SOMG-833, a novel, selective c-MET inhibitor, blocks c-MET...

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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.

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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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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Reference

Abounader R and Laterra J (2005) Scatter factor/hepatocyte growth factor in brain tumor growth and

angiogenesis. Neuro Oncol 7:436-451.

Bendell JC, Ervin TJ, Gallinson D, Singh J, Wallace JA, Saleh MN, Vallone M, Phan SC and Hack SP

(2013) Treatment rationale and study design for a randomized, double-blind,

placebo-controlled phase II study evaluating onartuzumab (MetMAb) in combination with

bevacizumab plus mFOLFOX-6 in patients with previously untreated metastatic colorectal

cancer. Clinical colorectal cancer 12:218-222.

Bertotti A, Burbridge MF, Gastaldi S, Galimi F, Torti D, Medico E, Giordano S, Corso S,

Rolland-Valognes G, Lockhart BP, Hickman JA, Comoglio PM and Trusolino L (2009) Only a

subset of Met-activated pathways are required to sustain oncogene addiction. Science

signaling 2:er11.

Birchmeier C, Birchmeier W, Gherardi E and Vande Woude GF (2003) Met, metastasis, motility and

more. Nat Rev Mol Cell Biol 4:915-925.

Boccaccio C and Comoglio PM (2006) Invasive growth: a MET-driven genetic programme for cancer

and stem cells. Nat Rev Cancer 6:637-645.

Broekman F, Giovannetti E and Peters GJ (2011) Tyrosine kinase inhibitors: Multi-targeted or

single-targeted? World journal of clinical oncology 2:80-93.

Comoglio PM, Giordano S and Trusolino L (2008) Drug development of MET inhibitors: targeting

oncogene addiction and expedience. Nat Rev Drug Discov 7:504-516.

Comoglio PM and Trusolino L (2002) Invasive growth: from development to metastasis. J Clin Invest

109:857-862.


at ASPE



ownloaded from


JPET #214817

24

Cooper CS, Park M, Blair DG, Tainsky MA, Huebner K, Croce CM and Vande Woude GF (1984)

Molecular cloning of a new transforming gene from a chemically transformed human cell line.

Nature 311:29-33.

Dietel M and Sers C (2006) Personalized medicine and development of targeted therapies: The

upcoming challenge for diagnostic molecular pathology. A review. Virchows Archiv : an

international journal of pathology 448:744-755.

Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, Lindeman N, Gale CM, Zhao X,

Christensen J, Kosaka T, Holmes AJ, Rogers AM, Cappuzzo F, Mok T, Lee C, Johnson BE,

Cantley LC and Janne PA (2007) MET amplification leads to gefitinib resistance in lung cancer

by activating ERBB3 signaling. Science 316:1039-1043.

Gherardi E, Birchmeier W, Birchmeier C and Vande Woude G (2012) Targeting MET in cancer:

rationale and progress. Nat Rev Cancer 12:89-103.

Grant DS, Kleinman HK, Goldberg ID, Bhargava MM, Nickoloff BJ, Kinsella JL, Polverini P and Rosen

EM (1993) Scatter factor induces blood vessel formation in vivo. Proc Natl Acad Sci U S A

90:1937-1941.

He CX, Ai J, Xing WQ, Chen Y, Zhang HT, Huang M, Hu YH, Ding J and Geng MY (2013) Yhhu3813 is

a novel selective inhibitor of c-Met Kinase that inhibits c-Met-dependent neoplastic phenotypes

of human cancer cells. Acta pharmacologica Sinica.35:89-97

Hill KS, Gaziova I, Harrigal L, Guerra YA, Qiu S, Sastry SK, Arumugam T, Logsdon CD and Elferink LA

(2012) Met receptor tyrosine kinase signaling induces secretion of the angiogenic chemokine

interleukin-8/CXCL8 in pancreatic cancer. PloS one 7:e40420.

Hong SW, Jung KH, Lee HS, Son MK, Yan HH, Kang NS, Lee J and Hong SS (2013) SB365, Pulsatilla


at ASPE



ownloaded from


JPET #214817

25

saponin D, targets c-Met and exerts antiangiogenic and antitumor activities. Carcinogenesis

34:2156-2169.

Hood L and Friend SH (2011) Predictive, personalized, preventive, participatory (P4) cancer medicine.

Nature reviews Clinical oncology 8:184-187.

Jeffers M, Rong S and VandeWoude GF (1996a) Enhanced tumorigenicity and invasion-metastasis by

hepatocyte growth factor scatter factor-met signalling in human cells concomitant with

induction of the urokinase proteolysis network. Molecular and Cellular Biology 16:1115-1125.

Jeffers M, Rong S and VandeWoude GF (1996b) Hepatocyte growth factor scatter factor Met signaling

in tumorigenicity and invasion/metastasis. J Mol Med-Jmm 74:505-513.

Kentsis A, Reed C, Rice KL, Sanda T, Rodig SJ, Tholouli E, Christie A, Valk PJ, Delwel R, Ngo V, Kutok

JL, Dahlberg SE, Moreau LA, Byers RJ, Christensen JG, Vande WG, Licht JD, Kung AL, Staudt

LM and Look AT (2012) Autocrine activation of the MET receptor tyrosine kinase in acute

myeloid leukemia. Nat Med 18:1118-1122.

Knowles LM, Stabile LP, Egloff AM, Rothstein ME, Thomas SM, Gubish CT, Lerner EC, Seethala RR,

Suzuki S, Quesnelle KM, Morgan S, Ferris RL, Grandis JR and Siegfried JM (2009) HGF and

c-Met participate in paracrine tumorigenic pathways in head and neck squamous cell cancer.

Clin Cancer Res 15:3740-3750.

Lemmon MA and Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell

141:1117-1134.

Li A, Dubey S, Varney ML, Dave BJ and Singh RK (2003) IL-8 directly enhanced endothelial cell

survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J

Immunol 170:3369-3376.


at ASPE



ownloaded from


JPET #214817

26

Ma PC (2012) Personalized targeted therapy in advanced non-small cell lung cancer. Cleveland Clinic

journal of medicine 79 Electronic Suppl 1:eS56-60.

Montesano R, Matsumoto K, Nakamura T and Orci L (1991) Identification of a fibroblast-derived

epithelial morphogen as hepatocyte growth factor. Cell 67:901-908.

Ren Y, Cao B, Law S, Xie Y, Lee PY, Cheung L, Chen YX, Huang X, Chan HM, Zhao P, Luk J, Vande

Woude G and Wong J (2005) Hepatocyte growth factor promotes cancer cell migration and

angiogenic factors expression: A prognostic marker of human esophageal squamous cell

carcinomas. Clinical Cancer Research 11:6190-6197.

Sattler M, Pride YB, Ma P, Gramlich JL, Chu SC, Quinnan LA, Shirazian S, Liang C, Podar K,

Christensen JG and Salgia R (2003) A novel small molecule met inhibitor induces apoptosis in

cells transformed by the oncogenic TPR-MET tyrosine kinase. Cancer Res 63:5462-5469.

Sequist LV, Akerley WL, Brugger W, Ferrari D, Garmey E, Gerber DE, Orlov S, Ramlau R, von Pawel J

and Schiller JH (2010) Final Results from Arq 197-209: A Global Randomized

Placebo-Controlled Phase 2 Clinical Trial of Erlotinib Plus Arq 197 Versus Erlotinib Plus

Placebo in Previously Treated Egfr-Inhibitor Naive Patients with Advanced Non-Small Cell

Lung Cancer (Nsclc). Annals of Oncology 21:122-122.

Smolen GA, Sordella R, Muir B, Mohapatra G, Barmettler A, Archibald H, Kim WJ, Okimoto RA, Bell

DW, Sgroi DC, Christensen JG, Settleman J and Haber DA (2006) Amplification of MET may

identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor

PHA-665752. Proc Natl Acad Sci U S A 103:2316-2321.

Straussman R, Morikawa T, Shee K, Barzily-Rokni M, Qian ZR, J D, Davis A, Mongare MM, Gould J,

Frederick DT, Cooper ZA, Chapman PB, Solit DB, Ribas A, Lo RS, Flaherty KT, Ogino S,


at ASPE



ownloaded from


JPET #214817

27

Wargo JA and Golub TR (2012) Tumour micro-environment elicits innate resistance to RAF

inhibitors through HGF secretion. Nature 487:500-504.

Torti D, Sassi F, Galimi F, Gastaldi S, Perera T, Comoglio PM, Trusolino L and Bertotti A (2012) A

preclinical algorithm of soluble surrogate biomarkers that correlate with therapeutic inhibition of

the MET oncogene in gastric tumors. International journal of cancer Journal international du

cancer 130:1357-1366.

Trusolino L, Bertotti A and Comoglio PM (2010) MET signalling: principles and functions in

development, organ regeneration and cancer. Nat Rev Mol Cell Biol 11:834-848.

Trusolino L and Comoglio PM (2002) Scatter-factor and semaphorin receptors: cell signalling for

invasive growth. Nat Rev Cancer 2:289-300.

Wang MH, Wang D and Chen YQ (2003) Oncogenic and invasive potentials of human

macrophage-stimulating protein receptor, the RON receptor tyrosine kinase. Carcinogenesis

24:1291-1300.

Wang Y, Ai J, Wang Y, Chen Y, Wang L, Liu G, Geng M and Zhang A (2011) Synthesis and c-Met kinase

inhibition of 3,5-disubstituted and 3,5,7-trisubstituted quinolines: identification of

3-(4-acetylpiperazin-1-yl)-5-(3-nitrobenzylamino)-7- (trifluoromethyl)quinoline as a novel

anticancer agent. J Med Chem 54:2127-2142.

Webb CP, Hose CD, Koochekpour S, Jeffers M, Oskarsson M, Sausville E, Monks A and Vande Woude

GF (2000) The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter

factor-met-urokinase plasminogen activator-plasmin proteolytic network. Cancer Res

60:342-349.

Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E, Peng J, Lin E, Wang Y, Sosman J, Ribas A,


at ASPE



ownloaded from


JPET #214817

28

Li J, Moffat J, Sutherlin DP, Koeppen H, Merchant M, Neve R and Settleman J (2012)

Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature

487:505-509.

Yoshida S, Ono M, Shono T, Izumi H, Ishibashi T, Suzuki H and Kuwano M (1997) Involvement of

interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor

necrosis factor alpha-dependent angiogenesis. Mol Cell Biol 17:4015-4023.

You WK and McDonald DM (2008) The hepatocyte growth factor/c-Met signaling pathway as a

therapeutic target to inhibit angiogenesis. BMB reports 41:833-839.

Zhang YW, Su Y, Volpert OV and Vande Woude GF (2003) Hepatocyte growth factor/scatter factor

mediates angiogenesis through positive VEGF and negative thrombospondin 1 regulation.

Proc Natl Acad Sci U S A 100:12718-12723.

Zhang YW and Vande Woude GF (2003) HGF/SF-met signaling in the control of branching

morphogenesis and invasion. Journal of cellular biochemistry 88:408-417.

Zou HY, Li Q, Lee JH, Arango ME, McDonnell SR, Yamazaki S, Koudriakova TB, Alton G, Cui JJ, Kung

PP, Nambu MD, Los G, Bender SL, Mroczkowski B and Christensen JG (2007) An orally

available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor

efficacy through antiproliferative and antiangiogenic mechanisms. Cancer Res 67:4408-4417.


at ASPE



ownloaded from


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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.


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