using the Drosophila expression system as described previously ( 18). The stable Schneider 2 Drosophila cells containing pMT/ERRP-V5-His plasmid were incubated with 0.5 mmol/L CuSO4 to stimulate the expression of V5-His-tagged ERRP fusion protein. Cells were lysed, and ERRP was purified by two sequential immunoaffinity columns: anti-ERRP, followed by a second column of antipolyhistidine antibodies ( 18).

Carcinogen and ERRP treatment. After 1 week of acclimatization, the mice were injected i.p. with 1,2-dimethylhydrazine (30 mg/kg bodyweight in 300 μL neutral buffered 10 mmol/L NaHCO3) once per week for 6 weeks. This dose has been successfully used in our previous studies ( 34, 35). Formation of ACF was confirmed in a few carcinogen-treated mice. One week after the last injection of 1,2-dimethylhydrazine, the animals were randomly divided into two groups of 10. They received daily i.p. injections of 50 μg ERRP in 100 μL HEPES buffer for 10 days, whereas the controls received vehicle. The dose of ERRP was chosen on the basis of our observation that s.c. injections of ERRP at doses 15 or 25 μg inhibited growth of SCID mice xenografts of colon or pancreatic cells with no signs of toxicity ( 18, 19, 22) and also that doses of ERRP up to 100 μg were well tolerated by SCID mice (data not shown). The mice were killed by CO2 asphyxiation, the colons were excised and opened longitudinally, and three randomly selected colons from each group were gently scraped with a glass slide, flash-frozen in liquid nitrogen and stored at −80°C for biochemical determinations. Colons from the other animals were fixed overnight in 10% neutral buffered formalin for ACF determination and immunohistochemical analyses.

ACF determination. The formalin-fixed colons were stained in 0.2% methylene blue for 20 min, and the number and size of ACF were recorded using a dissecting microscope with 10× or 20× magnification in a blinded manner. The tissues were then embedded into paraffin, and sectioned at 4 to 5 μg for immunohistochemical analyses.

Determination of tumor number and size in Min mice. Min mice were maintained on regular chow diet until they were 10 weeks old. At this time, all tumors have been formed but continue to grow in size ( 36). The mice were injected (i.p.) with ERRP or vehicle as described above. After 10 injections, they were killed by CO2 asphyxiation; the intestinal tract was excised, and 4 cm from the proximal (jejunum) and distal (ileum) small intestine were removed, opened longitudinally, and then rinsed with ice-cold PBS. They were fixed overnight in formalin, and the number and size of the tumors were recorded using a dissecting microscope with 4× to 10× magnification. Subsequently, the tissue was embedded in paraffin and sectioned for immunohistochemical analyses. From the rest of the small intestine, the epithelial layer was gently scraped with a glass slide and flash-frozen for further analyses. The number of colonic tumors was noted, and tumor volume was calculated by the formula V = {W2 × L}/2, wherein V is tumor volume, W is width, and L is length.

The tumors were excised and flash frozen, and 1 cm of the distal colons were fixed whereas the epithelial layer was harvested from the rest of the tissue. Tissues from the genetic background C57BL/J mice were processed exactly the same way and served as a baseline control.

Determination of proliferating cell nuclear antigen immunoreactivity. Determination of proliferating cell nuclear antigen (PCNA) immunoreactivity, as a measure of proliferative activity, was done as described previously ( 37). Briefly, sections were deparaffinized and microwaved in citrate buffer for antigen retrieval. The slides were incubated with anti-PCNA antibody. The immunocomplex was visualized with the labeled streptavidin-biotin system (Vector) according to the manufacturer's protocol. 3-Amino-9-ethylcarbazole was used as chromagen to localize PCNA-positive cells. All slides were counterstained with hematoxylin. At least 10 well-oriented crypts on each slide and five slides from each sample were examined in a blinded manner. At least 750 cells per slide were counted using 40× objective. The labeling index was calculated as the number of total labeled cells × 100/total cells per high power focus.

Determination of apoptosis by TUNEL assay. Intestinal sections were prepared as described above, and the TUNEL assay was done to detect apoptotic cells using the In situ Cell Death Detection kit from Roche Applied Science according to the manufacturer's instructions. 3-Amino-9-ethylcarbazole was used as chromagen, and the sections were counterstained with hematoxylin. Apoptotic cell nuclei appeared as red-stained structures against a blue-violet background. Mucosal areas of all stained tissues were measured with a 100-division eyepiece reticule and 10× objective. The apoptotic cells within the mucosa of each section were counted.

Immunoprecipitation and Western blot analysis. Western blot analysis was done using our standard protocol ( 14). Briefly, the tissues were solubilized in lysis buffer. After clarification at 10,000 × g for 15 min, the supernatant was used for Western blot analysis. Protein concentration was standardized among the samples, and immunoprecipitates of mucosal lysates containing the same amount of protein were separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Osmonics), blocked for nonspecific binding, and probed with one of the following antibodies (1:1,000 dilution): total EGFR, ErbB-2, ErbB-3 (Santa Cruz Biotechnology), phospho-EGFR (1173Tyr; Upstate Biotechnology), phospho–ErbB-2/HER-2 (1248Tyr), phospho–ErbB-3/HER-3 (1289Tyr), or anti–TGF-α antibodies (Cell Signaling). After incubation with appropriate secondary antibodies, the protein bands were visualized by enhanced chemiluminescence (Amersham). All Western blots were done at least thrice for each experiment. Densitometric measurements of the scanned bands (HP Precision Pro 3.13, Hewlett-Packard) were done using the digitized scientific software program UN-SCNAT. β-Actin or α-tubulin was used for normalization.

Nuclear protein extraction and electrophoretic mobility shift assay. Nuclear proteins were extracted from colonic mucosa as described earlier ( 38) with slight modifications. Briefly, tissues were homogenized on ice using a Dounce homogenizer in 0.05 mL of ice-cold buffer A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L KCl, 10 mmol/L MgCI2, 0.5 mmol/L DTT, 0.1% IGEPAL CA-630, and 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF)] followed by centrifuging at 5000 × g at 4°C for 10 min. The crude nuclear pellet was suspended in 25 μL of buffer B [20 mmol/L HEPES (pH 7.9), 25% glycerol, 1.5 mmol/L MgCl2, 420 mmol/L NaCI, 0.5 mmol/L DTT, 0.2 mmol/L EDTA, 0.5 mmol/L PMSF, and 4 μmol/L leupeptin] and incubated on ice for 30 min with intermittent vortexing. After centrifugation at 14,000 × g at 4°C for 30 min, the supernatant (nuclear proteins) was collected and protein concentration was determined using the bicinchoninic acid assay kit (Pierce Chemical Co.). Electrophoretic mobility shift assay (EMSA) was done by incubating 10 μg of nuclear extract with IRDye-700 labeled nuclear factor κB (NF-κB) oligonucleotide. The incubation mixture included 25 mmol/L DTT and 0.25% Tween 20 in a binding buffer. The DNA-protein complex formed was separated from free oligonucleotide on 8.0% native polyacrylamide gel using a buffer containing 50 mmol/L Tris, 200 mmol/L glycine (pH 8.5), and 1 mmol/L EDTA and visualized by Odyssey IR imaging system. Supershift analyseswere done by additional 30-min incubation at room temperature withspecific(p65) and nonspecific antibody (cyclin D1) before the addition of labeled probe.

Statistical analysis. Unless otherwise stated, data are expressed as means ± SD. Where applicable, the results were analyzed using a two-tailed Student's t test after ANOVA, taking P < 0.05 as the levels of significance.

Results

ERRP regresses early and intermediate stages of colon cancer. The objective of this investigation was to test the effectiveness of ERRP in eradicating existing colonic lesions using widely accepted rodent models for early and intermediate stages of colon cancer. Carcinogen-induced ACF, representing early stages of colon cancer, were confirmed in a separate control group before ERRP treatment began. Treatment with recombinant ERRP for 10 consecutive days significantly reduced the number of ACF (25.0 ± 3.02 versus 14.9 ± 1.57, for controls and ERRP-treated, respectively; P = 0.011; Fig. 1A ), indicating an eradication of existing early lesions in the colon by ERRP. However, at this time, we cannot exclude the possibility that ERRP also prevents the formation of new lesions. Most of the ACF were located in the distal and mid part of the colons, whereas very few were detected in the proximal colon. This distribution did not change between the two groups.

Figure 1.
Figure 1.

ERRP treatment (50 μg/mouse) for 10 days after carcinogen treatment reduced the number (A; *P < 0.01) and size (B; **, P < 0.001) of existing ACF in CF1 mice and caused the regression of existing tumors in the small intestine of Min mice (C; *P < 0.05). This reduction was seen also seen in the colon (D) but did not reach statistical significance. ERRP treatment also reduced tumor size in the small intestine (E; #P < 0.001) and in the tumor volume in the colon of Min mice (F; P = 0.074). Ctrl, control.

It has been postulated that the size of the ACF may be a better prognostic indicator of tumor formation. Treatment with ERRP significantly reduced the number of aberrant crypts per focus (1.81 ± 0.06 versus 1.42 ± 0.08, for control and ERRP-treated animals, respectively; P < 0.01; Fig. 1B). Furthermore, single-crypt ACF seemed to be less developed in the ERRP-treated group than the controls, as indicated by their size and staining intensity.

Next, we determined the therapeutic effectiveness of ERRP on intermediate stages of colon cancer by evaluating regression of adenomas in Min mice in response to recombinant ERRP. Treatment of Min mice began when most, if not all, tumors had already developed. As shown in Fig. 1C, ERRP caused a significant regression of adenomas in the small intestine (19.2 ± 10.06 versus 9.29 ± 3.99, for controls and ERRP-treated, respectively; P = 0.035). This was evident in both the jejunum (5.2 ± 1.79 to 3.0 ± 1.41; P < 0.05) and ileum (14.0 ± 8.46 to 6.29 ± 3.04; P < 0.05), wherein most of the tumors had formed. ERRP treatment also reduced the number of tumors found in the colon (0.8 ± 0.84 to 0.29 ± 0.49) but this did not reach statistical significance ( Fig. 1D). As expected, no tumors were detected in the C57BL/6J mice. We then evaluated the size of the flat small intestinal tumors and the volume of sessile colonic tumors. The size of the small intestinal tumors was significantly reduced by ERRP (control 1.36 ± 0.84 mm, ERRP-treated 0.95 ± 0.58 mm; P < 0.001; Fig. 1E). This was seen both in the jejunum (1.73 ± 0.82 to 1.12 ± 0.61; P = 0.007) and the ileum (1.26 ± 0.80 to 0.88 ± 0.55; P = 0.007). The volume of the colonic tumors was also reduced (control 14.19 ± 7.14, ERRP-treated 1.27 ± 0.75); however, this did not reach statistical significance ( Fig. 1F), which could be attributed to the small number of colonic tumors.

Neither weekly injection of 1,2-dimethylhydrazine nor daily injection of recombinant ERRP to 1,2-dimethylhydrazine–treated mice or Min mice produced signs of illness at any time during this study. In addition, no macroscopically visible morphologic changes (i.e., size, shape, or color) in any of the major organs were detected in any group. This confirms the observations from previous studies showing no apparent toxic side effects of the ERRP treatment.

ERRP inhibits proliferation and stimulates apoptosis in macroscopically normal colonic mucosa of 1,2-dimethylhydrazine–treated and Min mice. An increased proliferation has been identified as a driving force for carcinogenesis ( 39). Earlier studies have shown that 1,2-dimethylhydrazine treatment stimulates proliferation and inhibits apoptosis in the colonic mucosa of CF1 mice ( 40), and in Min mice; a significant increase of proliferation ( 41) and crypt fission ( 42) compared with the genetic background mice was shown. To determine whether ERRP-induced regression of ACF and adenomas could at least in part be due to inhibition of proliferation and stimulation of apoptosis, we analyzed the formalin-fixed intestinal tissues for changes in proliferative activity and apoptosis. Changes in proliferative activity were examined by measuring PCNA immunoreactivity. A significant reduction in proliferation (59%, P < 0.001) was observed in the ERRP-treated CF1 mice compared with the controls (control 155.8 ± 23.3/high power focus; ERRP-treated 63.6 ± 11.5/high power focus; Fig. 2A and B ). A comparison of proliferation in the small intestinal mucosa (where most of the tumors develop in Min mice) with their genetic background strain C57BL/6J showed that proliferative activity of the normal appearing Min mucosa is increased by 750%, confirming results published by other groups. Treatment with ERRP significantly reduced mucosal proliferation to ∼25% of the vehicle-treated controls ( Fig. 2E). In accordance to the inhibition of proliferation in the colonic tissue of CF1 mice, ERRP also reduced elevated proliferation in the Min colon (data not shown).

Figure 2.
Figure 2.

Effects of recombinant ERRP (50 μg/mouse) or vehicle (control) on proliferative activity (A and B) and apoptosis(C and D) in the colonic mucosa of 1,2-dimethylhydrazine–treated mice and in the small intestine of Min mice (E and F). *P < 0.001, compared with vehicle-treated C57BL6J Mice; #,P < 0.001, compared with vehicle-treated Min mice.

Apoptosis was determined using the TUNEL assay. ERRP treatment significantly increased the number of apoptotic cells in the colons of CF1 mice by 348% (P < 0.001) when compared with the corresponding vehicle-treated controls (control 4.2 ± 1.3 apoptotic bodies/high power focus; ERRP-treated 18.8 ± 2.8 apoptotic bodies/high power focus; Fig. 2C and D). Apoptosis in the small intestine was found to be significantly lower in Min mice by ∼40% when compared with the C57BL/6J littermates but up-regulated by ERRP by 350% compared with the vehicle controls (control 2.2 ± 0.05 apoptotic bodies/high power focus; ERRP-treated 10.2 ± 0.82; P = 0.001; Fig. 2F). This increase in apoptosis after ERRP treatment was also seen in the colon (data not shown).

ERRP inhibits activation of EGFR and its family members. We have previously shown that ERRP targets the expression and activity of members of the EGFRs family ( 14– 16). To determine whether ERRP-induced inhibition of proliferation and stimulation of apoptosis in the mouse intestinal tract and in turn regression of ACF and adenomas could be the result of attenuation of EGFR-activated signal transduction pathways, expression and constitutive activation (extent of tyrosine phosphorylation) of EGFR, ErbB-2, and ErbB-3 were analyzed. ERRP caused a marked reduction of activated EGFR, ErbB-2, and ErbB-3 in the colonic mucosa of 1,2-dimethylhydrazine–treated CF1 mice, as evidenced by the decreased levels of receptor tyrosine phosphorylation (P < 0.01; Fig. 3A ). ERRP also down-regulated EGFR but had little or no effect on the expression of ErbB-2 and ErbB-3. This suggests that, at least for ErbB-2 and ErbB-3, the reduction in tyrosine phosphorylation is not the consequence of down-regulation of the receptors.

Figure 3.
Figure 3.

Representative Western blots showing the levels of total and phosphorylated (p-) forms of EGFR, ErbB-2, and ErbB-3 in the intestinal mucosa of (A) 1,2-dimethylhydrazine–treated mice and in (B) Min mice after daily injection ERRP or vehicle (controls). Numbers represent percentage of the corresponding controls. All Western blots were done at least thrice using total mucosal lysates from different mice.

In Min mice, we found a higher expression of EGFR (77%), ErbB-2 (8–10%), and ErbB-3 (58%) in the small intestinal mucosa than in C57BL/6J mice ( Fig. 3B), indicating that changes in the expression of EGFRs is an early event in this model and detectable in the macroscopically normal appearing intestinal mucosa. However, the levels of activated receptors were not different between C57BL/6J and Min mice. As observed in CF1 mice, the levels of total and tyrosine phosphorylated forms of EGFR, ErbB-2, and ErbB-3 in the small intestine of Min mice were markedly decreased (50–80%) after ERRP treatment when compared with vehicle-treated Min mice or their genetic background C57 mice ( Fig. 3B). Similar changes were seen in the colon of Min mice (data not shown).

Given the importance of EGFRs in the development and progression of colorectal cancer, our current observation of inhibition of constitutive activation of EGFR, ErbB-2, and ErbB-3 suggests that they may partly be responsible for the ERRP-induced regression of ACF and adenomas in the mouse intestinal tract. This underscores the notion that ERRP functions as a pan-erbB inhibitor in vivo.

Sequestration of TGF-α by ERRP. Although the precise mechanisms responsible for ERRP-mediated inhibition of EGFR activation are still to be delineated, we speculated that ERRP with its substantial homology to the extracellular ligand-binding domain of EGFRs may sequester their ligands and, thus, rendering them unavailable to bind to and activate EGFRs. Ligand sequestration would lead to the formation of inactive monodimers and heterodimers and, thereby, to an attenuation of EGFR-activated signaling as depicted in Fig. 4A . This has been suggested by the results of our previous studies ( 16, 21). Thus, we speculated that ERRP will sequester TGF-α, the most common EGFR ligand synthesized in colonic mucosal cells. To test this possibility, we determined the amount of TGF-α bound to ERRP in the intestinal mucosa by immunoprecipitation. In CF1 mice, the amount of membrane-bound TGF-α precursors bound to ERRP was found to be significantly higher in ERRP-treated colonic mucosa compared with vehicle-treated controls (P < 0.001; Fig. 4B). In Min mice, the same increase in ligand sequestration by ERRP was observed ( Fig. 4B).

Figure 4.
Figure 4.

A, schematic representation of the proposed mechanism of action of ERRP. B,Western blot showing changes in the levels of membrane-bound precursor forms of TGF-α in the colonic mucosa from 1,2-dimethylhydrazine–treated mice (top) or small intestinal mucosa from Min mice (bottom) after 10 daily injections of ERRP or vehicle (controls). Mucosal lysates containing 1 mg protein were incubated with anti-ERRP antibodies at 4°C for 16 h. The immunoprecipitates containing ERRP were subjected to Western blot analysis with anti–TGF-α.

ERRP inhibits NF-κB activity. NF-κB plays a critical role in EGFR-Akt–mediated cell survival processes. To determine whether ERRP-induced stimulation of apoptosis in the colonic mucosa of 1,2-dimethylhydrazine–treated mice could partly be attributed to decreased activation of NF-κB; the DNA-binding activity of NF-κB was determined by EMSA. As expected, ERRP caused a marked inhibition of NF-κB binding activity in the colonic epithelium of 1,2-dimethylhydrazine–treated mice ( Fig. 5A ) and the small intestinal tissue of Min mice ( Fig. 5B). Interestingly, the DNA-binding activity of NF-κB was found to be already slightly higher in the small intestinal mucosa of Min mice than in the C57BL6 mice ( Fig. 5B), again indicating an early change in carcinogenesis in Min Mice.

Figure 5.
Figure 5.

EMSA showing reduction in DNA binding activity of NF-κB activity in the (A) colonic mucosa of 1,2-dimethylhydrazine–treated mice and (B) small intestinal mucosa from Min mice after 10 daily injections of ERRP or vehicle (controls).

To further determine the extent of the effect of the ERRP-induced inhibition of the DNA-binding activity of NF-κB to cyclooxygenase (COX)-2, a direct downstream target of NF-κB, whose expression is greatly increased in colon cancer ( 43) was determined. It was anticipated that ERRP-mediated regression of ACF would be associated with a concomitant reduction in COX-2 expression. Indeed, we observed that the levels of COX-2 in the colonic mucosa of ERRP-treated CF1 mice were decreased by 70% to 80% ( Fig. 6 ) and by ∼40% in the small intestinal tissue of Min mice, when compared with the corresponding controls (data not shown).

Figure 6.
Figure 6.

COX-2, a downstream target of NF-κB, is decreased in the colonic mucosa of 1,2-dimethylhydrazine–treated mice after 10 daily injections of ERRP when compared with vehicle-injected controls.

Discussion

The prevalence of overexpression or constitutive activation of EGFRs in different epithelial malignancies, including colorectal cancer, and their effect on disease etiology have identified this group of receptor tyrosine kinases as a prominent target for therapeutic intervention. ERRP, a recently identified pan-erbB inhibitor that targets multiple members of the EGFR family, inhibits growth of several colon, pancreatic and breast cancer cells that express varying levels of EGFRs ( 15). ERRP also inhibits in the growth of SCID mice xenografts of colon or pancreatic cancer cells ( 18, 19, 22). These studies suggest that ERRP could be a therapeutic agent for a number of epithelial cancers. Our current data, for the first time, show that ERRP treatment regresses existing carcinogen-induced ACF that represent the earliest visible changes in colon carcinogenesis as well as spontaneously formed adenomas in Min mice. In contrast to xenografts, these models mimic the multistage human disease in immunocompetent mice. These results indicate a clear therapeutic potential of ERRP for the early and intermediate stages of colon cancer. Our current data also set ERRP apart from other natural or endogenous compounds with preventive but not therapeutic functions. This together with an apparent lack of side effects identifies ERRP as a promising anticancer agent.

The mechanisms by which ERRP causes regression of ACF and adenomas are not fully understood. Our current studies suggest a combination of inhibition of proliferation and stimulation of apoptosis may be the underlying mechanisms. Increased proliferation and reduced apoptosis occur early in carcinogen-treated mucosa ( 35) that precedes ACF formation. Mucosal proliferation is increased in dysplastic ACF, which may represent an important advantage for progression of these lesions ( 44). Therefore, reversal of this hyperproliferation may be critical for the regression of ACF. This is not specific for the carcinogen induced colon cancer model because increased proliferation and decreased apoptosis have also been shown in the normal appearing intestinal mucosa of Min mice ( 33). Our current observations are in agreement with what has been noted by Moran et al. ( 33). Moreover, the fact that ERRP reverses these variables suggest that the regression of adenomas is partly the result of reduction in mucosal proliferation and stimulation of apoptosis.

Our previous studies have shown that ERRP-induced growth inhibition is associated with the attenuation of constitutive and/or ligand-induced activation of EGFRs and their subsequent signaling pathways. A similar phenomenon was also noted in the intestinal mucosa of 1,2-dimethylhydrazine–treated mice and Min mice after ERRP administration. ERRP caused a marked attenuation of constitutive activation of EGFR, ErbB-2, and ErbB-3, which could be attributed at least for EGFR, to the down-regulation of receptor expression. The structural similarity of ERRP with EGFR may be important for this difference: while there is an 85% to 90% homology to EGFR, there is only a 50% to 60% homology with other EGFRs ( 14). It remains to be determined if this difference is a time-dependent phenomenon, and whether a longer treatment period (more than the 10 days used in the present study) would result in down-regulation of other members of the EGFR family. However, the overexpression of EGFR in carcinogen-treated mucosa ( 29, 38), ACF ( 32), and Min intestinal tissue ( 33) suggests that this receptor plays a crucial role in the development of colon cancer.

The precise mechanism(s) by which ERRP attenuates activation of EGFRs and, in turn, cell growth have not been fully delineated. Previously, we reported that increased activation of EGFR in the colonic mucosa of aged or carcinogen-treated rodents was associated with elevated levels of membrane-bound precursor forms of TGF-α ( 29, 38), which was thought to be involved in activating the intrinsic EGFR activity through an autocrine/juxtacrine mechanism ( 45, 46). The substantial homology of ERRP to the extracellular ligand–binding domain of the EGFRs allows for sequestration of ligands, rendering them unavailable for binding to and activation of the receptors. The absence of the extracellular domain IV of EGFR has been suggested to increase the affinity of ligands to the receptor ( 47). The same could be true for ERRP as it also lacks the major part of the domain IV. In support of this postulation, we have observed an increased binding of TGF-α and HB-EGF to ERRP in ERRP-treated PC-3 cells leading to attenuation of EGFR signaling and inhibition of growth ( 21). Indeed, the attenuation of EGFR, ErbB-2, and ErbB-3 activation in the intestinal mucosa of CF1 and Min mice after ERRP treatment could be attributed to increased sequestration of the ligand(s) by recombinant ERRP. We have observed that the amount of precursor forms of TGF-α bound to ERRP in intestinal membranes from ERRP-treated mice is substantially higher than in controls.

Numerous studies have shown that NF-κB, a transcription factor, plays an important role in cell survival by its ability to block or decrease apoptosis ( 48). Activation of EGFRs has been shown to activate NF-κB. We predicted that attenuation of EGFRs activation will lead to a concomitant reduction in NF-κB activity. Indeed, we have observed that ERRP treatment for 10 days that caused an attenuation of EGFRs activation in the intestinal mucosa was associated with decreased NF-κB activity, as evidenced by decreased DNA binding activity of the transcription factor. This may partly be responsible for decreased expression of COX-2 in the colonic mucosa of 1,2-dimethylhydrazine–treated CF1 mice. Because NF-κB is critically involved in regulating cell survival, the increased apoptosis by ERRP could partly be due to the inhibition of NF-κB activity. Taken together the results suggest that ERRP could effectively inactivate both EGFR and COX-2 and as such could be useful for prevention and/or treatment of colon cancer.

In summary, our current data show that ERRP is effective in regressing ACF in the colon of CF1 mice that are formed in response to 1,2-dimethylhydrazine treatment as well as spontaneously developed adenomas in Min mice. This is thought to be the result of decreased activation of EGFR, ErbB-2, and ErbB-3, leading to inhibition of NF-κB activity and, in turn, inhibition of proliferation and stimulation of apoptosis. Based on our data, we conclude that ERRP could be an effective therapeutic agent for treatment of early and even advanced colon cancer.

Acknowledgments

Grant support: NIH/National Institute on Aging R01 AG014343 (A.P.N. Majumdar) and Department of Veterans Affairs VA Merit Review (A.P.N. Majumdar and A.K. Rishi).

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.

Footnotes

    • Received February 7, 2007.
    • Revision received March 20, 2007.
    • Accepted March 27, 2007.

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    Cancer Research: 67 (11)
    June 2007
    Volume 67, Issue 11

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    Regression of Early and Intermediate Stages of Colon Cancer by Targeting Multiple Members of the EGFR Family with EGFR-Related Protein
    Eva M. Schmelz, Hu Xu, Radha Sengupta, Jianhua Du, Sanjeev Banerjee, Fazlul H. Sarkar, Arun K. Rishi and Adhip P.N. Majumdar
    Cancer Res June 1 2007 (67) (11) 5389-5396; DOI: 10.1158/0008-5472.CAN-07-0536
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