Rat Prolactinoma cell growth regulation by Epidermal Growth Factor receptor ligands

Abstract

Epidermal growth factor (EGF) regulates pituitary development, hormone synthesis and cell proliferation. Although ErbB receptor family members are expressed in pituitary tumors, effects of EGF signaling on pituitary tumors are not known. Immunoprecipitation and Western blot confirmed EGFR and p185^c-neu protein expression in GH3 lacto-somatotroph but not in ACTH-secreting AtT20 pituitary tumor cells. EGF (5 nM) selectively enhanced baseline (~ 4-fold) and serum-induced (> 6-fold) PRL mRNA levels, while gefitinib, an EGFR antagonist, suppressed serum-induced cell proliferation and Pttg1 expression, blocked PRL gene expression, and reversed EGF-mediated somatotroph-lactotroph phenotype switching. Downstream EGFR signaling by ERK, but not PI3K or PKC, mediated the gefitinib-response. Tumors in athymic mice implanted sc with GH3 cells resulted in weight gain accompanied by increased serum PRL, GH and IGF-I levels. Gefitinib decreased tumor volumes and peripheral hormone levels by ~ 30% and restored normal mouse body weight patterns. Mice treated with gefitinib exhibited decreased tumor tissue ERK1/2 phosphorylation and downregulated tumor PRL and Pttg1 mRNA abundance. These results show that EGFR inhibition controls tumor growth and PRL secretion in experimental lacto-somatotroph tumors. EGFR inhibitors could therefore be useful for control of PRL secretion and tumor load in prolactinomas resistant to dopaminergic treatment, or for those prolactinomas undergoing rare malignant transformation.

INTRODUCTION

Pituitary adenomas account for ~ 15% of primary intracranial neoplasms and are discovered in up to 25% of unselected autopsy specimens. Despite their benign nature, tumor growth may lead to critical local compressive symptoms and altered hormone secretion leading to distinct endocrine syndromes, depending on the tumor cell type (1). Pituitary adenomas arise as clonal expansions of mutated somatic cells, but the sequence of initial transforming events are unclear and occur on a background of chromosomal instability, epigenetic alterations and mutations. Given the rich vascularisation of the gland, and tight hypothalamic control of hormone secretion, alterations within the pituitary microenvironment including paracrine or autocrine dysregulation and growth factor disruption may be permissive for accelerated growth (1).

Pituitary tumors arising from the lacto-somatotroph cell lineage which secrete growth hormone (GH) and or prolactin (PRL), usually respond to medical treatment with dopamine agonists and/or somatostatin analogues (1–3). Medical therapy may however be limited due to dopamine or somatostatin receptor resistance (2, 3), or drug intolerance. For ACTH-secreting and clinically nonfunctioning pituitary adenomas (NFPA), no effective drug therapies currently exist (4, 5). Success of transsphenoidal pituitary surgery is highly dependent upon the experience and expertise of the surgeon and the size and type of tumor. Radiotherapy, used after failed transsphenoidal surgery and or medical therapy, is associated with relatively high complication rates (2–6). Treatment alternatives are particularly required for recurring invasive macroadenomas and for infrequently encountered but aggressive pituitary carcinomas, which respond poorly to currently available therapies (7, 8).

Aberrant ErbB receptor activation is implicated in several human cancers, leading to the development of novel targeted therapeutics, including monoclonal antibodies and small compound tyrosine kinase inhibitors (9). Although ErbB receptors are expressed in pituitary adenomas (10, 11), and are particularly abundant in very rarely encountered pituitary carcinomas (12, 13), effects of EGFR signaling on pituitary tumors are unknown. EGFR inhibition in hormonally inactive pituitary folliculostellate cells resulted in reduced Pttg expression (14), a marker for pituitary tumor growth implicated in tumorigenesis and paracrine regulation (15).

Here we characterize EGFR-mediated pituitary signaling and report the impact of EGFR blockade on hormonally active pituitary tumor cells in vitro and in vivo. The results indicate that EGFR blockade controls experimental pituitary tumor growth and hormone secretion. EGFR inhibition could therefore serve as a molecular target for treating patients unresponsive to currently available forms of medical therapy.

MATERIALS AND METHODS

Materials

DMEM and RPMI media, fetal bovine serum, penicillin, streptomycin and amphotericin B were purchased from Invitrogen (Carlsbad, CA). EGF and NRG1-β1/HRG1-β1 were from Sigma (St. Louis, MO). GF109203X was purchased from Biomol (Plymouth Meeting, PA). Y-27632, LY294002, PP2, JAK inhibitor I, SB203580 and JNK inhibitor were from Calbiochem (San Diego, CA). U0126 was from Promega (Madison, WI) and gefitinib (iressa) was purchased from Biaffin GmbH & Co (Kassel, Germany).

Cell culture

AtT20 mouse corticotroph, GH3 rat lacto-somatotroph and MMQ lactotroph cells were purchased from American Type Culture Collection (ATCC). B104-1-1 cell line was a kind gift from Dr. Greene, University of Pennsylvania. After synchronization by serum starvation (GH3 cells in medium containing 0.2% BSA for ~ 24 hrs; AtT20 in 1% FBS medium for ~ 16 hrs), cells were plated in 100 mm dishes (~ 1.5 x 10⁶ cell density) or 6-well plates (~ 0.5 x 10⁶ cell density) and treatment agents were added with fresh serum-depleted medium (0.2% BSA) and samples collected at indicated times.

Templates for probes and Northern Blot Analysis

Probes for murine POMC, Pttg1, rat GH and PRL were generated as described (16–18). The β-actin probe was a 1.076 kb fragment of the mouse β-actin gene (Ambion, Austin, TX). RNA extraction was performed using TRIZOL^® reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For Northern blot analysis, 10 to 20 μg of total RNA were electrophoresed on a 1 % agarose, 6.4 % formaldehyde gel, transferred to a Hybond-N+^® membrane (Amersham, Arlington, IL) and UV-crosslinked. Probes were labeled with (α-³²P)CTP using the Prime-It^® Random Primer Labeling Kit (Stratagene, La Jolla, CA). Micro Bio-Spin^® Chromatography Columns (Bio-Rad, Hercules, CA) were used to purify probes. Membrane pre-hybridization and hybridization were performed using QuickHyb Solution^® (Stratagene) and then exposed to Hyperfilm^TM MP (Amersham) for 1 to 4 days at −70°C.

Quantitative PCR

Total RNA was extracted with Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions. The amount and the integrity of RNA were assessed by measurement of optical density at 260 and 280 nm. Before processing, RNA samples were treated with DNase I (Deoxyribonuclease I, Amplification Grade, Invitrogen) to eliminate genomic DNA contamination. Total RNA was reverse transcribed into first-strand cDNA using M-MLV Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. Q-PCR reactions were carried out in the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as previously described (19). Primer sequences (Invitrogen) for rat VEGF-A forward: 5’-TCCTGTGTGCCCCTAATG-3’, reverse: 5’-TGGCTTTGGTGAGGTTTG-3’, rat Pttg1 forward: 5’-CATAGGTGCTCTGGTCTCTGTTG-3’, reverse: 5’-GGCATTGAGGAAGGCTGGAAG -3’. Primer sequences for rat GH, PRL and β-actin have been described previously (19).

Immunoprecipitation and Western blotting

After completion of treatments, the cells were placed on ice and washed with cold PBS. For whole cell protein extraction, cells were lysed in 150 μl RIPA buffer (Sigma) containing complete protease inhibitor cocktail tablets (Roche Molecular Biochemicals). Lysates were centrifuged at 13,000 x g for 20 min at 4 °C and protein concentrations determined by Bradford’s method (Bio-Rad, Richmond, CA).

Immunoprecipitation (IP) with rabbit polyclonal anti-EGFR (3 μg; ab2430; Abcam, Cambridge, MA), polyclonal anti-Neu (C-18; sc-284) and anti-ErbB3 (sc-285) (2 μg; Santa Cruz Biotechnology, CA) was performed with an IP Kit (Roche Molecular Biochemicals) according to the manufacturer instructions. Cells cultured in 100 mm dishes were lysed in 1 ml Lysis buffer 1 containing protease and phosphatase inhibitor cocktail tablets (Roche Molecular Biochemicals). Pre-clearing was performed with agarose G beads (50 μl) overnight at 4ºC. IP with appropriate antibody titers was performed for 1 hr prior to addition of agarose G beads (50 μl) overnight at 4ºC. After extensive washes (2x wash buffer 1, 2x wash buffer 2 and 1x wash buffer 3 for 20 min each), samples were resuspended in 30 μl sample buffer (Invitrogen).

IP with monoclonal antibody 7.16.4 (20) (3 μg; a kind gift from Dr. Greene, University of Pennsylvania) which reacts specifically with rat p185 molecules was performed in cells lyzed with modified RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% NaDodSO4, 0.15M NaCl, 0.01M sodium phosphate pH 7.4, 2mM EDTA, 10mM sodium pyrophosphate, 400μM sodium orthovanadate) containing complete protease inhibitor cocktail tablets (Roche Molecular Biochemicals). Pre-clearing was performed with A/G PLUS-Agarose beads (20 μl; Sigma) overnight at 4ºC. IP with appropriate antibody titers was performed for 1 hr prior to addition of A/G PLUS-Agarose beads (20 μl) overnight at 4ºC. Immunoprecipitates were washed six times in washing buffer and resuspended in SDS sample buffer pH 6.8 as previously described (21).

Western blot analysis was performed according to the guidelines of NuPAGE^® electrophoresis system protocol (Invitrogen). In brief, whole cell lysates (~ 50 μg protein per lane) or IP samples were heated for 5 min at 100°C, respectively. Proteins were separated on NuPAGE^® 4–12% Bis-Tris gels and electro-transferred for 1 hr to PVDF (Invitrogen). Membranes were blocked for 1 hr in 2% nonfat dry milk (or 5% BSA) in TBS-T buffer, and incubated overnight with primary antibody. The following primary antibodies were used: mouse anti-pERK1/2, rabbit anti-ERK1/2 (1:200; Santa Cruz Biotechnology), mouse monoclonal anti-pTyr (PY99), rabbit polyclonal anti-EGFR (sc-03), anti-Neu, anti-ErbB3 (1:200; Santa Cruz Biotechnology), anti-pPDK1 (1:1000; Cell Signaling) and mouse anti-β-actin (1:1000; Sigma). After washing with TBS-T, membranes were incubated with peroxidase conjugated secondary antibody for 1.5 hrs (2% nonfat dry milk or 5% BSA in TBS-T buffer). Blots were washed and hybridization signals measured by ECL detection system (Amersham).

Flow cytometric cell cycle analysis

Treatments were added after synchronization in fresh serum-depleted medium and samples collected at indicated times. Cells were washed and fixed in 50% ice cold ethanol and cell cycle analysis was performed as previously described (14).

In vivo experiments

Female athymic NCR-NU mice (8–10 weeks of age) were purchased from Taconic Farms, Inc. (Hudson, NY, USA) and maintained in a laminar air-flow unit under aseptic conditions. The research protocol was approved and the care and treatment of experimental animals were in accordance with institutional guidelines. Mice were fed with a commercial pelleted diet and tap water ad libitum and were allowed to acclimatize for one week. For s.c. injections, rat GH3 cells (1.8 x 10⁶, 0.2 ml of suspension) were injected in both flanks of each mouse. When average tumor volumes reached 3–5 mm in diameter (day 1), mice were stratified by tumor volume into two groups of 12 mice (16 tumors per group). Although all 24 mice received two-sided injection of GH3 cells, in some cases tumor growth was not observed, so the groups were formed to contain equal numbers of mice harboring one tumor (8 mice per group) or two tumors (4 mice per group). Vehicle solution (1% Tween 80; 200 μl) vs. gefitinib (125 mg/kg) were administered via oral gavage daily (Monday through Friday) for a total of 14 weekday doses over 18 days. Maximum tolerated dose (MTD; 150 mg/kg) of gefitinib in NCR-NU mice has been determined previously (with similar administration schedules) (22, 23). A group of mice (n=12) with no injection of GH3 cells received vehicle solution per oral gavage daily to establish baseline values. Every 3 to 4 days mice were weighed and tumor volumes measured with a caliper and calculated using the formula, π/6 x large diameter x small diameter². On the last treatment day (day 18) within ~ 3h of drug administration, after determination of body weight and tumor volume, mice were euthanized; cardiac blood collected with 25G^5/8 syringes (Becton Dickinson) and primary tumors excised and weighed. Fragments of each tumor were fixed in formalin and embedded in paraffin for immunohistochemical staining, preserved in RNAlater solution (Qiagen) for subsequent RNA extraction and frozen in liquid nitrogen for subsequent protein extraction.

Immunofluorecence

Slides containing tissue sections were baked for 20 min at 60°C, washed in xylene, ethanol gradient (100%, 95%, 85%, 75%) and then ddH2O. For detection of EGFR expression, slides were placed into boiling EDTA (1mM, pH 8) and incubated in a steamer for 15 min (Black and Decker, Shelton, CT). Slides were blocked in 5% goat serum and then incubated with primary antibody overnight at 4°C. The following antibodies were used: Rabbit polyclonal anti-EGFR (1:50; ab2430; Abcam), guinea pig polyclonal anti-PRL (1:200; courtesy of Dr. Parlow, National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Following washes, slides were incubated with Alexa Fluor goat anti-rabbit 488 (H+L) secondary antibody (1:500; Invitrogen) for 1hr at RT. Nuclei were stained using 1:500 Topro-3 iodide 1mM solution (1:250 in PBS, Molecular Probes, Inc., Eugene, OR) for 15 min at RT, and following such, slides were mounted with Prolong Gold anti-fade reagent with DAPI (Molecular Probes, Inc). Confocal microscope images were obtained using a TCS-SP confocal scanner (Leica Microsystems, Mannheim, Germany). In order to detect contributions of autofluorescence in these paraffin embedded tissues, a spectral imaging approach was used. The confocal spectrophotometer was set to detect specific FITC fluorescence in the range from 505 to 540 nm. A second channel detecting autofluorescence with wavelength from 560 to 600 nm was used. Both channels were color coded and merged. Green color represents specific fluorescence from FITC and red color autofluorescence. The staining was strong and autofluorescence was very low in comparison to the specific signal. Only erythrocytes showed appreciable autofluorescence and appear dark orange in the images. A Leica PlanApo 20x 0.7 N.A. lens was used for overview images and a PlanApo 40x 1.2 N.A. for high magnification images.

Hormone assays

RIA for rat GH and PRL were performed in duplicate, using reagents provided by the National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases (Harbor-UCLA Medical Center, Torrance, California, USA). Iodination of GH and PRL (5 μg) with iodine-125 (500 μCi) (PerkinElmer Life & Analytical Sciences, Boston, MA, USA) mixed with 0.1 mg Iodo-Gen (Pierce, Rockford, IL) was performed using 10-ml columns prepared by G-75 Sephadex (Sigma Chemical Co.). Low interspecies cross-reactivity of the GH and PRL assays is shown in Fig. 5Bb. IGF-I was measured by RIA after acid-alcohol extraction using a kit specific for rat and mouse IGF-I (Diagnostic System Laboratories, Webster, Texas, USA). Thus in Fig. 5Bb, baseline IGF-I levels are also shown.

Fig. 5. Gefitinib attenuates lacto-somatotroph tumor growth and hormone secretion in vivo.

Subcutaneous inoculation of female athymic NCR-NU mice (8–10 weeks of age) with GH3 pituitary tumor cells (1.8 x 10⁶, 0.2 ml of suspension). When tumor volumes reached 3–5 mm in diameter (day 1), animals were stratified by tumor volume into two groups of 12 mice (16 tumors). Vehicle solution (1% Tween 80; 200 μl) vs. gefitinib (Gf; 125 mg/kg) were administered via oral gavage daily (Monday through Friday) for a total of 14 weekday doses over 18 treatment days. A group of mice (n=12) with no injection of GH3 pituitary tumor cells received vehicle solution per oral gavage daily to establish baseline values. Fold changes in tumor volume (Aa), average tumor volume (Ab) and tumor weight post mortem (Ac), average body weight (Ba) and serum GH, PRL, IGF-I (Bb) are shown.

C. Ex vivo fluorescent confocal microscopy images of tumor tissue formed by subcutaneous inoculation of GH3 cells in female athymic NCR-NU mice depicting EGFR expression in green (Alexa 488) and nucleic acid staining (TO-PRO-3) in blue. Ca, 20x magnification; Cb, 100x magnification of a peripheral area with strong EGFR expression; Cc, 100x magnification of marginal area depicting varying staining intensities; Cd, 100x magnification of central necrotic area with negative staining.

Statistical analysis

Cell cycle phases were analyzed by ModFit LT software (Version 2.0, Becton Dickinson). NIH Image 1.59 software was used for densitometric analysis of specific bands in Northern and Western blots and comparisons were evaluated using 2-tailed Student’s t test. Results are expressed as mean ± SE of independently performed experiments. Relationships between numerical variables were summarized by Pearson (for normally distributed variables) or Spearman (for non-normally distributed variables) correlation coefficients. Group differences in serum GH, PRL and IGF-1 levels and post-mortem tumor weights were assessed by Student’s t test (normally distributed variables) or the Wilcoxon rank sum test (non-normally distributed variables). Tumor volumes were transformed by cube roots for the purpose of linearization. Linear growth models (intercept and slope) were estimated for changes in tumor volume and weight across time, using maximum likelihood methods and a mixed models approach similar to repeated measures ANOVA. The covariance structure was estimated with an autoregressive model. Analyses were performed using SAS version 9.1. For all tests statistical significance was set at P<0.05.

RESULTS

EGFR expression and function in pituitary tumor cell lines

Immunoprecipitation (IP) with a specific EGFR antibody (ab2430) and subsequent immunoblotting (1005) revealed expression of the 170 kd EGFR in GH3 cells, whereas expression was not detected in MMQ and AtT20 pituitary cells (Fig. 1A). Similar to EGFR expression, IP and immunoblotting with Neu (C-18) antibody (sc-284) which detects ErbB2 and ErbB4, showed expression (~185 kd) of these related kinases in GH3 but not in MMQ or AtT20 cells (Fig. 1B). Mouse 3T3/A31 and human A431 cell lysate extracts served as positive immunoreactive controls (supplemental Fig. 1A).

Fig. 1. EGFR expression and time-dependent effects of EGF on hormone mRNA expression.

A and B. GH3, MMQ and AtT20 cells were serum-starved overnight and total protein extracted.

A. Immunoprecipitation (IP) was performed with an EGFR antibody (ab2430) and immunoblotting performed with a different EGFR antibody (sc-03).

B. IP and immunoblotting were performed with Neu (C-18) antibody (sc-284).

C. GH3 cells were serum-starved for 24 hrs and subsequently treated with EGF (5 nM). Total RNA was extracted at the times indicated and GH mRNA expression determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PRL and β-actin, respectively. The ratio of GH or PRL mRNA vs. β-actin mRNA was calculated by densitometric analysis of each treatment group. The GH / β-actin ratio and PRL / β-actin ratio of the control groups at 24 and 48 hrs were set as 1.0. Relative mRNA expression levels were normalized to these control groups. Relative mRNA expression levels (mean ± SE) (upper panel). *, p<0.05; ***, p<0.001. Experiment shown is representative of three independently performed experiments (lower panel).

In lacto-somatotroph GH3 cells, EGF treatment (5 nM) potently enhanced both baseline and serum-induced PRL mRNA abundance (Fig. 1C). EGF induced PRL mRNA gene expression ~ 4.1-fold (p<0.05) at 48 hrs. Serum alone also induced PRL expression ~ 3.2-fold, and co-treatment with serum and added EGF further enhanced this effect to > 6-fold (Fig. 1C). EGF modestly attenuated GH mRNA expression (Fig. 1C), and the growth factor did not induce S phase entry (supplemental Fig. 1B). In contrast to the observed effects in GH3 cells, EGF treatment for up to 48 hrs did not alter AtT20 cell POMC gene expression (not shown).

Effect of gefitinib on pituitary tumor cell proliferation and gene expression

Treatment with increasing concentrations of gefitinib (0.1 – 10 μM) dose-dependently attenuated serum-induced S phase entry in GH3 cells (Fig. 2B) and suppressed serum-induced Pttg1 mRNA expression (Fig. 2A). Gefitinib treatment modestly stimulated baseline and serum-induced GH, and inhibited PRL mRNA expression (Fig. 2A).

Fig. 2. Dose-dependent effects of gefitinib on GH, PRL and Pttg1 mRNA expression and cell proliferation.

A-D. GH3 cells were serum-starved for 24 hrs and pre-treated with gefitinib (Gf; 45 min) at indicated concentrations prior to induction.

A. Induction with serum (15% horse serum, 2.5% FBS). Total RNA was extracted at 48 hrs and indicated target mRNA expression determined as for Fig. 1C. A representative experiment of three independently performed experiments is shown (lower panel).

B. 22 hrs after induction with serum, cells were fixed and cell cycle analysis performed by flow cytometry. Percentage of cells in G0/1 phase is depicted in black bars, cells in S phase in white bars and cells in G2/M phase of the cell cycle in grey bars.

C. After overnight serum starvation, GH3 or AtT20 cells were pre-treated with gefitinib and subsequently cultured in the presence of complete medium for 4 days. Cell counts were performed with a hemocytometer.

D. Induction with EGF (5 nM). Total RNA was extracted at 48 hrs and indicated target mRNA expression determined as for Fig. 1C. *, p<0.05; **, p<0.01. A representative experiment of three independently performed experiments is shown (lower panel).

In contrast to GH3 cells, the percentage of AtT20 cells in S phase as well as serum-induced Pttg1 mRNA expression (>2.5-fold) was unaffected by increasing concentrations of gefitinib (0.1 – 10 μM; not shown). Gefitinib also did not alter POMC mRNA expression at 24 and 48 hrs in AtT20 cells (not shown).

Dose-dependent effects of gefitinib on GH3 and AtT20 cell proliferation are shown in Fig. 3C. Cells were incubated for 4 days with increasing doses of gefitinib. Doses as low as 0.1 μM led to a ~15 % (p<0.001) attenuation of GH3 cell number, with a dose-dependent decrease of ~ 72% (p<0.001) at 10 μM. AtT20 cell numbers were not altered at these doses.

Fig. 3. ErbB family expression and cell proliferation signaling pathways in GH3 cells.

A and B: GH3 cells were serum-starved overnight prior to stimulation.

Aa, EGF (5 nM) time course; Ab, pre-treatment with gefitinib (Gf) (dose response) prior to induction with EGF (5 nM) for 5 min; Western blot analysis was performed with a monoclonal pTyr (PY99) antibody. Subsequently, membranes were stripped and re-blotted with β-actin antibody as a loading control. The arrows indicate the approximate position of ErbB family receptors.

Ba-c. GH3 cells were pre-treated with gefitinib (Gf; 1 μM) for 45 min prior to induction with EGF (5 nM) for 10 min. Bb. B104-1-1 cells were serum-starved overnight and treated with EGF (5 nM) as indicated. Bd. Serum-starved GH3 cells were treated with EGF (5 nM) or HRG (6 nM) for 10 min. Immunoprecipitations were performed with EGFR, p185 (Ab 7.16.4) and ErbB3 antibodies as indicated. Immunoblotting was performed with pTyr antibody (upper panels). Subsequently, membranes were stripped with mild stripping buffer and re-blotted with EGFR, Neu and ErbB3 antibodies (lower panels).

C. GH3 cells were serum-starved for 24 hrs and pre-treated with gefitinib (7.5 μM) and/or U0126 (5μM), LY294002 (5 μM), GF109203X (1 μM) or H89 (1 μM) for 30 min prior to induction with serum (15% HS, 2.5% FBS). 22 hrs after induction with serum, cells were fixed and cell cycle analysis performed by flow cytometry. The amount of serum-treated cells in G0/1 phase (control) was set as 1. Relative levels of cells in G0/1 phase was normalized to this control group. Shown is –fold G0/1 phase induction in the treatment groups (mean ± SE) of five independently performed experiments. *, p<0.05; **, p<0.01; ***, p<0.001.

The observed EGF-induced lactotroph phenotype in GH3 cells (Fig. 1C) was reversed by pre-treating cells with gefitinib prior to induction with EGF (Fig. 2D). Dose-dependent effects of gefitinib on the EGF-mediated lacto-somatotroph phenotype were evident at concentrations (5 – 10 μM) which induced GH mRNA levels, whereas the lowest concentration tested (0.1 μM) selectively abrogated EGF-induced PRL gene expression.

ErbB family member expression in GH3 cells

Induction of GH3 cells with EGF (5 nM; Fig. 3Aa) or serum (not shown) rapidly (within 5 min) induced tyrosine phosphorylation of several proteins sized > 160, ~95, ~70 and ~ 40 kd. As shown in Fig. 3A, proteins sized ~170–180 kd were most prominently phosphorylated, corresponding to the approximate size of EGFR and related receptor members. EGF but not serum also induced tyrosine phosphorylation of ~55 kd-sized proteins, while phosphorylation by serum was more sustained (> 2 hrs, not shown), as compared to the transient EGF effects (5 – 15 min). The dose-dependency of gefitinib on EGF-induced tyrosine phosphorylation is shown in Fig. 3Ab.

Activation of ErbB receptor members was confirmed by immunoprecipitation with receptor-specific antibodies (Fig. 3B). EGF (5 nM) induced tyrosine phosphorylation of EGFR which was blocked by gefitinib pre-treatment (1 μM) (Fig. 3Ba). B104-1-1 murine fibroblasts transformed by constitutively active oncogenic rat neu variant p185^neu were used as a positive control for immunoprecipitation with the monoclonal antibody 7.16.4 (Fig. 3Bb). In contrast to B104-1-1 cells, GH3 cell neu tyrosine phosphorylation was only observed in response to ligand activation, suggesting that the cells express the proto-oncogenic cellular homologue p185^c-neu (Fig. 3Bb). Potent tyrosine phosphorylation of p185^c-neu was observed both in response to EGF (an EGFR receptor ligand) and HRG (6 nM; an ErbB3/4 ligand) (Fig. 3Bd), indicating p185^c-neu as the preferred ErbB family heterodimerization partner in this cell line. Gefitinib pre-treatment (1 μM) abrogated EGF-induced p185^c-neu activation (Fig. 3Bb). Potent ErbB3 tyrosine phosphorylation was detected in response to the receptor ligand HRG (Fig. 3Bd), whereas low levels of ErbB3 tyrosine phosphorylation were occasionally detected in response to EGF (shown in Fig. 3Bd), suggesting modest ligand-induced EGFR/ErbB3 heterodimer formation.

GH3 cell proliferation signaling pathways

To identify signaling pathways involved in GH3 cell proliferation, we blocked key signaling molecules prior to serum-induced release into the cell cycle. Inhibitors were screened at concentrations of 10 μM (not shown). Blockade of Rho-, Src- and JAK tyrosine kinases by Y-27632, PP2 or JAK inhibitor I, did not alter serum-induced cell proliferation. However, inhibition of MEK, PI3K and PKC with U0126, LY294002 and GF109203X, suppressed the number of cells entering S phase.

To analyze mechanisms for gefitinib-mediated inhibition of pituitary cell proliferation, the drug was tested together with specific pathway inhibitors (Fig. 3C). For these experiments gefitinib was employed at doses of 7.5 μM and the lowest active concentrations of U0126, LY294002 (5 μM) and GF109203X (1 μM) were used as derived from dose-response experiments (not shown).

Gefitinib inhibited serum-induced cell proliferation (induction of G0/1 phase vs. serum alone) ~ 1.2-fold (p<0.001). U0126, GF109203X or LY294002 also inhibited serum-induced cell proliferation ~ 1.1 (p<0.05), 1.2 (p<0.01) and 1.1 (p<0.001)-fold, respectively (Fig. 3C). Co-treatment with gefitinib and the aforementioned signaling blockers prior to serum induction led to further attenuation of cell proliferation. Results of combined EGFR and ERK or PI3K inhibition were within the expected additive range of single agent treatments, whereas greater-than-additive effects were observed for combined EGFR and PKC signaling blockade.

Effects of gefitinib on EGF and serum-induced ERK and PDK1 signaling

The time-dependent phosphorylation of EGFR, and subsequent ERK1/2 activation are depicted in Fig. 4. Similar to results observed in the total phospho-tyrosine blots (Fig. 3A), EGF-mediated ERK activation appeared to be transient, lasting for up to 60 minutes (Fig. 4A), whereas serum induced a more sustained effect (up to 4 hrs; Fig. 4C), and also modestly stimulated PDK1 phosphorylation (peaking at 5 to 10 min; Fig. 4C). Pre-treatment with gefitinib dose-dependently suppressed EGF-induced EGFR phosphorylation (Fig. 4B) and blocked both EGF and serum-induced ERK phosphorylation with a similar potency (Fig. 4B and D). EGF did not induce ERK phosphorylation in AtT20 cells (not shown), consistent with no detectable EGFR expression (Fig. 1A) and gefitinib did not alter serum-induced PDK1 phosphorylation (Fig. 4D).

Fig. 4. Effects of EGF or serum and gefitinib on EGFR, ERK and PDK1 activation.

GH3 cells were serum-starved overnight, and treated with EGF (5 nM; A) or serum (15% horse serum, 2.5% FBS; C) for indicated times or pre-treated with gefitinib (Gf) (indicated concentrations) prior to induction with EGF (B) or serum (D) for 10 min. Immunoprecipitation (IP) was performed with an EGFR antibody (ab2430) and immunoblotting was performed with pTyr antibody (first panels; A + B). Subsequently, membranes were stripped and re-blotted with EGFR (sc-03) antibody (second panels; A + B). ERK1/2 phosphorylation, total ERK1/2 load, PDK1 phosphorylation and total β-actin load as detected by Western blot in total protein extracts are shown (A – D). A representative experiment of 3 independently performed experiments is shown.

Effects of EGFR blockade on lacto-somatotroph tumor growth and hormone expression

To evaluate in vivo effects of gefitinib on lacto-somatotroph tumor growth and hormone regulation, GH3 cells were implanted subcutaneously into female athymic NCR-NU mice. In vehicle-treated animals, relative tumor volume increased progressively achieving 26-fold change from baseline at 18 days. In gefitinib-treated animals, relative tumor volume was attenuated by ~ 50% overall (Fig. 5Aa). Gefitinib treatment reduced absolute tumor volume from 865 ± 94 mm³ to 580 ± 110 mm³ vs. control by day 18 (Fig. 5Ab). Tumor volume correlated with post mortem tumor weight at day 18 (0.88, p<0.001). Analysis of all tumors (n=31) showed estimated slope values of 0.33 and 0.25, respectively, i.e. slower growth rate in the drug-treated group (p<0.05).

Vehicle-treated animals harboring tumors gained weight (~ 18%) as compared to age-matched mice not injected with tumor cells (slope for mean weight increase per day 0.27; Fig. 5Ba). Gefitinib treatment slowed the excessive weight gain, starting at day 8 and peaking at the end of the experiment (slope value 0.04; p<0.001). Differences in body weight (> 4 g) could not be ascribed to differences in tumor weight (~ 50 mg). Although anorexic effects of gefitinib cannot be excluded, at MTD (150 mg/kg) in NCR-NU mice, weight loss does not exceed 10% (22, 23) and weight loss was not observed compared to vehicle-treated animals not harboring tumors. Gefitinib treatment decreased serum GH, PRL and IGF-I levels to 74% (ns), 75% (p=0.036) and 66% (p=0.011), respectively (Fig. 5Bb).

Immunohistochemical analysis confirmed both EGFR and PRL expression in tumor tissue (Fig. 5C and 6B). EGFR immunoreactivity was especially prominent in peripheral areas of the tumor (Fig. 5Ca and b). Heterogeneous EGFR staining was also observed in tumor cells within the solid core area (Fig. 5Ca and c), while central necrotic tissue was not immunoreactive for EGFR (Fig. 5Cd), serving as an internal negative control.

Fig. 6. Tumor ERK phosphorylation, GH, PRL and tumor marker gene expression.

Aa, ERK1/2 phosphorylation and total ERK1/2 load as detected by Western blot are shown. Ab, the ratio of pERK vs. total ERK protein expression was calculated by densitometric analysis of each group. The pERK / ERK ratio of the vehicle group was set as 100%. Relative protein expression levels were normalized to this control group. Ac, Q-PCR analysis for GH, PRL, VEGF-A and Pttg1 mRNA expression in vehicle vs. gefitinib-treated groups is shown (n=12). Control values were set as 1.0 and relative mRNA expression levels were normalized to these control groups. Ad, GH mRNA expression was determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PRL and β-actin, respectively. *, p<0.05.

B. Ex vivo fluorescent confocal microscopy images of PRL expression in green (Alexa 488) and nucleic acid staining (TO-PRO-3) in blue. Ba, normal mouse pituitary; Bb, control tumor tissue; Bc, gefitinib-treated tumor tissue; 20x magnification.

Tumor protein and RNA samples were paired in vehicle and gefitinib-treated animals according to tumor volumes. Western blot of tumor protein samples revealed ~ 23% (p<0.05) decreased tumor ERK1/2 phosphorylation from gefitinib-treated animals (n=16; Fig. 6Aa and b). Quantitative (Q)-PCR also showed decreased PRL mRNA levels in 4/6 tumors, and decreased Pttg1 mRNA expression in 5/6 volume-matched paired tumor samples. Overall, intra-tumoral PRL and Pttg1 gene expression decreased by 35% (n.s.) and 37% (p<0.05), respectively (Fig. 6Ac). These results were confirmed by Northern blot of paired tumor extracts with sufficient RNA available (Fig. 6Ad). Intra-tumoral GH and VEGF-A mRNA levels assessed by Q-PCR (Fig. 6Ac) were not altered by treatment. In contrast, PRL immunoreactivity was suppressed in tumor cells after drug treatment (Fig. 6B).

DISCUSSION

EGFR expression has been detected in normal anterior pituitary hormone-secreting cells (24), and EGF induces cell proliferation of non-tumorous pituitary cells (25, 26). EGF induces lactotroph differentiation (27, 28) and targeted expression of a dominant negative EGFR in transgenic mice blocks both somatotroph and lactotroph development (29). Pituitary TGFα, an EGFR ligand, is up-regulated prior to development of estrogen-mediated lactotroph hyperplasia in rats (30) and lactotroph TGFα overexpression in transgenic mice results in hyperplasia and adenoma formation (31), suggesting a role for EGFR in lactotroph tumorigenesis. Varying levels of EGFR binding have been reported in human pituitary tumors (10, 32–34), particularly in invasive adenomas (34) or carcinomas (13).

Immunoprecipitation and Western blotting confirmed that EGFR protein is expressed in lacto-somatotroph GH3 cells (35). As neither MMQ or AtT20 corticotroph cells express EGFR family members (Fig. 1A and B), the non-responsiveness of these cell lines to tested ligands and drug was not surprising. In contrast, our results confirmed EGF-mediated induction of the lactotroph phenotype in GH3 cells, with marked induction of PRL transcription. The specificity of EGFR signaling was demonstrated by suppression of EGF-mediated PRL induction by gefitinib (as low as 0.1 μM). EGFR inhibition with gefitinib also reversed serum-induced lactotroph differentiation and cell proliferation, indicating a role for EGFR signaling in the mitogenic response. These results suggested that additional serum factors may lead to EGFR activation, which in turn is required for PRL gene transcription and induction of cell proliferation.

ErbB (HER) family receptor overexpression, ligand binding or oncogenic mutations lead to homo- or heterodimerization and subsequent induction of kinase activity. Several EGFR, ErbB3 and ErbB4 ligands have been identified, and ErbB2 is the preferred dimerization partner. The specific pattern of dimer formation determines specificity of downstream signaling (for review see) (9). Monoclonal antibodies directed against extracellular domains of ErbB receptors as well as small molecule tyrosine kinase inhibitors allow targeted inhibition of tumor ErbB receptor signaling (9).

As ErbB receptors are expressed in rat GH3 cells (Fig. 1A and B), we examined mechanisms for ErbB family expression and activation. The rat neu oncogene encodes p185^neu with intrinsic tyrosine kinase activity, which differs from its cellular homologue p185^c-neu by a single transmembrane amino acid substitution (Val-664 → Glu), resulting in constitutive activity of the intrinsic kinase (36). With the use of the monoclonal antibody 7.16.4, which reacts specifically with rat p185 molecules, we observed tyrosine phosphorylation of the precipitated receptor only after ligand induction, suggesting expression of proto-oncogenic cellular p185^c-neu in GH3 cells, compared to oncogenic and constitutively active p185^neu expression in B104-1-1 controls (Fig. 3B). Gefitinib prevented EGF-induced tyrosine phosphorylation of EGFR and p185^c-neu. In addition to activation of ErbB3, induction with the ErbB3 ligand heregulin also activated p185^c-neu, indicating p185^c-neu as the preferred dimerization partner in GH3 cells. However, the occasional detection of low level ErbB3 tyrosine phosphorylation in response to EGF (an EGFR ligand), also suggests the potential of EGFR/ErbB3 heterodimer formation in GH3 cells.

EGFR signaling involves diverse cell-specific intracellular pathways and patterns of dimer formation (9), and we show that EGFR-related pathways activated by serum-induced GH3 cell proliferation include ERK, PI3K and PKC. Combined treatment with gefitinib and the PKC inhibitor GF109203X resulted in marked induction of the G0/1 phase (Fig. 3C), suggesting that antiproliferative effects of EGFR and PKC inhibition are mediated by blockade of different signaling molecules. However, given the heterogeneity of PKC signaling (37) and the broad PKC isoform blockade achieved, specific involvement of particular PKC isoforms or novel isoform members cannot be excluded, as described for EGF-mediated regulation of the PRL promoter in GH4/GH4C1 cells (38).

As the results of combined EGFR and ERK or PI3K inhibition were within the expected additive range of monotherapies (Fig. 3C), we examined direct effects of EGF and serum on EGFR / ERK and PDK1 (which mediates PI3K-dependent Akt phosphorylation (39)) activation. Both EGF and serum induced rapid ERK phosphorylation, whereas serum activated PDK1, with sustained ERK activation, similar to patterns observed for tyrosine phosphorylation. Thus, transient EGFR-mediated ERK activation may be sufficient for induction of PRL gene expression, however, sustained ERK phosphorylation and associated nuclear accumulation (40–42) as well as stimulation of other signaling pathways such as PI3K are necessary for serum induction of pituitary cell mitogenesis. The duration and strength of ERK activation elicits different cellular growth responses i.e. proliferation versus differentiation (43). Concentrations of gefitinib as low as 0.1 μM effectively suppressed EGFR and ERK phosphorylation, but not PDK1. Thus, the results shown here suggest that the blockade of EGFR / ERK, and not PI3K signaling mediate gefitinib inhibition of GH3 cell proliferation.

Injection of nude mice with GH3 cells resulted in formation of large tumors and increased circulating serum PRL, GH and IGF-I levels. Vehicle-treated animals harboring tumors demonstrated increased weight gain during the course of the experiment, likely due to excessive GH and IGF-I levels. Treatment with gefitinib decreased tumor volume, serum hormone levels and an attenuated weight curve comparable to that of non-tumor bearing controls. In vivo antiproliferative activity of gefitinib was evidenced by lowered slope values for mean tumor volume increase in treated animals, and reduced Pttg mRNA expression in ex vivo tumor tissue. Failure of EGFR inhibition to completely block growth of already established tumors can be ascribed to dose-related tumor drug penetration, heterogeneity of EGFR expression, and possible receptor down-regulation in central tumor areas with high cell density, as shown in Fig. 5C. Similar to the in vitro results, ex vivo analysis showed down-regulation of PRL but not GH mRNA expression. Thus, reduced peripheral tumor GH/IGF-I levels was likely the result of tumor load reduction, whereas serum PRL reduction likely occurred consequent to intra-tumor PRL gene and protein suppression. In vivo involvement of EGFR / ERK signaling was confirmed by demonstrating reduction of ERK activity in treated tumor samples.

The results shown here suggest that EGFR inhibition could be useful for treating a subset of aggressive pituitary tumors with established EGFR expression, even if expression levels are relatively low, as EGFR expression levels do not necessarily predict gefitinib sensitivity (22, 44, 45). The results highlight the potential for screening post-operative pituitary tumor specimens for ErbB family expression. Inhibition of EGFR homo- and / or heterodimerization may block direct mitogenic signals by ErbB ligands or inhibit ErbB receptor trans-activation by other stimuli (46, 47) required for induction of cell proliferation. In addition to the beneficial effect of tumor load reduction, potent inhibition of EGFR-mediated PRL gene expression could abrogate dramatic PRL elevations seen in patients with malignant prolactinomas. These infrequent neoplasms usually exhibit a slowly progressive malignant phenotype with metastasis, while resisting most forms of conventional therapy including dopamine agonists, surgery and radiation (7, 8). In addition to recent reports using the alkylating compound temozolomide (48, 49), or new generation PRL receptor antagonists (50), targeted EGFR inhibition could represent an alternative treatment for patients with EGFR-expressing malignant prolactinomas.