Effects of Insulin-like Growth Factor Receptor Inhibition on Human Melanomas in Culture and in Athymie Mice1

The role of the insulin-like growth factor (IGF) receptor in regulating the growth of melanoma cells was evaluated by examining the effect of antibody-mediated IGF receptor inhibition on the growth of four human melanoma cell lines in culture and as xenotransplants in athymic mice. All four cell lines expressed typical type I IGF receptors and an antibody to this receptor (aIR-3) inhibited [I25I]IGF-I binding. However, the cell lines varied widely in their in vitro responsiveness to IGF-I and aIR-3: in the \\ M 373 and WM 852 cell lines, IGF-I stimulated cell replication and aIR-3 inhibited this response, whereas in the WM 239-A and WM 266-4 cell lines neither the growth factor nor the antibody affected growth. A wide variation was also observed in the effect of the antibody on the growth of the different cell lines as xenotransplants but this qualitatively correlated with the responses observed in vitro: aIR-3 treatment signifi cantly inhibited the growth of the WM 373 and WM 852 xenotransplants but did not inhibit the growth of the WM 239-A or WM 266-4 xenotrans plants and may even have had a slight stimulatory effect. These results indicate that the IGF receptor pathway is a functional regulator of the in vivo growth of some melanomas and that this is reflected in the activity of this pathway as determined in vitro. These findings suggest that therapies aimed at inhibiting the IGF pathway may be beneficial in treating some melanomas. INTRODUCTION IGF-I1 and IGF-II are polypeptide hormones which play a funda mental role in normal growth and differentiation (reviewed in Refs. 1 and 2). Functionally, the two IGFs have similar spectra of biological activities and mediate their effects through the same cell surface receptor, the type I IGF receptor (3, 4). The IGFs are synthesized by a variety of cell types and endocrine, paracrine. and autocrine actions have been described. In addition to their well recognized mitogenic activity, the IGFs also stimulate a wide variety of differentiated func tions, such as proteoglycan (5). myelin (6), and steroid hormone (7) synthesis in appropriate cell types. In stimulating both differentiated functions and mitogenesis, the IGFs often work synergistically with other trophic hormones and growth factors (7, 8). In view of their widespread anabolic effects on normal tissues, it is not surprising that a role for the IGFs in tumor growth has been postulated (9-11). The IGFs could stimulate the growth of trans formed cells either as a result of their normal mitogenic activity for a particular cell type or because of a specific alteration in the IGF receptor pathway in the transformed cell. In support of a role for the IGFs in the pathophysiology of tumor growth, it has been shown that a variety of tumor types express IGF receptors in vivo (12) and in vitro (9, 10, 13) and that some tumors aberrantly produce IGFs (14, 15). Moreover, type I IGF receptor inhibition has been shown to inhibit the Received 11/17/92; accepted 3/29/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by NIH Grant ROI-CA3898I. 2 To whom requests for reprints should be addressed, at Division of Endocrinology. Department of Pediatrics. University of Rochester School of Medicine and Dentistry, Box 777. 601 Elmwood Avenue. Rochester, NY 14642. 'The abbreviations used are: IGF-I. insulin-like growth factor I: IGF-II. insulin-like growth factor II; PBS. Dulbecco's phosphate buffered saline; SDS, sodium dodecyl sulfate. growth of Wilms' tumors ( 10) and some breast tumor cell lines in vivo (16). However, the role of these hormones in stimulating the growth of most tumor types remains unclear. Although a number of trans formed cell lines respond to the IGFs in vitro, many IGF independent cell lines have also been described (9, 17). Furthermore, it is not known whether the IGF responsiveness observed in vitro reflects an absolute dependence on these hormones or whether other growth factors could fulfill this role in vivo. Thus, the role which the IGFs play in the in vivo growth of specific tumor types remains largely undefined. The present studies were undertaken to define the role of the IGF pathway in stimulating tumor cell replication in vivo and to determine if the in vivo response correlates with that observed in vitro. To this end, we examined the effects of antibody-mediated type I IGF recep tor inhibition on the growth of four human melanoma cell lines in culture and as xenotransplants in athymic mice. This cell type was chosen because a variety of human melanoma cell lines that differ in their in vitro responsiveness to IGF are available and this cell type grows readily as s.c. tumors in athymic animals, thus allowing com parison of the in vivo and in vitro responses. The results indicate that the IGF receptor pathway is a functional regulator of the growth of some melanomas in vivo and that this is reflected in the activity of this pathway as determined in vitro. MATERIALS AND METHODS Cell Lines, Hormones, and Antibodies. The human melanoma cell lines used in these studies (WM 852. WM 373. WM 266-4. and WM 239-A) were provided by Dr. U. Rodeck (Wistar Institute. Philadelphia, PA) and have been described elsewhere (18, 19). All were obtained from metastatic lesions. The cells were cultured in RPMI 1640 containing 2c/c fetal bovine serum at 37°C in a humidified atmosphere containing 5<7cCOs. Powdered cell culture media (MCDB 104 and RPMI 1640) were purchased from GIBCO BRL (Grand Island. NY) and prepared as described previously (3). Human IGF-I was purified from Conn fraction IV-I as previously reported (3). All other reagents were obtained from Sigma Chemical Co. (St. Louis. MO). aIR-3, a monoclonal antibody to the human type I IGF receptor (20), was a generous gift of Dr. Steven Jacobs (Wellcome Research Laboratories, Re search Triangle Park. NC). This antibody is specific for the human type I IGF receptor and does not bind to the murine type I IGF receptor or the human insulin receptor; it is a competitive inhibitor of both IGF-I binding and IGF stimulated DNA synthesis in a variety of normal and transformed human cell types (3, 10, 16, 21). The aIR-3 used in these studies was produced as ascites fluid and purified on protein-A Sepharose (3). A mouse monoclonal antibody to the major surface glycoprotein of human Pneumocystis carini, «PC,which is of the same subclass as aIR-3 (IgG,). was used as a control in some of these studies. This antibody was a gift of Dr. Francis Gigliotti (University of Roch ester. Rochester. NY) and was purified as described for aIR-3. In Vitro Cell Replication Studies. To assess the role of the IGF-receptor pathway in stimulating the growth of human melanoma cell lines in vitro, a modification of the serum-free system previously described for WI-38 fibroblasts (3) was used. Cells from stock cultures were plated into 16-mm (24well) cluster dishes at a density of 20.000-80.000 cells/well in 1.0 ml RPMI 1640 supplemented with glutamine and 2% fetal bovine serum. After 24 h. the medium was removed and the cells were washed twice with PBS and 1.0 ml serum-free MCDB 104 medium was added. After 48 h, the medium was removed and 1.0 ml fresh MCDB 104 containing O.\7c bovine serum albumin, penicillin (100 units/ml), streptomycin (100 ug/ml). transferrin (1 ug/ml).