somg-833, a novel, selective c-met inhibitor, blocks c-met...
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JPET #214817
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SOMG-833, a novel, selective c-MET inhibitor, blocks c-MET dependent
neoplastic effects and exerts antitumor activity
Hao-tian Zhang, Lu Wang, Jing Ai, Yi Chen, Chang-xi He, Yin-chun Ji, Min Huang, Jing-yu
Yang, Ao Zhang, Jian Ding, Mei-yu Geng
Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road,
110016 Shenyang, PR China; Division of Anti-tumor Pharmacology, State Key Laboratory of
Drug Research and CAS Key Laboratory of Receptor Research and Synthetic Organic &
Medicinal Chemistry Laboratory (SOMCL), Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, P.R. China.
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JPET Fast Forward. Published on April 16, 2014 as DOI:10.1124/jpet.114.214817
Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: SOMG-833, a novel, selective c-MET inhibitor
Address correspondence to: Mei-Yu Geng, Division of Anti-tumor Pharmacology, State Key Laboratory
of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, P.R. China. Tel: (86) 21-50806072; Fax: (86) 21-50806072; E-mail: [email protected]
Number of the text pages: 31
Number of the tables: 1
Number of the figures: 5
Number of the references: 42
Number of the words in the Abstract: 236
Number of the words in the Introduction: 744
Number of the words in the Discussion: 706
Abbreviations: SOMG/SOMG-833,
(3-(4-Methylpiperazin-1-yl)-5-(3-nitrobenzylamino)-7-(trifluoromethyl) quinoline); HGF/SF, hepatocyte
growth factor/ scattering factor; ATCC, American Type Culture Collection; BSA, Bovine Serum Albumin;
DMSO, Dimethyl sulfoxide; EPH-A2, Ephrin type-A receptor 2; EGFR, Epidermal Growth Factor
Receptor; FBS, Fetal Bovine Serum; Flt-1, Fms-like Tyrosine Kinase; FGFR1, Fibroblast Growth Factor
Receptor 1; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase;IGF1R, Insulin-like Growth Factor 1
Receptor; PDGFR, Platelet Derived Growth Factor Receptor; KDR, Kinase insert domain receptor;
JCBR, Japanese Collection of Research Bioresources; DMSZ, Deutsche Sammlung von
Mikroorganismen und Zellkulturen; MAPK, Mitogen-Activated Protein Kinases; OPD,
O-Phenylenediamine; PBS, Phosphate Buffered Saline; PI, Propidium Iodide PI3K, Phosphoinositide
3-Kinase; RTK, Receptor Tyrosine Kinase; SDS, Sodium Dodecyl Sulfate;
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Abstract
The HGF/c-MET signalling axis plays an important role in tumor cell proliferation, metastasis, and tumor
angiogenesis, and therefore presents as an attractive target for cancer therapy. Notably, most
small-molecule c-MET inhibitors currently undergoing clinical trials are multi-target inhibitors with the
unwanted inhibition of additional kinases, often accounting for undesirable toxicity. Here we discovered
SOMG-833 as a potent and selective small-molecule c-MET inhibitor, with an average IC50 of 0.93 nM
against c-MET, over 10 000 fold more potent compared with 19 tyrosine kinases including c-MET family
member and highly homologous kinases. SOMG-833 strongly suppressed c-MET-mediated signalling
transduction regardless of mechanistic complexity implicated in c-MET activation including MET gene
amplification, MET gene fusion and HGF-stimulated c-MET activation. In a panel of 24 human cancer
or genetically-engineered model cell lines, SOMG-833 potently inhibited c-MET driven cell proliferation
whereas cancer cells lacking c-MET activation were markedly less sensitive (at least 15-fold) to the
treatment. SOMG-833 also suppressed c-MET-mediated migration, invasion, urokinase activity and
invasive growth phenotype. In addition, inhibition of primary HUVEC proliferation and down-regulation
of plasma pro-angiogenic factors IL-8 secretion resulted from SOMG-833 treatment suggested its
significant anti-angiogenic properties. These together led to the remarkable antitumor efficacy of
SOMG-833 in vivo, as demonstrated in c-MET-dependent NIH-3T3/TPR-MET, U-87MG and EBC-1
xenograft models. Collectively, our results suggested SOMG-833 as a promising candidate for highly
selective c-MET inhibitor, and a powerful tool to investigate the sole role of MET kinase in cancer.
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Introduction
The receptor tyrosine kinase c-MET was first discovered and identified as an oncogene in a fusion form
known as TPR-MET (Cooper et al., 1984). Downstream activation of mitogen-activated protein kinase
(MAPK) and phosphoinositide-3 kinase (PI3K) were well established signaling cascades responsible
for the diverse process exerted by HGF/c-MET axis, including proliferation, invasion, metastasis and
angiogenesis (Birchmeier et al., 2003; Lemmon and Schlessinger, 2010; Trusolino et al., 2010).
Several lines of evidence support the significant role of HGF/c-MET in cancer development (Gherardi et
al., 2012). Aberrant c-MET activation resulted from specific genetic lesions, transcriptional upregulation,
or ligand-dependent autocrine or paracrine mechanisms occurred in many types of cancers
(http://www.vai.org/met/) (Comoglio et al., 2008). Moreover, it has been shown that the propagation of
MET-dependent invasive growth signals is a general and remarkable feature of highly aggressive
tumors, which spawn ‘pioneer’ cells that move out, infiltrate adjacent tissues and establish metastatic
lesions (Comoglio and Trusolino, 2002; Trusolino and Comoglio, 2002; Boccaccio and Comoglio, 2006).
This, together with the observation that c-MET is expressed by endothelial cells and that HGF is a
potent angiogenic factor, implies that inhibition of HGF/c-MET signalling axis can potentially interfere
with cancer onset and metastasis (You and McDonald, 2008). In addition, c-MET signalling is
responsible for the resistance acquisition of approved therapies (Kentsis et al., 2012; Straussman et al.,
2012; Wilson et al., 2012). All these emphasize the HGF/c-MET as an attractive target for cancer
therapy, and several different therapeutic approaches are being clinically tested (Comoglio et al.,
2008).
Notably, most c-MET inhibitors currently undergoing clinical trials are multi-target inhibitors. The
unwanted inhibition of additional kinases often leads to undesirable toxicity (Broekman et al., 2011).
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The broad toxicity profile of multitarget kinase inhibitors also largely limited their chances in
combination regimens. In contrast, highly selective c-MET inhibitors could largely avoid off-target
toxicities at therapeutic doses, and favour their use in drug combinations. More importantly, in the new
era of personalized medicine, where cancer care relies on validated biomarkers to identify a patient
subpopulation harboring the specific molecular characteristics that is likely to benefit from a targeted
therapy (Dietel and Sers, 2006; Hood and Friend, 2011; Ma, 2012), there is a significant need for
targeted drugs with high specificity. As such, highly selective c-MET inhibitors represent the main
direction for the development of c-MET-targeted therapy.
Here, we described a novel, highly selective c-MET inhibitor, SOMG-833, that showed strong
potency against c-MET kinase, suppressed c-MET phosphorylation and the downstream signaling in
c-MET overactivated cancer cell lines, as well as inhibited c-MET-dependent cellular events in tumor
cells and primary endothelial cells. Moreover, SOMG-833 exhibited significant antitumor activity in
several c-MET-driven xenograft models. All these findings promise SOMG-833 as a potential candidate
for c-MET-driven human cancers.
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Materials and Methods
Compouds
SOMG-833 (3-(4-Methylpiperazin-1-yl)-5-(3-nitrobenzylamino)-7-(trifluoromethyl) quinoline) was
synthesized at Shanghai Institute of Material Medica, Chinese Academy of Sciences as reported by us
earlier (Wang et al., 2011). This compound was fully characterized and possessed a purity of 99%. The
compound was prepared as a 10 mM stock solution in 100% dimethyl sulfoxide and routinely stored at
room temperature.
ELISA kinase assay and ATP competitive assay
c-MET tyrosine kinase activity was evaluated in enzyme-linked-immunosorbent assay (ELISA) as
described before (He et al., 2013). Detail procedures were described in Supplementary Materials and
Methods. For ATP competitive assay, various concentrations of ATP were diluted for the kinase reaction.
The results were analyzed in Lineweaver-Burk plots.
Cell culture
Human cancer cell lines (SNU-1, SNU-5, AGS, DU-145, NCI-H661, NCI-H441, A549, H1581, Bx-PC3,
HepG2, DBTRG, MCF-7, MDA-MB-231, HT-29, HCT-116, A375, SK-MEL-28) were all purchased from
American Type Culture Collection (ATCC), human cancer cell lines (MKN-45, EBC-1) were purchased
from JCRB and routinely maintained according to ATCC’s or JCRB’s recommendations. BaF3 cell line
was purchased from DSMZ. MDCK (Madin−Darby canine kidney epithelial cell) cell line was kindly
gifted from Prof. H. Eric Xu. SMMC-7721, BGC-803, U-87MG, BEL-7404 cell lines were obtained from
the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, GES-1, SPC-A4 cell lines
were obtained from Shanghai Cancer Institute, Renji Hospital and Chest Hospital, Shanghai Jiaotong
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University School of Medicine, Shanghai, China. Primary human umbilical vascular endothelial cells
(HUVEC) were purchased from AllCells (H-001), Cells were cultured according to the suppliers’
instructions. BaF3/TPR-MET cell line was a genetically generated BaF3 cell line that stably expressed
a constitutively active oncogenic version of c-MET.
Western blotting
EBC-1, MKN-45, BaF3/TPR-MET cells were cultured under regular growth conditions to the
exponential growth phase and treated with SOMG-833 for 2 h. A549, NCI-H441, MDCK and HUVEC
cells were serum-starved for 24 h, then incubated with the compound for 2 h and HGF(PeproTech) 100
ng/ml were added for additional 15 min. Cells were then lysed in 1×SDS (sodium dodecyl sulfate)
sample buffer and subsequently resolved by 10% SDS−polyacrylamide gel electro-phoresis and
transferred to nitrocellulose membranes. The membranes were first probed with phospho-c-MET,
p-ERK, ERK, p-AKT, AKT (all from Cell Signaling Technology), c-MET (from Santa Cruz) or GAPDH
(KangCheng Biotech) antibody and then with anti-rabbit or anti–mouse IgG horseradish peroxidase
(Jackson). Immunoreactive proteins were detected using ECL Plus or Femto (Thermo Scientific), and
images were captured with ImageQuant LAS 4000 (GE Healthcare).
Cell proliferation/survival assays.
Tumor cells were seeded in 96-well 3000-8000/well in growth media over night and then exposed to
designated concentrations of SOMG-833 for 72 h. A SRB (sulforhodamine B, sigma) or MTT (hiazolyl
blue tetrazolium bromide, Sigma) assay was done to determine tumor cell proliferation. HUVEC cells
(passage 3) were first serum-starved in complete medium (ALLcells) media for 24 hours and treated by
SOMG-833 for 72 h in media containing 3% BSA and 100 ng/ml HGF. Appropriate controls were
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conducted (containing 100 ng/ml HGF (HGF+) or not (HGF-)). A CCK8 (Cell Counting Kit-8, Dojindo)
assay was done to determine the viability of HUVEC cells. HGF dependent proliferation inhibition % =
[1-(ODtreatment-ODHGF-/ODHGF+-ODHGF-)]×100%. IC50 values were calculated by concentration-response
curve fitting a four-parameter method.
Cell cycle and apoptosis assay
1×105 EBC-1 or MKN-45 cells were seeded in 6-well plates over night, the following day cells were
treated for different concentration of SOMG-833 for 24 h. After treatment, the cells were trypsinized,
fixed in 70% ethanol and incubated by 20 ng/mL RNase and 10 ng/mL propidium iodide and analyzed
using flow cytometer (FACS Calibur, BD). The data were analyzed using Modifit LT. Cell apoptosis were
determined by Annexin V- FITC/PI Apopotosis Detection kit (Vazyme).
Cell migration and invasion
For migration assays, NCI-H441 cells suspended in serum-free DMEM media at a density of 1.5×105
cells/mL were seeded (0.1 mL) in the plate inserts of the transwell chamber (pore size, 8 μm; Corning)
and serum-free DMEM (0.6 mL) containing HGF 100 ng/ml (only added to the lower well) or not with
designed concentrations of SOMG-833 were added. NCI-H441 cells were subsequently cultured for 24
h. Cells that migrated to the lower wells were then fixed by 90% ethanol, stained by 0.1% crystal violet
and photographed. The crystals stained on the lower side of the well were dissolved by 100 µL 10%
acetic acid, and the absorbance of the resulting solution was measured at 600nm using a multiwell
spectrophotometer (SpetraMAX 190, Molecular Devices). The group only stimulated by HGF was
designed as positive control (HGF+, 100% migrated/invasived). The relative migration/invasion =
(ODtreated/ODHGF+)×100%
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For the invasion assay, NCI-H441 cells were cultured in the top chambers containing
Matrigel-coated membrane inserts (Matrigel, BD). The ensuing procedure was identical to the migration
assay
Cell scattering assay
MDCK cells (1.5×103 cells per well) were plated into 96-well plate and grown overnight. Increasing
concentration of SOMG-833 and 100 ng/mL HGF were added to the appropriate wells and incubated at
37°C, 5% CO2 for 24 h. The cells were fixed with 4% paraformaldehyde, stained by 0.5% violet purple
and photographed under microscope.
Cell branching morphogenesis
Cells at a density of 20 000 cells/mL in serum-free DMEM medium were mixed with collagen I solution
(BD Biosciences) at a proportion 4:6 ( the PH was adjusted to alkaline), then plated at 0.1 mL/well of a
96-well culture plate, and incubated for 45 min at 37 °C, 5% CO2 to allow collagen gelling. Then 100
ng/mL HGF with or without SOMG-833 at various concentrations dissolved in the 100 μL of DMEM
were then added to each well. The medium was replaced with fresh growth medium every 2 days.
Pictures were taken under microscope after 4 days.
uPA activity detection assays.
uPA activity detection was carried out according to protocols reported before (Webb et al., 2000).
Detail procedures can be found in Supplementary Materials and Methods
In vivo studies
Animals. Female nu/nu mice (4-6 weeks old) were maintained under clean room conditions and housed
on particulate air–filtered ventilated racks. Animal experiments were performed according to
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institutional ethical guidelines of animal care.
S.c. xenograft models in athymic mice. Tumor cells at a density of 5×106 in 200 μL were first implanted
sc into the right flank of each nude mice and then allowed to grow to 700-800 mm3, defined as a
well-developed tumor. After that, the well-developed tumors were cut into 1 mm3 fragments and
transplanted sc into the right flank of nude mice using a trocar. When the tumor volume reached
100-150 mm3, the mice were randomly assigned into control and treatment groups (n=5 per group).
Efficacy studies. Control groups were given normal saline alone and treatment groups received
SOMG-833 via intraperitoneal injection once daily. The tumor volume (TV) was calculated as: TV =
[length (mm) ×width2 (mm2)]/2. RTV=TVDay N/TVDay 0 × 100%. Percent (%) inhibition values were
measured on the final day of study for drug-treated compared with vehicle-treated mice and were
calculated as (1 –((treated final day - treated day 0) / (control final day – control day0))) × 100%.
Signal transduction studies. At designated times (3 days) following SOMG-833 administration, mice
were humanely euthanized, and tumors were resected. Tumors were snap frozen in liquid nitrogen,
protein lysates were generated, and protein concentrations were determined using a BSA assay
(Pierce). The tissue total protein lyses were then conducted by western blot for detecting the
phospho-c-MET, phospho-ERK and phospho-AKT (Cell Technology Signaling ).
Cytokine secretion detection.
The serum from EBC-1 xenograft mouse were collected from vechicle and SOMG-833 treated group on
the final day (14 d) of experiment. Cytokine secretion was detected using ELISA assays (MultiSciences,
70-E-EK1081) and Bio-plex pro Human Cytokine 27-plex Assay (#M50-00031YV, Bio-rad).
Statistics
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Data were presented as the mean ± SD (in vitro) or mean ± SEM (in vivo). The two-tailed Student’s
T-test was performed to analyse statistical differences between groups and *P≤0.05, **P≤0.01,
***P≤0.001 were considered significant.
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Results
SOMG-833 is an ATP-competitive inhibitor of c-MET kinase with high selectivity
SOMG-833 was initially identified as a potent small-molecule inhibitor of c-MET with an IC50 of 0.93 ±
0.15 nM in a biochemical enzymatic assay (Fig. 1A) (Wang et al., 2011). We were prompted to
investigate whether this potency was specifically against c-MET. A panel of 20 human kinases were
profiled including c-MET family member RON and highly homologous kinase AXL. In contrast to its high
potency against c-MET, SOMG-833 barely inhibited other 19 tested kinases at a concentration up to 10
μM (Table 1), indicating SOMG-833 was a selective c-MET inhibitor.
Most kinase inhibitors discovered to date are ATP competitive. To examine whether SOMG-833
functioned in this manner, we evaluated the inhibitory potency of SOMG-833 on c-MET activity using an
ATP competitiveness assay. With the increasing concentration of ATP, the inhibitory activity of
SOMG-833 upon c-MET kinase was decreased. In a Lineweaver-Burk plot, the different concentration
curve of SOMG-833 intersected in the specific point (known as 1/Vmax) at y axis with a broad
concentration from 0.32 nM to 200 nM (Fig.1B), showing that SOMG-833 was a ATP competitive
inhibitor. Together, these data suggested that SOMG-833 is a potent and selective c-MET inhibitor
which blocks c-MET kinase activity in an ATP competitive manner.
SOMG-833 inhibits c-MET phosphorylation and blocks downstream signals
To confirm cellular effectiveness of SOMG-833 targeting c-MET kinase, four cell lines with different
mechanisms of c-MET activation were chosen, i.e., human non-small cell lung cancer (NSCLC) cell line
EBC-1 and gastric tumor cell line MKN-45 with MET gene amplification, a genetically engineered cell
line BaF3/TPR-MET stably expressing a constitutively active oncogenic version TPR-MET, and NSCLC
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cell line A549 responsive to HGF stimulation. These cells were treated with various concentration of
SOMG-833 and c-MET signaling was examined using western blot assays (Fig. 2A). The results
showed that SOMG-833 inhibited c-MET phosphorylation in a dose-dependent manner, with a
complete abolishment at 1 μM in all tested cells. Similar results were observed in EBC-1 and MKN-45
cells using immunofluorescence assay (Fig. 2B). ERK1/2 and AKT are the key downstream molecules
of c-MET and play important roles in c-MET functioning. In line with suppressed c-MET phosphorylation,
phospho-ERK and phospho-AKT were signifcantly inhibited by SOMG-833 in a dose-dependent
manner in all tested four cell lines (Fig. 2A). These data showed that SOMG-833 effectively suppressed
c-MET signalling in cancer cells, regardless of mechanistic complexity in c-MET activation across
different cellular contexts.
SOMG selectively inhibits c-MET dependent tumor cell proliferation
Sustained c-MET signaling elevation could trigger uncontrolled cell proliferation, one of the hallmarks of
cancer. We then evaluated the effect of SOMG-833 on c-MET dependent cell proliferation. SOMG-833
significantly inhibited the proliferation of EBC-1, MKN-45, SNU-5, BaF3/TPR-MET cell lines, whose
growth was driven by activated c-MET signaling arising from MET gene amplification or TPR-MET gene
fusion, with a mean IC50 value of 0.160 to 0.457 μM (Supplementary Table S1) (Fig. 3A). The inhibitory
effect of SOMG-833 on EBC-1 and MKN-45 cell proliferation were further confirmed in colony formation
assay (Fig. 3B). By expanding to a panel of cancer cell lines originated from different tissues with MET
low expression or activation, SOMG-833 showed at least over 15-fold less potency (Fig. 3A). These
data demonstrated that SOMG-833 specifically inhibited c-MET-dependent cancer cell growth.
SOMG-833 inhibits c-MET dependent cell proliferation through arresting cells at G1/S phase
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c-MET inhibition is known to block cell proliferation via cell cycle arrest and apoptosis (Sattler et al.,
2003; Smolen et al., 2006; Bertotti et al., 2009; Hong et al., 2013). To confirm whether the
anti-proliferative activity of SOMG-833 was associated with blockage of c-MET signalling, EBC-1 and
MKN-45 cells were treated with various concentrations of SOMG-833 for 24 h and cell cycle distribution
was analysed. SOMG-833 induced a G1/S phase arrest in the EBC-1 cells, with 82.05% of the cell
population in G1 phase in the presence of 1 μM SOMG-833 (versus 55.78% in the control group) (Fig.
3C, 3D). Similar results were recapitulated in MKN-45 cells (Fig. 3E, 3F). Consistently, the
cyclin-dependent kinase inhibitor p27 and p21 were significantly increased, while the expression of
G1/S modulators, CylinD1 and CyclinE1, were down-regulated by SOMG-833 (Fig. 3G). Meanwhile, no
obvious sub-G1 cell population was observed upon SOMG-833 treatment (Fig. 3C, 3E). Treatment with
SOMG-833 for up to 48 h displayed no apparent apoptotic cells as detected by Annexin V/PI dual
staining (Fig. 3H), suggesting the G1/S phase arrest contributed most in the proliferation inhibition
induced by SOMG-833 .
SOMG-833 potentially inhibits c-MET-mediated metastasis
HGF/c-MET axis activation promoted cell invasion and migration to allow cancer metastasis.(Jeffers et
al., 1996b). Then we tested the effects of SOMG-833 on these processes using transwell-based
migration and invasion assays. SOMG-833 inhibited HGF-induced NCI-H441 cells migration (Fig.
4A,4B). Similar results were observed in a wound healing assay using MDCK cells (Supplementary Fig.
S1). Further, SOMG-833 strongly suppressed HGF-induced NCI-H441 cell invasion (Fig. 4C,4D) under
a condition where no significant viability inhibition was observed (Fig. 4I).
Cell invasion and metastasis requires degradation of surrounding ECM. HGF/SF induced urokinase
plasminogen activator (uPA) plays a central role in catalyzing ECM/BM degradation mainly through
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cleavage of plasminogen into the broader specificity protease plasmin (Jeffers et al., 1996a). MDCK
cell line was a widely-used model to evaluate uPA-plasmin network expression upon HGF stimulation
(Webb et al., 2000). We found that SOMG-833 inhibited the activity of plasmin cleaved by uPA upon
HGF-stimulation in a dose dependent manner (Fig .4G).
Upon HGF stimulation, c-MET induces several biological responses that collectively give rise to a
program known as invasive growth, which is pivotal to drive cancer cell invasion and metastasis
(Boccaccio and Comoglio, 2006). Thus, we next examined whether SOMG-833 inhibited c-MET
associated invasive growth. In vitro, this morphogenetic program was recapitulated by stimulating
cultured MDCK epithelial cells with HGF in suspension in a three-dimensional extracellular matrix
(collagen) to form multicellular-branched structures, named morphogenesis (Montesano et al., 1991;
Jeffers et al., 1996b). In addition, induction of epithelial cell scattering is a unique feature of HGF and is
fundamental for HGF/c-MET signalling-elicited invasive growth (Birchmeier et al., 2003). We therefore
chose these two representative models, cell scattering and morphogenesis, to evaluate the impact of
SOMG-833 on c-MET-mediated invasive growth. MDCK cells were stimulated with HGF 100 ng/ml in
the presence of various centration of SOMG-833. At a concentration of 3 μM, SOMG-833 showed
strong inhibitory effects on cell-scattering (Fig. 4E) and morphogenesis (Fig. 4F), indicating SOMG-833
inhibited HGF-induced c-MET-mediated invasive growth.
In accordance with these effects, SOMG effectively blocked PI3K-AKT and MEK-ERK pathway (Fig.
4H), which mediated c-MET dependent survival, invasion and morphogenesis (Zhang and Vande
Woude, 2003). Together, SOMG-833 showed its potency against c-MET dependent migration and
invasion thus lowered the risk of tumor metastasis.
SOMG-833 inhibits c-MET dependent proliferation of human umbilical vein endothelial cells (HUVEC).
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In addition to its crucial role in cancer cells, HGF/c-MET signalling is a potent inducer of endothelial cell
growth and promoted angiogenesis (Grant et al., 1993; Abounader and Laterra, 2005; Ren et al., 2005;
You and McDonald, 2008). Hence, we also assessed the anti-angiogenesis potential of SOMG-833. As
shown in Fig. 4J, SOMG-833 dose-dependently inhibited HGF-stimulated growth of primary HUVEC
with an average IC50 of 0.1 μM. Consistently, HGF-dependent c-MET phosphorylation and its
downstream signaling were potently inhibited in HUVEC cells by SOMG-833 (Fig. 4K).
SOMG-833 shows strong antitumor activity in vivo
To assess the in vivo antitumor efficacy of SOMG-833, three representative tumor xenograft models
driven by dysregulated c-MET were chosen: A NIH-3T3/TPR-Met model, where tumor growth was
driven by constitutive active MET fusion independent of HGF stimulation; A U-87MG human
glioblastoma model with HGF and c-MET comprising an autocrine loop; An EBC-1 xenograft model
specifically driven by MET amplification.
In the NIH-3T3 model, upon 14-day SOMG-833 administration, tumor growth inhibition were
observed in SOMG-833 treated group, with an inhibitory rate of 79.0% (P<0.01) and 51.0% (P<0.05) at
the doses of 80 mg/kg and 40 mg/kg, respectively (Fig. 5A). In U-87MG glioblastoma model,
SOMG-833 showed a similar dose-dependent inhibition of tumor growth with inhibitory rate of 73.0%
(P<0.01) and 48.0% (P<0.05) respectively (Fig. 5B). In EBC-1 xenograft model, SOMG-833 strongly
inhibited tumor growth inhibition at doses of 40 mg/kg (56.0%, P<0.05) and 80 mg/kg (97.9%, (P<0.001)
(Fig. 5C). By collecting EBC-1 tumor tissue samples collected at different time point after SOMG-833
treatment on the day 3, we observed marked inhibition of intratumoral phospho-c-MET and its
downstream key effectors phospho-AKT and phospho-ERK levels in SOMG-833 treated group (Fig.
5F), suggesting the inhibition of tumor growth by the administration of SOMG-833 was associated with
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the blockage of c-MET signalling.
c-MET driven cancer malignancy are mediated by its impacts on both tumor cells and endothelial
cells . The intratumoral mitotic index (Ki67) was assessed using immunohistochemical (IHC) analysis.
A significant decrease in Ki67 expression level was observed at 80 mg/kg/day of SOMG-833 in the
EBC-1 xenograft models (Fig. 5D), indicating the potent inhibition of mitogenesis in vivo. Meanwhile,
the contribution of anti-angiogenic efficacy was assessed. SOMG-833 was evaluated for modulation of
microvessel density (MVD) assessed by immunostaining for CD31 (platelet endothelial cell adhesion
molecule 1). At the 80 mg/kg/day dose, SOMG-833 treated sample showed a significant reduction of
CD31-positive microvessels (Fig. 5D). Moreover, we found the levels of proangiogenic factor IL-8,
which is regulated by c-MET/HGF (Yoshida et al., 1997; Li et al., 2003; Zhang et al., 2003), in
SOMG-833 (80 mg/kg) treated group were down regulated compared to vehicle group using ELISA
assay (Fig. 5E). These results indicated that the antitumor activity of SOMG-833 is mediated by direct
effects on tumor cell growth as well as antiangiogenic mechanisms.
Together, SOMG-833 showed robust anti-tumor efficacy which was correlated with the inhibition of
c-MET mediated signaling in c-MET-dependent tumor models.
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Discussion
Aberrant c-MET activation has been frequently found in many human solid tumors and hematologic
malignancies. Overactivation of c-MET is known to initiate tumorigenesis and promote metastasis, as
well as causing therapeutic resistance (Engelman et al., 2007), underscoring the importance of
developing therapeutic strategies capable of interrupting c-MET signalling (Gherardi et al., 2012). In
fact, recent clinical trials of c-MET pathway-targeted agents have yielded convincing evidence for the
benefit of targeting c-MET in cancer therapy including monotherapy and combined therapy (Sequist et
al., 2010; Bendell et al., 2013). Previously, we conducted a screen to discover specific c-MET inhibitors
and SOMG-833 was selected for further characterization of c-MET-targeting antitumor efficacy (Wang
et al., 2011).
An important feature of SOMG-833 is its selectivity against c-MET. SOMG-833 presented an IC50
for c-MET in the nanomolar range and showed at least above 1000-fold selectivity over a panel of 19
human kinases, including c-MET family member Ron (Wang et al., 2003). Consistently, cancer cells
with low c-MET activity were markedly less sensitive (at least 15-fold) to SOMG-833 than c-MET
addicted cells. It is worth noted that most of the reported c-MET kinase inhibitors being clinically
evaluated are multi-target inhibitor, often resulting in unwanted broad nonspecific toxicity (Broekman et
al., 2011). Highly selective for c-MET kinase inhibition SOMG-833 could specifically achieve the
therapeutic potential of c-MET inhibition in patients harbouring c-MET aberrations and its use in
biomarker-directed drug combinations in personalized medicines becomes possible. In addition, the
feature of high selectivity makes SOMG-833 suitable for use as a tool inhibitor in preclinical models to
dissect the role of c-MET kinase activity in cancer progression.
Activation of c-MET drives a complex morphogenetic program termed invasive growth (Boccaccio
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and Comoglio, 2006). Under normal conditions, invasive growth is based upon a finely tuned interplay
between related phenomena including cell proliferation, motility, ECM degradation, and survival. In
transformed tissues, aberrant implementation of this interplay is responsible for cancer progression and
metastasis (Comoglio and Trusolino, 2002). In our study, using a series of cell model, we were able to
dissect the key biological steps of invasive growth, including cell proliferation, scattering, migration,
invasion and found potency of SOMG-833 against these individual steps. Moreover, SOMG-833
reversed the comprehensive three-dimensional branching morphogenesis phenotype stimulated by
HGF, further confirming the strong inhibitory effect of SOMG-833 on c-MET-mediated invasive growth.
All these indicated a potential role of SOMG-833 against tumor progression and metastasis.
HGF and its receptor c-MET have been implicated in the regulation of tumor angiogenesis through
multiple mechanisms (Zhang et al., 2003). In the present study, SOMG-833 showed the ability to inhibit
HGF-stimulated c-MET-mediated endothelial cell survival, and to reduce MVD in the EBC-1 model. In
addition to their reported direct role in regulating endothelial cell function, c-MET and HGF are also
implicated in the regulation of secretion of angiogenic factors by epithelial and tumor cells (Zhang et al.,
2003; Knowles et al., 2009; Hill et al., 2012). Zou et al. has reported that PF-2341066 could decrease
IL-8 and VEGF in c-MET-dependent gastric GTL-16 and glioblastoma U87-MG models (Zou et al.,
2007), and Davide et al has found that reduced secretion of IL-8 may served as a indicative biomarker
responding to c-MET inhibition by pharmacological inhibitors in c-MET-dependent gastric xenografts
(Torti et al., 2012). Consistently, we found SOMG-833 could down-regulate serum IL-8 level in EBC-1
xenograft, indicating that antiangiogenic activity observed with SOMG-833 may be mediated by direct
and indirect mechanisms. However, the serum VEGF in the vehicle group was very low in the EBC-1
xenograft (<20 pg/mL) and almost no changes were observed upon SOMG-833 treatment (data not
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shown), suggesting that the regulation of angiogenic factor types by HGF/c-MET axis are not common
to all tumor types.
In conclusion, we assessed the antitumor efficacy of SOMG-833, a novel selective c-MET inhibitor.
SOMG-833 showed strong potency against c-MET kinase, inhibited c-MET phosphorylation and the
downstream signalling across different oncogenic forms in c-MET overactivated cancer cells. In turn,
SOMG-833 inhibited c-MET-driven cellular phenotype in tumor cell and primary endothelial cell.
Furthermore, SOMG-833 treatment resulted in significant antitumor activity in several c-MET-driven
xenografts. And intratumoral inhibition of c-MET phosphorylation, proliferation index (Ki67), and IL-8
level suggested that the concurrent antiprolifertaive and anti-angiogenic activity of SOMG-833
accounted for its anticancer efficacy in vivo.
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Acknowledgements
We thank professor H. Eric Xu (Shanghai Institute of Materia Medica) for providing the MDCK cells and
the technical support in the in vitro metastasis experiments.
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Authorship Contributions
Participated in research design: Geng, Yang, Ai and Ding.
Conducted experiments: H.T.Zhang, Wang and Chen.
Contributed new reagents or analytic tools: H.T.Zhang, Wang and Ai.
Performed data analysis: H.T.Zhang, Wang, Chen, Ai and Geng.
Wrote or contributed to the writing of the manuscript: H.T.Zhang, Ai and Huang.
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Footnotes
This work was supported by the funds from National Program on Key Basic Research Project of China
[Grant 2012CB910704], the National Natural Science Foundation [Grants 91229205 and 81102461],
National S&T Major Projects [Grant 2012ZX09301001-007] and China Marine Commonweal Research
Project [Grant 201005022-5].
Hao-tian Zhang, Lu Wang and Jing Ai contributed equally to this work.
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FIGURE LEGENDS
Fig. 1. (A) Chemical structure of SOMG-833. (B) Lineweaver-Burk double-reciprocal plots depicted the
ATP-competitive nature of SOMG-833. Velocity versus ATP (5, 25, 625 μM) at varied concentrations of
SOMG-833 (0, 0.32, 1.6, 8, 200 nM) were shown. The data were a representitive of three independent
experiments.
Fig. 2. SOMG-833 inhibited c-MET phosphorylation and downstream signaling in various cells. (A)
SOMG-833 effectively inhibited the phosphorylation of c-MET and the c-MET pathway downstream
effectors ERK1/2 and AKT in EBC-1, MKN-45, BaF3/TPR-MET and HGF-stimulated A549 cells. EBC-1,
MKN-45, BaF3/TPR-MET cells treated for 2 h with SOMG-833 at the indicated concentrations were
lysed and subjected to western blot analysis. A549 cells treated with SOMG-833 for 2 h following 100
ng/ml HGF stimulation for 15 min were then lysed and subjected to western blot analysis (B) Inhibitory
effects of SOMG-833 on c-MET phosphorylation in EBC-1 and MKN-45 cells. Cells treated with
SOMG-833 0.3 μM for 2 h were subjected to immunofluorescence straining using specific p-c-MET
antibody (Scale bars, 200 μM). Representative data were shown from three independent experiments.
Fig. 3. SOMG-833 inhibited c-MET dependent cell proliferation through G1/S cell cycle arrest. (A) Cell
lines seeded in 96-well were incubated with a range concentration of SOMG-833 for 72 h and then the
cell proliferation were determined using MTT assays or the sulforhodamine B (SRB) assays. The IC50 of
SOMG-833 were plotted as mean ± SD (μM) or estimated values from three independent experiments.
(B) Proliferation inhibition on EBC-1 and MKN-45 cells treated by SOMG-833 (0.3 μM) were using
clone formation assay. (C-F) Induction of G1 phase arrest by SOMG-833. Cells were treated with
increasing concentrations of SOMG-833 or vehicle for 24 h. The DNA content was measured by FACS
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analysis(C, E). The percentage of EBC-1 (D) and MKN-45 (F) cells in different cell cycle phases
determined by FACS and analyzed with ModifitLT V3.0 were plotted. The data were shown as mean ±
SD and representative images were shown. (G) EBC-1 and MKN-45 cells were treated with SOMG-833
for 24 h and indicated G0/G1-S phase regulated proteins were analyzed by immunoblot. (H) Apoptosis
detection of MKN-45 cells treated by SOMG-833 0.3 μM for 48 h using annexin V and propidium
iodides (PI) double staining. The percentage of early apoptotic cells (low right corner) and late apoptotic
cells (top right corner) were shown as indicated. Representative data were shown from three
independent experiments.
Fig. 4. SOMG-833 potently inhibited HGF-induced c-MET-dependent cell metastasis and angiogenesis.
(A,C) The migratory ability (A) and invasive ability (C) of NCI-H441 cells induced by 100 ng/mL HGF
were impaired by SOMG-833. Representative pictures were shown (Scale bars, 200 μM). (B, D) The
relative quantitative determination of migrated and invasived cells were plotted. The data shown were
the mean ± SD from three independent experiments, assuming 100% migration or invasion of cells
stimulated with HGF. (E) Inhibition of HGF-dependent MDCK cell scattering by SOMG-833 at indicated
concentration for 24 h. Partial enlarged view of cluster of cells were shown (Scale bars, 200 μM). (F)
SOMG-833 inhibited the MDCK branching morphogenesis on collagen stimulated by HGF. The “-” and
“+” represented HGF (100 ng/mL) untreated and treated group. Images were photographed 4 d after
treatment. Scale bars, 100 μM. (G) SOMG-833 inhibited HGF-induced uPA activation in MDCK cells.
Cells stimulated or untreated with HGF were shown as HGF+ or HGF-. Data were presented as mean ±
SD. (H) SOMG-833 inhibited c-MET phosphorylation and downstream signaling in HGF-stimulated
NCI-H441 and MDCK cellls. NCI-H441 and MDCK cellls treated with SOMG-833 for 2 h following 100
ng/mL HGF stimulation for 15 min were then lysed and subjected to Western blot analysis. (I) The
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effects of SOMG-833 on NCI-H441 Cell viability. Cell viability of NCI-H441 treated with SOMG-833 for
24 h at indicated concentrations were determined by MTT assay. The data shown were the mean ± SD.
(J) SOMG-833 inhibited HGF-dependent cell proliferation of primary HUVEC cells. primary HUVEC
Cells were treated by SOMG-833 (0.1, 0.3, 1 μM) for 72 h with HGF (100 ng/mL). Cell viability were
measured by CCK8 assay. (K) SOMG-833 inhibited c-MET phosphorylation and downstream signaling
in HGF-stimulated primary HUVEC cellls. Primary HUVEC cellls treated with SOMG-833 for 2 h
following 100 ng/mL HGF stimulation for 15 min were then lysed and subjected to Western blot analysis.
Representative data were shown from three independent experiments.
Fig. 5. Antitumor efficacy of SOMG-833 in vivo (A-C). Tumor growth inhibition upon SOMG-833
treatment in NIH-3T3/TPR-MET (A), U-87MG (B) and EBC-1 (C) xenografts. SOMG-833 was
administered intra-peritonelly (i.p.) once daily for 14 to 21 days after the tumor volume reached 100 to
150 mm3. Results were expressed as mean ± SEM ( n=5 per group). The percent of tumor volume
inhibition values (Inh.) was measured on the final day of the study for the drug-treated mice compared
with the control mice. *, p<0.05, **, p<0.01, ***, p<0.001 vs vehichle group, using Student’s T-test. (D)
An IHC evaluation of Ki67, p-c-MET and CD31 expression at SOMG-833 80 mg/kg were determined of
the EBC-1 xengrafts on the d 14. Representative images were shown (scale bar, 1 μm) Partial enlarged
views were demonstrated at the upcorner. (E) Serum levels of human IL-8 at 80 mg/kg SOMG-833 was
determined by ElISA assay of EBC-1 xenografts on d 14. The data shown were the mean ± SD. (F)
Inhibition of c-MET signaling transduction upon SOMG-833 treatment of EBC-1 xenografts. Mice were
humanely euthanized on study day 3 at 0.5, 2 h post-administration of SOMG-833 and the tumors were
resected. Protein extracts from tumor tissues were analyzed for phospho-c-MET levels and its
downstream effectors p-ERK and p-AKT.
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Table 1. Kinase-selectivity profile of SOMG-833
Tyrosine Kinase IC50 (nM) Tyrosine Kinase IC50 (nM)
c-MET 0.93 ± 0.15 RET >10000
RON >10000 EGFR >10000
Axl >10000 ErbB2 >10000
Tyro-3 >10000 ErbB4 >10000
ALK >10000 c-Src >10000
Flt-1 >10000 ABL >10000
KDR >10000 EPH-A2 >10000
c-Kit >10000 EPH-B2 >10000
PDGFRα >10000 IGF1R >10000
PDGFRβ >10000 FGFR1 >10000
IC50s were shown as mean ± SD (nM) or estimated values from three separate experiments.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 16, 2014 as DOI: 10.1124/jpet.114.214817
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This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 16, 2014 as DOI: 10.1124/jpet.114.214817
at ASPE
T Journals on February 15, 2019
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This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 16, 2014 as DOI: 10.1124/jpet.114.214817
at ASPE
T Journals on February 15, 2019
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ownloaded from
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 16, 2014 as DOI: 10.1124/jpet.114.214817
at ASPE
T Journals on February 15, 2019
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This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 16, 2014 as DOI: 10.1124/jpet.114.214817
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ownloaded from