Gefitinib prevents cancer progression in mice expressing the activated rat HER2/neu

Authors

  • Marie P. Piechocki,

    Corresponding author
    1. Department of Otolaryngology-Head and Neck Surgery, Wayne State University and Karmanos Cancer Center, Detroit, MI
    • Department of Otolaryngology-Head and Neck Surgery, Wayne State University, Room 423 Prentis Building of KCI, 110 East Warren Avenue, Detroit, MI 48201, USA
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    • Fax: +313-833-7294

  • Susan K. Dibbley,

    1. Department of Otolaryngology-Head and Neck Surgery, Wayne State University and Karmanos Cancer Center, Detroit, MI
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  • Fulvio Lonardo,

    1. Department of Pathology, Wayne State University and Karmanos Cancer Center, Detroit, MI
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  • George H. Yoo

    1. Department of Otolaryngology-Head and Neck Surgery, Wayne State University and Karmanos Cancer Center, Detroit, MI
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Abstract

We tested the efficacy of gefitinib in the prevention of HER2/neu-mediated breast cancer development in BALB-NeuT transgenic mice. Oral administration of gefitinib to female transgenic mice from 5 to 14 weeks of age reduced tumor multiplicity from 9.6 ± 0.82 to 0.58 ± 1.1 (83%). We observed a decrease in the number and size of lobules and lobular nodules in treated mice with a reduction in the overall disease burden per gland. Normal duct development in the mammary glands was not affected by gefitinib. The development of acinic cell carcinoma in the parotid glands of these animals was also reduced coincident with decreased stromal involvement during progression. Gefitinib eliminated phosphorylation of HER2 and HER3 and signaling through MAPK and Akt in lobular hyperplasias and carcinomas. At the same time MAPK activity and cytokine production in splenocytes and lymph nodes was increased in gefitinib-treated animals coincident with an increase in lymph node size. Delaying gefitinib treatment until mammary glands exhibited atypical lobular hyperplasias reduced efficacy. These studies demonstrate the critical role of HER2 signal transduction in the onset and progression of HER2/neu-dependent breast cancer and suggest a role for specific inhibitors to prevent the outgrowth of early hyperplastic disease. © 2007 Wiley-Liss, Inc.

The development of targeted therapies for the specific inactivation of receptor tyrosine kinase oncogenes involved in tumor initiation and progression has lead to the ability to target signal transduction as a modality for preventing tumor development.1, 2 ZD1839 (gefitinib, Iressa®), an orally active, selective EGFR-tyrosine kinase inhibitor that blocks signal transduction pathways implicated in proliferation and survival of cancer cells and other host-dependent processes that promote cancer growth.3, 4 To date, we have already demonstrated the efficacy of gefitinib against mammary and salivary gland tumor cell lines derived from transgenic mice that over-express the activated rat HER2/neu.5, 6 We determined that signal transduction from the activated HER2/neu was more sensitive to inhibition by gefitinib in breast cancer cells when compared with salivary gland tumor cells. In these studies, gefitinib impaired MAPK and Akt signaling in breast cancer cells expressing HER2/neu and ErbB-3 leading to growth arrest and apoptosis6 while inducing only cytostasis in salivary gland tumor cells that do not over-express ErbB-3.5 Although gefitinib impaired MAPK signaling in salivary gland tumor cells and enhanced fas mediated apoptosis, survival signaling was not effected by gefitinib.

To determine the effectiveness of gefitinib in preventing breast tumor progression, we used the BALB-NeuT transgenic mouse model,7 which mimics the genotype of poor prognosis breast cancers.8 These mice harbor a transgene of the activated rat HER2/neu driven by the MMTV promoter and have been bred onto the BALB/c background. Over-expressed or mutated p185 leads toward the formation of homo- or heterodimers with other epidermal growth factor receptors. As these dimers transduce positive growth signals in a ligand-independent way,9, 10 they are involved in the initiation and progression of neoplastic transformation.9, 11 Expression of the activated neu oncogene in transgenic mice has been associated with both the synchronous (single-step) and the stochastic12 (multistep) transformation of mammary epithelium. Sequence analyses revealed that activation of neu occurs through a single amino acid change in the transmembrane (TM) portion of the protein.9 This single point mutation replaces the valine residue at position 664 in the TM domain of p185 with glutamic acid, favors p185 homo- and heterodimerization and transforms the Her-2/neu protooncogene into a dominant transforming oncogene.13, 14

The lobular mammary gland carcinomas that develop in BALB-NeuT transgenic virgin female mice have been described and characterized extensively in the pathology of their progression.7 These mice display no palpable lesion of the mammary gland until about 15 weeks of age. By 33 weeks of age all 10 mammary glands develop tumors. On histological exam, mammary tissue exhibits widespread hyperplasia of the small lobular ducts and lobules at 3 weeks of age. By 11 weeks of age ductules and acinar cells within the lobules appear distended by a solid occlusive growth of this epithelium, the morphology of these lesions is consistent with that of lobular carcinoma in situ (LCIS). Invasive growth occurs by 20 weeks and is characterized by the infiltration into the adipose tissue by alveolar groups of neoplastic cells in the absence of a myoepithelial lining. With a slight asynchronous but consistent progression, all of the mammary glands of female BALB/c mice transgenic for the transforming rat HER-2/neu oncogene progress to atypical hyperplasia and to invasive carcinoma.

In this aggressive mammary carcinoma model, prevention of mammary gland cancer has been achieved through immune modulation. Work from the Forni lab has established multiple protocols for immunizing BALB-NeuT mice that protect these animals from developing mammary tumors.15–21 The extent and duration of protection is contingent upon the vaccination schedule and modality. Previous studies have shown that chronic administration of interleukin IL-12 started at the 2nd week of age hampers this progression because of its ability to inhibit tumor angiogenesis and activate a nonspecific immune response.16 This protocol however presents considerable toxicity. DNA vaccination with a plasmid encoding the rat HER2/neu gene alone15, 20, 21 or in combination with systemic IL-1218 provided protection against mammary carcinogenesis via antibody, lymphocytic infiltration and suppression of tumor-elicited angiogenesis. Silencing HER2/neu signaling has not been evaluated as a potential therapeutic modality in this model. We examined the ability of gefitinib to inhibit signal transduction from the activated HER2/neu during early mammary gland hyperplasia and tumor progression to impair the development of lobular carcinoma.

Several groups have tested the efficacy of gefitinib against the development of carcinogen-induced and EGFR dependent cancers in the colon,22 bladder23 and lung24 as well as other EGFR-mediated pathologies associated with cirrhosis25 and injury-induced fibrosis.26, 27 Several of these reports suggest a role for the EGFR in the initiation and promotion of the pathological events. In a xenograft model using explants from EGFR positive DCIS patients, Chan et al.28 evaluated the effects of gefitinib on normal and premalignant breast and observed a 56% reduction in epithelial proliferation with ZD1839 in EGFR-positive DCIS, regardless of ER status. Studies by Lu et al.,29 further suggested that gefitinib could be beneficial in preventing the development of ER-negative mammary tumors via EGFR inhibition in transgenic animals. Our studies were designed to test the effectiveness of this agent in an aggressive HER2 over-expressing mouse model of lobular carcinoma and determine the extent to which targeting HER2 signal transduction prevents tumor progression.

Material and methods

BALB-NeuT transgenic animals

All animal research was conducted following an approved protocol filed with the Animal Investigation Committee at Wayne State University that oversees the Division of Laboratory Animal Resources (DLAR) at this institution and are in strict accordance with NIH guidelines. Two stock BALB-NeuT transgenic males were obtained through collaboration with Dr. Guido Forni. The BALB-NeuT strain originated from a transgenic CD1 random-bred breeder male mouse (no. 1330) carrying the mutated rat HER2/neu oncogene driven by the MMTV promoter.30 The mutated gene encodes a single point mutation that replaces the valine residue at position 664 in the TM domain of p185/neu with glutamic acid. This mutation promotes p185/neu homo- and heterodimerization and transforms the HER2/neu protooncogene into a dominant transforming oncogene. Transgenic breeder males were single housed with wildtype BALB/c females. Animals were maintained on a casein-based, phytoestrogen-free,31 D11243N modified AIN-76A diet with 5% corn oil (equivalent to 12% fat) and a 12 hr–12 hr light–dark cycle, 55% humidity, ambient 21°C temperature with water and pelleted diet ad libitum. At time of weaning, a 2-mm punch of ear tissue was collected from pups for PCR analysis. Littermates were randomly distributed into either the control or treatment arm of the study. Nontransgenic littermates were also included to monitor for toxic side effects due to treatment. Animals began therapy between 5 and 6 weeks of age. Gefitinib or diluent (0.1% Polysorbate 80) was administered by oral gavage daily for 5 consecutive days per week, followed by 2 days without treatment and repeated for 8–9 weeks until control animals showed evidence of mammary gland activity. Animals were weighed weekly and the dose increased every other week. Gefitinib dosing for the initial week of treatment was 75 mg/kg and increased by 15 mg/kg every other week, so that at end of the treatment period animals were receiving 135 mg/kg. Mice on study were provided with environmental enrichment (Cheerio rewards, and neslette) to enhance their ability to thrive and tolerate therapy. Animals were sacrificed at 14 weeks of age, necropsied and tissues harvested and paraffin embedded including fine inspection of each (of 10) mammary gland for the gross evidence of disease or tumor nodules. Representative mammary glands that were collected from each animal included the no. 9 and no. 10 from the inguinal area and the no. 1 and no. 6 from the cervical area in a composite resection that including all major and minor salivary glands and lymph nodes (LN). Tissues were formalin fixed, paraffin embedded and processed for histochemical analysis.

Gefitinib

Gefitinib “Iressa” (Zeneca Pharmaceuticals Macclesfield, Cheshire) ZD1839, 4-(3-chloro-4-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy) quinazoline was purchased from the Harper Hospital pharmacy (Detroit, MI). Immediately before use, tablets were crushed with mortar and pestle and suspended in sterile 0.1% Polysorbate-80 (Tween-80) at 10 mg/mL.

Immunohistochemistry

At time of necropsy, mammary glands were fixed in 10% neutral buffered formalin and paraffin embedded using standard histochemical techniques. Blocks were sectioned in 4-μm sections. Tissue sections were stained with Hematoxylin and Eosin for basic histological evaluation or processed for immunohistochemical analyses. For immunodetection of tissue antigens, histological grade primary antibodies were applied to the samples and incubated according to manufacturer's recommendation (HER2, PAD: Z4881, cat no. 08-1204 2nd Gen; Zymed, South San Francisco, CA; IL-1β H-153, sc-7884, Santa Cruz Biotech, CA). Samples were washed and labeled using the SuperPicTure™ Polymer Detection Kit, cat no.87-9263; Zymed), developed with DAB Substrate and counterstained with hematoxylin. Samples were evaluated using a Zeiss microscope and images were collected through a Sony 970 CCD camera interfaced with the MCID5+ imaging software package. Alternatively images were collected using a Nikon inverted microscope equipped with a SPOT digital cooled camera and imaging software. Stained sections were evaluated by a board certified pathologist, blinded to the treatment conditions and graded for extent of disease.

Western blot analysis

Lysates were prepared from mammary gland tissues in 1X Cell Lysis buffer (Cell Signaling Technology, no. 9803) supplemented with protease Inhibitor Cocktail Set III (cat no. 539134; Calbiochem, San Diego, CA), phosphatase inhibitor cocktail II (cat no. 524625; Calbiochem, San Diego, CA) and/or Halt™ Phosphatase Inhibitor Cocktail (cat no. 78420 Pierce, Rockford, IL) and PMSF. Lysates were incubated on ice for 30 min with occassional mixing, clarified by centrifugation at 16,000g for 20 min at 4°C and denatured by adding SDS sample buffer (no. 7722) supplemented with DTT from 3X stocks and boiling for 5 min. Lysates were stored at –20 to −80°C depending on frequency of usage. Fifty micrograms of tissue lysate were resolved in 4–12% or 4–20% PAGEr Duramide® Gels (Cambrex Bio Science, Rockland, ME) and electro transferred to Immobilon-P (Millipore, Bedford, MA) PVDF membranes. Membranes were fixed with methanol, then rehydrated and blocked for 2 hr prior to the addition of primary antibody at the recommended dilution in TBST buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.1% Tween-20) with 1–5% BSA or TBST supplemented with 5% nonfat dry milk according to the antibody manufacture. Blots were probed overnight at 4°C. Blots were washed and labeled for 90 min at room temperature with Peroxidase-conjugated AffiniPure Goat Anti-Mouse (cat no. 115-035-071) or Goat Anti-Rabbit (cat no. 111-035-046) secondary antibodies from Jackson ImmunoResearch Laboratories diluted at 1:2000 to 1:5000 in TBST + 5% Nonfat dry milk. Blots were developed with enhanced SuperSignal® West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL) and imaged Kodak-MR film. Blots were developed by ECL and then stained with Coomassie blue to ensure uniform loading between samples. Phosphospecific and mouse specific antibodies from Cell Signaling Technology (Beverly, MA) include: Her2 (no. 2242), pY1221/1222Her2 (no. 2249), p-p44/42 MAP kinase (Thr202/Tyr204, no. 9101), total p44/42 MAP kinase. Antibodies from Santa Cruz (Santa Cruz, CA) include: IL-1 beta (H-153, sc-7884) HSP 60 (N-20, sc-1052).

Statistical analysis

The software package associated with SLIDEWRITE PLUS V5 (Advanced Graphics Software, Encinitas, CA) was used for graphing, data statistics, 1- and 2-tailed t-test and ANOVA.

Results and discussion

Using littermates that were randomized to receive diluent or gefitinib, we conducted a total of 9 trials and observed excellent inter- and intratrial reproducibility. Twenty transgene positive females received control therapy and 26 transgene positive females received gefitinib. Animals received treatment from 5 to 6 weeks of age when lobular hyperplasia (LH) was present in the mammary glands, coincident with the onset of ductal morphogenesis (postpuberty) until 14 weeks of age when control animals exhibited LCIS and gross evidence of disease in all 10 glands. Treatment reduced tumor multiplicity by 83% (Fig. 1a) with minimal effect on the rate of weight gain (Fig. 1b). Specifically, tumor multiplicity was decreased from 9.6 ± 0.8 in control females (n = 20) to 0.6 ± 1.1 in gefitinib-treated females (n = 26). Furthermore, the extent of disease in the mammary glands found positive in gefitinib-treated animals was minimal relative to the controls.

Figure 1.

(a) Inhibition of tumor multiplicity by gefitinib in BALB-NeuT transgenic mice. The symbols in each grouping represent the number of mammary glands scored positive for disease for each animal in the trial. **Difference in tumor multiplicity between the control and treated animals is highly significant p < 0.0001. (b and c) Effect of gefitinib on the rate of weight gain. BALB-NeuT transgenic mice received (b) diluent or (c) gefitinib by oral gavage daily, 5 days per week from 5 to 14 weeks of Age. The weights (g) of individual animals (solid symbols) are plotted for each week. Box-Whisker plots represent the mean and standard error of the group. No significant difference in the rate of weight gain between the (b) Control 0.51 ± 0.03 and (c) gefitinib-treated 0.46 ± 0.02 animals was observed until 13 weeks of age when the gefitinib-treated animals failed to gain weight at the same rate p = 0.004. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Our effective dose range and treatment schedule is similar to that published by others,24, 28, 29 however, the animal models and the age at which animals began treatment is different. Side effects that correlated with gefitinib therapy occurred with variable frequency during the last 2 weeks of therapy included hair loss, (eyelashes and whiskers), dry eyes, brittle and broken nails, weight loss, hypothermia and enlarged LN. Similar side effects have been reported by other investigators using a similar treatment protocol.24, 28, 29 We observe that these side effects tend to be more pronounced in transgenic animals when compared with their nontransgenic littermates and often manifest in the last 2 weeks of therapy. When compared with the controls, gefitinib-treated animals tend to gain weight at a slower rate during the last 2 weeks of treatment (Fig. 1c). Nevertheless, animals appear to develop normally, and maintain normal eating, drinking and grooming behaviors.

The morphology associated with neoplastic progression that occurs in the mammary glands of BALB-NeuT mice has been described.7 We adapted the cartoon from Cifaldi et al.17 to illustrate and classify the diseased states and overall mammary gland morphology observed in our studies (Fig. 2a). As shown in Figure 2b, 78% of control cases were classified as LCIS and the remaining 22% were invasive carcinomas. In the treated cases, 36% were classified as normal and 43% were graded as LHs, suggesting that the disease progression was arrested or even regressed. The frequency of LCIS in treated animals was 7% and mammary gland atrophy was detected in 7% of cases. These 2 extremes exemplify the fine line between treatment success/failure and overt toxicity and will be discussed. One variable that contributes to our failure to prevent disease progression is due to the narrow treatment window in this aggressive model of breast cancer progression. Despite the fact that 36% of gefitinib-treated cases were described as morphologically normal, persistence of HER2/neu positive mammary epithelial cells was detected in focal areas of LH (not shown). Comparing mammary glands from the control (Fig. 2c) and gefitinib (Fig. 2d)-treated animals demonstrated the development of multiple extensive lobular carcinomas in control animals with extensive lobuloalveloar expansion and luminal obliteration of acini compared with few lobules involved focally with features of early hyperplasia and scarce luminal proliferation of individual acini in gefitinib-treated mice.

Figure 2.

(a) Illustration of HER2/neu mediated carcinogenesis and disease progression in the mammary gland of BALB-NeuT transgenic female mice [adapted from Cifaldi et al.17]. (b) Mammary gland disease burden in BALB-NeuT treated with gefitinib. Data represent the morphological analysis of mammary glands from control and gefitinib-treated mice harvested at 14 weeks of age. H&E stained cross-sections within 50 μm of the lymph node were evaluated and scored to generate an overall impression of the mammary gland morphology. The percentage of cases in each classification: Atrophy, Norm, normal; LH, lobular hyperplasia; ALH, atypical lobular hyperplasia; LCIS, lobular carcinoma in situ; inv. CA, invasive carcinoma were plotted in a continuum of bars to represent disease burden in the control (Black) and gefitinib-treated (White) animals. (c and d) Histological evaluation of mammary glands from (c) Control and (d) gefitinib-treated animals. Representative tissues were stained with H&E and photographed at (×1). Disease in the control was classified as LCIS and that in the treated was classified as LH. (Reproduced with permission of American Association for Cancer Research, from L. Cifaldi, 2001; permission conveyed through Copyright Clearance Center, Inc.)

In transgenic animals, mammary gland development and tumorgenesis displays a “signature” phenotype that is associated with the specific transgene construct.32 Rudimentary branching morphogenesis in the mammary gland is supported by the expansion of ductal outgrowth through the mammary fat pad and the formation of terminal end-buds. In general, the mammary gland epithelium reaches the end of the fat pad by 10 weeks of age. The role of ErbB-2 in this process and mammary gland tumor progression is in accelerating lobuloalveolar development,33, 34 while stromal EGFR is essential for ductal growth.35, 36 ErbB-3 functions with ErbB-2 in the malignant transformation of the mammary gland to promote survival and resist apoptosis.37 Epithelial-stromal signaling is required to orchestrate and coordinate these events by integrating ErbB signals upon ligand binding and activation.32, 38

Our goal was to specifically impair the “oncogenic signals” from the rat HER2/neu that drive tumor growth and progression. Since ErbB-2 is primarily responsible for lobuloavelolar development and proliferation and the EGFR is critical for the ductal development, we compared the effects of gefitinib on lobule and ductal formation. Luminal population of epithelial acini is controlled by ErbB2 activity39 thus our observation that gefitinib impairs luminal proliferation of acini is consistent with an effect on signal transduction from the rat HER2/neu. Duct formation, which requires activity of the stromal EGFR and input from erbB-2 was not effected by gefitinib.

When put into this perspective, background lobule formation in a 14-week-old Tg−/− female is around 3–5 and the number of ducts formed by this age is 18. Typically, lobule formation is not induced until pregnancy or oncogenic activation. HER2/neu activation in the mammary gland epithelium of BALB-NeuT results in extensive lobular development and expansion. At 14 weeks of age control females have 35 ± 11 lobules compared with 22 ± 9 in animals treated with gefitinib (Table I). Furthermore, the background of hyperplasia that exists in 5-week-old Tg+/− animals is similar to LH that persists (at 14 weeks) in the gefitinib-treated animals that began therapy at 5 week of age.

Table I. Effects of Gefitinib and Timing on Extent of Mammary Gland Disease Burden
 ControlGefitinib 
5 WOA>6 WOA
  • Mammary gland disease in 14-week-old BALB-NeuT transgenic females treated with diluent or gefitinib beginning at 5 WOA (weeks of age) or initiated after 6 WOA.

  • 1

    Average size range of lobules measured in 10−2mm.

  • 2

    Significance levels for comparisons between the Control and 5 WOA treatment groups. N.S., not significant; (+++), extensive.

No. of lobules35.6 ± 11.422.4 ± 6.8243 ± 9.0p = 0.006
Size range16.9–370.84.2–33.64.5–77.5p = 0.002
No. of ducts19.2 ± 15.517.7 ± 9.615 ± 5.2N.S.
Lobule/duct ratio3.5 ± 2.91.6 ± 0.93.1 ± 1.2p = 0.073
Size of lobules    
 <0.1 mm10.2 ± 7.011.3 ± 4.621.5 ± 10.1 
 >0.1<0.4918.2 ± 7.310.6 ± 5.717 ± 4.1 
 >0.5<0.94.6 ± 5.803.3 ± 6.5 
 >1.0<2.03.8 ± 4.800.25 ± 0.5 
 >2.00.9 ± 1.300 
Invasion22%00 
Necrosis22% (+++)025% focal 

Microscopic analysis of mammary glands from animals indicated that silencing HER2 neu signaling during this progression period effectively eliminated lobular disease development (Table I). Gefitinib produced significant differences in lobular morphology, lobule size, lobule to duct ratio, disease burden and the incidence of necrosis and invasion (Table I). Morphological analysis of mammary glands gave the overall impression that the disease burden in control animals was LCIS or invasive carcinoma (Fig. 2c) while gefitinib-treated animals presented with LH (Fig. 2d). Lobular nodules that developed in the control case of LCIS ranged in size from 10 to 500 × 10−2mm (Fig. 2c) and in the gefitinib case of LH ranged in size from 3 to 25 × 10−2mm (Fig. 2d). With optimal treatment initiated at 5 weeks of age, we observed a 10-fold reduction in the size range of lobules and a 40% reduction in the total number of lobules present without effecting development of the (non-HER2) number of ducts. This produced an overall reduction in disease burden in the gefitinib-treated animals that began therapy at 5 weeks of age. In cases where treatment was delayed beyond 6 weeks of age, we noticed a marked reduction in disease control.

We found it critical to initiate therapy when animals were 5 weeks of age, starting treatment earlier or later had compromised treatment with toxicity or disease progression, respective. Data comparing the morphological profile of mammary glands from animals that started therapy at 5 weeks versus >6 weeks demonstrates a significant difference in lobule number and the lobule to duct ratio observed at the end of treatment (Table I). Lobule numbers increase rapidly during early hyperplasia. When treatment is delayed, the number of lobules increases but the vast majority remains small in size suggesting that proliferation is still impaired despite the preexisting background of LH.

The biodistribution of gefitinib in rat tissues (especially mammary and salivary glands) and tumor xenografts has been reported to be very high.40, 41 Therefore it is likely that a high dose of the active metabolite reaches the target tumor cells.40, 41 This is critical since the dose required to effectively impair proliferation is considerably higher in tumor cells relative to the normal epithelium. Fortunately, proliferation of (most) normal cells does not appear to be further compromised28 with these increased doses. In our hands, lower doses of gefitinib (<100 mg/kg) failed to effectively control disease in this model.

Furthermore, starting treatment earlier than 5 weeks of age led to the onset of side effects and weight loss during the last 2 weeks of treatment as well as evidence of mammary gland atrophy. When atrophy presented with other side effects we concluded this could have manifested from a direct or indirect toxic effect on normal cells. However, since the number of ducts in atrophic glands was identical to the controls it is more likely that atrophy and lobular fibrosis was secondary to the effect of gefitinib on tumor cell survival/apoptosis and remodeling of the stroma upon the elimination of disease.

It is known that the genetic program changes dramatically during tumor progression. In this model an angiogenic switch that occurs at 12 weeks of age has been described.7 Comparing the profiles of mammary glands from 6 and 15 week old animals shows differences in several gene clusters8 representing genes involved in transcription and tumor-stroma interactions. Since the genetic program of tumor cells evolves to balance growth and survival capabilities, the qualitative and quantitative aspects of HER2/neu signaling during initiation and progression may be different. Nevertheless, we observed that HER2/neu signaling was sensitive to gefitinib during the entire treatment interval. We compared the effects of gefitinib on HER2/neu phosphorylation and signaling in Atypical LH and LCIS and found gefitinib to be equally effective in silencing HER2/neu, ErbB-3, pp44/42 MAPK and Akt in these stages of disease. Disabling signal transduction from the ErbB-2/ErbB-3 oncogenic unit decreases lobuloaveolar proliferation and survival. Representative mammary gland lysates from animals treated for 2 week intervals, at 10–12 or 13–15 weeks of age are shown (Fig. 3). Although we are able to impair phosphorylation of growth and survival mediators when we treat with gefitinib during advanced hyperplasia and LCIS, disease progression continues, albeit at a slower pace giving rise to flat, poorly vascularized fibrotic nodules. Thus, it is distinctly possible, that decreased efficacy of gefitinib when therapy is initiated at later stages of disease, represents a threshold in the intrinsic resistance that evolves during progression.

Figure 3.

Sustained suppression of HER2/neu and HER3 phosphorylation in mammary hyperplasias and LCIS in animals treated with gefitinib. Western blot analysis of mammary tissue lysates from BALB-NeuT transgenic mice to detect changes in HER2/neu phosphorylation and signal transduction. Animals were treated with diluent (lane 1) or 100 mg/kg gefitinib po daily on a schedule of 5 days on 2 days off per week (lanes 2 and 3) for 2 weeks from 10 to 12 weeks of age (lanes 1 and 2) or 13–15 weeks of age (lane 3). Fifty micrograms of whole tissue lysate was resolved in 4–12% SDS-PAGE, transferred to PVDF membranes and probed for the indicated Phosphospecific antigens as described in the methods.

Studies by Panellini et al.42 demonstrate that timing is critical in preventing the development of HER2/neu acinic cell carcinomas (ACA) in the parotid glands of BALB-NeuT that coexpress the mutated p53. Optimal prevention is contingent upon the disease being dependent on HER2/neu, thus early intervention is required as tumors gradually become independent of HER2 as they progress. We have already demonstrated the differential effects of gefitinib on HER2/neu expressing ACA and lobular carcinomas in vitro. Although gefitinib was a potent cytostatic agent in ACA by silencing the MAPK pathway, cells had intrinsic resistance to gefitinib-mediated apoptosis and sustained signaling through the Akt survival pathway.5 Breast cancer cells, on the other hand were highly sensitive to gefitinib mediated growth inhibition and apoptosis.6

To determine the relative efficacy of gefitinib in these 2 target tissues at the time of early hyperplasia, we evaluated parotid glands and adjacent cervical mammary glands in control and treated animals (Fig. 4). Parotid gland tumorgenesis in BALB-NeuT has been previously described.30 Briefly, disease emerges from HER2/neu expression in the salivary gland epithelium in the ductal-acinar structures giving rise to multiple ductal hyperplasias that coalesce into confluent disease and the establishment of multifocal ACA. We found gefitinib to be extremely effective in inhibiting the expansion of focal areas of ductal hyperplasias and preventing the development of the extensive acinic cell carcinomas observed in control animals (Fig. 4). This is the first demonstration that gefitinib impairs ACA development in BALB-NeuT females in parallel with inhibiting LCIS (Fig. 4). Thus during hyperplasia, the cells destined to become ACA and LCIS are both sensitive to gefitinib and reflects a window of opportunity when the disease in both target tissues is dependent on HER2/neu signals for progression. The high level of distribution of gefitinib to the salivary glands40 may also contribute to it efficacy in parotid hyperplasias.

Figure 4.

Comparison of the effect of gefitinib on mammary and salivary gland disease progression in BALB-NeuT female transgenic mice. Histological analysis of parotid glands (a and b) cervical lymph nodes (c) and mammary glands (d) of BALB-NeuT transgenic female mice. Photomicrographs compare the control (left) and gefitinib (right)-treated animals. In panel (a) parotid glands stained for HER2/neu; the control animal developed extensive ACA with diffuse positivity for HER2, while the gefitinib-treated animal showed small focal ductal hyperplasia of limited cellularity with prominent (+++) membrane HER2 staining as indicated by the arrows. Photos were taken under ×10 objective. In panel (b) parotid glands from the same animals stained for IL-1β demonstrating infiltration of parotid hyperplasia by IL-1β positive cells in the control surrounding ducts involved with hyperplasia (arrows) and a lack of infiltrating cells in an area of hyperplastic cells in the gefitinib treated animal (asterisk). Photos were taken under ×20 objective. In panel (c) cervical lymph nodes from the same animals and stained for IL-1β demonstrate a grossly enlarged lymph node with strongly reactive zones (arrows) in the gefitinib-treated animal compared to a smaller relatively inert lymph node in the control animal Photos were taken under ×4 objective. In panel (d) HER2/neu staining of mammary gland tumor burden in no. 6 cervical fat pads adjacent to the salivary glands in these same animals. Disease burden in the control is an extensive expansion of lobular carcinoma into adipose tissue. Focal atypical lobular hyperplasia was detected in the gefitinib-treated animal and demonstrates intense membranous staining for HER2/neu. Photos were taken under ×10 objective.

There was an obvious difference in tumor architecture between the ductal hyperplasias from gefitinib-treated animals and the early ACA in the controls. Tumors in control animals reflect confluent disease that is associated with a prominent fibrovascular support and considerable lymphocytic infiltration (Figs. 4a and 4b). In gefitinib-treated animals, there is an overt lack of fibro-pseudocapsule formation and lymphocytic infiltration (Figs. 4a and 4b). We hypothesized that this could be due to impaired production of growth factors and cytokines. In vitro, gefitinib decreased production of the inflammatory cytokine, IL-1β in ACA tumor cells.5In vivo, we were unable to detect reproducible changes in tumor cell associated IL-1β but did observe a distinct difference in the number of IL-1β positive lymphocytes associated with hyperplastic acini (Fig. 4b) in control animals that were not present in gefitinib-treated animals (Fig. 4b). The cervical LN in these same animals were dramatically different (Fig. 4c). LN in gefitinib-treated animals was enlarged and exhibited intense, distinct staining of IL-1β positive centers (Fig. 4c). These centers were also highly reactive for PCNA (not shown), suggesting active proliferation. The mechanism(s) responsible for accumulation of IL-1β positive cells in the LN of gefitinib-treated animals and their conspicuous lack of association with early hyperplasias is not known. It is possible that these lymphocytes may be unable to exit the lymph node and enter the tumor bearing tissues or that the tumor cells are not sending “signals” to recruit these cells to the microenvironment. LN from control animals were smaller and relatively inert (Fig. 4c). This observation is not restricted to the cervical neck LN, as all LN appeared to be larger in gefitinib-treated animals. Tumor burden in the cervical mammary gland was also less in gefitinib treated (Fig. 4d) when compared with the confluent disease present in the control (Fig. 4d).

Tissue lysates from LN and spleens of gefitinib-treated animals showed robust constitutive MAPK phosphorylation, which was negligible in controls (not shown). In collaboration we determined that splenocytes from gefitinib-treated animals generated 176 ± 8 IL-5-producing and 113 ± 11 IL-17-producing cells/106 splenocytes in response to ConA by ELISPOT assay, compared with 79 ± 4 IL-5-producing and 24 ± 4 IL-17-producing cells in controls (p < 0.05). No significant difference in the relative distribution of CD4 and CD8 positive cells was observed. We hypothesize that one mechanism by which gefitinib may impair tumor progression is through the inhibition of tumor-stroma interactions. This inhibition may result from altered ability of tumor cells to secrete factors that recruit cells into the microenvironment since gefitinib is known to alter tumor cell production of VEGF, IL-8 and VEGF-D.6 Overall, our impression of acinic cell disease burden in gefitinib-treated animals is that the number and size of tumor nodules that form is markedly reduced. Furthermore, tumors that develop in gefitinib-treated animals are flat and distorted without the three dimensional sphere-like architecture of tumor nodules that develop in control animals. Interestingly, hyperplasias that persist following gefitinib treatment exhibit a high level of HER2/neu expression accentuating the membrane (Figs. 4a and 4d). We observe a similar phenomenon in vitro in both ACA and LCIS derived tumor cells, within 4 hr of treatment with 1 μM gefitinib, a stable (2–3-fold) increase in cell surface HER2/neu is detected. Thus, in this setting, it may be possible to benefit from the (gefitinib-activated) immune system by treating with Neu-specific antibodies or vaccination43 to eliminate the persistent “Hi” HER2/neu expressing disease.

Since continuous production of neoplastic stem cells is likely in this model,44 continuous administration of gefitinib is probably required to maintain control (suppress) disease progression. Although therapy may have lead to the elimination of some cells and inhibited further proliferation required for the branching morphogenesis of the mammary gland we suspect that chronic treatment would be required to control for emerging cells in this transgenic background. Indeed, others have shown that cessation of kinase-inhibitor therapies in similar models allows for cancer outgrowth.45 Nevertheless, chronic therapy at the doses required to prevent tumor cell proliferation in the BALB-NeuT model would lead to the manifestation of additional side effects and potentially, the emergence of gefitinib-resistant tumors.6 In the immunoprevention setting, the transcriptome of the hyperplasias that persist following effective vaccination is identical to that of naïve animals (6 weeks) prior to therapy.44 This would imply that the genetic program of tumor does not progress as specific cells may proliferate or die during therapy. Whether this is the case following gefitinib is not known. Our data suggest that gefitinib can be effective in preventing the progression of mammary gland and salivary gland hyperplasias if administered at the early stages of the disease. Thus targeting HER2/neu signal transduction during early hyperplasia is an effective means of disease control.

Acknowledgements

Dr. Marie P. Piechocki is a Research Scholar of the American Cancer Society. The authors thank Dr. Guido Forni, (Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy and Center for Experimental Research and Medical Studies, Ospedale San Giovanni Battista, Turin, Italy) for generously providing the Balb-NeuT transgenic mice.

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