Activation of the Autocrine Transforming Growth Factor a Pathway in Human Squamous Carcinoma Cells1

[CANCER RESEARCH 51. 6254-6262. December 1. I991| Activation of the Autocrine Transforming Growth Factor a Pathway in Human Squamous Carcinoma Cells1

Michael Reiss,2 Ellen B. Stash, Vincent F. Vellucci, and Zhao-ling Zhou

Department of Medicine, Section of Medical Oncology, and Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06517

ABSTRACT presumably in the signaling pathway (2). The binding of growth factors to cell surface receptors initiates cellular proliferation Transforming growth factor a is an autocrine mitogen for nonneoplastic keratinocytes, which exerts its function by binding to the receptor for but also activates a number of counterregulatory mechanisms, epidermal growth factor. In order to determine whether this autocrine which provide negative feedback to the growth stimulus. These pathway is activated in squamous carcinoma cells, we analyzed the include the down-regulation of the receptor (5), the inhibition production of transforming growth factor a as well as the expression and of the tyrosine kinase activity of the receptor molecule itself regulation of epidermal growth factor receptors in a panel of human (6), and, perhaps, the induction of autocrine growth inhibitors. squamous carcinoma cell lines. Immunoreactive transforming growth factor a was detectable in squamous carcinoma cells as well as in Therefore, one might expect neoplastic cells to display the quiescent nonneoplastic keratinocytes. However, in the absence of exog simultaneous overproduction of an autocrine mitogen and a enous mitogens, only the squamous carcinoma cells secreted the growth defect in one or more of the mechanisms, which limit the factor into the medium, whereas untransformed keratinocytes did not. activity of its receptor. Such a situation might be termed "acti Each of the squamous carcinoma cell lines expressed significantly vation" of an autocrine growth factor pathway. greater numbers of cell surface epidermal growth factor receptors than The aim of the present study was to determine whether normal keratinocytes. The epidermal growth factor receptor gene was amplified and overexpressed in three of the squamous carcinoma cell activation of such an autocrine growth factor pathway occurs lines (A431, CaSki, SqCCA'l)- Two of the squamous carcinoma cell lines in human SqCCs. EGF and insulin are the 2 principal growth (C4-1 and CE-48) displayed a relative inability to down-regulate epider factors required to sustain the proliferation of primary keratin mal growth factor receptors in response to epidermal growth factor. The ocytes in vitro (7, 8). TGF-a stimulates DNA synthesis by mechanism of receptor overexpression in the remaining three cell lines activating the same cell surface receptors as EGF (9). Specific (A253, CaLu-1, FaDu) is unexplained. Thus, human squamous carcinoma TGF-a mRNA has been detected in nonneoplastic murine as cell lines frequently exhibit a combination of the constitutive secretion of transforming growth factor a and the overexpression of epidermal growth well as human keratinocytes (10, 11). In both cases, the expres factor receptors. Treatment of these tumor cells with an antibody directed sion of the TGF-a message was inducible in response to treat against the ligand-binding domain of the epidermal growth factor receptor ment of cells with EGF or TGF-a. Information from previous inhibited their growth by approximately 50%. These findings suggest studies suggested that the TGF-a pathway may be activated in that designing strategies to interrupt the transforming growth factor a SqCCs. (a) SqCCs (both cell lines and fresh tumors) frequently autocrine pathway might lead to new modalities to treat this class of overexpress EGF-Rs. In a small number of cases, the over- malignant tumors. expression of EGF-Rs by SqCC cell lines is the result of amplification and/or rearrangement of the EGF-R gene (12, INTRODUCTION 13). However, amplification of the EGF-R gene is not com Malignant or transformed cells frequently produce growth monly found in primary tumor specimen(s) (14). Thus, in most factors that they themselves require for proliferation (1, 2). cases, the mechanism of EGF-R overexpression is not known. Sporn and Todaro (1) proposed that such a constitutive pro (b) Although SqCCs often express TGF-a mRNA (14), it is not duction of an autocrine growth factor provides neoplastic cells known whether TGF-a protein is constitutively produced in with a proliferative advantage, which, in turn, contributes to greater amounts than in nonneoplastic keratinocytes. In addi malignant transformation. However, the experimental evidence tion, it is not clear whether or not tumor cells, which overpro that the overproduction of an autocrine growth factor alone is duce TGF-a, simultaneously express elevated numbers of EGF- sufficient for malignant transformation is limited. In fact, al Rs. though in vitro transformation of rodent fibroblasts has been achieved by inducing the constitutive production of platelet- In order to address these questions, we analyzed the produc derived growth factor (3), overexpression of TGF-a' in skin tion of TGF-a as well as the expression and regulation of the keratinocytes results only in the formation of hyperplastic EGF-Rs in the same panel of SqCC lines. We found that SqCCs epidermis but not of tumors (4). Full-fledged transformation as well as nonneoplastic keratinocytes all expressed and pro appears to require at least a second defect in an autocrine loop, duced TGF-a constitutively. However, in the absence of exog enous mitogens, only SqCCs secreted the growth factor into Received 6/19/91; accepted 9/23/91. The costs of publication of this article were defrayed in part by the payment the medium, whereas untransformed keratinocytes did not. of page charges. This article must therefore be hereby marked advertisement in Each of the SqCCs expressed significantly greater numbers accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'Supported in part by USPHS Grant CA-41556 of the National Cancer of EGF-Rs than normal keratinocytes. The EGF receptor gene Institute. was amplified and overexpressed in 3 of the SqCC lines. Treat 2To whom requests for reprints should be addressed, Section of Medical Oncology, Department of Medicine, Yale University School of Medicine, 333 ment of SqCCs with exogenous EGF resulted in incomplete Cedar Street, New Haven, CT 06510. down-regulation of EGF-Rs in 2 additional carcinoma lines. 'The abbreviations used are: TGF-a, transforming growth factor a; SqCC, squamous cell carcinoma; EGF, epidermal growth factor; EGF-R, EGF receptor; Overall, the number of receptors expressed on the cell surface HKc, primary human foreskin keratinocytes; DMEM, Dulbecco's modified Ea appeared to correlate better with the inability of the receptors gle's medium; TCA, trichloroacetic acid; PCA, perchloric acid; SSC, 150 m.\i to be down-regulated by EGF than with the degree of gene sodium chloride: 15 HIMsodium citrate, pH 7.0; SDS, sodium dodecyl sulfate; HPV, human papillomavirus. amplification or mRNA expression. 6254

MATERIALS AND METHODS (Biolmage-Millipore, Ann Arbor, MI), in order to determine the rela tive amounts of EGF-R-specific mRNA and DNA. The following DNA Materials. EGF, insulin, transferrin, and sodium selenite were ob probes were used. A 1.4-kilobase Â£coRIfull-length TGF-a complemen tained from Collaborative Research, Bedford, MA. Hydrocortisone, tary DNA fragment, cloned into plasmid pSP65C17N3 (a gift from triiodothyronine, ethanolamine, and phosphoethanolamine were pur Dr. R. Derynck, Genentech, Inc., South San Francisco, CA) (14), and chased from Sigma Chemical Co., St. Louis, MO. Bovine pituitary a 4.2-kilobase Sacll-Xhol full-length EGF-R complementary DNA extract was obtained from Hammond Cell Technology, Alameda, CA. fragment, contained in plasmid pCO12-EGF-R (a gift from Dr. T. The monoclonal antibody Ab528 directed against the ligand-binding Velu, National Cancer Institute, Bethesda, MD) (32), were used as site of the EGF-R was obtained from Oncogene Science, Manhassett, probes for TGF-a and EGF-R sequences, respectively. A l.I-kilobase NY. Xba-Pstl complementary DNA fragment was used to probe for human Cell Culture. The following SqCC lines were used in these studies. 7-actin mRNA, in order to ensure the integrity and to allow quantita- A253 submaxillary gland carcinoma cells (15), A431 vulvar carcinoma tion of the different RNA preparations. The DNA fragments were cells (16), C4-1 (17) and CaSki cervical carcinoma cells (18), CaLu-1 radiolabeled with [a-"P]dCTP (3000 Ci/mmol; New England Nuclear lung carcinoma cells (19), and FaDu buccal carcinoma cells (20) were Research Products, Wilmington, DE) by random primer extension to obtained from the American Type Culture Collection (Rockville, MD). a specific activity of > 10' cpm/^g of DNA (33). The esophageal CE-48 cells (21) were a gift from Dr. C-P. Hu, Taiwan, Detection of TGF-a by Radioimmunoassay. TGF-a in cell extracts Republic of China. SqCC/Yl cells were derived from a squamous and conditioned medium was measured by radioimmunoassay. For the carcinoma of the oropharynx (22). Cultures of HKc were established preparation of cell extracts, the medium of confluent cultures was and propagated as previously described (23, 24). Immortalized HKc/ entirely replaced with defined medium for 24 h. Cells were collected by HPV 16 human keratinocytes were a generous gift from Dr. L. A. Pirisi trypsinization and centrifugation. The pellets were washed twice with (University of South Carolina, Columbia, SC). This cell line was ice-cold phosphate-buffered saline and extracted in a buffer composed generated from a primary culture of human neonatal foreskin epidermal of 25 mM Tris (pH 7.4), 50 mM NaCl, 0.5% (w/v) deoxycholate, 2% cells by transfection with recombinant human papillomavirus type 16 (v/v) Nonidet P-40, 0.2% (w/v) sodium dodecyl sulfate, 1 mM phenyl- DNA as described previously (25). The spontaneously immortalized methylsulfonyl fluoride, 50 Mg/ml of aprotinin (Sigma), and l Â¿ig/mlof mouse epidermal cell line, BALB/MK, was obtained from Dr. B. E. leupeptin (Sigma) for 30 min on ice. Cells were then disrupted by Weissman, Chapel Hill, NC (26). passing them through a 30-gauge needle, and the suspension was All SqCC lines were maintained in monolayer culture using plastic clarified by centrifugation in a microfuge. For the collection of condi tissue culture flasks in standard medium, which was composed of 72.5% tioned medium, confluent cultures were fed fresh defined medium. (v/v) calcium-free DMEM, 22.5% (v/v) Ham's F-12 medium (Grand After 48 h, this medium was collected, frozen at â€”¿70Â°C,andreplaced Island Biological Co., Grand Island, NY), and 5% (v/v) fetal bovine with fresh defined medium that was collected after a second period of serum and which was supplemented with 50 Â»nnil of gentamicin 48 h and pooled with the first aliquot. For the radioimmunoassay, (GIBCO). HKc and HKc/HPV 16 cells were cultivated in MCDB153- aliquots of conditioned medium or cell extract were incubated with '"I- LB basal medium (see Ref. 25 for detailed description) that was sup labeled TGF-a tracer (Biotope, Inc., Seattle, WA) and latex beads plemented with EGF (10 ng/ml), insulin (5 ^g/ml), hydrocortisone (1.4 coated with anti-TGF-Â«(Biotope) for 16 h at 4Â°C.The beads were then x 10~7 M), ethanolamine (10~4 M), phosphoethanolamine (10~4 M), separated from the unbound TGF-a by centrifugation over a Spincu transferrin (10 Mg/ml), bovine pituitary extract (70 ng of protein/ml), and triiodothyronine (10~* M). BALB/MK cells were maintained in shion density gradient (Biotope), and the radioactivity associated with the latex beads was determined in a rack gamma counter (LKB, Upps standard medium supplemented with insulin (5 Mg/ml), transferrin (5 Mg/ml), selenium (5 ng/ml), and EGF (10 ng/ml). "Defined medium" ala, Sweden). The amount of TGF-a in the samples was determined by comparison with a standard curve generated using a set of standard refers to standard medium without EGF or serum. All cells were TGF-a preparations. routinely screened for and found to be free of Mycoplasma contamina EGF Binding. Binding of EGF to keratinocytes was determined as tion using the method described by Chen (27). described by Zendegui et al. (34), with minor modifications. The Stimulation of DNA Synthesis. For these experiments, the standard medium of confluent cultures in 24-well cluster dishes was replaced medium in confluent cultures was replaced with fresh defined medium. with defined medium (without EGF) 24 h prior to the assay. Mono- After 24 h, growth factors were added, and 20 h later, the cultures were labeled with 5 /Â¿Ci/mlof [mefA>'/-1H]thymidine (specific activity, 0.5 layers were washed twice with ice-cold binding buffer [calcium-free Ci/mmol) for 2 h at 37"C. The amount of cold TCA-precipitable, hot DMEM/50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (pH 7.5)/0.5% (w/v) bovine serum albumin] and incubated with I25I- PCA-soluble radioactivity was determined in a Beckman LS 7500 EGF at concentrations ranging from 0.075 to 3 n\i for 4 h at 4Â°C.The scintillation spectrometer in the presence of Optifluor (Packard Instru monolayers were then washed rapidly 6 times with ice-cold binding ments Co., Inc., Downers Grove, IL). Detection of EGF-R Gene Sequences, and TGF-a and EGF-R Gene buffer and solubÃlÃ/cdbyincubation for 30 min at room temperature with N NaOH/0.1% (w/v) sodium dodecyl sulfate. The radioactivity in Transcripts. Genomic DNA from SqCCs and HKc was isolated as described by Blin and Stafford (28). Equal amounts (5 to 10 ^g) of the suspension was determined in a gamma counter. Nonspecific bind ing was determined in control wells incubated with a 100-fold molar DNA from each cell type were subjected to electrophoresis on 1% (w/ excess of unlabeled EGF (CR-EGF, receptor grade; Collaborative Re v) agarose gels and then transferred onto nitrocellulose paper as de scribed by Southern (29). Total cellular RNA was extracted from cells search, Bedford, MA) and subtracted from total binding to obtain grown to confluency using guanidinium isothiocyanate, and centrifu- specific binding. The number of EGF-binding sites and the affinity of gation over a cesium chloride gradient, as described by Ullrich et al. the receptors for the ligand were calculated after transformation of the (30). After electrophoresis on a 1% (w/v) agarose/7% (v/v) formalde data as described by Scatchard (35). hyde gel (31), the RNA was transferred to nitrocellulose paper. In both cases, dried and baked filters were hybridized with radiolabeled DNA probes. Prehybridization and hybridization were carried out at 42Â°Cin RESULTS buffer containing 50% (v/v) formamide, 5x Denhardt's solution, 6x Previous studies in our laboratory had shown that human SSC, 0.1% (w/v) SDS, 50 mM Na2HPO4, and 100 Mg/ml of salmon SqCC cells were capable of exponential growth in defined sperm DNA. Filters were washed sequentially with 2x SSC/0.1% (w/ v) SDS at 20Â°C,Ix SSC/0.1% (w/v) SDS at 65Â°C,and 0.1% SSC/ medium that does not contain EGF, whereas, under these same 0.5% (w/v) SDS at 65Â°C.Hybridizing sequences were visualized by conditions, untransformed BALB/MK mouse keratinocytes autoradiography at -80Â°C, using Kodak XAR film with intensifying were arrested in the G0-G, phase of the cell cycle (see Refs. 23 screens. Densitometric analysis of the autoradiograms was performed and 36 and Footnote 4). These findings suggested that growth using a Visage 2000 gel scanner-computerized image analysis system of SqCCs in vitro is supported by autocrine factors, such as 6255

TGF-a. Fig. 1 shows that both primary keratinocytes and SqCCs expressed a 4.7-kilobase TGF-a-speeific mRNA species during exponential growth. Comparison of the hybridization _ 20 signal for TGF-a mRNA (normalized for the signal for the y- ÃŽ" actin transcript) by scanning densitometry revealed no signifi cant differences in the levels of TGF-a mRNA expression between keratinocytes and SqCCs. In order to determine whether this mRNA was being translated into protein, cell I extracts and conditioned media were analyzed for the presence of TGF-a by radioimmunoassay. Fig. 2A shows that cell ex Ãœ 5- tracts of all 8 SqCCs contained immunoreactive TGF-a. Inter estingly, cell-associated TGF-a was also found in nonneoplastic mill oo a " n A BALB/MK cells. However, in this case, no TGF-a was detect < -3 able in the conditioned medium, at least within the limits of detection of the Biotope assay used (approximately 1 ng/ml) Cell Line (Fig. IB). As BALB/MK cells are quiescent under the condi Fig. 2. TGF-a in cell extracts and conditioned media. Cell extracts and conditioned media of BALB/MK and SqCCs were prepared, and the amount of tions that we chose to obtain the extracts and conditioned TGF-a was determined by radioimmunoassay as described in "Materials and media (36), these findings suggest that the cell-associated TGI- - Methods." A, cell extracts; B, conditioned media. Columns, mean of determina a was unable to stimulate DNA synthesis. In contrast to BALB/ tions in 2 separate sets of extracts and media; bars, SE. MK cells, immunoreactive TGF-a was detectable in the condi tioned medium of each of the SqCC lines (Fig. 2B). As shown fold at concentrations between 1 and 10 ng/ml (see Fig. 3B). in Fig. 2B, 4 of the SqCC cell lines secreted approximately There was no evident quantitative relationship between the equal amounts of TGF-a, while A253, CaLu-1, and SqCC/Y1 amounts of TGF-a that the SqCC lines secreted and their cells produced significantly higher amounts of the peptide. Over response to exogenous EGF. a period of 96 h, concentrations of TGF-a between 0.67 and In order to determine whether the endogenous production of 6.74 ng/ml were achieved in the medium of SqCCs. These TGF-a by the SqCC cells was required for their proliferation values are within the range in which TGF-a is biologically in vitro, 6 of the cell lines were treated with the monoclonal active (11). antibody, Ab528, which blocks the binding of EGF/TGF-a to Based on the finding that SqCCs secreted endogenous TGF- its receptor. As shown in Fig. 4, treatment of these SqCCs with a to levels that were sufficiently high to stimulate proliferation, Ab528 inhibited their growth by between 45 and 60%. In we predicted that SqCCs would require variable but generally contrast, an immunoglobulin G2a mouse monoclonal control lower concentrations of exogenous EGF to stimulate DNA antibody did not affect the proliferation of the SqCC lines. synthesis than BALB/MK cells. Fig. 3A shows that this was These results provide strong evidence that these cells are, at indeed the case. Exogenous EGF stimulated DNA synthesis of least in part, dependent on the autocrine release of TGF-a for SqCCs either not at all (C4-1 and CaLu-1 cells) or only by 30 their growth. to 80%. In contrast, BALB/MK cells were exquisitely sensitive The EGF-R forms the second component of the EGF/TGF- to EGF, which stimulated DNA synthesis approximately 200- a autocrine pathway. Previous studies have shown that SqCC

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kb Fig. 1. TGF-a mRNA expression in SqCCs and HKc. Approximately 20 fig of total cellular RNA of SqCCs and HKc/HPV16 cells were re solved by formaldehyde/agarose gel electrophoresis, transferred to nitrocellulose paper, and hybrid ized to 32P-labeled TGF-a and -y-actin complemen 4.4 - â€¢¿TGFa tary DNA probes as described in "Materials and Methods." A 4.7-kilobase TGF-a-specific RNA transcript was expressed by HKc as well as each of the SqCCs. The 2.2-kilobase -y-actin transcript, which served as a control, is seen in the lower part of the autoradiogram. 2.4 - â€¢¿y-actin

6256

I 25Â° c-erbB protooncogene, which encodes the EGF-R (12, 13). Analysis of genomic DNA of both HKc and SqCCs (Fig. 6) showed that each of the cell lines contained the expected Â£coRI and ////it/Ill EGF-R-specific restriction fragments. Based on scanning densitometry of the autoradiograms and using HKc DNA as a control preparation, we estimated the EGF-R gene to be amplified approximately 60 times in A431 cells, 5 to 7 times in CaSki cells, and 2 to 4 times in SqCC/Yl cells. In order to determine whether the overexpression of EGF-binding sites was due to an increased expression of the EGF-R gene, we performed Northern blot analysis of RNA from SqCCs and HKc. As shown in Fig. 7, the HKc as well as each of the SqCCs expressed a predominant 10.5-kilobase, as well as a smaller, 0.6 1.3 2.5 5.0 10.0 0.1 1.0 10.0 100.0 EGF (ne/mi) 5.8-kilobase EGF-R-specific RNA species. In addition, A431 EGP (ng/ml) and CE-48 cells expressed an aberrantly sized specific RNA Fig. 3. Effect of EGF on DNA synthesis of SqCCs and BALB/MK cells. The medium of confluent cultures in 24 Â«ellcluster dishes was replaced with fresh species of 2.8 kilobases, as had been noted previously by others defÃnedmedium 24 h prior to the addition of EGF. After 20 h, cells were labeled (37). The level of expression of the EGF-R gene varied among with Imethyl-'H^hymidine for 2 h, and the amount of TCA-precipitable, hot PCA-soluble radioactivity was determined as described in "Materials and Meth the SqCC lines, as shown in Table 2. Compared to HKc, A431 ods." A, A431 (â€¢);C4-1 (A); CaLu-1 (D); CaSki (A); CE-48 (â€¢);FaDu (O); and SqCC/Yl cells expressed increased levels of both the 10.5- SqCC/Yl (Â»).B, BALB/MK (O) cells. Points, mean of at least 3 separate and 5.8-kilobase RNA transcripts. Although CaSki cells did experiments with duplicate wells per assay point; bars, SE. not express an elevated level of the 10.5-kilobase transcript, these cells did express increased amounts of the shorter, 5.8- OCâ€¢S kilobase, message. Thus, there was a good correlation between the degree of amplification of the EGF-R gene, the level of â€¢¿r(%ofcontrol)O RNA expression, and the number of EGF-binding sites detected on the cell surface of A431, CaSki, and SqCC/Yl cells. How ever, increased numbers of EGF-binding sites without amplifi cation of the receptor gene nor increased expression of EGF- ÃŒlii R-specific RNA was found in A253, C4-1, CaLu-1, CE-48, and 40-S \ ]fi FaDu cells. | Stimulation of the EGF-R by its ligand normally results in rapid down-regulation and internalization of the receptor (5). 20-n-ul\ Therefore, one might postulate that, in order for secreted TGF- a to continuously stimulate DNA synthesis, the expression of EGF-Rs on the cell surface would have to remain elevated. Fig. 8 shows that treatment of BALB/MK cells with EGF resulted a Nl *,03 in the loss of 90% of the cell surface EGF-Rs within 15 to 30 u B min. In contrast, down-regulation of the EGF-Rs on SqCC cells Cell Line was significantly reduced in 5 of 8 cell lines under the same Fig. 4. Effect of Ab528 anti-EGF-R antibody on the growth of SqCCs. SqCCs were plated in 24-well cluster dishes at a density of IO4cells/well. The following conditions (Fig. 8). These included the 3 cell lines that displayed day, the medium was entirely replaced with denned medium, and the AbS28 anti- increased transcription of the gene (A431, CaSki, SqCC/Yl), EGF-R antibody was added at concentrations of 1 (D), 10 (â€¢),or 20 (D) n\i. as well as C4-1 and CE-48 cells. Thus, besides gene amplifica Control wells received vehicle only. After 5 days, cells were detached from tion and mRNA overexpression, the relative inability of EGF- duplicate dishes by trypsinization and counted, using a Model ZM Coulter particle counter. Data are expressed as a percentage of untreated controls. Columns, mean of 2 separate experiments; Ann, SE. Table 1 Total '"1-EGF binding The medium of i-nntlm-m cultures was entirely replaced with denned medium for 24 h prior to the assay. Following incubation with 125I-EGF for 4 h at 4"C, cell lines often express high numbers of EGF-Rs but the rela the amount of cell-associated radioactivity was measured as described in "Mate tionship with TGF-a production was not determined. We stud rials and Methods." Values are derived from Scatchard transformation of the ied the characteristics of the EGF-Rs expressed by the same data. BALB/MK, A2S3, A431, CaLu-1, and FaDu cells expressed both high- and low-affinity EGF-Rs; C4-1, CaSki, and CE-48, only low-affinity binding sites; SqCCs that secreted TGF-a. Table 1 shows that, in the absence and SqCC/Yl cells, a single class of high-affinity EGF-Rs. of exogenous EGF, each of these cell lines expressed signifi receptors cantly higher numbers of EGF-Rs on their surface than BALB/ CelllineBALB/MKA253A431C4-1CaLu-1CaSkiCE-48FaDuSqCC/YlEGF(x lO'/cell)0.190.540.511.151.456.361.160.300.966.564.371.929.904.70Affinity(nM)1.5411.540.933.592.0810.569.740.282.486.633.774.1024.260.69 MK keratinocytes. A253, C4-1, and CaLu-1 cells expressed approximately twice the number of EGF-Rs as BALB/MK cells, whereas A431, CaSki, CE-48, FaDu, and SqCC/Yl dis played 4 to 10 times higher numbers of binding sites than BALB/MK cells (see Table 1). Fig. 5 shows that BALB/MK, A253, A431, CaLu-1, and FaDu cells express 2 classes of receptors. In contrast, C4-1, CaSki, and CE-48 cells expressed only a single class of low-affinity binding sites, whereas SqCC/ Yl cells displayed only high-affinity EGF-Rs (see Table 1). In a small number of cases of SqCC cell lines, the overexpression of EGF-Rs has been associated with amplification of the 6257

BALB/MK

l ' IÃ• io ',.

20 40 60 80 100 Bound (nnolei/108 ecu.)

ja 100 li il Â¡t soi

50 100 150 300 600 90O 1200 1500 Hound (fmole./io" cellÂ») Bound (fmolo/106 ecu.)

50 100 150 50 100 150 200 250 300 Bound (hnolÂ»/10H cellÂ») Bound (fmole./lO6 cell.)

200 400 GOO 800 1000 1200 200 400 600 800 1000 Bound tftnolei/106 cellÂ«] Bound (bnoleÂ»/IO6 cell.)

140 S^CC/Yl 120-

100-

1 Â»H

20-

0 200 400 600 800 1000 1200 1400 1600 Bound (fmole./106 cellÂ») 20 40 60 80 Bound (Imole./l OÂ«celi.) Fig. 5. Scatchard analysis of '"I-EGF binding to SqCCs and BALB/MK cells. Confluent cultures were incubated for 4 h at 4'C with 50 Ml/well of '"I-labeled EGF (range, 0.73 to 3 UM), and the amount of cell-associated radioactivity was determined as described in "Materials and Methods." Data were plotted as described by Scatchard (35). 6258

kb â€¢¿ Fig. 6. Detection of EGF-R gene sequences 21.8 â€”¿ in SqCCs and HKc. After genomic DNA from HKc and SqCCs was digested with /////

EcoRI Hindlll

Rs to be down-regulated in response to EGF may be contrib Table 2 EGF-R-specific RNA expression in SqCCs and HKc Expression of EGF-R- and -r-actin-specific RNA by HKc and SqCCs was uting to the increased expression of these receptors on the cell analyzed by Northern blotting as described in "Materials and Methods" and in surface of C4-1 and CE-48 cells. the legend to Fig. 7. The numbers in the table represent the ratio of the integrated intensity (absorbance x surface area) of the hybridization signal at the position In summary, the SqCCs that we examined produced and of the 10.5- and 5.8-kilobase bands to the signal obtained for the 2.2-kilobase y- secreted TGF-a constitutively, whereas nonneoplastic BALB/ actin transcript, as determined by scanning densitometry. normalized to a ratio MK mouse keratinocytes synthesized but did not release de of 1 for the HKc. tectable amounts of TGF-a into the medium. Furthermore, CelllineHKcA253A431C4-1CaLu-1CaSkiCE-48FaDuSqCC/transcript11.84.90.50.30.81.60.62.55.8-kilobasetranscript11.43.10.20.26.20.60.21.6 each of the SqCCs used in this study expressed higher numbers of EGF-Rs than untransformed keratinocytes. Thus, activation of the TGF-a/EGF-R autocrine loop is common in SqCC cells and is presumed to be important for maintaining the neoplastic phenotype of these cells.

Y1lO.S-kilobase kb DISCUSSION Our study indicates that the TGF-a autocrine pathway is activated in most SqCC lines, in the sense that the growth factor is constitutively produced, while its receptors are simul 9.5 - taneously overexpressed. These neoplastic cells apparently pro duce the growth factor constitutively and release biologically 7.5 - significant quantities of it into their microenvironment. In contrast, no TGF-a was detectable in conditioned medium of untransformed mouse keratinocytes. Previously, others had reported the presence of TGF-Â«-specific mRNA in primary 4.4 - keratinocytes (both murine and human) as well as SqCC biopsy specimens (10, 14). In the case of primary HKc, TGF-a is primarily produced and released into the medium in response to specific agents, such as EGF or phorbol esters (10, 38), although recent studies showed that dense cultures of HKc 2.4 - release small amounts of TGF-a into the medium in the absence of exogenous stimuli (39). Although we failed to detect any TGF-a in the conditioned medium of BALB/MK cells, we cannot exclude the possibility that small amounts of the growth Fig. 7. EGF-R-specific RNA expression in SqCCs and HKc. Approximately factor would have been detectable if the medium had been 20 jig of total cellular RNA of SqCCs and HKc were resolved by formaldehyde/ agarose gel electrophoresis, transferred to nitrocellulose paper, and hybridized to concentrated. In two previous studies, greater amounts of TGF- "P-labeled EGF-R and -y-actin complementary DNA probes as described in "Materials and Methods." EGF-R-specific RNA transcripts (10.5 and 5.8 kilo- a were detected in SqCC tumor specimens than in normal skin bases) were expressed by HKc cells as well as each of the SqCCs; in addition, an (40, 41). In aggregate, these studies and our own suggest that aberrantly sized 2.8-kilobase transcript was expressed by A431 and CE-48 cells. the production of TGF-a by normal keratinocytes is tightly 6259

presumably represents a feedback mechanism that shuts off cellular stimulation by TGF-a (or EGF) (47). A region of approximately 50 amino acids in the cytoplasmic portion of the EGF-R, which is required for receptor internalization, has recently been identified (47). Furthermore, NR6 fibroblasts, which express internalization-defective mutant EGF-Rs, dis play a transformed phenotype in the presence of low concentra tions of EGF (48). Therefore, we postulated that defective receptor internalization might represent the principal mecha nism that would allow continuous stimulation of SqCCs by the 0 15 30 45 60 90 TGF-a that is released into the medium. We observed a relative lime (min) inability of EGF-Rs to be down-regulated in 5 of the 8 SqCC Fig. 8. Down-regulation of EGF-Rs. The medium of confluent cultures in 24- lines. In the cases of A431, CaSki, and SqCC/Yl cells, which well cluster dishes of SqCCs or BALB/MK cells was replaced with fresh defined overexpress EGF-R RNA, the apparent decrease of internali medium without EGF 24 h prior to the experiment. Cells were then exposed to EOF (100 ng/ml) at 37'C for 0 to 90 min, incubated with acid buffer [50 HIM zation is probably the result of increased recycling of receptors glycine/100 mivi NaCl/0.02% (w/v) NaN3, pH 4.0] for 10 min at 20'C in order because of the increased production of receptor protein. to release bound EGF (53), and then exposed to 3 nM (20 ng/ml) '"I-EGF to Whether, in the case of C4-1 or CE-48 cells, the deficiency of determine the total number of cell surface EGF-Rs as described in "Materials and Methods." A, BALB/MK; Â»,A253; â€¢¿.A431;A, C4-1; T, CaLu-1; D, CaSki; receptor down-regulation is the result of a mutation of the O, CE-48; O, FaDu; â€¢¿,SqCC/Yl.Points, mean of 2 separate experiments; bars, EGF-R protein itself or reflects the presence of a regulatory SE. protein that prevents internalization and degradation of the receptor (see, for example, Ref. 49) remains to be determined. controlled, whereas the growth factor is constitutively released The mechanisms responsible for the increased expression of EGF-Rs on A253, CaLu-1, and FaDu cells are not known at by SqCC cells, and its production by SqCCs is greater than by HKc. Our findings also indicate that cell-associated (i.e., either this time, but it is apparently not due to an inability to down- intracellular or membrane bound) TGF-a does not act as a regulate the receptors. The autocrine production of TGF-a by the SqCC cell lines mitogenic stimulus, because even extracts of quiescent BAI .B/ MK cells contained approximately as much of the growth factor appears to be important to support their proliferation in vitro. as SqCC cells. These cells are capable of growth in the absence of exogenous EGF or TGF-a (Refs. 23 and 36; Footnote 4). Furthermore, Each of the SqCC lines that we examined expressed greater numbers of EGF-Rs on their cell surface than nonneoplastic the anti-EGF-R antibody Ab528 had been reported previously BALB/MK cells. This finding confirms earlier reports of in by others to inhibit the growth of A431 cells, both in vitro and creased EGF-R expression by SqCC cell lines as well as fresh i/i vivo (50, 51). We have now extended these observations to squamous carcinoma specimens (42-44). A small number of include 5 additional SqCC lines. It should be noted that Ab528 SqCC cell lines, which include A431 cells, have been described caused only about 50% inhibition of the growth of SqCCs. in which amplification of the EGF-R gene appears to be the Production of autocrine growth factors other than TGF-a by principal reason for overexpression of receptor protein (12,43). the SqCCs probably accounts for the residual growth of cells in Furthermore, in most cases of SqCC lines as well as primary the presence of Ab528. For example, we have recently found tumors, the EGF-R gene is not amplified, and other mecha that these same SqCC cell lines constitutively produce insulin- like growth factor I,4 which is also a mitogen for normal and nisms must be responsible for overexpression of the EGF-Rs (12,13,42,43). In our series, A431, CaSki, and SqCC/Yl cells neoplastic keratinocytes. The fact that malignant cells appar displayed amplification of the EGF-R gene. Two of these ently may produce multiple autocrine growth factors should be SqCCs (A431 and SqCC/Yl) expressed increased levels of both taken into account when designing forms of treatments aimed the 10.5- and the 5.8-kilobase EGF-R-specific RNA transcripts, at blocking autocrine pathways. whereas CaSki cells only overexpressed the 5.8-kilobase tran Exogenous EGF stimulated the DNA synthesis in SqCCs script. Both RNA species are believed to represent alternative significantly less well than in untransformed BALB/MK cells. splice products of the EGF-R gene and precursors of the mature Perhaps the most likely explanation for this discrepancy is the 3.9-kilobase mRNA that is translated into receptor protein fact that SqCC cells simultaneously release TGF-a into the medium, overexpress EGF-Rs, and are, therefore, constitutively (45). In addition to displaying gene amplification. A431 cells also expressed an aberrant sized EGF-R-specific RNA species maximally stimulated, whereas BALB/MK cells secrete no TGF-a (or very little) and are, therefore, more sensitive to of approximately 2.8 kilobases, as described previously (12). CE-48 cells expressed an aberrant message of similar size, exogenous growth factors. The stimulation of DNA synthesis although the EGF-R gene was not amplified in this case. of A431 cells by EGF is seemingly in contrast with previous Whether or not the aberrant mRNA species found in CE-48 reports that EGF inhibits the growth of this cell line (52). cells encodes a truncated form of the EGF-R, as is found in However, a minor degree of stimulation of DNA synthesis may A431 cells, remains to be determined (45). In any event, the not translate into actual promotion of cell proliferation. In increased level of EGF binding observed in all of the SqCCs addition, the responsiveness of A431 cells to EGF appears to can be attributed to overexpression of the EGF-R gene in only vary greatly among laboratories, suggesting the existence of 3 cases. Thus, another explanation is required for the increased different sublines with distinct phenotypes. This may well ex levels of EGF binding observed in the other SqCC cell lines, as plain the slight stimulation of DNA synthesis we observed. well as in tumor specimens. Under physiological circumstances, Finally, it is important to keep in mind that untransformed binding of EGF to its receptor is followed rapidly by receptor mouse keratinocytes may not represent the optimal control internalization and degradation (5, 46). Interhalization of the cells for comparisons with human SqCCs. The development of receptor is not required for initiation of DNA synthesis and 4Y. Brewer et al., submitted for publication. 6260

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. Activation of the Autocrine Transforming Growth Factor α Pathway in Human Squamous Carcinoma Cells

Michael Reiss, Ellen B. Stash, Vincent F. Vellucci, et al.

Cancer Res 1991;51:6254-6262.

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