We investigated the inhibitory effect of sunitinib, a newly approved multitargeted tyrosine kinase inhibitor, against the progression of renal cell cancer (RCC) bone metastases in vivo. In vitro cell proliferation was determined using the MTS assay. To investigate the inhibitory effects of sunitinib in vivo, we established luciferase-labeled ACHNLuc cells derived from papillary RCC. Mice in which ACHNLuc cells had been transplanted into the left ventricle to establish bone metastases were treated orally with 40 mg/kg/day sunitinib or vehicle control for 3 weeks. Growth of the cancer cells was monitored using an in vivo imaging system. In addition, 16 patients with metastatic RCC were treated with sunitinib, and serum and urine levels of amino-terminal telopeptide (NTx) were measured as markers of bone resorption. Sunitinib did not inhibit the growth of RCC cells in vitro at clinically or experimentally achievable serum levels (100 nM–1 μM). To investigate the inhibitory effect of sunitinib in vivo, we established luciferase-labeled human RCC cells (ACHNLuc). Sunitinib prevented the growth of ACHNLuc RCC cells in the bone metastatic mouse model. The number of osteoclasts in sunitinib-treated mice was significantly less than that in control mice. Serum and urine levels of NTx in patients with metastatic RCC declined significantly during the first 4 weeks of sunitinib treatment (p = 0.027). Sunitinib is a potent anticancer agent for RCC bone metastases, at least for papillary RCC.
Bone is a common site of metastasis, with the frequency of solitary or multiple metastases to bone ranging from 24 to 51% in patients with metastatic renal cell cancer (RCC).1–3 Although bone metastasis is not an independent prognostic factor associated with poor survival, the prognosis of patients with bone metastasis is not favorable when they are treated with cytokines, with an average life expectancy of 8–16 months.2–4 Moreover, bone metastases are associated with poor performance status due to intractable pain and pathological fractures.5 Because treatment options for RCC patients with bone metastasis are limited, appropriate treatment strategies are desired.
Sunitinib is a newly approved, multitarget, small-molecule tyrosine kinase inhibitor for the treatment of metastatic RCC. It inhibits various receptor tyrosine kinases, including vascular endothelial growth factor (VEGF) receptors 1, 2 and 3; stem cell factor receptor (KIT) and PDGF receptors α and β.6–8 Moreover, sunitinib has been known to inhibit the phosphorylation of colony-stimulating factor (CSF)-1R, resulting in the prevention of osteoclast function and CSF-1R-dependent osteolysis in an experimental breast cancer bone metastasis model.9, 10 These findings led us to propose the hypothesis that sunitinib may inhibit tumor growth and osteolysis in bone metastatic lesions in RCC patients.
Although establishing a treatment strategy for bone metastases from RCC is important for urologists, the assessment of inhibitory effects on the growth of bone metastases is often difficult in clinical practice. In this study, we show that sunitinib has anticancer as well as inhibitory activities against osteolysis in an experimental mouse model of bone metastasis of RCC cells.
Material and Methods
Animals, cell lines and reagents
Approval for these studies was obtained from the institutional review board at Akita University School of Medicine. Specific pathogen-free BALB/c nu/nu mice (CLEA, Kyoto, Japan) aged 7 weeks were used. The human RCC lines ACHN, CCFRC-1, CCFRC-2 and NC65 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and sunitinib was obtained from Pfizer (New York, NY).
A total of 16 native Japanese patients with metastatic RCC, who were treated at the Department of Urology at Akita University School of Medicine between 2008 and 2009, were enrolled, and the serum and urine levels of amino-terminal telopeptide (Serum NTx, normal range: 9.5–17.7 nmol/l) were measured as markers of bone resorption. The patients' characteristics are shown in Table 1. The median dose was 37.5 (25–50) mg/day and the median number of treatment cycles was 4.6 (1–21). Written informed consent was provided according to the Declaration of Helsinki. The study protocol was approved by the Ethics Committee of Akita University Graduate School of Medicine. The response was assessed by computed tomography (CT) after at least every two cycles of treatment, according to the Response Evaluation Criteria in Solid Tumors (RECIST ver. 1.0).11
Table 1. Patients characteristics
Growth inhibitory effects of sunitinib in vitro
Cell proliferation was determined by the MTS assay using CellTiter96 (Promega Corporation, Madison) as described previously.12
Generation of a stable luciferase-expressing cancer cell line
Among the RCC cell lines we tested (ACHN, CCFRC-1, CCFRC-2 and NC65), ACHN was the only line that was transplanted into the left ventricle and formed bone metastases successfully. Therefore, we used ACHNLuc in the in vivo experiment. ACHN cells were stably transfected with the pGL3 control vector (Promega Corporation, Madison) and with pSV2Neo (ATCC), as described previously.12 In brief, the cells were treated with 10 μg pGL3 control vector and 1 μg pSV2Neo vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM (Invitrogen) and selected using geneticin (400 μg/ml). Stable clones expressing luciferase were isolated and the clone with the highest level of luciferase expression (as determined by bioluminescence) was selected using luciferin (Xenogen, Alameda, CA) and an in vivo imaging system (IVIS; Xenogen).
In vivo effects of sunitinib
To produce bone metastasis models, RCC cell suspensions (3 × 106/100 μl phosphate-buffered saline) were injected into the left ventricle of mice under inhalation anesthesia with isoflurane (Abbott Japan, Tokyo, Japan). From 21 days after implantation, 14 mice with bone metastases were selected and divided into two matched groups on the basis of bioluminescence quantified by IVIS. On the same day, we started daily oral administration of 40 mg/kg (body weight) sunitinib or the solution used to dissolve sunitinib as vehicle control. According to the human 4 weeks on/2 weeks off schedule, mice were treated with sunitinib for 4 weeks before being sacrificed. Mice were observed by IVIS once per week.
Measurement of bone metastatic lesions by in vivo imaging
An aqueous solution of luciferin (150 mg/kg) was injected intraperitoneally 10 min before imaging. The animals were anesthetized with isoflurane and placed in the light-tight chamber of a CCD camera system (Xenogen) and photons emitted from the luciferase-expressing cells within the animal were quantified for 5 min using the software program Living Image (Xenogen) as an overlay on Igor (Wavemetrics, Seattle, WA). Using this in vivo imaging system, we evaluated the efficacy of sunitinib by measuring the photon counts of the metastatic lesions in the mandible and both hip joints in a blinded manner as described previously.13
Measurement of serum VEGF and M-CSF in the mouse bone metastasis model in vivo
The serum concentrations of VEGF and M-CSF in mice were determined using Quantikine ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. To investigate the serum concentrations of VEGF and M-CSF, sera from each of seven treated and seven untreated mice were collected and analyzed 4 weeks after ACHNLuc inoculation.
After imaging studies, the femora of the mice were removed, frozen immediately and stored at −80°C. To detect osteoclasts, 4-μm-thick sections were stained with tartrate-resistant acid phosphatase (TRAP) using the TRAP and ALP double-stain kit (Takara Bio, Otsu, Japan), as described previously.14 Three sections were examined in each femur. The number of TRAP-positive osteoclasts was counted per ten high-power microscope fields by two blinded examiners, as described previously.14
The influence of sunitinib on the growth of bone metastases was analyzed by Student's t test. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS version 13.0; SPSS, Chicago, IL), and two-sided p values <0.05 were considered statistically significant.
Effect of sunitinib on RCC growth in a mouse bone metastasis model in vivo
Injection of cancer cells via the left ventricle is an established method of inducing bone metastases, as reported previously.12, 13 In the present study, all mice that were successfully implanted with ACHNLuc cancer cells developed bone metastases 3 weeks after injection. Of these mice, we excluded those that showed brilliant bioluminescence in the lungs. The remaining 14 mice were then divided into two matched groups according to bioluminescence quantified by IVIS, and we administered either sunitinib or vehicle control for 4 weeks and monitored the growth of bone metastases in the lesions in the maxilla and bilateral hip joints, as described previously12, 13 (Fig. 1b). Metastatic bone lesions in the control group progressed during the 3 weeks. On the other hand, photon emission was significantly suppressed in the sunitinib treatment group (p < 0.001) (Figs. 1a and 1c). The mean body weights of the mice did not differ significantly between the two groups.
Serum VEGF and M-CSF in a mouse bone metastasis model in vivo
To examine the indirect antitumor effect of sunitinib, we measured the concentrations of VEGF and M-CSF. However, no significant difference was present in the serum concentrations of these growth factors between the two groups (Fig. 1d).
Effect of sunitinib on osteoclasts in a mouse bone metastasis model
Next, we investigated the efficacy of sunitinib against osteoclasts in the tumor-bearing mice. Femoral bone sections were stained with TRAP to enable counting of the number of osteoclasts, as described previously.13 The mean number of TRAP-positive osteoclasts in mice treated with sunitinib was significantly lower than that in mice treated with vehicle control (23.1 ± 4.7 vs. 33.2 ± 7.9 osteoclasts/100 high-power fields, respectively; p = 0.013).
Sunitinib did not inhibit cell proliferation in vitro at a clinically achievable serum concentration
To assess the direct antitumor effect of sunitinib, four RCC cell lines (ACHN, CCFRC-1, CCFRC-2 and NC65) were cultured in the presence of various concentrations of sunitinib (0.1 nM–10 μM). Sunitinib inhibited the proliferation of these cell lines in a concentration-dependent manner (Fig. 2). However, sunitinib was not effective in vitro at the clinically achievable serum concentration (∼80 nM), as demonstrated previously.8 On the other hand, the serum concentration of sunitinib was reported to be ∼100 nM on administration to mice at 40 mg/kg/day.14 The IC50s of sunitinib for these cell lines were estimated to be >1 μM. These results suggest the involvement of an indirect growth inhibitory mechanism of sunitinib, at least partially, for bone metastatic lesions in mice.
Effect of sunitinib on serum and urine levels of NTx in patients with metastatic RCC
The characteristics and demographic data of the patients are shown in Table 1. As shown in Figure 3, both serum and urine levels of NTx significantly declined during the first 4 weeks of treatment with sunitinib (p = 0.027). During the holiday period when the administration was discontinued following 4 weeks of administration of sunitinib, the serum and urine levels of NTx showed gradual recovery (Fig. 3). Of these 16 patients, five had bone metastatic lesions, but we could not evaluate the efficacy of sunitinib quantitatively. Regarding the extraosseous sites, nine of 14 patients demonstrated a partial response (PR) or stable disease (SD) whereas the remaining five demonstrated progressive disease (PD). The reduction rate of the serum NTx level from the baseline in patients with favorable efficacy (PR/SD; 30.8%) was higher than that in patients with poor efficacy (PD; 22%), although the difference was not significant (p = 0.6404).
In patients with metastatic RCC, bone is the major metastatic organ, second only to the lung.1–3 Bone metastases were shown to be associated with severe bone pain, pathological fractures, spinal cord compression and a short survival period.4, 11 Several studies have demonstrated that bone metastasis is one of the risk factors for poor prognosis in the cytokine era, although it was not identified as an independent prognostic factor.1–4 Négrier et al. investigated the prognostic factors of 782 metastatic RCC patients treated with cytokines and found that 32% (248/776) had bone metastases, and that these patients had a significantly worse prognosis than those without bone metastases (p = 0.008).2 Recently, Naito et al. retrospectively analyzed the prognosis of 1,463 Japanese metastatic RCC patients in the cytokine era and demonstrated that 24.6% (320/1,302) had bone metastases, and that these patients also had a significantly worse prognosis than those without bone metastases (p = 0.003).3 Accumulated evidence suggests that systemic immunotherapy is not effective in the management of bone metastasis of RCC.
The efficacy of sunitinib against RCC bone metastasis, however, remains to be established and is difficult to evaluate in clinical practice. Thus, we sought to investigate the efficacy of sunitinib against bone metastatic RCC in the preclinical setting. The dose of sunitinib used in this study (40 mg/kg/day) was intended to provide a serum level of sunitinib similar to that attained in the clinical setting.8, 15 Pharmacokinetic and pharmacodynamic analyses showed that the clinical dose of 50 mg/day led to plasma concentrations ranging from 50 to 100 ng/ml in humans.8 This dose is equivalent to the plasma concentration in mice administered sunitinib at 40 mg/kg/day.15 Data from VEGF-induced vascular permeability assays also support 50–100 ng/ml as the range, including the minimum plasma concentrations required to inhibit VEGFR and PDGFR in vivo.8 Therefore, our results obtained in the RCC bone metastatic model used in this study might be reflective of those obtained in the clinical setting.
Similar to several other in vitro analyses, our results showed that sunitinib at concentrations of 50–100 ng/ml did not inhibit the proliferation of RCC cells in vitro.10, 16 Therefore, we sought an indirect mechanism for this in vivo growth inhibition of RCC bone metastases. Bone is an abundant repository for immobilized growth factors, including transforming growth factor beta, fibroblast growth factor, insulin-like growth factors I and II, PDGF and bone morphogenetic proteins.17 When osteoclasts absorb bone by secreting protons and proteases, these growth factors are released and they provide fertile ground for the growth of cancer cells. Therefore, osteoclasts are a suitable therapeutic target in the treatment of bone metastases. In this study, there were significantly fewer TRAP-positive osteoclasts in the mice treated with sunitinib than in those treated with vehicle control (Fig. 1e). This observation is consistent with previous reports.10, 18 Zwolak et al. reported that treatment with sunitinib decreased the percentage of active osteoclasts to 45.6% ± 5.8% compared with the percentage in untreated tumor-bearing mice (79.4% ± 8.6%), suggesting that sunitinib treatment (40 mg/kg/day) may inhibit osteoclast maturation.18 Murray et al. reported that sunitinib inhibited osteoclast development and function mediated by M-CSF, which is one of the differentiating factors for osteoclasts and is a target tyrosine kinase of sunitinib, both in vitro and in vivo.10 Our clinical observation of decreases in serum and urine NTx is also in line with these reports (Fig. 3).
NTx is a degradation product of Type I collagen and is often used as a marker of bone resorption both in serum and urine. Some clinical studies have suggested that levels of NTx correlate with the presence and extent of bone metastases, prognosis and response to treatment.19, 20 Although our data did not show an association between the reduction rate of NTx and the efficacy of sunitinib, further investigation is necessary to clarify this association, especially in bone metastatic lesions.
During the completion of this manuscript, we found that ACHN originated from papillary renal cancer in a 22-year-old patient (Reference21 and by personal communication from Dr. Ernest Borden). Recent studies have suggested the possible clinical efficacy of sunitinib for patients with clear and non-clear cell cancer.22, 23 However, there are no prospective Phase 2 or Phase 3 studies clarifying this question. We therefore have to wait for the results of a large prospective study on the use of sunitinib for non-clear cell cancer. Since bone is the second most common site of metastases for RCC, we reported an indirect mechanism that may partly help to elucidate the reasons for the clinical efficacy of sunitinib.
Mesenchymal-epithelial transition factor (MET) and fumarate hydratase (FH) are considered to be the genes responsible for Type 1 and Type 2 papillary RCC, respectively.24, 25 MET, which is a proto-oncogene, encodes a tyrosine kinase membrane receptor, and activation of MET can indirectly promote angiogenesis and tumor growth through overexpression of VEGF.26, 27 FH is an enzyme in the mitochondrial tricarboxylic acid (TCA) cycle. Loss of FH leads to a state of pseudohypoxia through overexpression of hypoxia-inducible factor (HIF), resulting in an increase in downstream targets, including VEGF.26, 28 Therefore, activation of MET and loss of FH, which are considered to be responsible for Type 1 and Type 2 papillary RCC, lead to angiogenesis. Clinically, Ljungberg et al. demonstrated that the mRNA levels of VEGF, VEGF-receptor Type 1 and VEGF-receptor Type 2 above the median were related to adverse survival in papillary RCC.29 Therefore, it is relevant to measure VEGF in a clear or non-clear cell RCC model.
To elucidate whether sunitinib has any other indirect effects, we measured the concentrations of VEGF and M-CSF. However, we found no significant difference between the two groups in the serum concentrations of these growth factors. This observation is consistent with previous findings. Ebos et al. reported a significant increase in the serum VEGF level on administration of 60–120 mg/kg sunitinib.30 While it has been shown that sunitinib is a multikinase inhibitor that inhibits Class III and Class V RTKs, including PDGF receptors, VEGF receptors, KIT and FLT3, with low nanomolar potency,30 other growth factor-mediated signals might be inhibited by sunitinib. Further investigation is necessary to clarify the precise mechanism of action of sunitinib and its clinical efficacy against bone metastases.
In conclusion, we demonstrated that oral administration of a clinically achievable dose of sunitinib prevented the growth of RCC bone metastases in vivo. Because RCC cell lines are resistant to clinically and preclinically achievable plasma concentrations in vitro, prevention of osteoclast activity and/or maturation is one of the mechanisms of growth inhibition in metastatic bone lesions. Our study supports the use of sunitinib as an initial treatment for RCC patients with bone metastasis.
The authors thank Ms. Yuka Izumida and Ms. Tomomi Kawamura for their technical assistance.