Dovitinib preferentially targets endothelial cells rather than cancer cells for the inhibition of hepatocellular carcinoma growth and metastasis
- Zhi-Yuan Chen†1, 2, 3,
- Ming Shi†1, 2,
- Li-Xia Peng2, 3,
- Wei Wei1, 2,
- Xin-Jian Li2, 4,
- Zhi-Xing Guo1, 2,
- Shu-Hong Li1, 2,
- Chong Zhong5,
- Chao-Nan Qian2, 3Email author and
- Rong-Ping Guo1, 2Email author
© Chen et al.; licensee BioMed Central Ltd. 2012
Received: 19 August 2012
Accepted: 19 November 2012
Published: 10 December 2012
Dovitinib is a receptor tyrosine kinase (RTK) inhibitor targeting vascular endothelial growth factor receptors, fibroblast growth factor receptors and platelet-derived growth factor receptor β. Dovitinib is currently in clinical trials for the treatment of hepatocellular carcinoma (HCC).
In this study, we used five HCC cell lines and five endothelial cell lines to validate molecular and cellular targets of dovitinib.
Tumor growth and pulmonary metastasis were significantly suppressed in an orthotopic HCC model. Immunoblotting revealed that among known dovitinib targets, only PDGFR-β was expressed in two HCC cell lines, while four of five endothelial lines expressed PDGFR-β, FGFR-1, and VEGFR-2. Dovitinib inhibited endothelial cell proliferation and motility at 0.04 μmol/L, a pharmacologically relevant concentration; it was unable to inhibit the proliferation or motility of HCC cells at the same concentration. Immunohistochemical analyses showed that dovitinib significantly decreased the microvessel density of xenograft tumors, inhibiting proliferation and inducing apoptosis in HCC cells.
Our findings indicate that dovitinib inhibits HCC growth and metastasis preferentially through an antiangiogenic mechanism, not through direct targeting of HCC cells.
Hepatocellular carcinoma (HCC) is characterized by highly vascularized and rapid tumor progression, a high recurrence rate after surgical resection, and an extremely poor prognosis. It is the fifth most common cancer in the world, and the third most frequent cause of cancer death [1–4]. The highly vascularized nature of HCC has been considered as the main reason for its devastating outcome, because of intrahepatic and distant metastases [5–7]. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) are three important pro-angiogenic factors involved in hepatocarcinogenesis, and they participate in the neovascular, invasive, and metastatic potentials of HCC [8–11].
VEGF expression is detected in dysplastic nodules and correlates with histological grades; VEGF is increased during hepatocarcinogenesis . Sorafenib, an inhibitor of several kinases, including Raf-1 and VEGF receptor (VEGFR), is currently the first-line therapy for advanced or recurrent HCC. It has a modest survival benefit, but patients develop subsequent drug resistance [12–14].
Dovitinib (TKI258; formerly CHIR258) is a potent inhibitor of receptor tyrosine kinases (RTKs). It inhibits VEGFR-1, VEGFR-2, and VEGFR-3; fibroblast growth factor receptors (FGFR1, FGFR2, and FGFR3); and platelet-derived growth factor receptor β (PDGFR-β) [15, 16]. Dovitinib is reported to directly inhibit the proliferation and survival of colon cancer and leukemia cells, which harbor either activating mutations or translocations in the target RTKs or their ligands, at pharmacologically relevant concentrations of 0.01–0.3 μmol/L [17–19].
In preclinical studies, dovitinib has been able to inhibit xenograft HCC growth in immunodeficient mice and even overcome sorafenib resistance [20, 21]. However, the lack of somatic mutations of RTK genes in HCC has caused doubt about whether HCC cells are the primary cellular target of dovitinib . It has been reported that endothelial cells and perivascular cells (pericytes) can express VEGFR, PDGFR, and/or FGFR [8, 23, 24]; thus, these cells are theoretical targets of dovitinib, and the drug might act as an angiogenesis inhibitor in vivo. However, the ability of dovitinib to suppress tumor angiogenesis has not been established.
In the present study, our aim was to reveal the cellular targets of dovitinib in HCC treatment at pharmacologically relevant concentrations, which is crucial for the future development of this treatment strategy.
Materials and methods
Dovitinib (CHIR-258, TKI258) [4-amino-5-fluoro-3-[6(4-methyl-1-piperazinyl)-1H- benzimidazol-2-yl]-2(1H)-quinolinone], with a molecular weight of 392.4, was provided by Novartis Pharma AG (Novartis Institutes for Biomedical Research, Basel, Switzerland).
Cells and cell culture
The human HCC cell lines MHCC-97H, QGY-7703, SMMC7721, Hep3B, and CRI2234, as well as a human bone marrow endothelial (HBME) line, were maintained in DMEM or RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) in a humidified incubator containing 5% CO2 at 37°C. Human umbilical vascular endothelial cells (HUVECs), human dermal microvascular endothelial cells (HMVECs), human umbilical artery endothelial cells (HUAECs), and human lung microvascular endothelial cells (HLMVECs) were maintained in Clonetics Endothelial Basal Medium-2 (EBM-2) supplemented with essential growth factor supplements EGM-2 SingleQuots or EGM-MV SingleQuots (Lonza). All the cell lines were used within 50 passages.
Cell viability assay
Cell viability was assessed using an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit (Sigma) with dye conversion at 490 nm, following the manufacturer’s instructions. Briefly, cells were seeded 3 × 103/well in a 96-well flat-bottomed plate and starved in no serum for 18 h, and were then treated with increasing concentrations of dovitinib and stimulated with 30 ng/mL recombinant human VEGF or PDGF-BB (Sigma V7259 or SRP3138). At 72 h, 20 μl of MTS was added to each well. After 1.5 h of incubation at 37°C, the results were analyzed by a plate reader at 490 nm. The sample data was normalized to background readings of medium only.
In vitro migration and invasion assays
For Transwell migration assays, 5.0 × 104 HCC cells or endothelial cells in 500 μl of serum-free DMEM or EBM were added to the cell culture inserts with an 8-μm microporous filter without an extracellular matrix coating (Becton Dickinson Labware). To the bottom chamber was added 800 μL of DMEM or EGM containing 10% FBS. After 24 h of incubation, the cells on the lower surface of the filter were fixed, stained, and counted under a microscope (×100 magnification). The number of migrated cells in five random optical fields from triplicate filters was averaged. For invasion assays, the inserts of the chambers to which the cells were seeded were coated with Matrigel (Becton Dickinson Labware). The number of invaded cells in five random optical fields (×100 magnification) was averaged from triplicate inserts.
For the wound healing assay, the cells were plated in 6-well plates (3 × 105 cells/well) and allowed to attach and reach confluence. A scratch was made using a sterile 100-μl pipette tip and detached cells were removed by washing with PBS. Phase contrast images were taken at specified time points. The scratched wound was evaluated at 18 h (endothelial cells) or at 48 h (HCC cells) after scratching.
Efficacy of dovitinib in an orthotopic HCC model
Male athymic mice between 4 and 5 weeks of age were purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). All the animal studies were conducted in accordance with the principles and procedures outlined in the guidelines of the Institutional Animal Care and Use Committee at Sun Yat-sen University Cancer Center. Mice were anesthetized by continuous inhalation of isoflurane (Baxter Healthcare, NJ).
For orthotopic xenografts, an upper abdominal midline incision was made. MHCC-97H, SMMC7721 or QGY-7703 HCC cells (1 × 106) in 30 μl of culture medium with 33% Matrigel were injected into the left lobe of the liver using an insulin syringe with a 31-gauge needle (Becton Dickinson, NJ). Two weeks later, the nude mice were randomized into three groups of 20 mice each and were treated either with 0.9% sodium chloride or 25 or 50 mg/kg of TKI258 for 14 days. On day 30 after tumor cell inoculation, the animals were weighed, euthanized, and autopsied. The liver and lungs were weighed and sampled for tissue sectioning. To examine lung metastases, 100 sequential lung sections (4 μm) were cut from the lungs of each mouse and every tenth section was stained with hematoxylin and eosin (H&E).
Formalin-fixed and paraffin-embedded sections 4 μm thick were dewaxed in xylene and a gradient of alcohols, hydrated, and washed in PBS. After pretreatment in a microwave oven (30 min in citrate buffer, pH 6.0), endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide for 10 min, and the sections were incubated with 10% normal goat serum for 15 min. Primary antibodies—rabbit polyclonal anti-CD34 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-Ki67 (Abcam, Cambridge, UK) and rabbit polyclonal anti-PARP (Abcam, Cambridge, UK)—were applied overnight in a moist chamber at 4°C. A standard avidin-biotin peroxidase technique (DAKO, Carpinteria, CA) was applied. Briefly, biotinylated goat anti-rabbit immunoglobulin, goat anti-rat immunoglobulin, and avidin-biotin peroxidase complex were applied for 30 min each, with 15-min washes in PBS. The reaction was finally developed using the DAKO Liquid DAB+ Substrate-Chromogen System. The methods for quantification of microvessel density (MVD), proliferation index, and apoptosis index were reported previously [25, 26]. Briefly, the largest section of each xenograft tumor was analyzed by randomly capturing images of microscopic fields at low magnification, and the microvessels or stained cells were counted and averaged. The final results were the mean value of each case from two independent referees.
Immunoprecipitation and immunoblotting
Cells were lysed in cold RIPA buffer (100 mM Tris HCl, 300 mM NaCl, 2% NP40, 0.5% sodium deoxycholate) supplemented with a proteinase inhibitor cocktail (Roche, Indianapolis, IN) and a phosphatase inhibitor cocktail (Merck, Darmstadt, Germany). Protein concentration was determined using a detergent-compatible protein assay according to the manufacturer’s instructions (Bio-Rad). Protein (1 mg) from each sample was immunoprecipitated overnight at 4°C with an anti-VEGFR-2, anti-PDGFR-β, or anti-FGFR-1 (Cell Signaling Technology) antibody plus protein G/A agarose beads (Pierce). Immune complexes were washed with cold RIPA buffer containing proteinase inhibitors and phosphatase inhibitor. Proteins were eluted by boiling in SDS sample buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Millipore). Membranes were probed with an anti-phosphotyrosine antibody (Cell Signaling Technology) and then stripped with stripping buffer (Abcam). To detect total VEGFR-2, PDGFR-β, and FGFR-1 levels, membranes were re-probed with the same anti-VEGFR-2, anti-PDGFR-β, and anti-FGFR-1 antibody that was used for the immunoprecipitation. Immunoblotting of phospho- ERK1/2 and ERK1/2 (Cell Signaling Technology) was performed on whole-cell lysates (40 μg) with β-actin (Abcam) as a loading control.
Continuous data were expressed as median and range and were compared between groups using one-way ANOVA and Dunnett t test. Categorical variables were compared using the chi-square test, or Fisher’s exact test, where appropriate. All data were analyzed using the SPSS 13.0 computer program, and significant difference was defined as P < 0.05.
Dovitinib inhibited HCC xenograft tumor growth and metastasis
Dovitinib inhibited RTK signaling pathways in vitro
Based on the combined data of the mice and the cell lines, we focused our study on VEGFR-2, FGFR-1 AND PDGFR-β signaling in the cells. As expected, dose-dependence was found in the inhibitory effects of dovitinib on the phosphorylation of PDGFR-β, VEGFR-2, and FGFR-1, as well as their major downstream effector, the phosphorylation of ERK, on these cells (Figure 2C–D), but not the phosphorylation of Akt (Additional file 2: Figure S2B). While the levels of cleaved PARP and cleaved caspase 3 were also readily detected in dose-dependence of dovitinib (Additional file 2: Figure S2A).
The proliferation of endothelial cells (but not the HCC cells) was inhibited by dovitinib
Dovitinib inhibited the migration of endothelial cells but not of HCC cells
Dovitinib inhibited tumor angiogenesis in vivo
Dovitinib is currently in Phase II studies for the treatment of advanced hepatocellular carcinoma (NCT01232296), but the underlying mechanism of dovitinib in targeting HCC is not known. Our results showed that dovitinib preferentially targeted endothelial cells by inhibiting their proliferation and motility and inhibiting angiogenesis in vivo. At pharmacologically relevant concentrations, dovitinib did not affect HCC cells.
Other groups have reported that dovitinib concentrations lower than 1 μmol/L are sufficient to inhibit RTK signaling [17, 18]. In cellular assays, Andrew and colleagues found that dovitinib inhibited FGFR signaling in Ba/Fs cells lines of myeloproliferative syndrome with IC50 values of 0.09–0.15 μmol/L , consistent with our studies on endothelial cells. Finally, both clinical and preclinical pharmacodynamic studies showed that pharmacologically and clinically relevant plasma concentrations of dovitinib are 0.01–0.3 μmol/L [17, 18].
A recent study using a high concentration (1.727 μM) of dovitinib reported that anchorage-independent growth and FGF-induced motility of HCC cells was inhibited . Unfortunately, this study did not evaluate the effect of dovitinib on endothelial cells, and the concentration used was much higher than a pharmacologically relevant dose. In our study, dovitinib at 0.1 μmol/L did not affect the viability or proliferation of HCC cell lines in vitro. In contrast, it did inhibit endothelial cell proliferation and motility at concentrations that also inhibited VEGFR and FGFR signaling in these cells. Studies of HCC xenografts treated with pharmacologically relevant concentrations of dovitinib showed more effect on the inhibition of tumor angiogenesis in vivo than on proliferation or apoptosis. Taken together, these data indicate that dovitinib acts preferentially to target tumor vasculature rather than cancer cells in the treatment of HCC.
Some previous studies have reported that high expression of the angiogenic factors VEGF, basic FGF, and platelet-derived growth factor receptor are detected in patients with HCC, suggesting that VEGFR, FGFR, and PDGFR are likely targets of dovitinib. Our analysis of HCC and endothelial cell lines found that, of the known dovitinib-sensitive RTKs, only PDGFR-β was expressed by SMMC7721 and MHCC-97H cells, where VEGFR-2 and FGFR-1 were highly expressed by endothelial cells. Although high PDGFR-β expression has been correlated with HCC progression , our in vitro studies showed that dovitinib inhibition of PDGFR signaling was not sufficient to inhibit the proliferation of HCC cells. Thus, PDGFR signaling in HCC cells is likely through redundant growth signaling pathways. In contrast, dovitinib inhibited the phosphorylation of VEGFR-2 and FGFR-1 in endothelial cells at similar concentrations, indicating the important role of VEGFR and FGFR signaling in the proliferation of endothelial cells.
The endothelial cells recruited to the tumor tissue are not only related to blood perfusion of the tumor, but they are also believed to be involved in the cancer–stromal cell interaction favoring tumor growth . However, some believe that normal endothelial cells may be the barrier to hematogenous metastasis . Blocking the adhesion of tumor cells to endothelial cells prevents the implantation of tumors cells in capillaries, thus inhibiting the migration of tumor cells in the circulation . In our previous study, we found in HCC tissues that a distinct pattern of endothelial structures, endothelium-coated tumor clusters, was an independent predictor for survival and recurrence in patients with HCC . Endothelial cells facilitate the efficiency of metastasis, irrespective of the invasiveness of tumor cells . In our study here, a pharmacologically relevant concentration of dovitinib, 0.1 μmol/L, did not affect the migration or invasion of HCC cell lines in vitro. In contrast, dovitinib did inhibit endothelial cell migration and invasion and did so at concentrations that also inhibited VEGFR and FGFR signaling in these cells. Moreover, studies of orthotopic HCC models treated with pharmacologically relevant concentrations of dovitinib inhibited lung metastasis in vivo. These findings suggest that the migration of endothelial cells could be an important step in HCC metastasis by providing an envelope that protects the tumor cells in circulation .
In summary, we found that at pharmacologically relevant concentrations, dovitinib targeted endothelial cells, but not HCC cells, in inhibiting HCC growth and metastasis. To our knowledge, this is the first study to clarify the cellular target of dovitinib in the treatment of HCC, which might be helpful for future development of targeted therapy in HCC.
The novelty and impact of the work
Dovitinib is a receptor tyrosine kinase (RTK) inhibitor in clinical trials for the treatment of hepatocellular carcinoma (HCC). We do not have a full understanding of the cellular target(s) of dovitinib in HCC treatment. By using five HCC cell lines and five endothelial cell lines in validating targets, we found that dovitinib inhibited HCC growth and metastasis preferentially through an antiangiogenic mechanism, not through direct targeting of HCC cells.
This work was supported by grants from the National Natural Science Foundation of China (No. 81172037/H1606; No. 81030043), Guang Dong Province Science Foundation of China (No. 2009B080701012; No. 2008B030301322) and the Van Andel Foundation. We thank David Nadziejka, Grand Rapids, Michigan, for critical reading of the manuscript.
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