A Third-Generation Bisphosphonate, Minodronic Acid (YM529), Augments the Interferon A/B-Mediated Inhibition of Renal Cell Cancer Cell Growth Both in Vitro and in Vivo

Vol. 11, 853–859, January 15, 2005 Clinical Cancer Research 853

A Third-Generation Bisphosphonate, Minodronic Acid (YM529), Augments the Interferon A/B-Mediated Inhibition of Renal Cell Cancer Cell Growth Both In vitro and In vivo

Takeshi Yuasa, Masaki Nogawa, Shinya Kimura, INTRODUCTION Asumi Yokota, Kiyoshi Sato, Hidekazu Segawa, Renal cell cancer (RCC) is the most lethal of the urologic Junya Kuroda and Taira Maekawa malignancies and its incidence is currently on the increase (1). At the time of initial diagnosis, one third of patients with RCC Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto, Japan exhibit visceral metastasis, and half of the remainder eventually develop distant metastases (2, 3). Currently, the only effective therapeutic and preventive agents for distant metastases and local ABSTRACT recurrence are IFN and interleukin 2, although these agents have Purpose: Minodronic acid (YM529) is a third-generation achieved response rates of only 15% (2). Therefore, the discovery nitrogen-containing bisphosphonate. Here, we have investi- of a novel, more effective therapeutic agent is urgently needed. gated the therapeutic efficacy of YM529 against renal cell Bisphosphonate is an inhibitor of bone resorption and has cancer (RCC) alone or in combination with IFN both in vitro been shown to directly and indirectly prevent proliferation and and in vivo. inhibit metastasis of various types of cancer cells (4–9). Experimental Design: One murine and eight human RCC Bisphosphonates inhibit farnesyl pyrophosphate synthase, which cell lines were used for the in vitro studies and were subjected serves in the mevalonate pathway (9). Consequently, they inhibit to a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- the activation of small G-proteins such as Ras, Rap1, and Rho, zolium bromide assay and Western blotting. Luciferase- reduce the signals they mediate, and thereby prevent the growth, labeled murine RCC cells (RENCALuc) were transplanted adhesion/spreading, and invasion of cancer cells (4–9). In into the s.c. tissue or the renal subcapsule of syngeneic BALB/c addition, bisphosphonate-induced inhibition of the mevalonate mice. These mice were treated with YM529 and/or murine pathway increases the levels of the middle metabolic products of IFN and the growth of the cancer cells was monitored by an isopentenyl pyrophosphate, which stimulate g/y T lymphocytes in vivo imaging system. (10). g/y-T lymphocytes exhibited marked cytotoxicity against Results: YM529 inhibited the growth of RCC cells in a various tumor cells including RCC (11). Moreover, bisphosph- dose- and time-dependent manner and enhanced the growth onates inhibit angiogenesis by reducing the levels of vascular inhibitory potential of IFN in vitro. In the in vivo mouse endothelial growth factor (VEGF) that are produced by the models, YM529 did not markedly inhibit the RCC cell cancer cells (12). growth on its own but it augmented the anticancer effect of RCC is an attractive target for both angiogenesis inhibitors IFN (P < 0.05). The YM529-treated mice (with or without and immunomodulators because angiogenesis is crucial for IFN) did not alter the ;/D T-lymphocyte numbers. The tumor development and RCC responds well to immunotherapy. various treatment regimens were also not associated with any The third-generation bisphosphonate zoledronic acid delays the adverse effects. However, YM529 combined with IFN onset of skeletal-related events and the progression of skeletal reduced the serum vascular endothelial growth factor levels. disease in patients with advanced RCC (13). However, the Conclusions: Our study suggests that YM529 may be a antitumor effect of bisphosphonates against RCC remains potent anticancer agent for RCC. The efficacy and safety of unknown. In this study, we show that minodronic acid (also IFN plus YM529 as a therapy for RCC should be verified by known as YM529), another third-generation bisphosphonate, has early-phase clinical trials. anticancer activity and synergistically augments the growth inhibitory effects of IFN against RCC both in vitro and in vivo.

Received 8/9/04; revised 10/7/04; accepted 10/21/04. Grant support: Uehara Memorial Foundation, Foundation for Promo- MATERIALS AND METHODS tion of Cancer Research, Public Trust Haraguchi Memorial Cancer Reagent, Cell Lines, and Animals Research Fund, Ichiro Kanehara Foundation, Fujiwara Memorial Foundation, COE program of the Ministry of Education, Culture, Sports, YM529 (3-amino-1-hydroxy-propylidene-1.1-bisphospho- Science and Technology, Japan, and Grants-in-Aid for Scientific nate) was obtained from Yamanouchi (Tokyo, Japan). Natural Research from the Ministry of Education, Culture, Sports, Science, and human IFN-a and mouse IFN-a/h were kindly provided by Technology, Japan. Hayashibara Biochemical Laboratories, Inc. (Okayama, Japan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked The human RCC cell lines 293, ACHN, CCFRC-1, CCFRC-2, advertisement in accordance with 18 U.S.C. Section 1734 solely to CAKI-1, CAKI-2, NC65, and RPMI-SE were obtained from indicate this fact. the American Type Culture Collection (Rockville, MD). The Requests for reprints: Takeshi Yuasa, Department of Transfusion murine RCC cell line RENCA was stably transfected with Medicine and Cell Therapy, Kyoto University Hospital, 54 Kawahara- Luc cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751- luciferase (Luc) to produce RENCA cells, as described 3630; Fax: 81-75-751-3631; E-mail: [email protected]. previously (8). Approval for these studies was obtained from #2005 American Association for Cancer Research. the institutional review board at Kyoto University Hospital.

Specific pathogen-free 6- to 8-week-old BALB/c mice were RESULTS used in this study (SLC, Kyoto, Japan). Effect of YM529 on the Prenylation of Ras and Rap1A and VEGF Production in RCC Cells Western Blot Analysis We investigated whether YM529 prevented the prenylation Western blot analysis was done as described previously (5). of Rap1A (which was activated after geranylgeranylation) and Goat polyclonal anti-unprenylated Rap1A antibody (diluted Ras (which was mainly activated after farnesylation). YM529 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse clearly inhibited the prenylation of Rap1A as it resulted in a monoclonal anti-Ras antibody (diluted 1:1,000; Becton Dick- dose- and time-dependent increase in unprenylated Rap1A levels inson, San Jose, CA) were used as the primary antibodies. in both human and murine renal cancer cell lines (ACHN and Determination of Cell Proliferation In vitro RENCA, respectively; Fig. 1A). The prenylation status of Ras Cell proliferation was determined by a modified 3-(4, was slightly inhibited in YM529-treated ACHN cells but its 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) prenylation status was not altered in treated RENCA cells. Thus, similar to other cancer cells, bisphosphonates can inhibit assay, as described previously (5). The IC50 was obtained using the nonlinear regression program CalcuSyn (Biosoft, Cambridge, United Kingdom).

Flow Cytometry Two-color flow cytometric analyses were conducted by fluorescence-activated cell-sorting analysis (BD Biosciences, Mountain View, CA), as described previously (5). The antibodies used were FITC-conjugated anti-mouse TCR g/y (Immunotech, Marseille, France), Cychrome-conjugated anti- mouse TCRh (BD PharMingen, San Diego, CA), and phyco- erythrin-conjugated anti-mouse CD3 (BD PharMingen).

Production of VEGF by RENCA Cells In vitro and In vivo The concentrations of VEGF of the culture supernatants or the sera of the mice were determined by using ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. To investigate the serum concentrations of VEGF, the sera of treated mice (see below) were collected and analyzed.

Mouse Models To generate s.c. or renal orthotopic models of RCC, RENCALuc cells (1 Â 105 per 100 AL PBS) were injected into the s.c. tissue of the lateral flank or the renal subcapsule after exposing the kidney, respectively. Tumor growth was monitored by using an in vivo imaging system (IVIS, Xenogen, Alameda, CA) with an aqueous solution of luciferin (150 mg/kg i.p., Xenogen), as described previously (8).

In vivo Effects of YM529 and/or IFN Mice were given the indicated number of RENCALuc cells on day 0. One week later, the mice were observed by IVIS and the bioluminescence from the implanted cancer cells was measured. The mice were then divided into four groups of seven mice in such a way that each group had almost the same average bioluminescence. The mice were then either left untreated or were treated thrice a week for 2 weeks with 80 Ag/kg YM529 and/or 1 Â 104 Fig. 1 Effect of YM529 on the prenylation of Ras and Rap1A in RCC units of mouse IFNa/h. The YM529 and mouse IFN were given cells and on VEGF elaboration. In vitro effect of YM529 on the s.c. Three weeks after RENCALuc cell inoculation, all mice were prenylation of Rap1A and Ras in ACHN and RENCA cells (A). The cells were treated with various concentrations of YM529 for 24 or 48 hours killed humanely and their sera were collected. Serum levels of the and then their lysates were immunoblotted for unprenylated Rap1A, following were then determined: aspartate aminotransferase, Ras, or h-actin. Top and bottom arrows, unprenylated and prenylated alanine aminotransferase, lactate dehydrogenase, total protein, forms of Ras, respectively. In vitro effect of YM529 (B) or IFN (C)on creatinine, blood urea nitrogen, calcium, and VEGF. the release of VEGF into the supernatant by cultured RENCA cells. The cells (1 Â 105 per well) were plated in six-well plates and incubated for Statistical Analysis 24 hours, after which medium was removed and replaced with medium containing various doses of YM529 or IFN. After 96 hours of incubation, Statistical significance was determined by the Student’s the culture supernatants were collected and the concentrations of VEGF t test (P < 0.05) using Excel (Microsoft, Redmond, OR). were determined by ELISA. Columns, means; bars, SE.

Fig. 2 Effect of YM529 alone and in combination with IFNa/h on the in vitro growth of RCC cells. A, growth inhibitory effect of YM529 alone on RCC cells. Various RCC cell lines were plated at 3,000 per well in 96-well plates, incubated for 24 hours, and then treated with various doses of YM529-containing medium. After 48 or 96 hours of incubation, relative cell growth was measured by a modified MTT assay. Columns, means; bars, SE. B, effect on RCC cell proliferation of combining YM529 and IFN. CI is plotted as a function of the affected fraction, which represents the percentage of growth inhibition and was evaluated by using the modified MTT assay (0.5 = 50%). This allows the combination of multiple equi- potent drug concentrations to be analyzed for synergistic (CI <1), additive (CI = 1), or antagonistic (CI >1) effects. Columns, means of three in- dependent experiments; bars, 1 SE.

prenylation in RCC cells, although they prevented geranylger- cell lines and RENCA by the MTT assay. YM529 inhibited the anylation more than farnesylation. growth of these cells in a time- and dose-dependent manner To examine the indirect effects of YM529 and IFN on RCC, (Fig. 2A). The IC50 values of YM529 with the nine RCC cell we investigated the concentrations of VEGF in the culture lines are summarized in Table 1. These results indicate that supernatant of YM529-treated and untreated RENCA cells. The bisphosphonates can inhibit the growth of RCC cells as well treatment reduced the elaboration of VEGF in a dose-dependent as other cancer cells. manner, with 50% reduction being achieved with 28.0 Amol/L YM529 (Fig. 1B). In contrast, IFN at concentrations below Effect on RCC Growth of Combining YM529 and IFN 10,000 units had no effect on the production of VEGF by the Bisphosphonates are known to augment the effects of RCC cells (Fig. 1C). several anticancer drugs (6, 7) but it has not yet been reported whether bisphosphonates enhance the anticancer effect of Inhibitory Effect of YM529 and IFN on RCC Cell Growth IFN. Thus, we investigated the effect on in vitro RCC cell In vitro growth of combining YM529 and IFN. The data are plotted as Next, we investigated the ability of YM529 and human and combination index (CI) versus the fraction affected (Fa). At mouse IFNs to inhibit the growth of eight human renal cancer growth inhibition levels exceeding Fa 0.2 and 0.4, YM529 plus

Table 1 IC50 values of YM529 and IFNs against renal cancer cells 293 ACHN CCFRC-1 CCFRC-2 CAKI-1 CAKI-2 NC65 RPMI-SE RENCA YM529 20.3 F 6.08 12.3 F 5.13 7.28 F 1.87 29.7 F 6.12 59.9 F 3.65 45.4 F 4.16 55.4 F 12.6 77.2 F 1.18 18.2 F 0.62 (Amol/L) IFN-a >1 Â 104 9620 F 50.9 944 F 72.7 >1 Â 104 979 F 89.1 >1 Â 104 >1 Â 104 >1 Â 104 (IU/mL) Murine IFN a/h 3.25 Â 104 (units/mL) F 1.98 Â 104 NOTE. Values are means F SE (n = 3).

IFN acted synergistically (CI <1.0) with two renal cancer Effect of YM529 and/or IFN on RCC Growth in a cell lines, ACHN and RENCA, and acted additively with Subcutaneous Mouse Model CCFRC-1 cells (Fig. 2B). At Fa 0.50 and 0.80, the CIs of In vivo growth of the RENCALuc cells can be monitored by ACHN, CCFRC-1, and RENCA were 0.681 F 0.172 and 0.880 their extracorporeal bioluminescence, as we have observed an F 0.161, 0.806 F 0.209 and 0.910 F 0.422, and 0.481 F 0.0763 excellent correlation between the cancer cell bioluminescense and 0.604 F 0.0596, respectively. These results suggest that and the tumor volume (8). We used this model to investigate the at least, combining YM529 and IFN does not reduce their effect of YM529 alone or in combination with IFN on the in vivo respective anticancer effects and that they can act synergistically growth of the tumor cells. The photon counts of the mice were to inhibit the growth of RCC cells. measured every week and the average of the groups was plotted

Fig. 3 Effect on in vivo RCC cell growth of YM529 alone or in combination with IFNa/h using the s.c. mouse model. Real time growth curves show the mean photon counts of RENCALuc cells in the s.c. implanted mouse model (A). n, untreated mice; 5,mice treated with YM529; ., mice treated with IFN; o,mice treated with both YM529 and IFN. On day 21, a significant inhibition of in vivo tumor growth was seen in mice treated with both IFNa/h andYM529 (B). Effect of IFN and YM529 on the serum levels of VEGF in RENCA-bearing mice (C). Af- ter 2 weeks of treatment, sera of these mice were collected on the day after the last treatment and the VEGF levels were assayed by ELISA. In vivo effect of IFN and YM529 on the a/h and g/y T lymphocyte populations of RENCA bearing mice (a/h T lymphocytes, D; g/y T lymphocytes, (E). After 2 weeks of treatment, the blood of these mice was collected on the day after the last treatment and the T-lymphocyte populations were investigated by two-color flow cytometry.

augments the direct growth inhibitory effect of IFN as well as indirect effects by reducing the elaboration of VEGF by the tumor.

Effect of YM529 and/or IFN on RCC Growth in a Renal Orthotopic Mouse Model After administering bisphosphonates, their concentrations in the kidney are substantial because it is the only eliminating organ for the drugs (14). Although the pharmacokinetics and pharmacodynamics of bisphosphonates may differ between humans and mice, this observation suggests that bisphosphonates may be of value in treating the primary renal lesion. To investigate this, we established a renal orthotopic model by implanting 1 Â 105 RENCALUC cells into the left renal subcapsule. Shortly after the implantation, bioluminescence was detected in all of the mice. The photon emissions were not detectable the following day but were detected again 1 week later, after which the photon emissions increased substantially over time (Fig. 4A). Using this orthotopic mouse model, we investigated the effect of YM529 alone or in combination with IFNa/h on the in vivo growth of the RENCALuc cells. As with the s.c. model, YM529 did not significantly inhibit the tumor cell growth on its own. However, it did augment the growth inhibitory effect of IFN (Fig. 4B). Figure 4C shows that by day 21, the inhibitory effect of combining IFNa/h and YM529 was statistically significant compared with the untreated mice. To investigate the safety of this combination, we examined the body weight during the treatment period and the serum concentrations of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, total protein, creatinine, blood urea nitrogen, and calcium. Combining the treatments did not have an adverse effect on body weight or any of the serum values tested (data not shown). Fig. 4 Effect on in vivo RCC cell growth of YM529 alone and in combination with IFNa/h using the renal orthotopic mouse model. Luc-labeled RENCALUC cancer cells implanted orthotopically were monitored by IVIS (A). Images were obtained extracorporeally 1 day DISCUSSION (A), 1 week (B), 2 weeks (C), and 3 weeks (D) after the injection. The respective photon counts of each mouse are represented by the color This study shows that the third-generation bisphosphonate scales. The real-time growth curves show the mean photon counts of YM529 has a direct effect on the in vitro proliferation of RCC the cancer cells in the renal orthotopic mouse model (B). n, untreated cells and that it augments the effects of IFN on RCC cells both 5 o mice; , mice treated with YM529; ., mice treated with IFN: , mice in vitro and in vivo. IFN has been widely used in immuno- treated with both YM529 and IFN. Analyses of the photon counts on day 21 shows that combining IFNa/h and YM529 significantly pre- therapy for RCC (2, 3), but there is still a paucity of effective vented the tumor growth in the renal orthotopic model (C). medical treatments for RCC, which is one of the reasons RCC is the most lethal urologic malignancy (1, 2). To improve the current therapies for this disease, clinical trials with several (Fig. 3A). On day 21 after cell implantation, when we sacrificed new attractive agents such as anti-VEGF antibody bevacizumab all of the mice humanely, we found that combining IFN and (15), the small molecule inhibitor of VEGF-mediated signaling YM529 significantly inhibited the growth of the RENCALuc SU5416 (16), and thalidomide (3, 17) have been carried out. cells compared with the growth of these cells in the untreated However, a well-established medical therapy for patients with mice (Fig. 3B). Furthermore, this combination significantly RCC with or without IFN is still lacking. reduced the serum VEGF levels (Fig. 3C). We have focused on bisphosphonate as a therapeutic partner We examined the proportion of blood cells that consists of for IFN because, as with other cancer cells, it may exert a direct a/h and g/y T lymphocytes in the RENCA tumor-bearing mice anticancer effect on RCC as well as act as an immunomodulator after 2 weeks of treatment. The a/h T lymphocytes increased in that induces g/y T lymphocytes (10) and prevents angiogenesis the IFN and IFN plus YM529 treatment groups but this was not (12). It is known that bisphosphonates inhibit protein prenylation statistically significant (Fig. 3D). The g/y T-lymphocyte levels in various cancer cells (5–9), but the effects of bisphosphonates also did not alter between the different groups (Fig. 3E). Thus, it on RCC have not yet been reported. Here we found that YM529 seems that these agents do not alter the proportion of a/h or g/y blocked the prenylation of Rap1A in ACHN and RENCA cells T lymphocytes in the blood, which suggests that YM529 (human and murine RCC cell lines, respectively; see Fig. 1) and

inhibited the in vitro growth of RCCs (Fig. 2A) in a dose- and was several times higher than the recommended dose for time-dependent manner. This indicates that YM529 may be a osteoporosis, which is 4 mg given as a 15-minute i.v. infusion at potent anti-RCC agent. intervals of 3 to 4 weeks (22). Treatment of patients with high When we examined a broader range of RCC lines, we levels of bisphosphonates can cause renal toxicity (23, 24). found that YM529 inhibited the in vitro growth of five of eight However, Chen et al. (25) have reported that the type, frequency, human RCC lines and the one mouse RCC line that we evaluated or severity of adverse events and changes from baseline vital with IC50 values ranging from 7 to 50 Amol/L (Table 1). How- signs and clinical laboratory variables are unrelated to the dose ever, three of the human lines required more than 50 Amol/L of zoledronic acid (4, 8, and 16 mg) or the pharmacokinetic YM529 to inhibit their in vitro growth. One multidrug resis- parameters that they examined. In the present study, we tance mechanism of RCC is the cellular overproduction of examined the body weight of the mice and their serum variables. P glycoprotein, which acts as an efflux pump for various We did not detect any harmful effects, even in the mice that were anticancer drugs (18). Differences in the sensitivity of the RCC treated with a combination of YM529 and IFN. Thus, we lines to YM529 may depend on their bcl-2, P glycoprotein, or speculate that the dosage we used to treat the RCC-grafted mice farnesyl pyrophosphate synthase activities. However, our pre- may be safe for patients with RCC as well. vious studies indicated that there is no correlation between We also investigated the effect of YM529 on production of sensitivity to zoledronic acid and bcl-2 expression in small-cell VEGF by the RCCs and the T-lymphocyte subset levels in the lung cancer cell lines and that the growth inhibitory effect of blood to determine how YM529 imposed its anticancer effects. zoledronic acid does not involve the P glycoprotein–related We did not detect an inducing effect on g/y T lymphocyte multidrug resistance system (5, 6). Salmo et al. have reported numbers in YM529-treated mice, but we did find that combining that bisphosphonate-resistant cells show increased farnesyl YM529 and IFN depleted the VEGF levels in the serum. VEGF pyrophosphate synthase activity without up-regulation of its is an important therapeutic target, as described above (3, 15–17). gene transcription (19). Therefore, it may be that the differences These observations suggest that the growth inhibitory effect of in RCC line sensitivity to YM529 that we observed is due to YM529 plus IFN in vivo is due to direct synergism between varying farnesyl pyrophosphate synthase activity. the antiproliferative activities of the two drugs in combination When we assessed the effect on in vitro RCC cell growth with an indirect effect, namely, the reduction of VEGF levels. of combining YM529 with IFN, we found that it had synergic In conclusion, YM529 is a third-generation bisphosphonate or at least additive antiproliferative effects on two human cell with anticancer activity against RCC that acts synergistically lines, ACHN and CCFRC-1, and the murine cell line, RENCA with IFN both in vitro and in vivo. This indicates that IFN plus (Fig. 2B). Therefore, we investigated the growth inhibitory effect YM529 may be a promising therapeutic strategy for RCC. of YM529 in combination with IFN in two mouse models, Recently, the U.S. Food and Drug Administration approved namely, a s.c. implanted model and a renal orthotopic model. several bisphosphonates for treating not only osteoporosis but In both models, treatment with YM529 or IFN alone yielded also cancer-related bone complications (26). The efficacy and a marginal anticancer effect. However, combining IFN and safety of IFN plus bisphosphonate as a therapy for RCC should YM529 had a significant antiproliferative effect in both models be verified by early-phase clinical trials. (Fig. 3B and 4C). The low efficacy of YM529 alone may be because it is difficult to achieve therapeutically effective serum concentrations of YM529 in vivo. 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Takeshi Yuasa, Masaki Nogawa, Shinya Kimura, et al.

Clin Cancer Res 2005;11:853-859.

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