Expression and activation of erbB-2 and epidermal growth factor receptor in lung adenocarcinomas.

ErbB-2 and EGFR (epidermal growth factor receptor) are expressed in lung adenocarcinomas and associated with a poor prognosis. Immunocytochemical analysis revealed erbB-2 and EGFR coexperession as a characteristic feature of most lung adenocarcinomas, and at levels of receptor expression present in bronchial epithelial cells. In primary lung tumours and cell lines, erbB-2 detected using Western blot analysis demonstrated low-level phosphotyrosine staining of the 185 kDa band, as compared with breast cancer cell lines. A549 and A427 lung adenocarcinoma cells treated with neu differentiation factor (NDF) showed increased erbB-2 phosphotyrosine staining, but to a much lesser extent than breast cancer cells. The lung cells were examined for expression of the potential autocrine growth factors NDF and transforming growth factor alpha (TGF-alpha) by Northern blot analysis. Both NDF and TFG-alpha mRNA were abundantly expressed in the A549 cells. NDF mRNA was highest during active cell proliferation and decreased in confluent cells or after treatment with the growth-inhibitory steroid dexamethasone. Primary tumours and cell lines expressed EGFR, showing higher basal level phosphotyrosine staining than erbB-2. Treatment with NDF and EGF (epidermal growth factor) stimulated cell growth, and in A549 cells the presence of both factors provided an additive increase in cell growth. The growth stimulus that ligand-activated erbB-2 and EGFR provides to lung adenocarcinoma cells may establish a background of continued cell proliferation over which other critical transforming events may occur. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5

ErbB-2 and EGFR are homologous membrane-bound receptors with tvrosine kinase activity which are expressed in many fetal and adult epithelia and are thought to play a role in the growth and differentiation of these normal tissues (Press et al.. 1990: Suda et alt. 1990Madtes, 1993). Overexpression of erbB-2 and EGFR has been identified in a variety of epithelial tumours and has been extensively studied in breast and lung cancer. In lung adenocarcinomas, erbB-2 overexpression relative to normal alveolar lung tissue has been found to correlate with a worse prognosis . EGFR overexpression in conjunction with autocrine ligand expression may also be associated with a worse prognosis in lung adenocarcinoma (Tateishi et al.. 1990: Diattadi et al.. 1991: Pavelic et al., 1993: Veale et al.. 1993. The association between overexpression of erbB-2 and EGFR with poor prognosis suggests that their overexpression may contribute to lung adenocarcinoma tumorigenesis and metastatic potential. Despite the association with expression and poor prognosis, the exact role that erbB-2 or EGFR overexpression plays in lung adenocarcinoma development remains unclear. Malignant transformation by transfection of the EGFR alone has not yet been demonstrated, but it can induce mitogenesis in vitro after binding either EGF or transforming growth factor a (TGFa) (Velu et al., 1987;Di Marco et al.. 1989). The transfection and overexpression of erbB-2 can confer tumorigenicity on immortalised fibroblasts (Chazin et al., 1992) but is insufficient to induce malignant transformation in transfected immortalised human bronchial epithelial cells (Noguchi et al.. 1993) or in transgenic mice (Stocklin et al.. 1993). However. transfection and overexpression of erbB-2 have been show%n to enhance lung cancer cell metastatic potential (Yu et al.. 1994). It appears that erbB-2 and EGFR overexpression contributes to. but may be insufficient for, tumorigenesis in lung adenocarcinoma.
Signal regulation and tumorigenesis of erbB-2 and EGFR are dependent on their tyrosine kinase activity (Segatto et al., 1990). ErbB-2 and EGFR are thought to interact by transphosphorylation via heterodimer formation, resulting in enhanced ligand affinity (Kokai et al., 1988;Connelly and Stern, 1990;Wada et al., 1990). Expression of both receptors in a tumour cell may allow interaction between the two receptor types, modifying signal transduction in a way that could contribute to tumorigenesis. We hypothesise that erbB-2 and EGFR coexpression may provide a cooperative growth stimulus and therefore would be a selected characteristic of lung adenocarcinomas. This study was undertaken to establish the frequency of erbB-2 and EGFR coexpression in lung adenocarcinomas. In addition. established lung adenocarcinoma cell lines were used to examine the potential role of erbB-2 and EGFR in lung tumorigenesis by determining the expression of these receptors. their steady-state activation status and the effect of erbB-2 and EGFR ligand-mediated activation on cell growth.

Materials and methods
Human tissues Samples of normal lung and lung tumour were obtained after informed consent from patients undergoing operation for lung cancer at the University of Michigan between August 91 and April 94. Additionally, samples of bronchial epithelium free of tumour were obtained from a subset of the same patients. Analyses were performed on tissues from patients with the final diagnosis of primary lung adenocarcinoma. Immediately upon resection. specimens were divided into thirds. The middle third was embedded in OCT compound (Miles. Elkhart, IN, USA), frozen in isopentane cooled to the temperature of liquid nitrogen and used for cryostat sectioning and subsequent immunocytochemistry. The other two portions were frozen in liquid nitrogen and used for RNA and protein isolation. Samples were stored at -70'C until analysed.
Histology and staging The final hospital pathology reports of all patients were reviewed and used to establish the histology and the stage of the tumours. Tumours were staged according to the AJCCS system (American Joint Committee on Cancer Staging. 1992).
Cells used for immunocytochemical analysis were cytospun onto poly-L-lysine-coated slides.
The effect of cell confluence on NDF, erbB-2 and EGFR mRNA expression was determined by splitting a nearconfluent plate of A549 or A427 cells approximately 1:20 into 100mm culture plates, incubating cells in appropriate medium supplemented with 10% FBS, and then harvesting cells on days 1, 2, 4 and 7 for RNA isolation. Cells were noted to be confluent on day 4. The effect of dexamethasone treatment on NDF and erbB-2 mRNA was determined by splitting near-confluent plates of A549 or A427 cells into 100mm plates with 1 x 106 cells per plate, and incubating cells in appropriate medium supplemented with 10% FBS overnight. Medium was removed, cells washed with PBS, and medium was replaced in duplicate plates with steroid-stripped 10% FBS medium (Hanson et al., 1991) with either 0, 100 or 1000 nM dexamethasone (Sigma). Duplicate plates were prepared for each experimental group. Cells were allowed to grow until plates were approximately 90% confluent, and then harvested for Northern blot analysis as described below.
.Northern blot anal vsis Total RNA was isolated from tissues and cell lines using Tri-Reagent (Molecular Research Center, Cincinnati, OH, USA) following the manufacturer's protocol. Ten micrograms of total cellular RNA per sample was separated by electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde and then vacuum transferred to nylon membranes (Gene Screen Plus: NEN, Wilmington, DE, USA). Membranes were prehybridised in 5 x SSPE (0.9 M sodium chloride. 50 mM sodium phosphate. pH 7.7, and 5 mM EDTA), 5 x Denhardt's, 50% formamide. 3% SDS, 5% dextran sulphate. 5 mg ml -' heat-denatured salmon sperm DNA and 3 ;tg ml-1 yeast tRNA for 1 h at 48'C. Probes were labelled with [3'P]dCTP by the random primer labelling method (Prime-It II, Stratagene. La Jolla. CA. USA) and purified by Sephadex G-50 exclusion chromatography. Membranes were hybridised with 1.5 x 1o6 c.p.m. ml-' heat-denatured, labelled probe for 16-18 h in a 48'C shaking water bath. Membranes were washed according to the manufacturer's recommendations and autoradiograms prepared (Hyperfilm-MP; Amersham. Arlington Heights. IL. USA). Loading and transfer of RNA were normalised using a probe for 28S rRNA as previously described (Hanson et al.. 1991).
EGFR and erbB-2 in lungtumours WJ Rachwal et al 57 cDNA probes DNA probes used included: a 2.5 Kb Clal fragment of the human EGFR cDNA (Xu et al.. 1984). a 1.6 Kb EcoRI fragment of the human erbB-2 cDNA (Di Fiore et al.. 1987).

Immunocv tochemistrv
Immunocytochemistry was performed on 5 gm cryostat sections of normal lung, lung adenocarcinoma and bronchial epithelium as well as using cytospun cell lines. Monoclonal antibody to erbB-2 (Ab-2. clone 9G6) was obtained from Oncogene Science Inc.. (Uniondale. NY. USA) and used at a dilution of 1:100. Monoclonal antibodv to EGFR was obtained from Triton Diagnostics (Alameda. CA. USA) and used at a dilution of 1:5. Control reactions consisted of incubations without the primary antibodies. A standard avidin-biotin-peroxidase complex method With 3.3'diaminobenzidine as the chromagen was used according to the manufacturer's recommendations (Vecta-Stain-Elite. Vector Laboratories Inc., Burlingame. CA. USA). All sections were examined by two observers and classified as either present or absent, and when present. as either homogeneously expressed in all cancer cells or variably expressed.
W'estern blot anal!ysis Membrane protein was isolated from tissues and cell lines bv homogenising near-confluent cell plates or approximately 1 g tissue samples in membrane lysis buffer (20 mm Hepes. 5 mm sodium orthovanadate. 10 mM sodium pyrophosphate and 1 mM phenylmethylsulphonyl fluoride). These supernatants were ultracentrifuged at 100 000 g for 30 min. and the pellet resuspended in Western lysis buffer (10 mM sodium phosphate, 100mM sodium chloride. 1% Triton X-100. 0.50'o sodium deoxycholate, 0.1% SDS, 5mM sodium orthovanadate. 10 mM sodium pyrophosphate and 1 mM phenylmethylsulphonyl fluoride). Protein concentrations were quantified using the colorimetric micro-Lowry method (Sigma). Either 75 or 100 pg of sample protein was loaded per lane. or the total sample protein isolated was divided equally between compared samples. In all cases, compared samples contained equal amounts of protein. Samples were boiled for 5 min. loaded and separated by electrophoresis on a 7.5% SDS-PAGE gel along with high-range molecular weight markers (Bio-Rad Laboratories. Richmond, CA. USA). The gel was then electrotransferred to a PVDF membrane for immunoblotting. Membranes were blocked in 0. 1% Tween 20. 100 mM Tris base, 0.9% sodium chloride and 3% powdered milk solution for 1 h. Avidin-biotin complex staining was performed using polyclonal erbB-2 (9.3 anti-erbB-2. a generous gift from Dr Beatrice Langton. Berlex Biosciences. Richmond. CA. USA). monoclonal EGFR (Triton Diagnostics. Alameda. CA, USA) or monoclonal phosphotyrosine antibody (PY20, ICN Biomedicals, Costa Mesa. CA. USA). Immunoprecipitation Western blots were performed in a similar fashion except that cells or tissue membrane protein was incubated in primary antibody (polyclonal erbB-2) for 2 h. and then incubated in 50 ytL of protein Aagarose (Sigma) for 1 h. Protein A beads were then spun out, the precipitate resuspended. and gels and blots were prepared as described above.

Growth assays
Cell lines were plated at low density using three 60-mm plates for each experimental group and then incubated in F 12 Ham's with 10% FBS overnight. Three plates were then counted to determine initial plating density (time zero). The F12 Ham's with 10% FBS was then removed and triplicate plates were treated with one of the following: F12 10% FBS. SFM (serum-free medium). SFM plus 10 ng ml-' NDF P3 (a generous gift from Amgen. Thousand Oaks. CA. USA). -Ff Ji h2 g bi_s Wi P et a1 SFM plus 10 ng ml epidermal growth factor (EGF) and SFM plus NDF and EGF. Cells were grown for 1 week, nuclei isolated using Bretol solution containing ethyihexadecyldimethyl ammonium bromide (Eastman Kodak, Rochester, NY, USA) and glacial acetic acid, and then counted a Coulter Counter as by the manufacturer (Coulter Electronics, Hialeah, Fl, USA). Counts were coincidence corrected and analysed by analysis of variance with Fisher's protected least signifiant difference post-hoc testing (StatView statistl program, Abacus Conceptions, Berkeley, CA, USA).
Rest Expreswion oferbB-2 and EGFR Immunocytochemical analysis was used to determine the frequency of erbB-2 and EGFR protein coxpresson in 43 prinmry human lung adenocarcinomas as well as the thre cell lines (A549, A427 and SKLU-1) derived from human hmg adenoarcinomas. The staining patterns of normal alveolar tissues and bronchial t samples were also determined from patients with hmg adenocarcinoma. All histologically normal alveolar tissues examined were negati for erbB-2 and EGFR protein e on. All 43 tumours examined demonstrated a membranous erbB-2 protein staining pattern (Figure la). EGFR protein was de d in 36 out of the 43 tumours (Figure lb). ErbB-2 and EGFR coexpression was therefore det in 83% of lung adeocarcinomas. Seven of the 36 coexprssing tumours demonstrated non-uniform staning for EGFR, in which saining was not observed on all tumour cells. Several coexpressing tumour sections contained adjacent normal alveolar tissue, highlighting the relative overexpression of erbB-2 or EGFR in the tumour as compared with the absent expresion in the alveolar tissue (not shown). These results are consistent with previous reports of the low klvel or absent epson of these a er qr > er :F C receptors in normal lung alveolar tissue (Rusch et al., 1993;Bongiorno et al., 1994). In contrast, five out of five histologically normal bronchial epitheium samples coexpressed erbB-2 and EGFR at levels similar to that seen in the primary tumours ( Figure Id and e). The staining of EGFR in the normal bronchial epithelium was limited to the basal cell layer, while erbB-2 was present throughout the pseudostratified architecture ( Figure Id). ErbB-2 and EGFR expression status relative to tumour stage at time of resection is shown in Table I. Tumours with variable or absent expresson of EGFR tended to be of a higher stage than tumours showing uniform erbB-2 and EGFR expression. The distibution of tumour stages in these surgical patients, however, is not representative of that seen in all lung cancer patients, since most patients with either known stage m or IV disease are excluded from pulmonary resection.
Human lung adenocarcinoma cell lines A549, A427 and SKLU-1 were evaluated for erbB-2 and EGFR expresson usng immunocytochemical techniques, as was done in the primary tumours Each cell line demonstrated erbB-2 staining similar to the prinary lung mas, but less than SKBR3, a breast a arcmoma cell ie containing amplified and overexpressed erbB-2 which served as a positive control ( Figure Ig). EGFR was also xprssa in the lung lines and SKBR3 cells. These results are consistet with Northern blot analysis, which demonstrated relatively similar levels of erbB-2 and EGFR mRNA levels in the cell lines and in a lung adenocarcinoma, with the exception of A549 cells, which expressed higher levels of EGFR mRNA (Figure 2a). SKBR3 demonstrated substantially higher levels of erbB-2 mRNA than the lung tumours or cell lnes. A similar level of erbB-2 expression was obwrved in all cells of the primary tumour specmens eamined by immunocytochemcal analysis. Further, A549 cells analysed by Northern blot also demonstrated similar levels of erbB-2 mRNA in both growing and confluent cells (Figure 2b), suggesting that erbB-2 expresson is independent of rate of cell growth within the tumour. A549 cells treated with dexamethasone, which is known to inhibit A549 cell growth and induce some differentiated features (Speirs et at., 1991;Croxtall et al., 1993), also did not alter the levels of erbB-2 mRNA (data not shown). In contrast, EGFR protein expression was found to be variable in some primary tumours, suggesting that its expression may be affected by the growth state of individual cells. Similarly, A549 cells grown to different degrees of confluence demonstrated increased EGFR mRNA levels during subconfluent cell growth and decreased levels with cell confluence (Figure 2b). These results are consistent with EGFR protein expresson in bronchiolar epithelium, w the highest expression is present in the prolferatng basal cells (Figure le).
Autocrie ligand expression Tumour cells may maintain constant levels of the erbB-2 receptor protein, but the expression of autocrine lgands for erbB-2 and EGFR may be major determinants for receptor activation and cell growth. Thefore, lung adenocarcinoma cell lines A549, A427 and SKLU1 were evaluated by Northern blot analysis for the expression of NDF and TGF-4 mRNA, the ligands capable of activating erbB-2 and EGFR respUecively. A549 cells expressed abundant mRNA for both NDF and TGF.<a, while A427 expressed only small amounts of TGF-a mRNA (Figure 3a). HT-29 colon cancer cells a '.# Ac'.t. f expressed abundant TGF-x mRNA and SKBR3 breast cancer cells expressed potentially truncated forms of TGF-x mRNA. Interestingly, expression of NDF mRNA was highest in rapidly dividing, subconfluent A549 cells, and decreased as cells became confluent (Figure 2b). Similarly, treatment of A549 cell lines with growth-inhibitory concentrations of dexamethasone (Speirs et al., 1991;Croxtall et al., 1993) also inhibited NDF mRNA in a dose-dependent manner (Figure 3b). The association between high NDF mRNA expresson and rapid cell growth would be consistent with NDF acting as an autocrine growth factor in these cells.
Activation of erbB-2 and EGFR If erbB-2 and EGFR are contributing to tumorigenesis in lung adenocaranomas it would be expected that these reptors would show evidence of activation. Activation of erbB-2 and EGFR in cell lnes and tumours was therefore assessed by xamining receptor tyrosne phosphorylation using a PY-20 antiphosphotyrosine antibody and Western blot analysis.
Tyrosne kinase activity is known to be assocated with autophosphorylation of these receptors (Segatto et al., 1990).
The SKBR3 breast cells served as a positive control. The A549 lung crcinoma cells and five primary lung adenocarcinoma specim were examined. Western blot analysis demonstrated a 185 kDa erbB-2 protein in the A549 ells and prinary tumours, however little corresponding phosphotyro staining of 185 kDa proteins was present ( Figure  4a). The SKBR3 cells contained a strong 185 kDa staining band with both erbB-2 and PY-20 antibodsie, demonstrating high-level constitutive activation of erbB-2 in these cells. A 175 IDa protein corresponding to the size of the EGFR was detectd using an anti-EGFR antibody in three out of four tumours. Unlike the erbB-2 protein, the EGFR was associated with a corresponding phosphotyrosine staining band, or possibly a doublet band, at 175 kDa. To detrmine whether freezing and thawing of the primary tumours before preparing protein extracs affected phosphotyrosine staining, A549 cell pellets were frozen at -70-C, and phosphotyrosine stain- ing was compared with that of freshly harvested A549 cells.
No change in phosphotyrosine staining pattern or intensity was observed. Thus, in primary lung adenocarcinomas, phosphotyrosine staining was more pronounced in the EGFR protein than in the erbB-2 protein.
To examine further the factors affecting erbB-2 and EGFR activation in the three lung adenocarcinoma cell lines, the A549, A427 and SKLU-1 (not shown) were analysed using Western blots and compared with the breast cell lines MCF-10 and SUM52. All three lung adenocarcinoma cell lines expressed the erbB-2 protein at 185 kDa, but little basal level phosphotyrosine staining at 185 kDa was observed ( Figure  4b). Instead, A549 cells expressed more EGFR protein and relatively higher levels of the corresponding phosphotyrosine staining protein at 175 kDa than A427 cells. A549 cells showed little difference in phosphotyrosine staining of the EGFR band in either the presence or absence of FBS. The increased 175 kDa phosphotyrosine staining of the EGFR band in the A549 vs the A427 cells is consistent with the higher level of EGFR protein (Figure lj and 1) and EGFR mRNA (Figure 2a) in these cells. The SUM52 cells do not express the 175 kDa EGFR band, but contain a strong 185 kDa band that is associated with phosphotyrosine staining. SUM52 and SKBR3 breast cancer cells demonstrated higher basal-level erbB-2 phosphotyrosine staining than either the lung adenocarcinoma cell lines or the primary lung tumours. Western blots of immunoprecipitated erbB-2 from the A549 cells was also unable to detect phosphotyrosine staining, while SKBR3 cells were positive (data not shown). This may be due to the lower level of total erbB-2 protein present in the lung cells. ErbB-2 and EGFR activation in the lung adenocarcinoma cell lines was therefore similar to that present in the primary tumours.
To determine if erbB-2 or EGFR phosphotyrosine staining could be enhanced above basal levels, lung cell lines were treated with NDF and/or EGF. NDF treatment of A549 and A427 resulted in a slight increase in the phosphotyrosine staining of the 185 kDa band corresponding to the erbB-2 protein (Figure 5a). This increase was much less than that seen in the breast cell line MCF1O, which had marked enhancement of the 185 kDa phosphotyrosine band following NDF treatment (Figure 5a). Treatment of A549 cells with the antibody Tab-250, which has erbB-2 ligand-like properties in some cells, did not increase PY-20 staining of erbB-2. Addition of EGF to A549 cells did increase PY-20 phosphotyrosine staining associated with EGFR, as compared with either NDF treatment or under serum-free conditions ( Figure  5b). Treatment with both NDF and EGF did not increase the extent of PY-20 staining of either the 175 kDa EGFR or the 185 kDa erbB-2 bands over that observed with either factor alone.
Growth of cells with NDF and EGF treatment A549 cells grown in SFM demonstrated a 14-fold increase in cell number after 1-week ( Figure 6). This suggests that these cells produce autocrine growth factors, and thus is consistent with the presence of TGF-a and NDF mRNA expressed in these cells (Figure 3a). A427 cells grown in SFM demonstrated a 5-fold increase in cell number after 1 week (data not shown). The increased autocrine ligand expression in A549 cells corresponded with greater growth under serum-free conditions observed with these cells as compared with A427 cells (Figure 3a). SKLU1 cells did not express significant levels of either ligand, and demonstrated little capacity to grow under serum-free conditions (data not shown). A549 cells treated with SFM plus NDF or EGF had a statistically significant increase in cell growth over SFM alone. NDF plus EGF treatment in A549 cells had an additive effect on cell growth, equal to that obtained with 10% FBS-supplemented medium.
EGFR an wbB-2 i hun btmows Growth of A427 cells in SFM plus NDF was also significantly greater than in SFM alone (data not shown). EGF treatment of A427 cells, however, added little to the growth stimulation provided by NDF alone, and was variably stimulatory or inhibitory. The reasons for this are unclear but may relate to the expression of the EGFR in cells grown at different plating densities.

Dosi
Overexpression without gene amplification of erbB-2 and EGFR has been described in a subset of lung adenocarcinomas (Slamon et al., 1989;Kern et al., 1990;Rusch et al., 1993). In vitro studies suggest that erbB-2 and EGFR may interact through heterodimer formation or transphosphorylation (Kokai et al., 1988;Wada et al., 1990). In breast cancer, erbB-2 and EGFR coexpression correlates with a poor prognosis (Osaki et al., 1992), and amplification of these genes is more commonly observed. Information that these receptors may interact to contnibute to tumorigenesis led us to evaluate their coexpression in primary lung adenocarcinomas. Interestingly, all 43 (100%) lung adenocarcinoma tumours examined expressed erbB-2 protein, and 36 (83%) of these also expressed EGFR protein. All three lung adenocarcinoma cell lines examined also expressed both receptors. ErbB-2 and EGFR coexpression is therefore characteristic of most lung adenocarcinomas. The frequency of erbB-2 and EGFR coexpression reported in this study is higher than previously reported (Scagliotti et al., 1993), possibly reflecting the enhanced sensitivity from using frozen rather than paraffin sections (Press et al., 1994). Our studies suggest that continued expression of these receptors, and not necessarily overexpression, may be the most significant feature in lung adenocarcinomas.
To determine if the pattern and degree of erbB-2 and The effect of serum. EGF and NDF on A549 cell growth. A549 cells were grown for 1 week in either medium containing 10% FBS (Serum), serum-free medium (SFM), 10 ng ml-NDF, 10 ng ml-' EGF or 10 ng ml-' NDF plus 10 ng ml-' EGF. The number of cells plated at time zero for each condition is indicated (Start). Mean cell number of triplicate plates is shown. Error bar represents the confidence interval of the mean cell number. *Statistically significant (P<0.05) difference in the mean cell number as compared with SFM using analysis of variance. *Statistically significant (P <0.05) difference in the mean cell number as compared with either SFM, NDF or EGF treatments.
EGFR expression found in lung adenocarcinomas was different from that found in normal tissues, we examined their expression in normal bronchiolar and alveolar epithelial tissues. All alveolar lung tissue examined was negative for erbB-2, as we have previously reported (Bongiorno et al., 1994). EGFR expression was very low, with only scattered cellular staining in this tissue. consistent with previous reports (Rusch et al., 1993). Bronchial epithelium, however, was found to express both erbB-2 and EGFR, consistent with previous reports (Dazzi et al., 1989;Weiner et al., 1990), and suggests that the bronchial epithelium may be the cells of origin for lung adenocarcinomas. ErbB-2 and EGFR expression in the lung adenocarcinomas may therefore represent a normal level of expression present in bronchial epithelial cells rather than overexpression from transformed alveolar tissue levels. One must consider, however, that the bronchial epithelium from cancer patients examined in this study may in fact be abnormally expressing erbB-2 or EGFR. Evidence suggests that some bronchial epithelium from cancer patients may have cytogenetic abnormalities (Sozzi et al., 1991). In addition, other normal tissues, such as pharyngeal epithelium, have been shown to have increased expression of EGFR directly adjacent to tumour tissue (Shin et al., 1994). A field effect of premalignant change in the bronchial epithelium therefore may affect erbB-2 and EGFR expression, which in turn could stimulate growth and predispose these cells to additional genetic events.
EGFR protein staining was observed only in the proliferative basal cell layer of the bronchial epithelium, while erbB-2 immunoreactivity was expressed throughout the pseudostratified architecture. Staining of EGFR in the basal layer, which is decreased in more luminal layers, suggests that this receptor may play a role in normal growth and differentiation of the bronchiolar cells. Expression of EGFR in primary tumours may vary depending on the growth state of individual cells. ErbB-2 expression may be independent of the growth or differentiated state of either bronchial epithelial cells or tumour cells since it is expressed throughout the bronchial epithelium and uniformly in tumours. A similar phenomenon was observed in vitro with A549 cells, in which the levels of erbB-2 mRNA were similar during active cell growth or confluence. In contrast, EGFR mRNA levels were highest in rapidly growing cells (Figure 3), and this is consis-62 a ErbB-2 PY-20 1 40 0000- 4VT Vi 'Wiv'V W'*I' I . We*, I . Iv. tent with the higher EGFR protein levels in the proliferating basal cells of the bronchiolar epithelium. Signal regulation by erbB-2 and EGFR is dependent on their tyrosine kinase activity. When lung adenocarcinoma tissues or cell lines were examined for activation by Western blot analysis, low-level EGFR and very little erbB-2 activation was detected. This suggests that the transforming potential of erbB-2 in lung adenocarcinomas does not involve constitutive activation of intrinsic tyrosine kinases in the absence of ligand as may occur in breast cells with amplified erbB-2 genes. Even in A549 cells, which produce both NDF and TGF-a mRNA (Figure 3a), ligands potentially capable of activating erbB-2 and EGFR respectively, only low-level erbB-2 activation is detected. Treatment of A549 and A427 cells with NDF increased the phosphotyrosine staining of the 185 kDa erbB-2 band in these cells, indicating that the receptors can be activated, however the level of activation was always less than that observed in the breast cell lines. Thus erbB-2 activation in lung adenocarcinoma tumours and cell lines is distinctly different from that of breast tissue. MCF1O benign mammary epithelial cells have low basal-level activation of erbB-2 similar to lung adenocarcinomas, but demonstrate a much greater capacity for activation by NDF. The SUM52 breast cancer cell line, which overexpresses erbB-2 protein without gene amplification, demonstrates greater basal-level activation of erbB-2 than is seen in lung adenocarcinoma cells. Amplification of erbB-2 in SKBR3 is associated with constitutive activation of greatly overexpressed erbB-2 protein. Amplification of erbB-2 expression is present in up to 30% of breast cancers and is associated with a worse patient prognosis independent of stage (Slamon et al., 1987), possibly related to constitutive activation of erbB-2 tyrosine kinase. These differences between the lung and breast systems may relate to the absolute amounts of erbB-2 expressed, or may be related to the absence or presence of co-factors necessary for the activation of erbB-2 by NDF, such as erbB-3 or erbB4 (Akita et al.. 1994;Plowman et al., 1994) or cell specific factors (Peles et al., 1993). We are currently examining whether the lung adenocarcinoma cells express either erbB-3 or erbB-4. It appears, therefore, that the role of erbB-2 in lung tumorigenesis differs from that of breast in both the frequency of amplification with constitutive activation and the capacity to be activated by NDF.
Growth assays of A549 and A427 demonstrate the capacity of these cells to grow under serum-free conditions. Autocrine growth factors NDF and TGF-a, produced by both lines, probably play a contributory role in their serumfree growth. Treatment of A549 and A427 with NDF further enhanced activation and stimulated cell growth above that of serum-free medium, demonstrating functional yet low-level activation in these cells. The relatively greater activation of EGFR relative to erbB-2 in both the lung cell lines and primary tumours, as demonstrated by Western blot staining for phosphotyrosine, may suggest that expression of erbB-2 may facilitate EGFR activation. ErbB-2 and EGFR are thought to interact by transphosphorylation via heterodimer formation, resulting in enhanced ligand affinity for the EGFR (Kokai et al., 1988;Connelly et al., 1990;Wada et al., 1990). A549, interestingly, demonstrated an additive growth stimulus with NDF and EGF, suggesting a cooperative role between erbB-2 and EGFR, one that may exist in tumours coexpressing these receptors. Other transforming characteristics such as p53 and K-ras mutations are present in these cell lines (Lehman et al., 1991), potentially contributing to their serum-free growth. Similarly, primary lung adenocarcinomas also contain alterations such as p53 and K-ras mutations (Bongiorno et al., 1994), which may affect cell growth, invasive and metastatic properties. ErbB-2 and EGFR expression may therefore provide a cooperative ligand-dependent growth stimulus to bronchial epithelial cells, which acquire other critical genetic changes necessary for lung tumorigenesis.