Introduction
Gastric cancer is the fifth most common malignancy worldwide, with an estimated 950,000 new cases in 2012; approximately two thirds of cases occur in men [
1,
2]. Mortality statistics are even more striking, with more than 720,000 deaths due to gastric cancer estimated to occur each year, making it the third most common cause of cancer-related death [
1,
2]. Only 30 % of cases occur in developed countries, while the highest incidence is in Eastern Asia, particularly China, accounting for 50 % of patients [
1,
2]. A number of risk factors for the development of gastric cancer have been identified, the most important of which is infection with
Helicobacter pylori [
3].
Surgical resection is the first choice of treatment for early-stage gastric cancer [
4]; however, many cases are locally advanced or metastatic at the time of diagnosis and are thus unresectable [
5]. Although a number of cytotoxic agents have been found effective in this indication, treatment options for patients whose disease progresses on chemotherapy are limited [
4], resulting in an overall 5-year survival rate of just 28 % [
5].
The presence of gain-of-function mutations affecting receptor tyrosine kinases (RTKs) is associated with poor prognosis in patients with gastric cancer [
6,
7]. In an effort to provide additional treatment options in this patient group, a variety of targeted therapies have been investigated. Potential molecular targets identified include RTKs involved in angiogenesis and tumor proliferation, such as vascular endothelial growth factor (VEGF), angiopoietin, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) receptors, and HER2/neu [
6,
8‐
10]. Interestingly, some of the receptors identified as potential targets have overlapping intracellular signal transduction cascades, notably the PI3K/AKT/mTOR and MAPK pathways [
11‐
14]. Activation of these signaling cascades is associated with increased tumor-cell proliferation and survival, as well as inhibition of apoptosis [
15,
16].
Clinical trials of targeted therapies in gastric cancer have met with varying levels of success. Studies of bevacizumab, cetuximab, panitumumab, and everolimus have failed to show a significant survival benefit versus varying control treatments, and a phase II trial of sunitinib failed to meet its primary endpoint [
17‐
22]. However, the anti-VEGF receptor 2 (VEGFR2) antibody ramucirumab improved survival compared with placebo in a phase III trial [
23] and has been approved for advanced gastric cancer by the US Food and Drug Administration and the European Medicines Agency. Addition of the anti-HER2/neu monoclonal antibody trastuzumab to chemotherapy has also been shown to provide benefit versus chemotherapy alone in patients with HER2/neu-positive tumors [
24].
Regorafenib is a multikinase inhibitor with activity at a range of protein kinases involved in oncogenesis (KIT, RET, and RAF), angiogenesis (VEGFR1–3 and TIE2), and maintenance of the tumor microenvironment (PDGFR and FGFR) [
25]. Regorafenib has demonstrated efficacy in phase III trials in patients with metastatic colorectal cancer (CRC) [
26,
27] and advanced gastrointestinal stromal tumors (GIST) [
28] and has been approved in these indications in a number of countries. Given the wide range of kinases inhibited by regorafenib and its clinical efficacy in other gastrointestinal tumors, we investigated its antitumor activity in patient-derived xenograft (PDX) models of gastric cancer.
Methods
Reagents
Antibodies against Bim, cleaved poly(ADP ribose) polymerase (PARP), AKT, p-Ser473 AKT, p-Thr202/Tyr204 ERK1/2, p-Ser10 histone H3, S6R, p-Ser235/236 S6R, Rb, p-Ser780 Rb, p-Ser807/811 Rb, VEGFR2, p-Tyr951 VEGFR2, p90RSK1–3, p-Thr359/Ser363 p90RSK, p70S6K, p-Thr421/Ser424 p70S6K, p-Tyr15 CDC-2, p-Thr14/Tyr15 CDK-2, 4EBP1, p-Thr70 4EBP1, and TIE2 were obtained from Cell Signaling Technology. Antibodies against BAD, p21, CD-31, CDK-2, CDK-4, CDC-2, cyclin B1, ERK1/2, p27, survivin, and α-tubulin were obtained from Santa Cruz. Triton X100, NaCl, and NP-40 were obtained from Merck KGaA. EDTA, sodium orthovanadate, and Tris-base were from Sigma-Aldrich. Tween-20 was purchased from Promega Corporation.
Regorafenib was dissolved in dimethyl sulfoxide to create a stock solution with a concentration of 100 mg/mL. To achieve the solution with the final concentration for administration, 0.1 mL of the regorafenib stock solution (or dimethyl sulfoxide for the control group) was further diluted in vehicle (4 mL of polyethylene glycol 300 and 3.9 mL of 30 % Captisol [purchased from CyDex] in water).
Patient-derived xenografts
Animal experiments were approved by the ethics board at the National Cancer Centre of Singapore and Singapore General Hospital. All mice were maintained according to the Guide for Care and Use of Laboratory Animals, published by the US National Institutes of Health [
29]. Animals were provided with sterilized food and water
ad libitum, and were housed in negative-pressure isolators with 12-h light/dark cycles.
Xenograft experiments were performed with male severe combined immunodeficiency (SCID) mice (Animal Resources Centre). Eight patient-derived gastric cancer PDX models (GC09-0109, GC28-1107, GC22-0808, GC30-0309, GC10-0608, GC17-0409, GC05-0208B, and GC23-0909) were used to establish subcutaneous tumors in mice aged 9–10 weeks. Tumor model histology and mutation status are shown in Additional file
1: Table S1.
Antitumor activity in vivo
For dose response and tolerability analyses, mice bearing GC09-0109 and GC28-1107 tumor xenografts were given oral vehicle or regorafenib 5, 10, or 15 mg/kg/day. Each treatment group comprised 15 or 16 mice. Treatment was started when tumors reached approximately 150 to 200 mm3. Tumors were measured bidimensionally and their volume was calculated using the formula: (length) × (width2) × (π/6). Mice were killed at the end of the study; tumor weight and bodyweight were recorded, and tumors were preserved for further analysis.
For the assessment of antitumor activity in additional tumor models, xenografts were grown subcutaneously in mice (14 to 20 mice per group) to a size of approximately 200 to 300 mm3. Mice were then given daily oral doses of either 200 μL of vehicle or regorafenib 10 mg/kg, with the last dose given 2 h before death. Tumor volumes were determined as in the dose–response experiments. A portion of each tumor was fixed in paraformaldehyde and embedded in paraffin, with further portions snap frozen for tumor lysate generation and cryopreserved for immunohistochemistry (IHC).
Western blot analysis
To investigate changes in levels of phosphorylated and total proteins identified as targets of regorafenib or with roles in tumor cell proliferation, apoptosis, cell cycle regulation, and survival, three to four randomly selected independent tumors from vehicle and drug-treated mice were combined and homogenized in lysis buffer (0.5 % Triton X100; 150 mMol/L NaCl; 10 mMol/L EDTA; 2 mMol/L sodium orthovanadate; 0.5 % NP-40). Protein concentration was determined by Bio-Rad protein assay (Bio-Rad Laboratories). Eighty micrograms of total lysate per tumor sample preparation were analyzed by Western blot. Blots were incubated with primary antibodies diluted in TBST (20 mMol/L Tris, pH 7.6, 150 mMol/L NaCl and 0.1 % Tween-20) containing 1 % nonfat dry milk and a 1:7500 dilution of horseradish peroxidase-conjugated secondary antibodies. All primary antibodies were then visualized with a chemiluminescent detection system (Amersham, Pharmacia Biotech).
Immunohistochemistry and histological staining
Fifteen micron sections of optimal cutting temperature compound-embedded (Tissue-Tek; Sakura Finetek) tumors were immunostained with anti-CD31 antibodies to assess microvessel density (MVD). To quantify MVD, the number of immunostained vessels in ten 0.159 mm2 fields at a magnification of × 100 from ten randomly selected tumors in each group was counted. Five micrometer sections of paraffin-embedded tumor tissue were immunostained with anti-p-Ser10 histone H3 or cleaved PARP antibodies to assess tumor-cell proliferation and apoptosis, respectively, based on the percentage of p-Ser10 histone H3-positive and cleaved PARP-positive cells per ≥500 cell region, respectively. Three tumors per treatment and four regions per tumor were analyzed for tumor cell proliferation and apoptosis. Induction of apoptosis was defined as a two-fold or greater increase in the proportion of cells identified as cleaved PARP-positive in tumors from regorafenib-treated mice compared with tumors from vehicle-treated animals. Images were recorded using an Olympus BX60 microscope equipped with an Olympus DP11 camera. All experiments were performed in triplicate.
Tumor necrosis was assessed by microscopic examination of hematoxylin and eosin (H&E)-stained tumor sections, with ten random fields examined at a × 100 magnification. Tumor necrosis was only qualitatively assessed.
Statistical analysis
Differences in tumor weight at death, p-Ser10 histone H3 index, mean MVD, and cleaved PARP-positive cells were compared by analysis of variance or Student’s t-test. Significance was established at p < 0.05 for all statistical analyses.
Discussion
Expression of VEGF is strongly correlated with tumor progression and poor prognosis in gastrointestinal malignancies, including gastric cancer [
31], with an association between VEGF expression, increased MVD, and decreased survival established in previous studies [
10,
32]. Preclinical studies of VEGFR-targeting agents in gastric cancer have shown significant antitumor effects [
33,
34], and a clinical trial with the VEGFR2 antibody ramucirumab monotherapy has demonstrated survival benefits over placebo for patients with advanced gastric cancer, validating VEGFR2 as a relevant therapeutic target in gastric cancer [
23]. However, overall survival gains after ramucirumab treatment were moderate and the response rate was low [
23], which indicates a need for additional antiangiogenic approaches.
This study was performed to assess the antitumor activity of the multikinase inhibitor regorafenib, a known potent inhibitor of VEGFR kinases in gastric cancer xenografts, and to investigate the underlying antitumor mechanisms. Our findings show that all eight patient-derived xenograft models investigated in the current study respond favorably to regorafenib, with tumor growth inhibition of 72 to 96 % at a dose of 10 mg/kg/day in a variety of histological subtypes. At this dose, regorafenib exposure and C
max in mice are comparable to those observed in humans after 21 days of treatment with regorafenib 160 mg/day [
35], a dose which has demonstrated efficacy in patients with CRC and GIST [
26‐
28].
Analysis of the mechanisms by which regorafenib inhibited tumor growth inhibition showed a pronounced antiangiogenic effect in xenografts from all regorafenib-treated mice, as measured by MVD reduction versus vehicle-treated animals. Tumors from vehicle-treated animals were well vascularized, as judged by both measured MVD and visual appearance (Figs.
2 and 3 and Table
2). Elevated MVD has previously been detected in clinical samples of diffuse- versus intestinal-type tumors [
10,
32], which was not apparent in our vehicle-treated xenografts. A stronger antiangiogenic effect with regorafenib treatment was noticed in intestinal than in diffuse-type tumor models, but did not translate into differences in antitumor activity. Consistent with the antiangiogenic effects detected by IHC, levels of phosphorylated or total VEGFR2 protein were reduced in pooled tumor lysates from some models (Fig.
4).
Regorafenib also inhibited cell proliferation, as shown by the significant decrease in the proportion of p-Ser
10 histone H3-positive cells in all but two models, both of which were of intestinal origin. However, there was no correlation between the antiproliferative and antitumor effects, similar to what was observed with the antiangiogenic effects. Ser
10 of histone H3 is phosphorylated by mitogen- and stress-activated kinase 1, Aurora B, or checkpoint kinase 1 [
36]; none of these kinases is significantly inhibited by regorafenib in biochemical assays [
25], precluding a direct antiproliferative activity of regorafenib by inhibition of Ser
10 histone H3 phosphorylation. In Western blots, no effects were observed on proteins associated with the cell cycle, such as cyclin-dependent kinases 2 (Fig.
4), cyclin-dependent kinases 1, 4, and 6 (data not shown), and the RB protein (Fig.
4). Although not systematically analyzed, regulatory proteins such as cyclin B1 and the cyclin inhibitors p21 and p27 were not affected (data not shown). Given these findings, more detailed research is required to provide a molecular explanation for the antiproliferative effect of regorafenib.
Gastric cancer cell apoptosis was induced by regorafenib through the caspase-mediated mitochondrial pathway, demonstrated by the increased proportion of caspase-cleaved PARP-positive tumor cells and elevated levels of cleaved PARP in tumor lysates (Fig.
4 and Table
2). The extent of apoptosis induction varied widely and was strongest in diffuse- and mixed-type tumor models; however, there was no correlation with tumor growth inhibition. Small reductions in levels of the antiapoptotic protein survivin were consistently observed in all models investigated (Fig.
4 and data not shown), but levels of the proapoptotic protein BAD were not affected. High survivin expression has been correlated with poor prognosis in gastric cancer [
37], suggesting that regorafenib-induced reductions could contribute to the antitumor activity of regorafenib. Expression of another proapoptotic protein, PUMA (p53 upregulated modulator of apoptosis), has also been found to be downregulated in gastric cancer [
38]. PUMA expression was recently shown to be upregulated by regorafenib in CRC cells [
39], raising the possibility that regorafenib may have a similar effect in gastric cancer. In light of the multiple pathways that appear to play a role in apoptosis, it may be necessary to take an integrated systems approach covering the entire apoptosis network, as was used by Lindner et al
. [
40], to better understand the role of apoptosis in the antitumor activity of regorafenib.
The effects of regorafenib on gastric cancer xenografts in the current study are consistent with the findings of previous preclinical studies of regorafenib, including xenograft studies in other gastrointestinal tumor types. At doses of 10 to 30 mg/kg/day, regorafenib inhibited tumor growth by up to 75 % versus vehicle in various CRC tumor models, including subcutaneous xenografts of the tumor cell line Colo-205, five of seven CRC PDX models, and an orthotopic CRC model derived from the murine cell line CT26 [
25,
30,
41]. Tumor regression was also observed in a GIST PDX model [
42]. Analysis of angiogenesis in the CT26, Colo-205, and Co5896 CRC PDX models and the GIST PDX model showed a significant reduction in tumor vessel area or vessel number in regorafenib-treated xenografts, assessed by CD31 staining, while no significant change in microvessel area was observed in the regorafenib-refractory Co8541 CRC model [
25,
30,
41,
42]. These previous results, in addition to those from the current study, suggest that antiangiogenesis is one of the main drivers of the antitumor activity of regorafenib in gastrointestinal tumors such as CRC, GIST, and gastric cancer, with further support from the observation of central necrosis in all of the gastric cancer models of this study (Fig.
3 and data not shown) and in a GIST PDX model [
42]. Although not specifically investigated here, induction of hypoxia could lead to apoptosis, which would also explain the apoptotic events observed in this study. Regorafenib has been shown to induce apoptosis in a murine CT26 CRC model, with an approximately 18-fold increase in apoptosis observed in regorafenib-treated mice compared with controls [
41]; however, no effects were observed in the GIST PDX model [
42].
Competing interests
This study was supported by Bayer Pharma AG and a grant to HH from The National Medical Research Council Singapore (NMRC/MOHIAFCAT1/0004/2014). HH and RO have no other conflicts of interest to disclose. DZ is a full-time employee of Bayer Pharma AG.
Authors’ contributions
HH was involved in the design of the study, acquisition of the data, analysis and interpretation of the data, writing, reviewing, and revision of the manuscript. RO was involved in the acquisition of the data, material support, analysis and interpretation of the data, and review of the manuscript. DZ was involved in the design of the study, as well as writing and review of the manuscript. All authors read and approved the final manuscript.