(CANCER RESEARCH 57. 4517-4522. October 15. I9>)7| Induction of Parathyroid Hormone-related Peptide by the Ras Oncogene: Role of Ras Farnesylation Inhibitors as Potential Therapeutic Agents for Hypercalcemia of Malignancy1 Fasika Aklilu, Morag Park, David Goltzman, and Shafaat Ahmed Rabbani2 Department of Medicine, McGill University and Calcium Research Laboratory and Molecular Oncology Group. Royal Victoria Hospital. Montreal. Quebec, H3A IAI Canada ABSTRACT into the circulation and acts at the level of bone to stimulate bone résorptionand Ca2^ release and on the kidneys to increase Ca2 + Parathyroid hormone related peptide (PTHRP) is the major causal reabsorption (19). The outcome of these actions is the development of agent in the syndrome of malignancy-associated hypercalcemia (MAH). hypercalcemia, which may be fatal. Several studies have shown that PTHRP production is increased in re sponse to growth factors and oncogenes, such as Tpr-Met, that are asso Increasing number of studies on the regulation of PTHRP gene ciated with the tyrosine kinase signaling pathway. Using site-directed expression are beginning to shed light on the molecular mechanisms mutagenesis of Tpr-Met and chemical inhibitors of phosphotidylinositol-3 responsible for PTHRP overproduction by cancer (20, 21 ). We have kinase and Ras isoprenylation, we demonstrated previously that induction previously shown the critical role played by growth factors, acting on of PTHRP is mediated via the Ras signaling pathway. In the present study, tyrosine kinase receptors, in stimulating PTHRP production (22). We we have directly investigated the role of the Ras oncogene in MAH. As a subsequently demonstrated the effect of the oncogenic derivative of model system, we used Fisher rat 3T3 fibroblasts stably transfected with the hepatocyte growth factor/scatter factor receptor, Tpr-Met, on a Ras oncogene (Ras-3T3). Ras transfection enhanced PTHRP production increasing PTHRP production and demonstrated that the mechanism 5-10-fold in these cells, and inoculation of this cell line into nude mice led of this stimulation involves the intracellular Ras signaling pathway to the development of hypercalcemia within 2 weeks. We used this system (22). Previous studies by us and others, examining the relationship to evaluate the effect of a potent inhibitor of Ras processing, B-1086, on between tyrosine kinases and PTHRP, have indeed indicated that Ras cell growth, PTHRP production, plasma calcium, and tumor growth. Treatment of Ras-3T3 cells in vitro with B-1086 at 0.1-10 ¿ig/mlproduced is at the cross-roads through which the signal for PTHRP induction is a significant reduction in PTHRP mRNA expression and PTHRP secre propagated. tion and a significant decrease in cell proliferation. Treatment in vivo of Ras genes play a fundamental role in basic cellular functions (23). BALB/c/nu/nu mice bearing Ras-3T3 tumors with B-1086 resulted in a Ras proteins are GDP/GTP-regulated switches that in their mature significant inhibition in tumor growth. In addition, this treatment pro functional form are anchored to the cell membrane by an isoprenyl tail duced near normalization of serum C'a2*, a significant decrease in plasma (24, 25). In normal physiology. Ras functions in transducing devel PTHRP, and a reduction in tumoral PTHRP mRNA levels. These results opmental and proliferative signals from the cell surface to the nucleus. show that the Ras pathway is involved in PTHRP production by tumors, In addition to its role in growth and differentiation. Ras is also identifies Ras as a potential target for treatment of MAH, and demon important in controlling cytoskeletal protein expression and in regu strates Ras processing inhibitors as candidate therapeutic agents against lating vesicle trafficking within the cell (26). Perhaps owing to its this syndrome. central role within the cell, alteration of this gene is often associated with the initiating steps that lead toward carcinogenesis. Indeed, INTRODUCTION activating mutations in the Rax gene account for at least 40% of Hypercalcemia of malignancy is a major complication in a signif human neoplasia and play a significant role in progression of these icant portion of cancer patients and PTHRP1 has been identified as the malignancies (27). In light of this, Ras is an ideal target for therapeutic major pathogenic factor for this disease (1-5). PTHRP shares a high intervention to combat both the establishment of the primary cancer degree of homology in its NHrterminal region with PTH and because and for the prevention of secondary syndromes (28). One approach to of this sequence homology is able to mimic the actions of PTH by target Ras has been the development of peptidomimetic inhibitors of interacting with a common PTH/PTHRP receptor (6. 7). In normal Ras, which mimic the farnesylation site of the Ras protein, a CAAX physiology, PTHRP is widely expressed, acts in an autocrine or consensus sequence at the COOH-terminal, and act by competitively paracrine manner, and has been shown to have a broad spectrum of inhibiting Ras farnesylation (29, 30). Farnesylation is essential for functions especially related to modulating cell growth and differenti subsequent modifications of Ras, for membrane localization, and ation (8-11 ). PTHRP appears to be a key regulator of skeletal devel ultimately for functioning of this oncogenic protein (31). opment in the fetus but has also been implicated in transepithelial In the present study, using Fisher rat 3T3 fibroblasts transfected calcium transport in such sites as mammary epithelium, kidney, and with the Ras oncogene, we have examined the role of Ras in PTHRP placenta (12-16). Furthermore, PTHRP has been shown to be a potent production and in the development of hypercalcemia in vivo. We have muscle relaxant acting on the smooth muscle of the uterus, urinary also tested the ability of a Ras farnesylation inhibitor B-1086 to alter bladder, and vascular wall (17). In malignancy-associated hypercal the course of the tumor and its production of PTHRP and assessed the cemia, PTHRP acts primarily as an endocrine factor (18). When large effects on the hypercalcémiesyndrome in vivo. quantities of PTHRP are produced by cancer cells, PTHRP is secreted MATERIALS AND METHODS Received 5/29/97; accepted 8/18/97. The costs of publication of this article were defrayed in part by the payment of page Ras Farnesylation Inhibitor B-1086. The Ras farnesyl transterase inhib charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. itor. B-1086 (29). was kindly provided by Dr. Michael Lewis of Eisai Research 1This work was supported by Medical Research Council of Canada Grants MT-10630. Institute (Andover. MA). MT-12609. and MT-5775 and by a grant from the National Cancer Institute of Canada. 2 To whom requests for reprints should be addressed, at Calcium Research Laboratory. Animal Protocols. Female BALB/c/iui/nu mice (Charles River Breeding Laboratories, Wilmington. MA), weighing 15-20 g (4-5 weeks of age) were Room H4-72. Royal Victoria Hospital. 687 Pine Avenue West. Montreal. Quebec. H3A injected on day 0 s.c. into the left and right flanks with Ras-3T3 cells ( 1 X IO5) 1A1 Canada. Phone: (5141843-1632; Fax: (514)843-1712. 1The abbreviation used is: PTHRP. parathyroid hormone-related peptide. suspended in 0.1 ml of sterile saline. Animals were housed individually and fed 4517 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1997 American Association for Cancer Research. INDUCTION OF PTHRP BY THK A,i.i ONCOGENE ad lihitiim on autoclaved standard rodent chow (rat chow 5012: Ralston-Purina ':P|dCTP as described previously (22. 33). After a 24-h incubation at 42°C, Canada. Inc.. Lasalle. Quebec. Canada) containing \r/c calcium and 0.74% filters were washed twice under low stringency conditions ( 1X SSC and phosphorus and sterilized tap water (32). On day 5, vehicle. 50 mg/kg B-1086. 1% SDS: at room temperature for 40 min) and under high stringency or 100 mg/kg B-1086 was administered daily i.p. into anaesthetized animals. conditions (0.1 X SSC, 0.1% SDS; at 55°Cfor 40 min). Autoradiography of All animals were examined for the development of tumors daily for up to 15 filters was carried out at —¿70°CusingXAR film (Eastman Kodak Co., days. On the day of sacrifice, animals were bled by intracardiac puncture, Rochester. NY) with two intensifying screens or by using a phosphorim- and tumor volume of control and experimental animals was measured in ager screen (Eastman Kodak Co.. Rochester. NY). The level of PTHRP two dimensions by calipers, and the tumor volume was calculated expression was quantified by densitometric scanning using the Mac BAS ((width + length/2)1]. On the day of sacrifice, animals were bled by intracar VI.01 alias program. diac puncture, and tumor tissue was extracted and immediately frozen in an Ras Processing Assay. Ras-3T3 cells were plated in medium containing ethanol bath for RNA analysis. 10% calf serum, and 24 h later, treated with 0. 10, or 50 ¿ig/mlof B-1086 for Cells and Cell Growth. The Fisher rat (Fr) 3T3 fibroblasts expressing the 72 h and harvested in lysis buffer (PBS. 1% NP40, 0.5% sodium deoxycholate. Ras oncogene were generated by ecotropic retroviral infection as described 0.1% SDS, 10 ptl/ml PMSF. 30 /Ml/ml aprotinin, and 10 /il/ml sodium or- previously (22). Transient transfection into COS-1 cells of the pLXSN retro- thovanadate). The lysates (25 fj.g) were electrophoresed on a 15% SDS- viral vector encoding the Kux gene and G418 resistance and of the pSV-Y-E- polyacrylamide gel. transferred to nitrocellulose membranes, and immuno- MLV plasmid encoding retroviral packaging proteins was performed by the blotted with anti-ras antibody (Santa Cruz Biotechnology).