Preclinical evaluation of combined TKI-258 and RAD001 in hepatocellular carcinoma
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- Chan, S.L., Wong, C., Lau, C.P.Y. et al. Cancer Chemother Pharmacol (2013) 71: 1417. doi:10.1007/s00280-013-2139-4
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RAD001 targets at the mammalian target of rapamycin (mTOR), while TKI-258 is a potent tyrosine kinase inhibitor targeting at fibroblast growth factor receptor, vascular endothelial growth factor receptor, platelet-derived growth factor receptor and c-kit. We aim to study the activity of combined RAD001 and TKI-258 in cell lines and xenograft model of hepatocellular carcinoma (HCC), with reference to the parallel and upstream pathways of Akt–mTOR axis.
A panel of 4 human HCC cell lines HepG2, Hep3B, PLC/PRF/5 and Huh7 and the Hep3B-derived xenograft were treated with TKI-258 or/and RAD001, respectively. Related mechanistic studies (including apoptosis and angiogenesis) were conducted.
There was an enhanced increase in suppression of cell proliferation with combined TKI-258 and RAD001 compared with either drug alone. The combination could significantly suppress the phosphorylation of mTOR, MEK1/2 and p38 MAPK. Although the addition of the TKI258 only slightly suppressed the phosphorylation of AKT induced by RAD001, the pi-mTOR and its downstream signaling pathways including pi-p70S6K, pi-S6 and pi-4EBP1 were lowered in the combination. In Hep3B-derived xenograft, TKI-258 and RAD001 had shown an enhanced inhibition of tumor growth without impact on the weight of animals. There was a reduction in microvessel density in the xenograft with the combination, which indicated an enhanced inhibition on angiogenesis. Pro-caspases-3 and PARP cleavage were slightly detected at 48 h after treatment, suggesting that the combination mainly increased the cytostatic arrest ability.
The combination of RAD001 and TKI-258 was active in HCC via inhibition of both mTOR-mediated signaling and its parallel pathways.
Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer and the third most lethal cancer globally.  More than 50 % of patients present with inoperable disease and require systemic therapy for treatment [2, 3]. Following positive results of clinical trials on sorafenib, inhibition of signaling pathways by kinase inhibitors has been the main focus of development of targeted agents for HCC . Genomic analyses showed that phosphatidylinositide 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is deregulated in approximately half of HCC samples [5, 6]. RAD001 (everolimus) is principally an inhibitor of mTOR complex and has been found to exert anti-tumor activity on HCC preclinical models . The agent is being tested in a phase III clinical trial comparing with placebo in patients with HCC which is refractory to sorafenib (ClinicalTrials.gov identifier: NCT01035229). Despite the effective inhibition of mTOR activity following the administration of RAD001, there exists a negative feedback loop involving activation of p70Sk6 and Akt leading to activation of mTOR and undermines the clinical activity of RAD001 [8, 9].
For HCC, overexpression of fibroblast growth factors (FGFs) has been associated with tumor vascularization and proliferation . TKI-258 (dovitinib) is a broad spectrum tyrosine kinase inhibitor of membranous receptors including FGF receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), c-kit and FMS-like tyrosine kinase3 (FLT3) [11, 12]. TKI-258 exhibits potent anti-tumor activity in a board range of preclinical models [11, 13, 14] and phase I clinical trials . In orthotopic models of HCC, TKI-258 could also potently inhibit the growth of primary tumor and occurrence of lung metastases . Preclinical studies also showed that TKI-258 could overcome the resistance of sorafenib in HCC cell lines and downregulate STAT3-related signaling . TKI-258 is currently evaluated in a randomized phase II clinical trial comparing to sorafenib as the first-line treatment for advanced HCC (NCT01232296).
For development of novel therapeutics for HCC, as a result of relatively limited overall survival of patients, there is a strong rationale for drug combination so that suitable patients can be treated with effective regimen before occurrence of liver failure or terminal disease [18–20]. Because of the activity of TKI-258 on upstream membrane receptors, the addition of TKI-258 to RAD001 could potentially diminish the feedback loop induced by mTOR inhibition as well as inhibit the parallel intracellular pathways thereby exerting enhanced effects on HCC. In the current study, we aim to study the effects and the related mechanism of the combination of RAD001 and TKI-258 in both cell lines and xenograft model of HCC.
Materials and methods
Drugs, chemicals and antibodies
RAD001 and TKI-258 were obtained from Novartis International AG, Basel, Switzerland. DMEM medium and fetal bovine serum (FBS) were from Hyclone, Thermo Fisher Scientific (Logan, Utah, USA). Penicillin (50 IU/mL) and streptomycin (50 μg/mL) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Gibco, Invitrogen (Gaithersburg, MD, USA). Amersham ECL Western blotting detection reagents were from GE Healthcare Biosciences (Pittsburgh, PA). Antibodies recognizing FGFR1 (#3472), total mTOR (#2972), pi-mTOR (Ser2448)(#2971), pi-AKT (Ser473)(#9271), pi-AKT (Thr308)(#9275S), total AKT (#4691), pi-MEK1/2 (Ser217/221)(#9121), total MEK1/2 (#9122), pi-p38 MAPK(Thr180/Tyr182)(#9211), total p38 MAPK(#9212), pi-4E-BP1 (ser65)(#9451), total 4E-BP1 (#9452), pi-p70S6K (thr389)(#9234), total p70S6K (#9202), pi-S6 (Ser240/244)(#2215), total S6 (#2217), Caspase 3 (#9668) and cleaved PARP (Asp214)(#9541S) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibody (CP01) was from Calbiochem, Merck (Gibbstown, NJ, USA). FGFR2 (sc-122) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
For in vitro study, RAD001 and TKI-258 were dissolved in DMSO at 10 mM and stored in aliquot at −20 °C as recommended by the manufacturer. Aliquots were thawed and diluted in corresponding medium just before addition to cell cultures. For in vivo study, RAD001 solution (20 mg/ml), which is stored in aliquot at −20 °C, and TKI-258 powder were freshly diluted and dissolved in water just before use as recommended by the manufacturer.
Human HCC cell lines, Hep3B, HepG2 and PLC/PRF/5 were obtained from ATCC (Manassas, VA, USA). HuH-7 was obtained from Health Science Research Resources Bank, Osaka, Japan. All cells were cultured in DMEM with 10 % fetal bovine serum (FBS).
Assay of cytotoxicity
Cytotoxicity was assessed by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Tumor cells were cultured in 48-well plates (8,000–20,000 cells per well) in respective culture medium. RAD001 and/or TKI-258 in complete medium was added at 24 h after cell plating and incubated at 37 °C with 5 % CO2 for 48 and 72 h. Cell growth inhibition was expressed as the percentage of the absorbance of control cultures measured at 570 nm with a microplate reader (PerkinElmer 1420 Multilabel Counter VICTOR3, Waltham, Massachusetts, USA) and the 50 % of the maximum growth inhibition (IC50) was calculated (GraphPad PRISM; Intuitive Software for Science, San Diego, CA). In each experiment, triplicate wells were performed for each drug concentration (n = 3), and assay was repeated in three independent experiments.
Western blot analysis
Cells were treated with various drug doses according to the individual study. Cells were lysed with the western lysis buffer (150 mM NaCl, 1 mM EDTA, 1 % NP40, 10 mM sodium phosphate pH 7.2, 0.6 μg/mL aprotinin, 4 μg/mL leupeptin, 4 μg/mL PMSF and 0.2 mM DTT) for 10 min at 4 °C. The lysate was then centrifuged at 4 °C, 12,000 rpm for 10 min. Supernatant was collected for protein quantitation. Protein quantitation was performed using the Protein Assay Solution (BioRad Laboratories, Hercules, CA) and bovine serum albumin of known concentration as the standard. Twenty-five μg of total protein was resolved on SDS–PAGE gel and transferred onto the Trans-Blot nitrocellulose membrane (BioRad Laboratories, Hercules, CA) using the wet transfer machine (BioRad Laboratories, Hercules, CA). After protein transfer, the membrane was blocked with 5 % non-fat dry milk, 0.2 % Tween 20 in 1× TBS (TBST) for 2 h at room temperature. The membrane was incubated with the primary antibody at 4 °C overnight and washed 3 times with TBST for 15 min. The membrane was incubated with secondary antibody for 1 h at room temperature and then washed 3 times for 15 min. The blot was developed with GE Amersham ECL chemiluminescent substrate by autoradiography. Assay was repeated in three independent experiments.
Analysis of apoptosis by cleaved PARP
2 × 105 Hep3B cells were plated in 50-mm2 Petri dishes and treated with 200 pM RAD001 and/or 0.3 μM TKI-258. Cells were lysed at 24 h in lysis buffer and cleaved PARP immunoblotting was performed as described above. Assay was repeated in three independent experiments.
Tumor xenograft studies
Four-week-old male athymic nude mice (nu/nu) were supplied and housed under pathogen-free conditions by the Laboratory Animal Services Centre of the Chinese University of Hong Kong. All experiments were conducted under license from the Hong Kong Department of Health and according to approval given from the University Animal Experimentation Ethics Committee, CUHK. Hep3B cells (3 × 106 cells resuspended in 200 μl Serum-Free medium) were injected subcutaneously into the dorsal flank region of nude mice. Once palpable tumors were established (tumor volume reaching 20–40 mm3), animals were randomized into 6 groups, each containing 4–6 mice, for the following treatments: vehicle control, TKI-258 (15 mg/kg/day, orally gavaged; twice per week for 2 weeks), RAD001 (2.5 mg/kg/day; orally gavaged; three times per week for 2 weeks) and their combination. The body weight of the mice and tumor size were measured and recorded every 3 days. Tumor volume was calculated using the equation V = (length × width × depth)/2. At the end of the experiment, all the mice were killed, and their tumors were excised out and fixed with 10 % buffered formalin for later use.
Tumor microvessel densities (MVD) were evaluated in tumor xenograft. Tumor sections were stained with anti-mouse CD34 (1:100 dilution, sc-18917) (Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA). The mean microvessel count of the five most vascular areas was taken as the MVD, which was expressed as the absolute number of microvessels per 1.06 mm2 (100× field) (area = 1.19 × 0.89 mm2).
Statistical analyses were performed using PRISM4 Software (GraphPad, La Jolla, CA). Unpaired T-test with Welch Correction was used unless specified. Findings were considered as statistically significant when P value < 0.05.
Effects of TKI258 and RAD001 on cell viability in HCC cell lines
The IC50 of TKI-258 and RAD001 in respective cell lines of HCC
2.727 ± 0.429
1.200 ± 0.226
5.900 ± 0.360
3.660 ± 1.040
4.223 ± 0.839
0.892 ± 0.044
4.510 ± 0.910
2.995 ± 0.763
16.120 ± 4.001
3.110 ± 0.337
9.275 ± 4.305
0.781 ± 0.750
15.007 ± 7.334
3.980 ± 0.803
3.890 ± 1.010
1.458 ± 0.802
Combined treatment of TKI-258 and RAD001 interferes with tyrosine kinase downstream signaling
Pro-caspase 3 cleavage was not observed and cleaved PARP was only slightly detected at 24 h after treatment with single agent alone or combined treatment (Fig. 3c). This suggested that the effects of these chemicals are not directly apoptotic but rather cytostatic by affecting the overall translation process and protein synthesis, as shown in Fig. 3a, b.
In vivo activity of combined RAD001 and TKI-258 in HCC xenograft
The multi-targeted tyrosine kinase inhibitor is a new promising class of therapeutic agents in treating many types of malignancies. TKI-258 is a broad-range tyrosine kinase inhibitor that targets FGFR family, which also cross-reacts with a number of tyrosine kinases. In this study, we evaluated the growth inhibitory activity of TKI-258 in combination with RAD001, which is an inhibitor of mTOR signaling. We found that the combination of these two agents exerted an effective growth inhibition on HCC cells in vitro, whereas more than additive effects were observed with a relatively low dose of combination. The enhanced effects of the combination are further supported by the xenograft model in which the Hep3B-derived tumor size and weight were significantly reduced compared with the use of single agent alone.
Because of low radiologic response rate observed in most of clinical trials on targeted therapy for HCC, combination of effective targeted agents has been postulated as a strategy to improve the response rate and clinical benefits [4, 21]. However, more than 80 % of patients with HCC suffer from comorbidity of liver cirrhosis thereby resulting in impaired hepatic metabolism . Therefore, although the combination of the therapeutics could enhance the efficacy of treatment, additional toxicity is a frequent concern in HCC patients with comorbid cirrhosis. In addition, the drugs cannot be administered at their maximum dosages during combination. In current study, it is observed that the combination of TKI-258 and RAD001 does not induce weight loss or added toxicity in treated mice. It also noted that lower dose of each drug can be used during combination for effective anti-neoplastic effects. Given these findings, it is worthwhile to evaluate the efficacy and safety of the combination using reduced dose of TKI-258 and RAD001 in patients with advanced HCC in a setting of clinical trial.
Activation of AKT is a well-described effect of treatment of rapamycin analogs , and in nasopharyngeal carcinoma, our group previously reported administration of RAD001 could induce activation of AKT via the negative feedback loop related to inhibition of mTOR . In current study, we similarly observed AKT activation after administration of RAD001–HCC cell lines. For breast cancer models, TKI-258 could inhibit the PI3K/AKT signaling in cell lines, 676NR and 4T1, which in turn induced apoptosis and impaired tumor metastasis . However, in current study, the addition of TKI-258 can only slightly reduce the RAD001-induced AKT phosphorylation, which shows that it is ineffective in counteracting the RAD001-induced AKT feedback loop. Therefore, the enhanced anti-tumoral effects of the drug combination of RAD001 and TKI-258 are unlikely due to modulation of AKT. This finding is in line with previous reports that the degree of activation of AKT does not predict the anti-proliferative effects of mTOR inhibitor . On the other hands, the other tyrosine kinase receptor downstream signaling pathways were significantly suppressed by the co-treatment of TKI-258 and RAD001. In particular, the phosphorylation of mTOR, MEK1/2 and p38 MAPK was significantly reduced under the co-treatment, which indicated a better anti-proliferative effect compared with the use of single agent alone. This is probably achieved by the inhibition of the mTOR pathway, where the downstream molecular targets such as p70S6K, S6 and 4E-BP1 were further inactivated under co-treatment.
HCC is a highly vascular tumor, and anti-angiogenic-targeted agents have been shown effective in inhibiting tumor proliferation and stabilizing the size of tumor [2, 4, 21]. In current study, the MVD was significantly reduced compared with single agent treatment, indicating an improved anti-angiogenic ability with the combined treatment of RAD001 and TKI-258. In addition, both TKI-258 and RAD001 were not effective in inducing apoptosis, and no significant caspase 3 cleavage was observed. Only small amount of cleaved PARP was detected in TKI-258-treated cells, and similar result was observed in the sample treated with the combination. This suggests that the combined effects of these two chemicals do not induce cell death in short. Instead, the mechanism appears to be enhancement of the cytostatic effect by altering the translation and protein synthesis. Further studies are warranted to explore the best administration dose and fully utilize their potential in treating HCC.
In the current study, it has to be pointed out that the four HCC cell lines are different in terms of origin and genetic makeup. For examples, HepG2 is derived from an American Caucasian, while Huh-7 is derived from Japanese, and both Hep3B and PLC/PRF5 cell lines harbor hepatitis B virus genome. Despite the heterogeneity in origin and genetic makeup, the additive effect of TKI-258 and RAD001 is universally found in the four HCC cell lines. The Hep3B cell line was chosen for in vivo study for three reasons: First, the additive inhibition on cell viability is most apparent in the Hep3B in the in vitro model. Second, the Hep3B is related to hepatitis B virus, which is the most frequent etiology of HCC seen in Asian world. Third, Hep3B-derived xenograft is a well-established in vivo preclinical drug test model in our laboratory [9, 26].
In summary, we demonstrated a combination treatment by TKI-258 and RAD001 in HCC is significantly more effective in both in vitro and in vivo growth inhibition compared with single agent. No additional toxicity was observed in mice, and the results of this study support further clinical evaluation of the drug combination in HCC.
The study receives funding from Novartis. The authors would like to thank The Charlie Lee Charitable Foundation for the generous support of this study.
Conflict of interest
Dr. Stephen L. Chan has attended advisory board meeting for Novartis.