Potential Role of the Epidermal Growth Factor Receptor in Human Pancreatic Cancer Murray Korc Departments of Medicine and Biochemistry, University of Cahfornia, Irvine, CA 92717

Summary The epidermal growth factor (EGF) receptor is a transmembrane protein that has.tyrosine kinase activity~ It is activated by both EGF and transforming growth factor-alpha (TGF-c0. Human pancreatic cancer cells overexpress the EGF receptor and exhibit a parallel increase in EGF receptor mRNA without a detectable increase in the number of gene copies coding for the receptor. These cells also produce TGF-c~ and are capable of binding exogenous TGF-c~. They often recycle EGF, but markedly and rapidly degrade TGF-c~. However, TGF-c~ is 10-100-fold more potent than EGF in enhancing their anchorage-independent growth. Both growth factors induce EGF receptor down-regulation, but EGF is more efficient than TGF-c~ in this regard~ The concomitant overexpression of the EGF receptor and production of TGF-cq the recycling of EGF, and the attenuated ability of TGF-c~ to down-regulate the EGF receptor may combine to provide a distinct growth advantage to human pancreatic cancer cells. Key Words: Epidermal growth factor; transforming growth factor-alpha; pancreatic cancer. INTRODUCTION Carcinoma of the pancreas is a m a j o r cause of cancer death in the United States. In spite of considerable progress in our understanding of normal pancreatic physiology, the factors that regulate pancreatic cancer cell proliferation are still not clearly delineated, and the reasons for the aggressiveness of this cancer are not known. Previous work has established that several cultured h u m a n pancreatic cancer cell lines overexpress the epidermal growth factor (EGF) receptor by comparison with normal rat pancreas (1). Overexpression of the E G F receptor is known to be associated with enhanced metastatic potential and t u m o r invasiveness (2) and has been reported in a variety o f cultured human carcinoma cell lines and in primary biopsy speciInternational Journal of Pancreatology

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mens in several human malignancies (3-6). Therefore, the observation that human pancreatic cancer cell lines overexpress the EGF receptor suggested that this receptor may be important in providing a growth advantage to these cells~ The present review will provide an update on our understanding of the EGF receptor and its ligands, and describe its potential role in the regulation of pancreatic cancer cell growth. THE EPIDERMAL GROWTH FACTOR RECEPTOR

The EGF receptor is a 170-kdalton glycosylated phosphoprotein (7,8) that is coded for by a gene that is located on the short arm of chromosome 7 (9). The receptor consists of an extracellutar domain, a transmembrane domain, and an intracetlular domain (10). The extracellular portion of the receptor is cysteine-rich, and contains the ligand binding region (10). The transmembrahe domain is very hydrophobic, and allows the receptor to anchor itself within the cell membrane (10,11). The intracellular domain contains three important components: a kinase region that catalyzes the autophosphorylation process, tyrosine phosphorylation sites, and an ATP binding domain (7,8, 10-14). Tyrosine phosphorylation of the EGF receptor is essential for the proliferative response to EGF (11-13), and this type of reaction has been implicated in the regulation of cell proliferation by a variety of growth-promoting proteins. The potential importance of the overexpression of the EGF receptor in conferring an abnormal growth advantage to cancer cells is underscored by the observations that this receptor exhibits a strong sequence homology with the protein product of the avian erythroblastosis virus v-erb B oncogene (15) and that an increase in the number of EGF receptors during transfection experiments induces the transformed phenotype (16). Following binding of EGF to its receptor, the ligand-receptor complexes translocate through the membrane and internalize into the cell by a process termed receptor-mediated endocytosis (17). In most cell types, internalized EGF undergoes rapid degradation within lysosomes or other acidic organelles (18). It is generally accepted that EGF degradation is initiated by the sequential removal of carboxy-terminal amino acids, allowing EGF to dissociate from its receptor (19). In most instances the receptor is then also susceptible to rapid degradation. Consequently, EGF binding is decreased by incubating cells with EGF (20). The binding and subsequent endocytosis of EGF are also decreased by a variety of other signals, including 12-O-tetradecanoylphorbol-13-acetate (TPA), platelet-derived growth factor (PDGF), fibroblastderived growth factor (FGF), cholecystokinin (CCK), carbachol, caerulein, bombesin, vasopressin, interleukin-1, and 1,25-dihydroxy-vitamin D3 (21-28). The inhibitory effect of CCK is probably mediated through a rise in cytosolic free calcium (25) as well as through the activation of protein kinase C (29), a calcium-activated, phospholipid-dependent enzyme (30). TPA directly activates protein kinase C (21-23), thereby phosphorylating the receptor on a threonine residue at position 654, close to the inner surface of the plasma membrane (31). This leads to a decrease in the affinity of the EGF receptor for EGF (32).

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Insulin deficiency is associated with a decrease in EGF binding in the pancreas and liver (33,34). Insulin deficiency is also associated with alterations in cellular calcium homeostasis in the pancreas (35), and calcium is known to regulate EGF binding and endocytosis (25). Furthermore, hyperinsulinemic diabetes is associated with decreased binding of EGF (36). It is probable, therefore, that either the hyperglycemia per se or the metabolic changes that occur in diabetes cause the observed decreases in EGF binding. It is also conceivable that certain tissues are especially susceptible to diabetes-induced alterations in EGF binding and action. Thus, these findings raise the possibility that the increased incidence of pancreatic cancer in patients with diabetes mellitus may be caused, in part, by perturbations in the regulatory actions of the EGF receptor within this tissue.

TRANSFORMING GROWTH FACTOR-ALPHA In addition to binding EGF with high affinity, the EGF receptor also binds transforming growth factor-alpha (TGF-o~), vaccinia virus growth factor, Shope fibroma virus growth factor, myxoma virus growth factor, and amphiregulin (37-43). Although these growth factors share only a 30-35% homology with EGF, they possess six cysteine residues in the same relative positions as E G F (42,43). Therefore, the three-dimensional configuration of their receptor binding domain is believed to be very similar to that of EGFo There is no evidence for the existence of a distinct receptor for TGF-c~. Therefore, the biological activities of this polypeptide are believed to result from its ability to bind to the EGF receptor and to phosphorylate it on tyrosine residues (44,45). Although EGF and TGF-c~ exert similar biological effects in a variety of cell types (43,49), there are several exceptions to this rule. Thus, TGF-c~ exerts a greater stimulatory effect than EGF with respect to calcium mobilization from fetal rat bones (50), angiogenesis in the hamster cheek pouch model (51), arterial blood flow in the dog (52), skin wound healing (53), formation of keratinocyte megacolonies (54), and induction of cell ruffling (55). TGF-c~ is also a more potent inhibitor of the proliferation of RL95-2 human endometrial carcinoma cells than is EGF (56). In addition to these quantitative differences, there are qualitative differences between the actions of the two growth factors. The inhibitory effect of TGF-c~ on norepinephrine-induced contraction in arterial strips is diminished following repeated exposure of the strips to EGF, but not to TGF-o~ (52). In primary lung carcinoma cells, TGF-c~ enhances but EGF inhibits cell proliferation (57). The chicken EGF receptor exhibits a higher affinity toward TGF-c~ than toward EGF (58). Furthermore, TGF-e~ is 100-fold more potent than EGF in stimulating DNA synthesis in 3T3 cells that express the chicken EGF receptor by comparison with 3T3 cells that express the human homolog (58). These observations raise the possibility that the two ligands acting through the same receptor may activate different effector pathways and second messengers. A variety of cancer cell lines have been found to express large quantities of TGF-o~ (59). Furthermore, overexpression of TGF-~ following transfection experiments confers unto cells the ability to proliferate in an anchorage-inde-

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pendent manner and to form tumors following implantation into nude mice (60)~ TGF-c~ has also been implicated in chemical-induced transformation and in oncogene-induced tumorigenicity (,61,62). Although TGF-c~ is also produced by normal cells and may enhance cell growth in certain benign hyperproliferative states, such as psoriasis (63,64), the production of this factor by malignant cells may represent an important mechanism whereby these cells obtain a growth advantage. AUTOCRINE REGULATION OF THE EGF RECEPTOR IN PANCREATIC CANCER

EGF enhances both the anchorage-independent and anchorage-dependent growth of cultures human pancreatic carcinoma cells (65, 66). Although these cells overexpress the EGF receptor, none are growth-inhibited by EGF. In contrast, certain cell types that overexpress the EGF receptor are growthinhibited by EGF (67). The mechanisms whereby pancreatic cancer cells overexpress the EGF receptor are not known, and the reasons why this overexpression may confer a growth advantage to these cells have not been clearly delineated. However, it is established that these cells exhibit increased levels of EGF receptor mRNA (1). Some of these cell lines exhibit either structural or numerical alterations of chromosome 7 (1). However, none demonstrates either a measurable increase in the number of copies of the gene coding for the receptor or abnormal EGF receptor m R N A transcripts (1,68). EGF is not readily degraded in normal pancreatic acini (25), nor in PANC-I, T3M4, ASPC-1, or COLO-357 (ref. 69,70; Fig. 1) human pancreatic carcinoma cells. In the case of PANC-I and T3M4 cells, several different types of experiments have demonstrated that there is considerable recycling of the ligand between the intracellular and extracellular compartments (69, 70). It is possible that the attenuated degradation of EGF by these cancer cells is a reflection of their tissue of origin. In the case of both normal and cancerous pancreatic cells, this phenomenon may allow one EGF molecule to induce the internalization and degradation of numerous EGF receptors. Furthermore, the release of large amounts of biologically active EGF by pancreatic cancer cells may also allow for its local accumulation, resulting in its enhanced availability to a variety of cell types that are present within a pancreatic tumor mass. In contrast to EGF, TGF-c~ is extensively degraded in cultured h u m a n pancreatic carcinoma cells (70). The enhanced degradation of TGF-~ is illustrated in Fig. 2. In the case of PANC-I, T3M4, and ASPC-1 cells, the lysosomal inhibitor methylamine completely inhibits the degradation of both EGF and TGF-o~ (69, 70). In the case of COLO-357 cells, the degradation of EGF is completely inhibited by methylamine (Fig. 1B), whereas that o f TGF-~ (Fig. 2B) is markedly but incompletely attenuated. This observation suggests that the processing of both growth factors occurs mainly within lysosomal or

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