Background
Hepatocellular carcinoma (HCC) is the sixth most common cancer and one of the leading causes of cancer-related deaths in the world [
1]. HCC is an aggressive cancer with a very dismal prognosis due to a high incidence of metastasis at diagnosis and lack of effective medicinal treatment. Induction of angiogenesis has been recognized as a crucial step and one hallmark of cancer progression [
2]. Given the key importance of VEGF and its receptor VEGFR in angiogenesis, hopes were raised that blocking this pathway would eradicate the tumor vasculature and provide cancer patients maximal survival benefit. Currently sorafenib, a VEGFR inhibitor which also counters the activity of platelet-derived growth factor receptor β (PDGFR-β), the cytokine receptor c-KIT, Raf-1 and B-Raf, is the first line treatment that was approved by the U.S. Food and Drug Administration (FDA) for the advanced-stage HCC patients [
3,
4]. However, the survival benefit of sorafenib is limited, and preclinical studies have shown that the initial suppression of tumor vasculature and tumor growth by VEGFR inhibitor treatments (such as sorafenib and sunitinib) succumbs to rapid revascularization and leads to more invasive and metastatic behavior of cancers [
5,
6]. So, it is urgent to explore the involved mechanisms and develop novel strategies to enhance their efficacy and neutralize the side-effects on cancer invasion and metastasis.
MET, a transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF), has been observed to contribute to cancer metastasis, resistance to chemotherapeutic agents, and dismal outcomes of patients with solid cancers including HCC [
7‐
10]. Within tumor environments, VEGFR and MET signaling pathways have synergistic effects on tumor growth [
11,
12]. Emerging evidences suggest that HGF/MET pathway plays important role in the development of resistance to antiangiogenic therapy [
13]. Therefore, dual inhibition of MET and VEGF pathways may critically disrupt angiogenesis, tumorigenesis and progression of cancers. Although multiple therapies targeting the MET and VEGFR2 pathways have been described to have clinical benefits in HCC treatment [
14,
15], it is unclear whether simultaneous inhibition of MET and VEGFR2 signaling is necessary and sufficient to inhibit HCC invasiveness and metastasis.
In the present study, first we identified the contribution of MET signaling induced by inhibition of VEGF signaling to promote malignancy of HCC in preclinical models. Then we tested if NZ001, a novel ATP-competitive multi-targeted kinase inhibitor that simultaneously inhibits both MET and VEGFR2, could suppress both tumor growth and metastasis. Finally, we found MET amplification and overexpression were useful in subgrouping the HCC patients that might get the optimal benefit from MET inhibitor treatment.
Methods
Reagents and antibodies
For in vitro assays, NZ001 was obtained from Nanjing Zhongrunyuan Pharmaceutical Company (Nanjing, Jiangsu, China). XL184, sorafenib and PF-04217903 were purchased from Selleck Chemicals (Houston, TX, USA). Goat anti-mouse VEGF antibody (AF-493-NA) was purchased from R&D Systems (Minneapolis, MN, USA). NZ001, XL184 and PF-04217903 were prepared as a 20-mM stock solution in DMSO (Sigma-Aldrich, St. Louis, USA) for in vitro studies. For in vivo studies, NZ001 was formulated in sterile ddH2O and administered via oral gavage at 10 mg/kg or 30 mg/kg. Sorafenib was dissolved in Cremophor EL/ethanol (50:50; Sigma Cremophor EL, 95% ethyl alcohol) at 4-fold (4×) the highest dose. The final dosing solutions were prepared on the day of use by diluting to 1× with ddH2O and were administered via oral gavage at 30 mg/kg. VEGF antibody was injected intraperitoneally 3 times per week at 7.5 mg/kg. Recombinant human HGF, mouse HGF and human VEGF were obtained from R&D Systems (Minneapolis, MN, USA). All information of primary antibodies used for Western blot and immunoprecipitation were shown on Additional file
1: Table S1. All secondary antibodies were purchased from Jackson ImmunoResearch (Philadelphia, PA, USA).
Patients and specimens
For prognostic analysis, frozen tumor and peritumor tissues were obtained from 109 patients who underwent hepatectomy for HCC at the authors’ institute between January 2005 and December 2006. For the evaluation of MET expression and microscopic vascular invasion(MVI) in HCC patients, formalin fixed and paraffin embedded tissue samples were collected from 122 patients who received the curative liver resection for HCC at the authors’ institute between September 2014 to December 2016. The entire area of the cut surface containing the greatest tumor dimensions and noncancerous liver tissue was submitted for the histologic examination. The clinicopathological characteristics of these patients were presented in Additional file
1: Table S2, S3. All patients were diagnosed with HCC, and none had received any preoperative cancer treatment. Clinical samples were collected from patients after obtaining informed consent in accordance with a protocol approved by the Ethics Committee of Fudan University (Shanghai, China).
To evaluate the MVI in HCC patients, the specimens were stained with hematoxylin and eosin. Microscopic vascular invasion(MVI) was defined as the presence of clusters of cancer cells floating in the portal vein, hepatic vein, or bile duct of the tumor and surrounding noncancerous tissues that were visible only on microscopy. All of the measurements were performed by two pathologists at Huashan Hospital, Fudan University (Shanghai, China) with more than 10 years of experience in hepatic pathology.
Cell lines
Cell lines were described in the Additional file
2: Materials and Methods. The clinicopathological characteristics of patients, whose tissue samples were used to establish patient-derived HCC cell lines were presented in Additional file
1: Table S4.
Western blot
Western blot was performed as previously described [
16]. Briefly, the cells or the isolated independent tissues were lysed with RIPA Lysis Buffer (Santa Cruz Biotechnology, CA, USA) containing protease inhibitor (Roche Corp., Basal, Swiss) and phosphatase inhibitor (Roche Corp., Basal, Swiss). The proteins were separated by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked and blotted with the relevant antibodies. Antibody binding was detected by enhanced chemiluminescence reagent (Millipore Corp., MA, USA).
Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence analysis (IF)
Enzyme-Linked Immunosorbent Assay (ELISA) and immunofluorescence analysis (IF) were described in the Additional file
2.
RNA isolation and real-time quantitative reverse-transcription PCR
RNA isolation and real-time quantitative reverse-transcription PCR were described in the Additional file
2.
RNA interference
Short interfering RNA (siRNA) sequences specifically targeting human HIF-1α (5’-GGGUAAAGAACAAAACACA-3′) and mouse HIF-1α (5’-CCCATTCCTCATCCGTCAAAT-3′) were purchased from shanghai GenePham (Shanghai, China). Cells were transfected with either target-specific siRNA or a scramble control siRNA using Lipofectamine RNAi MAX reagent (Life Technologies, MD, USA) according to the manufacture’ instructions.
DNA mutation analysis
DNA mutation analysis was described in the Additional file
2.
MET copy number variation analysis
MET copy number variation analysis of 12 HCC cell lines and 16 Chinese patient-derived HCC cells were produced using the Affymetrix Genome-Wide Human SNP Array 6.0. The MET copy number variation analysis of mouse cell lines were produced using Affymetrix Mouse Diversity Genotyping Array. All raw data were processed with the PICNIC software program and are presented as the number of MET copies.
Cell proliferation, colony-formation assays and capillary tube formation analysis
Cell proliferation, colony-formation assays and capillary tube formation analysis were described in the Additional file
2.
Cell invasion and wound-healing assays
Cell invasion assay and wound-healing assays were performed as previously described [
17]. The detail of cell invasion and wound healing assays was described in the Additional file
2.
Immunohistochemical analysis and diagnostic scoring system
The detail of evaluation of immunohistochemical analysis and diagnostic scoring system were described in the Additional file
2.
Evaluation of in vivo tumor growth and metastasis in mice models of HCC
The detail of evaluation of in vivo tumor growth and metastasis in mice models of HCC was described in the Additional file
2. Tumor volume (mm
3) was calculated by the following formula: ab
2/2 (where a and b refer to the largest and smallest dimensions collected every 3 days after treatment). Tumor growth inhibition (TGI%) [
18] was calculated using {1-[(Tt/T0)/(Ct/C0)]/1-[C0/Ct]} × 100, where Tt is the tumor volume of the treated group at indicated time t; T0 is the original tumor volume of the treated animal; Ct is median tumor volume of untreated mice at time t; and C0 is the median original tumor volume of the control group.
Statistical analysis
Statistical analyses were performed using SPSS 15.0 for Windows (SPSS, Inc., Chicago, IL). Quantitative data between groups were compared using the one-way ANOVA; Student t test was used to compare data between 2 groups. Categorical data were analyzed by the chi-square test or Fisher exact test. OS and cumulative recurrence rates were calculated by the Kaplan–Meier method and differences were analyzed by the log-rank test. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. A p-value < 0.05 was considered statistically significant.
Discussion
Suppressing neo-angiogenesis has become an important cancer therapeutic approach. However, accumulating preclinical and clinical evidences indicate that there are several limitations with this approach, including the limited survival benefit, lack of specific biomarker for identifying the patients likely to be advantageous, primary or acquired resistance, and inducing more invasive or metastatic behavior of cancers [
19]. So, it is urgent to explore the involved mechanisms and develop novel strategies to overcome the side effects of anti-angiogenic therapy such as cancer invasion and metastasis.
Agents that block the actions of VEGF not only cause vascular pruning but also result in hypoxia [
24]. Intratumoral hypoxia, which is known as a major stimulator of VEGF and consequent angiogenesis, is associated with a greater risk of metastasis and less favorable prognosis [
25]. One of crucial mechanisms is that hypoxia increases MET expression in tumor cells through HIF-1α binding to the MET promoter and induces transcriptional activation of MET, which drives cell motility and invasion [
26]. These open issues led us to study about whether MET activation under hypoxia inducing a tumor-invasive switch in HCC is a mechanism of refractory to antiangiogenic treatment and whether this form of evasive resistance can be prevented or reversed by inhibition of MET in HCC. Our in vivo experiment demonstrated that application of VEGF signaling monoclonal antibody slowed tumor growth but promoted invasion of tumor cells into the adjacent normal tissue and increased the number of intrahepatic metastases. We illustrated that intratumoral vascular pruning induced by VEGF Ab resulted in hypoxia, HIF-1α nuclear accumulation, MET activation and conversion to a more mesenchymal tumor cell phenotype. In vitro assays demonstrated that hypoxia caused by VEGF signaling inhibition induced HIF-1α nuclear accumulation leading to elevated total-MET expression, which synergized with HGF to enhance the invasion of HCC cells. We also observed that patients with MET overexpression had more vascular-invasive tumors and shorter survival. Importantly, the exaggerated aggressiveness of tumors was circumvented when the selective MET inhibitor, PF-04217903, was co-administered with the VEGF Ab. PF-04217903 given in combination with VEGF antibody also attenuated the MET phosphorylation and the epithelial to mesenchymal transition. Taking together, these findings indicated that VEGF inhibition resulted in hypoxia and activated MET signaling enhancing the metastatic potential of HCC, and MET signaling inhibition attenuated the metastasis-promoting effects of HCC induced by VEGF inhibition.
Because of these favorable effects of the combination treatment, we designed a chemical compound, NZ001, which could simultaneously block both MET and VEGFR2 signaling. The structure of NZ001 had less similarity to existed VEGFR or MET selective inhibitors and the manner of kinase inhibition by NZ001 was shown to be ATP antagonism with the IC50 values against MET and VEGFR2 in the low nanomolar or subnanomolar range. We examined the effect of NZ001 on the spontaneous and cytokine-induced invasion in HCC cell lines under normoxia and hypoxia condition. Our results showed that NZ001 had minimal impact on the spontaneous invasion of Huh7 and HepG2 cells but strongly reduced HGF-stimulated invasion in those cells under both normoxia and hypoxia condition, which was accompanied by a marked inhibition of HGF-stimulated phosphorylation of MET and its downstream effectors STAT3, AKT and ERK. However, in non-cytokine stimulated Huh7 and HepG2 cells, NZ001 had no demonstrable effect on the phosphorylation of STAT3, AKT or ERK, which indicated that the invasion ability of those cells was depended on the downstream signaling of MET. In hepa1-6 orthotopic mice model, we found that inhibition of VEGF signaling by multitargeted RTK inhibitor sorafenib slowed tumor growth but promoted invasion of tumor cells into the adjacent normal tissue and increased the number of intrahepatic metastases. These effect also accompanied with vascular pruning, hypoxia, HIF-1a accumulation, MET activation and conversion to a more mesenchymal tumor cell phenotype. However, NZ001 caused greater inhibition of hepa1-6 tumor growth and invasion than control- and sorafenib-treatment. Administration of NZ001 blocked MET activation and epithelial to mesenchymal transition caused by VEGF antibody and sorafenib. In experimental metastases model, mice treated with NZ001 showed fewer HCC metastatic foci in lung and liver tissues compared with control treated groups. These suggested that NZ001 profoundly inhibited the tumor growth and metastasis of HCC indicating advantages over sorafenib.
Despite significant preclinical data supporting the role of MET as a potential oncogenic driver in HCC, the clinical data obtained with application of MET inhibitors in HCC was not appreciable [
27,
28]. The hidden reasons behind this still unclear, but consensus exists on distinguishing patients who could get the optimal benefit from MET targeted therapy. To identify the predictive biomarkers for MET targeted therapy in HCC, we employed SNP 6.0 assay, sanger sequencing and IHC. We then characterized the MET gene copy numbers, mutation status and quantified HGF/MET/P-MET protein levels in a panel of HCC cell lines and HCC patient-derived cells. We found that HCC cells with
MET amplification and MET/P-MET overexpression exhibited higher sensitivity to MET inhibitors in vitro. However, those with mid-level of MET/P-MET expression (IHC 2+), which have been used as selection criteria for MET targeted therapy in clinical trials of lung carcinoma [
29], showed poor response to MET inhibition. Notably,
MET exon 14 alterations, which resulted in increased MET protein levels due to disrupted ubiquitin mediated degradation and considered as a viable therapeutic target in NSCLC [
20,
21], was not detected in those sensitive HCC cell lines. Furthermore, though elevated circulating HGF levels were observed in patients with HCC [
30,
31], the levels of HGF were also not significantly different in sensitive and non-sensitive HCC cell lines. Consistent to in vitro study, NZ001 treatment to MET amplified MHCC-97H xenografts resulted more pronounced TGI compared with non-MET amplified Huh7 xenografts. Both of MHCC-97H and Huh7 xenografts treated with NZ001 showed no significant difference in vessel pruning, but it exerted higher apoptosis and suppression of cell cycle on MHCC-97H xenografts than on Huh7 xenografts. Based on our findings, we proposed that the antitumor effect of NZ001 on MET amplified MHCC-97H xenografts was likely to be mediated by inhibiting tumor angiogenesis through VEGFR2 inhibition (anti-angiogenesis effect) and by directly inhibiting tumor cell proliferation (anti-proliferation effect). For non-MET amplified HCC such as Huh7 tumors, impeding stromal angiogenesis through VEGFR2 inhibition (anti-angiogenesis effect) contributed to the dominant abrogation of tumor growth. Importantly, NZ001 also showed more profound TGI than sorafenib in MET-amplified MHCC-97H xenografts. These findings suggested that MET amplification and overexpression, rather than MET mutation and HGF expression, could be used to identify the subgroup of HCC patients most likely to get the optimal benefit from NZ001 treatment.
Moreover, both in vitro and in vivo assays demonstrated that NZ001 selectively inhibited MET and VEGFR2, and their downstream effectors, such as P-STAT3, P-AKT and P-ERK, in a dose-dependent manner only to HCCs with MET amplification and MET/P-MET overexpression. These further supported interrelation between NZ001 sensitivity and MET-amplification/MET overexpression and verified NZ001 as a simultaneous inhibitor of both MET and VEGFR2 signaling.