Cancer Letters, 65 (1992) 189 - 199
Elsevier Scientific Publishers Ireland Ltd.
Expression of epidermal growth factor receptor and transforming growth factor cy in human larynx carcinoma Eddi Di Marco”, Enrico Albanese”, Stefano Bensoa, Fabio Beatriceb, Ranieri Cancedda” and Salvatore Tomaa ‘Ystituto Nazionale
per la Ricerca sul Cancro,
and b10 Clinica ORL Uniuersitir di Torino,
(Received 4 February, 1991) (Revision received 7 May 1992) (Accepted 18 May 1992)
Summary Altered expression of growth factors and growth factor receptors is frequently described in human tumors and human tumor cell lines. This further supports the hypothesis that oncogenesis is due to the subversion of mitogenresponsive pathways. The aim of this study was to investigate the expression of epidermal growth factor receptor (EGFR) and transforming growth factor CI (TGFcY)in 13 larynx carcinomas and 2 carcinomas of the oral cavity. We found receptor ouerexpression in 7 out of 15 tumors at mRNA and/or protein leuel but low expression in the majority of the normal adjacent tissues. TGJ% was expressed only in 1 case, but no tyrosine kinase activity of the receptor was detected by antiphosphotyrosine antibody.
Keywords: epidermal growth factor; epidermal growth factor overexpression; transforming growth factor a; larynx carcinoma Correspondence to: Eddi Di Marco, Laboratorio di Differenziamento Cellulare, lstituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV no. 10, 16132, Genova, Italy.
Abnormalities involving growth factors and growth factor receptors have been found in several human tumors and human tumor cell lines [l&26 - 28,30,52,60]. In addition to the abnormalities described in gene structure these alterations involve gene amplification and/or overexpression of normal RNAs and proteins 127 - 28). In particular amplification and/or overexpression of epidermal growth factor receptor (EGFR) have been observed in a number of epithelial malignancies including breast , head and neck [23,43,62], genito-urinary [6,17,20,24], lung  gastric carcinomas [5,57] and in glioblastomas . Several reports showed that high levels of EGFR have a prognostic value in breast cancer [11,49], gastric carcinoma  and head and neck squamous cell carcinomas [40,54,62]. Altered expression of EGFR can be associated to major risk of early recurrence in bladder cancer  and glottic squamous cell carcinoma . Yano et al. found that esophageal squamous cell carcinoma with strong expression of EGFR  metastasize to lymphonodes. In ovarian carcinomas  and more recently in head and neck squamous cell carcinomas  a high
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level of EGFR has been linked with a better response to chemotherapy, suggesting that EGFR can be used as a biological marker in several malignancies. In parallel the TGFcr, which shares structural and functional homology with the EGF and binds to the same receptor [34,35,58], has been found to be expressed in neoplastic tissues or cell lines derived from spontaneous human tumors [1,2,12,41,56] or chemically induced animal tumors [31,33]. In addition TGFa activity has been isolated from the urine of patients with a variety of disseminated cancers including head and neck carcinomas [l, 161. By in vitro studies it has been demonstrated that the overexpression of human EGFR can confer an EGF-dependent transformed phenotype to
NIH-3T3 cell [13,46,61], while the coexpression of high levels of EGFR and TGFcr in the same cells leads directly to the transformed phenotype . Moreover the TGFa expression by some EGFR overexpressing tumor cell lines resulted in tyrosine kinase receptor protein activation [ 141. The TGFa by itself shows weak, if any, transforming activity when transfected in cells displaying a low or moderate number of receptors [15,37,47]. On the contrary in transgenic mice the expression of TGFa can induce several changes in growth and differentiation of adult tissue leading in some cases to neoplastic transformation [25,36,50]. In the present study we have examined 15 head and neck carcinomas localized in the
Table 1. Characteristics of head and neck carcinomas analyzed for EGFR overexpression Pt
EGFR mRNA” NC T’
EGFPproteinb NC T’
1 2 3 4 5 6 7 8 9 10 11 12 13 14
67 81 66 75 69 69 68 63 54 58 64 70 69 47
2 2 1 1 2 2 2 2 2 2 2 2 2 2
G2 G2 G2 Gl G2 G3 G3 G3 G2 G2 G2 G2 G2 G2
T2NlMO TSNOMO TlNOMO T3NOMO TZNOMO T2N 1MO T3N3MO T4NlMO T3NlMO T3NlMO T3NlMO T2N3MO T3NOMO T4NlMO
III III I III II III IV IV III III III III III IV
n/ad 1 n/a 0.5 0.1 n/a 0.5 1 0.5 1 5 2.5 1 2
0.1 50 0.5 1 0.1 1 0.5 2.5 5 1.5 5 5 12.5 10
n/a 1 n/a 1 0.1 n/a 0.1 1 1 4 6 1 0.4 1
n/a 60 0.5 40 0.1 0.1 0.1 4 10 10 4
Larynx Larynx Oral cavity Tongue Larynx Larynx Larynx Larynx Larynx Larynx Larynx Larynx Larynx Pharynx/ larynx Larynx (relapse)
n/a 3 3
Tumor stage and grade, nodal status and size of the primary tumor were determined according to the TNM and G classification of malignant tumor . “EGFR mRNA level is expressed as multiples of normal tissue values of case 2 after correction for the amount of RNA present in the filter. bEGFR protein relative amount, detected with anti-EGFR antibody is indicated in comparison to the signal observed in normal tissue of case 2 after long autoradiographic exposure. ‘N: normal tissue, T: tumoral tissue. dn/a: not available.
larynx and the oral cavity for EGFR overexpression at mRNA and protein level. All samples were also screened for TGFa mRNA expression. Parallel evaluations were performed in specimens of healthy tissue derived from a non-tumoral area of the same patients. Tyrosine-kinase activity of the EGFR was also examined by Western blot analysis using an antiphosphotyrosine antibody. Materials and Methods
Cell lines and tissue samples A431 human squamous cell carcinoma cell line and Jurkat human T cell line were obtained from ATCC (American Type Culture Collection). NIH-EGFR c1.A and NIH-EGFR cl.A-TGFcr cell lines were provided by Dr. S.A. Aaronson. The cell lines were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Flow) supplemented with 10% fetal calf serum at 37OC in a 5% CO2 humidified air atmosphere. Tumor samples were obtained from 15 patients with head and neck squamous cell carcinoma. Thirteen were larynx carcinomas and 2 carcinomas of the oral cavity. Control biopsies of 11 out of 15 cases were also obtained from a symmetrical non-tumoral area. All samples, biopsed at the I” Clinica ORL, University of Turin, weighed between 0.1 and 0.2 g. They were frozen in liquid nitrogen and stored at -8OOC until used for biological evaluations. Tumor stage and grade, nodal status and size of the primary tumor were performed according to the TNM and G classification of malignant tumors (UICC 1988) . The above clinical parameters together with performance status and tumor localization are summarized in Table I. The mean age of the patients was 65 (range 47 - 81). RNA extraction and dot blot/slot blot analysis Frozen tissues were minced in liquid nitrogen using a mortar and pestle and homogenized in 4 M guanidine thiocyanate.
RNA was extracted by cesium chloride gradient [lo]. RNA dot blot and slot blot analyses were performed by spotting serial dilutions of total RNA in 15 x SSC (1 x SSC = 0.15 M sodium chloride, 0.015 M sodium citrate) onto Gene Screen plus TM membranes (Du PontNew England Nuclear) in a dot blot or slot blot manifold after RNA denaturation in 6 x SSC and 7.4% w/v formaldehyde at 65OC for 15 min. The RNA was immobilized on the nylon membrane by UV light cross linking and prehybridized at 42OC for 3 h in 50% deionized formamide, 0.75 M sodium chloride, 25 mM sodium phosphate, 5 mM EDTA, 0.2 mg/ml salmon sperm DNA, 0.5% SDS. The hybridization was performed at 42OC for 16 h in the above buffer after the addition of 32Plabeled probes (2 x lo6 counts/min per ml) and 10% dextran sulfate. The filters were extensively washed in 50% formamide and 3 x SSC at 42OC for 30 min, twice in 0.75 x SSC at 65OC for 15 min and once in 0.1 x SSC and 0.1% SDS at 65OC for 30 min. All filters were autoradiographed on Xray films (Hyperfilm-MP, Amersham) with intensifying screens at -8OOC. The EGFR probe consisted of the cDNA insert including part of the extracellular domain from the Sma I site to the Sal I site. The completed coding sequence of the TGFa used as a probe was obtained by Cla I digestion of the pMTE42 plasmid . The RNA amount present in the filters was determined by hybridization with pXCR7 plasmid  specific for ribosomal RNA (rRNA). lmmunoblotring Minced tissues and cell lines were lysated in 1 x hot SDS-PAGE sample buffer and sonicated. This treatment ,does not affect protein phosphorylation and blocks protein degradation (Di Marco, unpublished). When indicated the in vivo EGFR auto-phosphorylation assay was performed by adding 300 ng/ml EGF for 10 min at 37OC after 2 h of cell starvation in serum free medium. Samples were clarified at 12 000 x g for 15 min at 4OC and size fractionated on a 7.5% SDS-PAGE after protein
determination by BCA Pierce method. Proteins were transferred to Immobilon-P membranes (Millipore)  and filters treated for 1-2 h in TTBS (20 mM Tris, pH 7.5, 0.9% sodium chloride and 0.05% Tween) with 3% non-fat dry milk. Blots were incubated for 1 h with either antiphosphotyrosine polyclonal antibody (2 pg/ml) or EGFR polyclonal antibody crC (1 - 200 dilution) in TTBS with 0.5% BSA, then washed three times with TTBS solution and incubated with 2 x lo5 counts/min per ml 1251protein A (Amersham) in TTBS with 0.5% BSA for 30 min at room temperature. After extensive washing in TTBS, blots were autoradiographed with intensifying screens zf - 80’ .
RNA dot blot and slot blot analysis To determine the level of EGFR and TGFa transcripts we extracted mRNA from 15 headneck squamous cell carcinomas. Thirteen were larynx carcinomas and two carcinomas of the oral cavity. Degree of differentiation, stage and nodal status are reported in Table 1. Most of the tumors were advanced non-metastatic carcinomas. In 11 out of 15 cases fragments of healthy tissue, taken from a non-tumoral area and histologically tested, have been examined and compared to the corresponding neoplastic specimen.
Fi2. 2. Slot blot analysis of 10 pg total RNA extracted from normal and neoplastic samples of patient 13, 14, and 15 from NIH-EGFR &A, NIH-EGFR cl.A-TGFar and Jurkat cell lines. the filter was hybridized with EGFR and TGF OL c-DNA probes.
By dot blot analysis (Fig. 1) we found altered EGFR mRNA expression in 6 cases (patients 2,8,9,12,13,14), while the corresponding norma1 tissue showed a low level of transcript. In 4 cases (patients 4,5,7,10) the EGFR mRNA level was uniformly low and we could not
detect major differences between normal and neoplastic specimens. In one case (patient 11) both normal and neoplastic tissues presented high levels of EGFR mRNA. The 4 unpaired neoplastic samples (patients 1,3,6,15) showed a low amount of EGFR transcript. The level of a