Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 15, Num. 2, 1998, pp. 65-69

Jorge Berlanga,¹ Silvia Álvarez,¹ José de la Fuente¹ and Pedro López-Saura²

1Mammalian Cells Genetics Division, ²Clinical Trials Division. Center for Genetic Engineering and Biotechnology. PO Box 6162, Havana 10600, Cuba. Telef: (53-7) 21 8164; Fax: (53-7) 21 8070; 33 8008; E-mail: jorge.berlanga@cigb.edu.cu

Code Number: BA98009
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ABSTRACT

The epidermal growth factor (EGF) is a potent mitogen for a variety of epithelial and mesenchyma derived cells. Cells treated with EGF exhibit transient metabolic changes and a morphologic phenotype which mirror those found in malignant counterparts. EGF binds to a specific membrane receptor with intrinsic tyrosine kinase activity coded by a cellular proto-oncogene, whose mutated form has been largely implicated in malignancy. Experimental evidence indicates that EGF potentiates chemical carcinogenesis in vivo as well as viral transformation in vitro. Thus EGF has been historically considered as a tumor promoter. Nevertheless, recent experimental findings, mostly derived from preclinical models, strongly suggest that EGF treatment is insufficient to initiate benign or malignant transformation. On the contrary, in certain scenarios EGF may act as a potent cytoprotective agent for normal cells, and sensitizes cancer cells to anti-tumor therapy. All these experimental data provide fundamental basis for the clinical use of EGF in terms of safety. The clinical experience accumulated so far suggests that EGF does not seem to initiate carcinogenesis.

Key words: EGF, carcinogenesis, malignancy, toxicity

El factor de crecimiento epidérmico (FCE) es un potente mitógeno para células epiteliales y de origen mesenquimatoso. Las células tratadas con FCE muestran cambios metabólicos y morfológicos de carácter transitorio que son comparables con aquellos encontrados en células malignas. La respuesta celular al FCE está condicionada a la interacción con un receptor específico de membrana, modelo de actividad tirosina-cinasa, codificado por un proto-oncogen celular y cuya forma mutada ha sido vista asociada a la transformación maligna. Algunas evidencias experimentales han demostrado que el FCE potencializa el efecto transformante de carcinógenos químicos y virus oncogénicos, por lo que ha sido considerado como un agente promoter de la carcinogénesis. No obstante, hallazgos recientes, mayormente obtenidos en modelos animales, sugieren que el tratamiento a largo plazo con FCE es insuficiente para iniciar eventos premalignos o malignos. Contrariamente, el FCE ha mostrado ser un potente factor citoprotector para células normales, que además sensibiliza las células malignas al tratamiento con agentes antitumorales. Los datos aquí revisados son argumento para el uso clínico del FCE en términos de seguridad toxicológica. El seguimiento a largo plazo de pacientes que han recibido FCE, evidencia que este factor de crecimiento no parece ser un agente iniciador de carcinogénesis.

Palabras claves: FCE, carcinogénesis, malignización, toxicidad

The epidermal growth factor (EGF), is a 53-amino acid polypeptide that was originally isolated from the mouse submandibular glands as a concomitant of the nerve growth factor and recognized by its ability to stimulate precocious incisor eruption and eyelid opening in newborn mice (1). EGF is synthesized in a variety of tissues in mammals as a large precursor (prepro-EGF) of 1217 amino acids, which includes at least seven EGF-like sequences (2).

Mature EGF binds to a specific cellular receptor exerting a potent mitogenic activity on most epithelial tissues, fibroblasts, and endothelial cells (3). Besides, profound biochemical events are induced upon EGF-receptor interaction including protein phosphorylation, diacyl glycerol, inositol phosphate, prostaglandin and cyclic nucleotide generation, as alterations in intracellular ion concentrations. These events bring about cytoskeletal and morphological changes, alterations in the metabolic pathways of glucose and protein synthesis, which eventually culminate in cell-cycle progression (4). These EGF-induced metabolic effects mirror certain biochemical features found in tumor cells (5).

Several lines of evidence appeared more than ten years ago suggesting that the EGF-stimulated cell regulatory system may play a role in carcinogenesis. In brief, this evidence indicated that EGF elicits transformation-associated phenotypes in certain target cells, that EGF potentiates chemical transformation in vivo and viral transformation in vitro, and that polypeptide growth factors included in the EGF-like peptide family are secreted by transformed cells, thus enhancing tumor development (for review see 6).

This has, for years, brought concern on the clinical use of EGF. Recent data, mainly derived from animal models, suggest that EGF does not initiate cell transformation. Furthermore, it is likely that its role in carcinogenesis is only as a promoting agent by acting in an epigenetic manner when there is a prone genetic cell type toward malignancy.

Here we briefly mention classical findings related to the effect of EGF on cell metabolism, which have been considered as responses associated to neoplastic transformation. Accordingly, other data are in conflict with the first line of evidence:

1. EGF induces a partial loss of the dependence of growth on density inhibition and the dependence on serum growth (7-9) and increases the level of phosphotyrosine in proteins (10, 11). These examples describe only transient effects which are common to other growth factors and cytokines, that do not necessarily imply neoplastic transformation. Furthermore, these effects are reversible, so normal cells regain their functional pattern upon EGF removal. The fact that EGF increases the level of phosphorylation in tyrosine is, on the other hand, an ordinary and standard mechanism in cell signal transduction, which takes place in both normal or transformed cells (12).
2. EGF induces the expression of some cellular proto-oncogenes (13). Proto-oncogenes play a critical role in normal cell physiology (mitosis, migration, anchoring, differentiation), and they are depicted as membrane receptors, signal transducers, and transcription factors (14). Furthermore, the indispensability of certain proto-oncogenes for normal mammal organogenesis and development has been clearly illustrated by knock out animal models (15).
3. Growth of cells in soft agar and thereby colony formation is potentiated by EGF (16, 17). This is a phenotype-associated event, and not a gene-commanded behavior. It has been proved to be a transient and reversible event upon EGF removal (18).
4. EGF enhances viral transformation in cultured cells (19, 20). The effect of EGF on these cells mirrored those observed by 12-O-tetradecanoylphorbol13-acetate, a well-known tumor promoter. In other experiments, the presence of EGF was interpreted as a mandatory condition to perpetuate the transformed phenotype. However, EGF alone was unable to induce phenotypic changes in noninfected cells. Classical tumor promoters do not have or show very weak tumorigenicity by themselves, but can enhance the carcinogenic potential of chemical substances (21). Thus, EGF may be considered a tumor promoter, but as judged by these experiments it was insufficient to initiate malignant transformation.
5. EGF enhances the carcinogenic potential of methylcholanthrene in rodent skin (22, 23). However, it should be highlighted that repeated EGF intravenous administrations in rats, provoked a marked hypercellularity in several epithelial organs, including the skin, in a dose-dependent fashion. These changes were not associated to cell indifferentiation, benign or malignant tumorigenesis, nor they were detrimental to animal health (24).

1. According to preclinical models, EGF administration does not induce mutagenicity, clastogenicity nor cytotoxicity (24). Rather it was seen that EGF antagonizes the effects of some well-known mutagenic drugs as cyclophosphamide, cisplatin and thiotepa (Bello JL, National Institute of Oncology and Radiobiology, Havana, Cuba. Unpublished data).
2. EGF inhibits the growth of several lines of mammary, liver, pituitary, and cutaneous cancer cells (25-30).
3. Certain concentrations of EGF transiently inhibit tumor growth in vivo (31).
4. EGF is a cytoprotective agent for some cell systems (i.e. digestive tract). In this context, the role of EGF is linked to mucosal damage prevention against irritating and necrotizing agents (32).
5. EGF protects some peripheral tissues against radiogenic treatment secuelae (33).
6. Some recent experimental evidence showed that EGF sensitizes in vitro and in vivo cancer cells to anti-tumor therapy (34).
7. EGF was shown to prevent abnormal healing in animal models by preventing excess fibrous proliferation and scarring in the esophagus (35).
8. The fact that EGF stimulates the proliferation of basal epithelial cells has been recently interpreted as of therapeutic value in the treatment of cancer-chemotherapy associated mucositis (36).
9. Recent data from in vivo models suggested that EGF might prevent lipid peroxidation (37). Indeed, free radicals are considered as carcinogenic agents (38).
10. A constitutive over-expression of the EGF receptor and of a potent receptor ligand, transforming growth factor alpha (TGF-a ) is seen in psoriasis (39). However psoriasis is not a malignant disease. The excess production of TGF-a does not seem to be a determinant requisite to initiate carcinogenesis. The TGF-a transgenic mouse further supports this assumption. In these animals, the growth factor over-expression was targeted to the epidermal suprabasal layer by a specific keratin gene promoter. The consequences of the excess production of the TGF-a paracrine in relation to malignant transformation, were mostly limited to the appearance of papillomas while most of these tumors appeared in skin areas subjected to traumas (40). The presence of benign tumors primarily at wound sites suggests that factors produced in wounds must act together with TGF-a overexpression to cause papilloma formation.
11. The consequences associated to long term administrations of EGF in laboratory animals have recently been described. These data are briefly summarized here:

• Male Wistar rats received EGF at 150 µg/kg/day for 4 weeks. At the end of the treatment period, the animals exhibited a significant increase in mucosal weight and surface area of the functioning small intestine (41).
• A similar experiment was done in mini pigs receiving EGF at 30 µg/kg/day for 4 or 5 weeks. The experiment showed a significant size increase of the ureters and the urothelium. These findings were associated to the enhancement of the mitotic activity in the basal cells of the urothelium (42).
• In mini pigs and rats, the subcutaneous administration of EGF at 30 and 150 µg/kg/day, respectively during 4 weeks provoked an increase in the thickness of the esophageal epithelium. However, this hypercellularity was accompanied by a normal pattern of differentiation as judged by the characterization of the lectin binding affinity (43).
• A consistent ovarian growth was observed in Wistar rats receiving EGF at 150 µg/kg/day for 4 weeks. The ovarian growth was due to an increased number and size of follicular cysts and an increase in the quantity of luteinizing cells (44).
• Finally, we (Bello JL, Berlanga J, and López-Saura P, unpublished data) treated Beagle dogs (both sexes) with human recombinant EGF (hr-EGF) at 900, 180, and 54 µg/kg/day during 47 days. After seven days of treatment the animals treated with the largest EGF dose, exhibited systemic alopecia, sebaceous dermatitis, constant sialorrea and corneal illness. Days later, these changes appeared in the other dose-treatment groups. Ascitis and a marked increase in cutaneous thickness were onset at about day 20 of administration. At autopsy we found: (i) increase of the dermal thickness, (ii) a dramatic fibrotic proliferation of the connective tissue surrounding thoracic and abdominal organs, (iii) up to 100 mL of peritoneal liquid, and (iv) several visceral adherences to cavity walls. The microscopic study revealed a consistent thickness in kidneys and liver capsules and the corresponding organ stroma, hair follicle apoptosis, hyperplasia of several epithelial organs (salivary glands, thyroid epithelium, duodenum, etc.). The intensity of these changes was expressed in a dose and sex dependent fashion, where the most dramatic effects were observed in males.

All these experiments have confirmed the potent mitogenic capacity of EGF on a wide variety of epithelial responding tissues, as well as for some mesenchymally derived cells. They have also demonstrated that, at least under the experimental conditions established so far, the EGF-mediated hypercellularity is not associated to malignancy in animals with a conceivable normal cellular substrate. Indeed, it should be taken into account that these experiments were done in different animal species, sexes, and using a varied range of doses of the growth factor.

1. As judged by pharmacokinetic studies, EGF is rapidly cleared from the central bloodstream, and rapidly eliminated via urine after its parenteral administration (45). Furthermore, there is no data to indicate that EGF may be stored in any body compartment after a bolus infusion.
2. An illuminating experiment (46) showed that at least the events involved in skin healing wounds (cell migration, mitosis, angiogenesis, etc.) demand a sustained release of the peptide to achieve a significant clinical effect. In a similar context, we also observed that for EGF in semisolid vehicles to enhance wound healing, a frequent treatment schedule is required (47).
3. Wounding is considered a tumor promoter event at least in the skin (for review see 48). However, tumorigenesis has never been reported as a consequence of EGF treatments in wounded skin areas in humans (49, 50).
4. There are some mechanisms for the control of EGF availability in the pericellular milieu, in regard to cellular uptake and signaling pathways, so that cells may become insensitive to further EGF activation. These include cellular and extracellular peptidases (51, 52) and molecular events involving signal transduction as a "down-regulation" receptor, PKC-mediated tyrosine kinase regulation, etc. (for review see 53, 54).

EGF is identified as a tumor promoter according to in vitro and in vivo models. However, the experimental evidence obtained so far, indicate that EGF does not initiate carcinogenesis. The appearance of recent data indicating that EGF may be beneficial by preventing mutagenicity, lipid peroxidation and by sensitizing tumors to anti-cancer therapy, support the concept that EGF is, however, a potent cytoprotective polypeptide for certain cell systems.

The fact that: (i) EGF has contributed in saving lives under threatening condition, (ii) for such entities the treatment period is short, (iii) EGF is rapidly cleared from body compartments, and (iv) the clinical use of certain drugs is presided by a risk-benefit ratio analysis, in a case-by-case manner, provides a fundamental basis for a wider clinical use of EGF in terms of safety.

1. Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J Biol Chem 1962;237: 1555-1562.
2. Scott J, Urdea MS, Quiroga M, Sánchez-Pescador R, Fong N, Selby M et al. Structure of a mouse submaxillary messenger RNA encoding epidermal growth factor and seven related proteins. Science 1983;221:236-240.
3. Bennet N, Schultz G. Growth factors and wound healing: biochemical properties of growth factors and their receptors. The Am J of Surgery 1993;165:728-737.
4. Pusztai L, Lewis CE, Lorenzen J, McGee JOD. Review article- Growth factors: regulation of normal and neoplastic growth. J Pathol 1993;169:191-201.
5. Tannock IF, Rotin D. Acidic pH in tumors and its potential for therapeutic exploitation. Cancer Research 1989;49:4373-4384.
6. Stoscheck CM, King LE. Role of epidermal growth factor in carcinogenesis. Cancer Research 1986;46:1030-1037.
7. Carpenter G, Cohen S. Human epidermal growth factor and the proliferation of human fibroblasts. J Cell Physiol 1976;88:227-237.
8. Kirkland WL, Yang NS, Jorgensen T, Langley C, Furmanski P. Growth of normal and malignant human mammary epithelial cells in culture. J Natl Cancer Inst 1979;63:29-39.
9. Westmark B. Density dependent proliferation of human glia cells stimulated by epidermal growth factor. Biochem Biophys Res Commun 1976;69:304-309.
10. Hunter T, Cooper JA. Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A-431 human tumor cells. Cell 1981;24:741-752.
11. Nakamura KD, Martnez R, Weber MJ. Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts. Mol Cell Biol 1983;3:380-390.
12. Miyajima A, Kitamura T, Harada N, Yokota T, Arai KI. Cytokine receptors and signal transduction. Annu Rev Immunol 1992;10: 295-331.
13. Carpenter G, Wahl MI. The epidermal growth factor family. Handbook of Experimental Pharmacology: peptide growth factors and their receptors. Vol. 95. Berlin: Springer-Verlag, 1990:70-173.
14. Bishop MJ. Molecular themes in oncogenesis. Cell 1991;64:235-248.
15. Hilberg F, Aguzzi A, Howells N, Wagner E. c-Jun is essential for normal mouse development and hepatogenesis. Nature 1993;365:179-181.
16. Carpenter G, Stoscheck CM, Preston YA, De Larco JE. Antibodies to epidermal growth factor receptor block the biological activities of sarcoma growth factor. Proc Natl Acad Sci USA 1983;80:5627-5630.
17. Roberts AB, Anzano MA, Lamb LC, Smith LM, Frolik CA, et al. Isolation from murine sarcoma cells of novel transforming growth factors potentiated by EGF. Nature (Lond) 1982;295:417-419.
18. Roberts AB, Frol CA, Anzano MA, Sporn MB. Transforming growth factors from neoplastic and nonneoplastic tissues. Fed Proc 1983;42:2621-2625.
19. Fisher PB, Bozzone JH, and Weinstein IB. Tumor promoters and epidermal growth factor stimulate anchorage-independent growth of adenovirus-transformed rat embryo cells. Cell 1979;18:695-705.
20. Harrison J, Auersperg N. Epidermal growth factor enhances viral transformation of granulosa cells. Science (Wash. DC) 1981; 213:218-219.
21. Greenhalgh DA, Roop DR. Dissecting molecular carcinogenesis: Development of transgenic mouse models by epidermal gene targeting. Advances in Cancer Research 1994;64:247-296.
22. Reynolds VH, Bohem FH, Cohen S. Enhancement of chemical carcinogenesis by an epidermal growth factor. Surg Forum 1965; 16:108-109.
23. Rose SP, Stahn R, Passovoy DS, Herschman H. Epidermal growth factor enhancement of skin tumor induction in mice. Experientia (Basel) 1976;32:913-915.
24. Maraschin R, Bussi R, Conz A, Luciana O, Pirovano R, Nyska A. Toxicological evaluation of u-hEGF. Toxicologic Pathologic 1995; 23(3):356-366.
25. Hamburger AW, White CO, Brown RW. Effect of epidermal growth factor on proliferation of human tumor cells in soft agar. J Natl Cancer Inst 1981;67:825-830.
26. Imai Y, Leung CKH, Friesen HG, Shiu RPC. Epidermal growth factor receptor and effects of epidermal growth factor on growth of human breast cancer cells in long-term tissue culture. Cancer Res 1982;42:4394-4398.
27. Johnson LK, Baxter JD, Vlodasvdy I, Gospodarowicz D. EGF and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response. Proc Natl Acad Sci USA 1980;77: 394-398.
28. Knowles AF, Salas Prato M, Villela J. Epidermal growth factor inhibits growth while increasing the expression of an ecto-calcium-ATPase of a human hepatoma cell line. Biochem Biophys Res Commun 1985;126:8-14.
29. Schonbrunn A, Krasnoff M, Westendorf JM, Tashjian AH. EGF and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. J Cell Biol 1980;85:786-797.
30. Barnes DW. Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free culture. J Cell Biol 1982;93:1-4.
31. Lage A, Lombardero J, Pérez R. Estudios sobre el factor de crecimiento epidérmico (EGF). IV. Efecto del EGF sobre la incorporación de timidina en células tumorales in vivo. Interferón y Biotecnología 1987;4(2):115-120.
32. Brzozowski T, Kont PC, Kont SJ, Konturek P. Mucosal irritant, adaptive cytoprotection, and adaptation to topical ammonia in the rat stomach. Scand J Gastroenterol 1996;31(9): 837-846.
33. Alert J, Rodríguez J, Lombardero J, Pérez R. Acción radioprotectora local del factor de crecimiento epidérmico humano recombinante: reporte preliminar. Interferón y Biotecnología 1989;6(1):62-66.
34. Christen RD, Horn DK, Porter DC, Andrews PA, Macleod CL, Howel SB. Epidermal growth factor regulates the in vitro sensitivity of human ovarian cancer cells to cisplatin. J Clin Invest 1990;86:1632-1640.
35. Juhl CO, Vinter Jensen L, Jensen LS, Dajani EZ. Preventive effect of recombinant human epidermal growth factor on the oesophageal epithelium in pigs subjected to sclerotherapy. Eur J Gastroenterol Hepatol 1995;7(9):823-829.
36. Sonis ST, Costa JN, Evitts SM, Lindquist LE, Nicolson M. Effect of epidermal growth factor on ulcerative mucositis in hamsters that receive cancer chemotherapy. Oral Surg Oral Med Oral Pathol 1992;74:749-755.
37. Berlanga J, Caballero M, Ramírez D, Torres A, Valenzuela C, Playford RJ. Evidence for an EGF-dependent hepatic cytoprotection in rats. A novel therapeutic avenue for EGF. Clin Sci 1998;94:219-223.
38. Nilsson R, Beije B, Preat V. On the mechanism of the hepatocarcinogenicity of peroxisome proliferators. Chem Biol Interact 1991;78:235-250.
39. Elder JT, Fisher GJ, Lindquist PB et al. Overexpression of transforming growth factor-a in psoriatic epidermis. J Invest Dermatol 1989;243:811-814.
40. Vassar R, Fuchs E. Transgenic mice provide new insights into the role of TGF-a during epidermal development and differentiation. Genes and Development 1991;5:714-727.
41. Vinter-Jensen L, Smerup M, Kissmeyer-Nielsen P, Poulsen SS. Chronic systemic treatment with epidermal growth factor in the rat increase the mucosal surface of the small intestine. Regul-Pept 1995;60(2-3):117-124.
42. Vinter-Jensen L, Juhl CO, Poulsen SS, Djurhuus JC, Dajani EZ, Nexo E. Chronic administration of epidermal growth factor to pigs induce growth specially of the urinary tract with accumulation of epithelial glycoconjugates. Lab Invest 1995;73(6):788-793.
43. Juhl CO, Vinter-Jensen L, Poulsen SS, Orntoft RF, Dajani EZ. Chronic treatment with epidermal growth factor causes esophageal epithelial hyperplasia in pigs and rats. Dig-Dis-Sci 1995;40(12):2717-2723.
44. Christiansen JJ, Vinter-Jensen L, Nielsen K. Systemic treatment in the rat with epidermal growth factor causes polycystic growth of ovaries. APMIS 1996;104:147-152.
45. Kuo BS, Kusmik W, Pole J, Elsea S, Chang J, Hwang KK. Pharmacokinetic evaluation of two human epidermal growth factors (hEGF51 and hEGF53) in rats. Drug Metab Disposition 1992;20(1):23-30.
46. Buckley A, Davidson J, Kamerath CD, Wolt TB, Woodward SC. Sustained release of epidermal growth factor accelerates wound repair. Proc Natl Acad Sci USA 1985;82: 7340-7344.
47. Berlanga J, Lodos J, Labarta V, Hayes O, Mulet J, López-Saura P. The effect of epidermal growth factor treatment schedule on the healing process of full-thickness wounds in pigs. Biotecnología Aplicada 1997;14: 163-168.
48. Sieweke MH, Bissell MJ. The tumor-promoting effect of wounding: A possible role for TGF-b -induced stromal alterations. Critical Reviews in Oncogenesis 1994;(2&3):297-311.
49. Brown GL, Nanney LB, Griffen J, Schultz GS. Enhancement of wound healing by topical treatment with epidermal growth factor. N Engl J Med 1989;321:76-79.
50. Cohen IK, Crossland MC, Garret A, Diegelmann RF. Topical application of epidermal growth factor onto partial-thickness wounds in humans volunteers does not enhance reepithelialization. Plast Reconst Surg 1995;96(2):251-254.
51. Mast BA, Schultz GS. Interactions of cytokines, growth factors and proteases in acute and chronic wounds. Wound Repair and Regeneration 1996;4(4):411-420.
52. Okumura K, Kiyohara Y, Komada F, Iwakawa S, Hirai M, Tohru F. Improvement in wound healing by epidermal growth factor (EGF) ointment. I. Effect of Nafamostat, Gabexate, or gelatin on stabilization and efficacy of EGF. Pharmaceutical Res 1990;7(12): 1289-1293.
53. Earp H S, Dawson LT, Li X, Yu H. Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research. Breast Cancer Research and Treatment 1995;35:115-132.
54. Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell 1990;61:203-212.

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