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. 2004 Nov;6(6):846–853. doi: 10.1593/neo.04379

Phosphorylation of Both EGFR and ErbB2 Is a Reliable Predictor of Prostate Cancer Cell Proliferation in Response to EGF

Soha Salama El Sheikh *, Jan Domin †, Paul Abel ‡, Gordon Stamp *, El-Nasir Lalani *
PMCID: PMC1531689  PMID: 15720812

Abstract

Despite multiple reports of overexpression in prostate cancer (PC), the reliance of PC cells on activated epidermal growth factor receptor (EGFR) and its down-stream signaling to phosphoinositide 3′-kinase/Akt (PI3K/Akt/PTEN) and/or mitogen-activated protein kinase (MAPK/ERK) pathways has not been fully elucidated. In this study, we compared the role of EGF-mediated signaling in nonmalignant (BPH-1, PNT1A, and PNT1B) and PC cell lines (DU145, PC3, LNCaP, and CWR22Rv1). EGF-induced proliferation was observed in all EGFR-expressing PC cells except PC3, indicating that EGFR expression does not unequivocally trigger proliferation following EGF stimulation. ErbB2 recruitment potentiated EGF-induced signals and was associated with the most pronounced effects of EGF despite low EGFR expression. In this way, the sum of EGFR and ErbB2 receptor phosphorylation proved to be a more sensitive indicator of EGF-induced proliferation than quantification of the expression of either receptor alone. Both Akt and ERK were rapidly phosphorylated in response to EGF, with ERK phosphorylation being weakest in PC3 cells. Extrapolation of these findings to clinical PC suggests that assessment of phosphorylated EGFR + ErbB2 together could serve as a marker for sensitivity to anti-EGFR-targeted therapies.

Keywords: EGF, PI3K, Akt, MAPK, prostate cancer

Introduction

Growth factors and their receptors have received much attention as potential targets for the treatment of prostate cancer (PC) [1,2]. Epidermal growth factor (EGF) and the related transforming growth factor-α (TGF-α) are two autocrine/paracrine growth factors that have been identified in PC specimens and cell lines (reviewed in Refs. [3,4]). Their cognate receptor, the epidermal growth factor receptor (EGFR/ErbB1), is the most extensively studied member of the ErbB family, which includes ErbB2 (HER-2/Neu) [5], ErbB3 (HER-3) [6], and ErbB4 (HER-4) [7]. Many cells coexpress multiple ErbB receptors that homodimerize and heterodimerize upon stimulation with growth factor ligands [8].

Activated ErbB receptors trigger a number of important intracellular signaling pathways, including the phosphoinositide 3′-kinase (PI3K) and mitogen-activated protein kinase/extracellular-related kinase 1/2 (MAPK/ERK1/2) pathways. Class IA PI3K is recruited through an 85-kDa adapter subunit to phosphotyrosine residues on cytoplasmic domains of activated ErbB receptors [9], particularly ErbB3 [10]. The recently identified class II PI3K enzymes also lie downstream of EGFR, where they bind adaptor proteins through proline-rich regions present within their N-terminal sequences [11,12]. The 3′-phosphoinositide (PtdIns) products of class I and class II PI3K activity act as membrane-associated second messengers that bind and recruit a variety of cytosolic signaling enzymes to the cell membrane [9]. One of these, the serine/threonine kinase Akt, becomes phosphorylated at Thr308 and Ser473 residues, and promotes survival through multiple mechanisms [13]. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor-suppressor gene that downregulates Akt activity by acting as a PtdIns (3,4,5) P3 3-phosphatase [14]. The phosphorylation status of Akt (P-Akt) in vitro and in vivo has been widely used to monitor aspects of malignant behavior such as proliferation, resistance to chemotherapy, irradiation, invasion, and metastasis [15,16].

The MAPK cascade constitutes a functional signaling unit that links surface receptor-mediated signals to nuclear events affecting cellular processes of growth, division, differentiation, and death [17]. Phosphorylation of the most downstream elements, p44 and p42 MAPKs (also called ERK1 and ERK2), is a hallmark of MAPK activation and is increased in many human tumors including PC [18,19].

With the advent of novel therapies that specifically target ErbB receptors, there is a need to establish rational methods to stratify tumors that are dependent on these receptors for growth and survival. This would allow selective targeting of patients whose tumors were most sensitive, and identify those who might be resistant to these treatments. In this report, we used immortalized nonmalignant prostatic cells (BPH-1, PNT1A, and PNT1B) and PC cell lines (CWR22Rv1, LNCaP, DU145, and PC3) to identify molecular markers that would correlate best with an EGF-induced proliferative response. We compared cell proliferation, ErbB/PI3K/Akt/PTEN expression, ErbB receptor phosphorylation, PI3K/Akt, and MAPK enzyme activation in these cells under basal conditions and with EGF stimulation to ascertain potential differences between responsive and nonresponsive cells.

Materials and Methods

Cell Culture

BPH-1 [20], PNT1A, and PNT1B [21,22] were gifts from Dr. S. Hayward (Vanderbilt University School of Medicine) and Prof. N. Maitland (York, UK), respectively. PC cell lines CWR22Rv1 [23], LNCaP [24], DU145 [25], and PC3 [26] were obtained from the American Type Culture Collection (ATCC; Rockville, MD). BPH-1, PC3, LNCaP, and CWR22Rv1 cells were routinely maintained in RPMI 1640, whereas PNT1A, PNT1B, and DU145 were maintained in Dulbecco's modified Eagle's medium (DMEM). Media were supplemented with 10% fetal bovine serum (FBS) and cultures were grown at 37°C and 10% CO2.

Reagents and Antibodies

FBS was obtained from Gibco Europe Ltd. (Paisley, Scotland, UK). RPMI 1640 and DMEM culture media were obtained from Cancer Research UK (CRUK, London, UK). Recombinant human EGF was obtained from Peprotech (London, UK). The PI3K inhibitor, wortmannin, was purchased from Sigma-Aldrich Company Ltd. (Dorset, UK) and LY294002 from Calbiochem (CN Biosciences, Nottingham, UK). Antibodies to EGFR (sc-03), PTEN (sc-7974), and PY99 (sc-7020) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to c-ErbB2 (Ab-17), c-ErbB3 (Ab-7), and c-ErbB4 (Ab-4) were obtained from Neomarkers (Fremont, CA). Antibodies to EGFR (Ab-1 Mab-528) and c-ErbB4 (Ab-1) were obtained from Oncogene (Nottingham, UK). Anti-PI3K antibody (anti-p85) and antiphosphotyrosine antibody PY20 (610000) were obtained from BD Biosciences (Lexington, KY). Total Akt (9272) and Ser473-Akt (9271) were obtained from New England Biolaboratories (Beverly, MA). The PI3K-C2α and PI3K-C2β antisera were previously characterized [11,27]. Anti-phospho-MAPK was obtained from Promega (Southampton, UK). β-Actin antibody was obtained from Sigma-Aldrich Company Ltd.

Cell Proliferation Assay

Cells were dispersed by trypsin-EDTA, resuspended in serum-supplemented medium, and seeded into 24-well culture plates at a density of 5 x 103 cells/ml. Following an overnight incubation, cells were washed and treated with serum-free medium for 24 hours. At the end of this time, cells were either treated with fresh serum-free medium (control), or EGF was applied at ascending concentrations of (0.01–10 ng/ml) (day 0). Cell growth was monitored either by: 1) adding alamarBlue (Biosource, Nivelles, Belgium) at a concentration of 10% on day 0, assessed daily for the reduced product by reading fluorescence (Cytoflour2300; Millipore, Watford, UK) (emission filter 530; excitation filter 590); or 2) counting the cell number in triplicate cultures daily using a Coulter Counter (Luton, UK).

Western Blot (WB) Analysis and Immunoprecipitation (IP)

Asynchronous cultures were lysed in Triton X-100 lysis buffer (10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, and 1% Triton X-100) supplemented with protease inhibitors [5 mg/ml aprotinin, 1 mM pepstatin, 1 µg/ml antipain, 1 ng/ml leupeptin, and 100 mM phenylmethylsulfonyl fluoride (PMSF)] at 4°C for 20 minutes. Alternatively, cultures were serum-starved overnight then stimulated with EGF (100 ng/ml) for 10 minutes at 37°C, or pretreated with wortmannin (50 nM) then lysed as described above. Lysates were clarified by centrifugation (13,000g for 20 minutes at 4°C). Protein samples were either fractionated directly or immunoprecipitated for 3 hours at 4°C. Immune complexes were collected on protein A Sepharose beads for 1 hour (Amersham Pharmacia Biotechnology, Buckinghamshire, UK), washed in lysis buffer, and extracted in sample buffer [200 mM Tris-HCl, 6% sodium dodecyl sulfate (SDS), 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol, pH 6.8] then fractionated in an 8% to 10% gel. Proteins were electro-transferred to polyvinylidene difluoride (PVDF) membranes and Western-blotted with the optimally diluted primary antibodies. Horseradish peroxidase (HRP)-conjugated secondary antibodies (sheep antimouse/donkey antirabbit) (Amersham Pharmacia Biotechnology) and enhanced chemiluminescence (ECL; Amersham Pharmacia Biotechnology) were used to visualize immunoreactive proteins. Densitometric analysis of optimally exposed radiographic film using a high-resolution scanner and the UN-SCAN-IT gel analysis software (Version 5.1; Silk Scientific, Inc., Orem, UT) was used to quantify the relative band intensities. Results were expressed as average pixel values.

Statistical Analysis

Cell proliferation data were statistically analyzed using Student's t test. A value of P < .05 was considered as statistically significant. The GraphPad Prism statistical package was used to perform the analysis (version 3.02 for Windows; GraphPad Software, San Diego, CA).

Results

EGF-Induced Cell Proliferation

The difficulty in establishing primary PC cell lines from explants of prostate tissues has resulted in a scarcity of PC cell lines compared to the clinical prevalence of PC or the number of cell lines available for other solid tumors [23]. Our initial assessment of the effect of EGF on PC cell proliferation involved treatment of four widely available PC cell lines DU145, CWR22Rv1, LNCaP, and PC3 with ascending doses of EGF, and monitoring their proliferation over a 5-day period using alamarBlue assay. Addition of EGF (1–10 ng/ml) increased the proliferation of LNCaP, CWR22Rv1, and DU145 cells compared to untreated cells (Figure 1). This effect was observed earlier and at lower doses of EGF (0.1–10 ng/ml) in LNCaP cells. It was least notable in DU145 cells where an EGF-induced growth response was observed after 3 days of treatment with 10 ng/ml EGF compared to untreated cells. Growth of PC3 cells treated with up to 10 ng/ml EGF did not show any increase above the control. To confirm these observations and to analyze the effects of EGF on immortalized cell lines, cultures were treated with a concentration of 10 ng/ml EGF for 96 hours followed by trypsinization and counting. A significant increase in cell number was observed in BPH-1 (P = .01), DU145 (P = .01), LNCaP (P < .001), and CWR22Rv1 (P < .001), but not in PNT1A, PNT1B, or PC3 cells (P = 0.62, P = .45 and P = .386, respectively) (Figure 2). The greatest change in EGF-induced proliferation was seen in the LNCaP cells where proliferation nearly doubled compared to control.

Figure 1.

Figure 1

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Growth curve of PC cells following EGF stimulation. Serum-starved cultures incubated in the absence (-●-) or presence of ascending concentrations of EGF (-◯-, 0.01 ng/ml; -▲-, 0.1 ng/ml; -x-, 1 ng/ml; -□-, 10 ng/ml) were monitored daily by fluorometric measurement of the rate of reduction of alamarBlue for 5 days following treatment. Error bars represent standard error of the mean.

Figure 2.

Figure 2

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EGF-induced cell proliferation in prostate cell lines. Cells were serum-starved for 24 hours then treated with fresh serum-free medium or 10 ng/ml EGF. Cells were trypsinized and counted after 4 days of treatment. Changes in cell number in response to EGF were represented as fold increase over control. A two-fold change in cell number equals 2.0 fractional change, whereas 1 represents identity with control cells. Data obtained from three experiments are represented as mean with error bars representing standard error of the mean. *P < .05 was considered significant.

Expression of ErbB Receptors in PC Cell Lines

We then explored the relationship between EGF-induced mitogenesis and levels of ErbB receptor expressed as analyzed by Western blot analysis. Of the immortalized cell lines, only BPH-1 cells expressed high levels of EGFR. All expressed low levels of ErbB2 but none expressed ErbB3 or ErbB4 (Figure 3). Of the PC cell lines, only CWR22Rv1 expressed all four ErbB receptors, whereas LNCaP, DU145, and PC3 expressed EGFR, ErbB2, and ErbB3 but not ErbB4. Regarding ErbB3, high levels were observed in DU145 and CWR22Rv1 cells with markedly lower levels in PC3 and LNCaP (Figure 3). These data are consistent with our observation that EGF-induced proliferation only occurred in cells that express EGFR. Surprisingly, cellular levels of EGFR did not predict the magnitude of the proliferative response. For example, PC3 cells expressed more EGFR than LNCaP, and yet no difference in proliferation was observed between PC3 cells grown in the presence or absence of EGF. The data also indicate that high ErbB2 expression is not required for EGF-induced mitogenesis because BPH-1 cells, which expressed low levels of ErbB2, proliferated in response to EGF treatment (Figure 2).

Figure 3.

Figure 3

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Expression of ErbB receptors in prostate cell lines. Protein lysates from an equal number of cells were fractionated by SDS polyacrylamide gel electrophoresis (PAGE) and Western-blotted with indicated antibodies. Proteins were visualized with HRP-labeled secondary antibody and ECL detection. Equal loading was confirmed by blotting for β-actin.

EGF-Induced Receptor Dimerization and Phosphorylation

To identify indicators, besides the expression of EGFR that could favor the proliferation of one PC line and not another, we analyzed the degree of ErbB receptor dimerization and phosphorylation following EGF stimulation. Quiescent EGFR-expressing prostatic cells were stimulated with EGF, lysed, and immunoprecipitated using antibodies to EGFR, ErbB2, ErbB3, ErbB4, or antiphosphotyrosine (PY20) (Figure 4). Following EGF stimulation, EGFR became phosphorylated in each cell line. EGF-induced heterodimerization of EGFR and ErbB2 was consistently observed. Levels of phosphorylated ErbB2 were higher in LNCaP cells than those typically seen in other PC cell lines (data for CWR22Rv1 and LNCaP are shown in Figure 4). Unlike EGFR and ErbB2, no detectable levels of ErbB3 and ErbB4 tyrosine phosphorylation were observed under experimental conditions used in this study. In this way, ErbB3 and ErbB4 do not appear to be significant dimerization partners of the EGFR in prostatic cells.

Figure 4.

Figure 4

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EGF induces ErbB receptor phosphorylation. CWR22Rv1 (panel A) cells and (panel B) LNCaP cells were incubated in serum- free medium overnight then stimulated with 100 ng/ml EGF for 10 minutes at 37C. Cell lysates were prepared with protein A Sepharose beads in the absence (-) or presence of ErbB antibodies or PY20. The resultant immune complexes were fractionated by SDS-PAGE and Western-blotted. The pattern of ErbB phosphorylation shown in CWR22Rv1 (panel A) was typical of that seen in other EGFR-expressing PC cell lines (except LNCaP cells).

To compare receptor-mediated tyrosine phosphorylation across the different cell lines, EGF-stimulated cells (3 x 106 cells/ml) were lysed, immunoprecipitated, then Western-blotted using an antiphosphotyrosine antibody (Figure 5A). EGF had no effect on PNT1A cells (data not shown) but rapidly increased tyrosine phosphorylation of several proteins, most prominently those corresponding to the ErbB receptors (170–185 kDa). This phosphorylation was highest in BPH-1 and LNCaP, followed by DU145, and lowest in PC3 cells. Under starved conditions, an elevated phosphotyrosine content in BPH-1 and DU145 cells indicated that these cells were perhaps not rendered completely quiescent.

Figure 5.

Figure 5

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EGF induces strong tyrosine phosphorylation in responsive prostatic cells. Panel A: Confluent cultures of prostatic cells were serum-starved overnight then incubated in the absence (-) or presence (+) of EGF (100 ng/ml) for 10 minutes before lysates were prepared. These were immunoprecipitated with antiphosphotyrosine antibody (PY20) and the resultant immune complexes were fractionated by SDS-PAGE and Western-blotted using the antiphosphotyrosine antibody (PY99), EGFR (panel B), and ErbB2 (panel C). Radiographs shown in panels B and C were scanned and digitalized to semiquantify EGFR and ErbB2 phosphorylation (panel D).

To evaluate the relative amounts of tyrosine-phosphorylated EGFR and ErbB2 incorporated within this phosphotyrosine immune complex, the blot was reprobed for each receptor (Figure 5, B and C) and the data were scanned and quantified (Figure 5D). Marked EGFR phosphorylation was observed in BPH-1, whereas EGFR/ErbB2 heterodimerization was evident in DU145, LNCaP, and CWR22Rv1 cells. In PC3 cell lysates, EGFR and ErbB2 were hardly detectable in antiphosphotyrosine immunoprecipitates. Quantitatively, the total amount of phosphorylated EGFR + ErbB2 was highest in EGF-responsive cells. The values obtained with PC3 cells were at least less than one third of those seen with other cells. These data indicate that relative expression levels of EGFR do not correlate with the degree to which the individual ErbB receptors are activated in response to EGF, and that the sum of phosphorylated EGFR and phosphorylated ErbB2 is a more sensitive indicator of EGF-induced proliferation than total levels of EGFR expressed.

Expression and Activation of PI3K/Akt in PC Cell Lines

We first evaluated the expression of the class IA adaptor p85 and class II PI3K enzymes (PI3K-C2α and PI3K-C2β), Akt and PTEN. We observed that p85, PI3K-C2α, PI3K-C2β, and Akt were each expressed in all seven cell lines in equivalent amounts (Figure 6). PTEN was expressed in all cells except LNCaP and PC3. Reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis confirmed the expression of wild-type PTEN mRNA in BPH-1, PNT1A, PNT1B, DU145, and CWR22Rv1, and the previously reported PTEN mutation/deletion in LNCaP and PC3 cells [28,29] (data not shown).

Figure 6.

Figure 6

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Expression of PI3K/Akt/PTEN in prostate cell lines. Protein lysates from an equal number of cells were fractionated by SDS-PAGE and Westernblotted with indicated antibodies. Proteins were visualized with HRP-labeled secondary antibody and ECL detection. Equal loading was confirmed by blotting for β-actin.

To confirm that Akt activation is dependent on PI3K-generated phosphoinositides, we treated PTEN-expressing prostatic cell lines (BPH-1, PNT1A, DU145, and CWR22Rv1) with the PI3K inhibitor, wortmannin. Cultures were stimulated with EGF, and Akt phosphorylation was quantified in the resultant lysates using an anti-phospho-Ser473 Akt antibody. Wortmannin attenuated or abolished Akt phosphorylation when added simultaneously with or prior to EGF, respectively (Figure 7A). PNT1A cells, which do not express EGFR, showed no Akt phosphorylation in response to EGF (data not shown). This confirms that EGF-induced Akt phosphorylation is dependent on EGFR expression and PI3K activation in these cells.

Figure 7.

Figure 7

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Inhibition of PI3K/Akt signals in prostate cell lines. Panels A and B: Indicated cells were serum-starved overnight then incubated with medium (-) or wortmannin (W) (50 nM) for 20 minutes followed by EGF (E) or EGF + more wortmannin (EW). Lysates from stimulated cells were included as a positive control (P). Panel C LNCaP cells were treated with indicated doses of LY294002 followed by EGF stimulation (10 ng/ml). Wortmannin and LY294002 reduced Akt phosphorylation but had no effect on total Akt expression levels. Panel D: PC cells were serum-starved overnight then incubated in the absence (-) or presence of 10 ng/ml EGF (E) for 10 minutes at 37C. Lysates were probed for phosphorylated Akt, phosphorylated MAPK/ERK, and β-actin as a loading control.

She et al. [30] have recently reported that loss of PTEN function and overactive phosphoinositide signaling uncoupled EGFR and PI3K/Akt signaling pathways, making cells insensitive to extracellular stimuli. They also suggested that PI3K inhibition reestablishes EGFR-stimulated Akt activation by reducing constitutive basal activation of Akt. In PTEN-null PC3 and LNCaP cells, Akt phosphorylation was elevated under serum-starved (basal) conditions compared to PTEN-expressing CWR22Rv1 cells (Figure 7B). However, contrary to that study [30], wortmannin reduced constitutive Akt phosphorylation in LNCaP and PC3 cells and rendered them insensitive to further EGF stimulation similar to PTEN-expressing CWR22Rv1 cells. Another PI3K inhibitor, LY294002, also reduced Akt phosphorylation in a dose-dependent manner in LNCaP cells (Figure 7C). Taken together, these findings indicate that phosphorylated Akt is a robust indicator of PI3K activation in both PTEN-positive and PTEN-null PC cells.

We then analyzed the impact of ErbB expression and phosphorylation on PI3K/Akt activation. Despite variations in EGFR and PTEN expression, Akt phosphorylation in response to EGF (Figure 7D) was increased in each cell line compared to control cells, including PC3 cells, which do not proliferate in response to EGF. Cells that expressed more ErbB3 (CWR22Rv1 and DU145) did not show higher Akt phosphorylation compared to cells with lower ErbB3 expression (LNCaP and PC3). Taken together, our data indicate that the degree to which EGF stimulates the PI3K/Akt pathway is similar in prostate cell lines that express EGFR regardless of their derivation (i.e., immortalized or PC), level of either EGFR or ErbB3 expression, amplitude of EGF-induced ErbB phosphorylation, and the presence or absence of a functional PTEN.

p42/p44 MAPK Activation Correlates with ErbB Receptor Phosphorylation in PC Cells

From the above experiments, it became clear that early events of signal transduction (i.e., differences in ErbB receptor expression, phosphorylation, and dimerization) were more relevant physiologically to EGF-induced proliferative responses than PI3K/Akt activation. To analyze the role of an alternative signaling cascade, the MAPK pathway was examined using the phosphorylation of ERK as readout. PC cells were incubated in serum-free medium overnight before stimulation with EGF. Cells were then lysed, and proteins were fractionated and Western-blotted using an antibody against phosphorylated—and thus active—forms of MAPK/ERK1/2 (p44/p42). Consistent with a previous report [31], DU145 cells exhibited high basal ERK phosphorylation, which was not reduced by 24-hour serum withdrawal. EGF stimulation rapidly increased ERK phosphorylation in all PC cells compared to control cultures. This increase was more evident in DU145, CWR22Rv1, and LNCaP cells compared to PC3 cells, where phosphorylation was weaker, especially ERK2 (p42) (Figure 7D).

Discussion

Over the last decade, data have accumulated to suggest that aberrant expression of EGFR and ErbB2 was involved in the development of PC [32–37]. Although providing important prognostic information, these immunohistochemical studies often examine the expression of a single receptor and neglect the fact that up to four ErbB receptors could play a role in transducing the signaling response. They also do not address the influence of ErbB receptor expression/coexpression on the cellular response to a growth factor such as EGF and on downstream signaling pathways. In vitro models, which reflect the heterogeneity of PC in the clinical setting, have allowed us the opportunity to correlate cellular growth kinetics with specific activated biochemical pathways.

Consistent with the aforementioned reports, EGFR, ErbB2, and also ErbB3 were frequently coexpressed in PC cell lines compared to nonmalignant cells. Immortalized nonmalignant prostatic cell lines (BPH1 and PNT1A/PNT1B) may generally be considered equivalent to prostatic intraepithelial neoplasia, which could progress to PC under certain conditions [38]. PNT1A and PNT1B cells are thought to be derived from luminal prostatic epithelium [22] and were found to be EGFR-negative and insensitive to EGF stimulation. BPH-1 cells were originally reported to be of similar derivation, but they were found to express basal cell markers including EGFR (this report) and cytokeratin 14 [39]. This suggests that BPH-1 cells are derived from either basal or intermediate prostatic cells. Their strong EGF-induced responses could contribute to their transformation/malignant potential, which was demonstrated on exposure to carcinogenic doses of testosterone and estradiol or in PC-associated fibroblast coculture [38].

The relative abundance of ErbB receptors within a given cell type is likely to influence the nature and intensity of growth factor-induced signals and the signaling cascades they initiate. EGF was a mitogen for all prostatic cell lines (nonmalignant or malignant) that express EGFR with the exception of PC3 cells. The expression of either EGFR or ErbB2 alone failed to predict the magnitude of EGF-induced mitogenic responses, ErbB phosphorylation, or the degree of PI3K/Akt or MAPK activation. Tyrosine phosphorylation in serum-starved DU145 and BPH-1 cells may result from autocrine production of either TGF-α or EGF [40–42].

Equivalent levels of PI3K/Akt expression in prostatic cells (whether of benign or malignant origin) and loss of PTEN expression in two of four malignant PC cell lines suggest that the increased activity, rather than overexpression, of PI3K/Akt enzymes is important in PC development. Cells with an intact PTEN expressed higher levels of EGFR and ErbB3 than those with mutated PTEN, but when deprived of serum, the latter expressed increased levels of phosphorylated Akt. This suggests that, if PTEN is intact, elevated expression of the ErbB receptors alone is insufficient to activate the PI3K/Akt pathway under basal conditions. The absence of PTEN activity did not preclude a rise in Akt phosphorylation following stimulation. These findings denote that PTEN is the critical regulator of phosphorylated Akt under basal conditions. In cells lacking PTEN, other mechanisms (including other phosphoinositide 3′-phosphatases) [43–45] may be involved in downregulating acutely elevated PtdIns levels to allow signal amplification and propagation in response to new exogenous stimuli.

In PC3 cells, both Akt and MAPK were rapidly activated in spite of a low EGF-induced tyrosine phosphorylation. The absence of functional PTEN could allow phosphoinositide products of PI3K to accumulate after EGF stimulation and to amplify Akt phosphorylation signals. However, this was insufficient to drive PC3 cells into mitosis because they did not proliferate in response to EGF treatment. On the other hand, greater phosphorylation of ERK was observed in LNCaP cells, which are also PTEN-null but do proliferate in the presence of EGF. Activation of the MAPK pathway thus correlated well with EGF-induced phosphotyrosine and mitogenic responses in PC cells. If PTEN-null, phosphorylation of ERK becomes a more reliable marker of proliferative potential than Akt phosphorylation in these cells.

Establishing the relative contribution of individual members of the ErbB receptor family and activated downstream pathways in PC proliferation is far from complete. Our data support previous in vivo observations of upregulated expression of ErbB receptors in PC compared to nonmalignant prostate cells. They also suggest that, with appropriate controls, quantifying the sum of EGFR and ErbB2 phosphorylation could identify those PCs that grow in response to EGF and are likely to benefit from anti-EGFR-directed therapies. More work is required to assess the utility of quantifying receptor expression ratios and to establish standards and limits that associate ErbB receptor phosphorylation with mitogenesis.

Acknowledgements

S. El Sheikh is a receiver of the Overseas Research Students Award granted by the Committee of Vice Chancellors and Principals of the Universities of the United Kingdom. The authors thank Anil Chandrashekran for reviewing the manuscript and for his constructive comments.

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