A HGF/cMET Autocrine Loop Is Operative in Multiple Myeloma Bone Marrow Endothelial Cells and May...

Published OnlineFirst September 11, 2014.Clin Cancer Res Arianna Ferrucci, Michele Moschetta, Maria Antonia Frassanito, et al. therapeutic targetbone marrow endothelial cells and may represent a novel A HGF/cMET autocrine loop is operative in multiple myeloma

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1

A HGF/cMET autocrine loop is operative in multiple myeloma bone marrow 1

endothelial cells and may represent a novel therapeutic target 2

3

Arianna Ferrucci1, Michele Moschetta1, Maria Antonia Frassanito2, Simona Berardi1, Ivana 4

Catacchio1, Roberto Ria1, Vito Racanelli1, Antonella Caivano3, Antonio Giovanni Solimando1, 5

Daniele Vergara4, Michele Maffia4, Dominga Latorre4, Antonia Rizzello4, Alfredo Zito5, Paolo 6

Ditonno6, Eugenio Maiorano7, Domenico Ribatti8, and Angelo Vacca1 7 8 1Internal Medicine Unit, Department of Biomedical Sciences and Human Oncology, University of 9 Bari Medical School, Bari, Italy; 2General Pathology Unit, Department of Biomedical Sciences and 10 Human Oncology, University of Bari Medical School, Bari, Italy; 3Laboratory of Pre-Clinical and 11 Translational Research, IRCCS-CROB, Rionero in Vulture, Italy; 4Laboratory of General 12 Physiology, Department of Biological and Environmental Sciences and Technologies, University of 13 Salento, Lecce, Italy; 5Pathological Anatomy Unit, and 6Hematology Unit, Di Venere Hospital, 14 Bari, Italy; 7Pathological Anatomy Unit, University of Bari Medical School, Bari, Italy; 8Section of 15 Human Anatomy and Histology, Department of Basic Medical Sciences, Neurosciences, and 16 Sensory Organs, University of Bari Medical School, and National Cancer Institute “Giovanni 17 Paolo II”, Bari, Italy 18 19

Correspondence: Abstract word count: 216 20 Angelo Vacca, MD, PhD Words of text: 4100 21 Department of Biomedical Sciences and Human Oncology, 22 Section of Internal Medicine “G. Baccelli”, 23 University of Bari Medical School, 24 Policlinico, Piazza Giulio Cesare 11, 25 I-70124 Bari, Italy 26 Phone: +39-080-5478057 27 Fax: +39-080-5478895 28 E-mail: [email protected] 29 30

Running Title: HGF/cMET pathway and MM angiogenesis. 31

Keywords: angiogenesis, bone marrow endothelial cells, HGF/cMET pathway, cMET inhibitor, 32

multiple myeloma. 33

Conflicts of interest: no conflicts of interest were declared. 34

Acknowledgements: this work was supported by Associazione Italiana per la Ricerca sul Cancro 35

(AIRC, Milan), Investigator grant 2013 (n. 14095) and the 5 per thousand Molecular Clinical 36

Oncology Special Program (grant n. 9965) to AV, the European Commission's 7th Framework 37

programme 2007-2013 (EU FPT7) under grant agreement n. 278706 (OVER-MyR) to AV, and EU 38




2

FPT7 under grant agreement n. 278570 to DR, and grants from MIUR PRIN 2009WCNS5C_004 to 1

RR, and 2010NECHBX to AV. The sponsors of this study are public or non-profit organizations 2

that support science in general. They had no role in gathering, analyzing, or interpreting the data. 3

The authors are fully responsible for the content and editorial decisions for this manuscript. 4

5

Authors’ contributions 6

AF designed research, performed experiments and wrote the manuscript; MAF supervised 7

experiments and analyzed data; MM, AC, SB, IC, AGS, DV, MMa, DL, AR, DR performed 8

experiments; AZ, PD, EM contributed material; RR and VR commented on the manuscript; and AV 9

supervised the experiments, provided financing and wrote the manuscript. 10

11

List of abbreviations 12

2-DE, two-dimensional gel electrophoresis; 7-ADD, 7-aminoactinomycin D; Abs, antibodies; 13

ANXA2, annexin A2; ANXA4, annexin A4; BM, bone marrow; BMSCs, BM stromal cells; CAM, 14

chorioallantoic membrane; CFSE, carboxyfluorescein succinimidyl ester; CM, conditioned 15

medium; cMET, mesenchymal-epithelial transition factor; CPNS1, calpain small subunit 1; Ct, 16

threshold cycle; CXCL16, chemokine ligand 16; DAPI, 4',6-diamidino-2-phenylindole; DMEM, 17

Dulbecco’s modified eagle’s medium; ECs, endothelial cells; FAK, focal adhesion kinase; FITC, 18

fluorescein isothiocyanate; FGF-2, fibroblast growth factor-2; HGF, hepatocyte growth factor; IEF, 19

isoelectric focusing; IL, interleukin; MALDI-TOF, matrix-assisted laser desorption/ionization-time 20

of flight; MGECs, ECs of MGUS patients; MGUS, monoclonal gammopathy of undetermined 21

significance; MM, multiple myeloma; MCP-1, monocyte chemotactic protein-1 (CCL2); MMECs, 22

ECs of MM patients; MS, mass spectrometry; MVD, microvessel density; OD, optical density; p-23

cMET, phospho-cMET; PCs, plasma cells; PE, phycoerythrin; PHB, prohibitin; PI, proliferation 24

index; PI3K, phosphatidylinositol 3-kinase; PMF, mass fingerprinting; PRDX6, peroxiredoxin-6; 25

RT-PCR, reverse transcriptase-PCR; SC, scatter factor; SERPIN E1, serpin peptidase inhibitor, 26




3

clade E (nexin, plasminogen activator inhibitor type 1), member 1; SERPIN F1, serpin peptidase 1

inhibitor, clade F (α2 antiplasmin, pigment epithelium derived factor), member 1; SFM, serum-free 2

medium; TRITC, tetramethylrhodamine isothiocyanate. 3

4

\STATEMENT OF TRANSLATIONAL RELEVANCE 5

Bone marrow (BM) angiogenesis is enhanced in multiple myeloma (MM) vs monoclonal 6

gammopathy of undetermined significance (MGUS) and entails an attractive target for treatment. 7

HGF/cMET pathway is implicated in the MM pathogenesis and progression. Here we show that 8

BM derived endothelial cells (ECs) from patients with active MM (MMECs) but not those from 9

patients with MGUS (MGECs) or with benign anemia (controls) present the HGF/cMET pathway 10

constitutively activated and operative in an autocrine fashion as an angiogenesis amplifier. In vitro 11

and in vivo studies of a novel selective cMET inhibitor, i.e., SU11274, tested singularly and in 12

combination with bortezomib or lenalidomide, suggest that the HGF/cMET pathway of MMECs 13

may be envisaged as a new therapeutic target for the antiangiogenic management of active MM 14

patients. 15

16

ABSTRACT 17

Purpose: To investigate the angiogenic role of the HGF/cMET pathway and its inhibition in bone 18

marrow (BM) endothelial cells (ECs) from patients with multiple myeloma (MM) vs those with 19

monoclonal gammopathy of undetermined significance (MGUS) or benign anemia (controls). 20

Experimental Design: The HGF/cMET pathway was evaluated in ECs from MM patients 21

(MMECs) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies or on 22

refractory phase to these drugs, in ECs from patients with MGUS (MGECs), and in those from 23

controls. The effects of a selective cMET tyrosine kinase inhibitor (SU11274) on the MMECs 24

angiogenic activities were studied in vitro and in vivo. 25




4

Results: MMECs express more HGF, cMET, and activated cMET (phospho (p)-cMET) at both 1

RNA and protein level vs MGECs and control ECs. MMECs are able to maintain the HGF/cMET 2

pathway activation in absence of external stimulation, while treatment with anti-HGF and anti-3

cMET neutralizing antibodies (Abs) is able to inhibit the cMET activation. The cMET pathway 4

regulates several MMECs activities including chemotaxis, motility, adhesion, spreading, and whole 5

angiogenesis. Its inhibition by SU11274 impairs these activities in a statistically significant fashion 6

when combined with bortezomib or lenalidomide, both in vitro and in vivo. 7

Conclusions: An autocrine HGF/cMET loop sustains MM angiogenesis, and represents an 8

appealing new target to potentiate the antiangiogenic management of MM patients. 9

10

INTRODUCTION 11

Multiple myeloma (MM) is a clonal expansion of plasma cells (PCs) in the bone marrow (BM), 12

where they proliferate and acquire resistance to apoptosis and drugs (1). MM angiogenesis results 13

from interactions between PCs and BM microenvironment cells, and is a constant hallmark of 14

disease progression since it correlates with tumor growth, relapse and drug resistance (2). The 15

angiogenesis is enhanced in MM patients compared to those with monoclonal gammopathy of 16

undetermined significance (MGUS) and normal controls (3); and it correlates with prognosis (4). 17

MM remains an incurable malignancy, despite important advances in conventional as well as high-18

dose chemotherapies supported by autologous stem cell transplantation. To overcome drug 19

resistance and to improve clinical response, novel therapeutic approaches halting both PCs and 20

angiogenesis are under experimental and clinical studies (5). 21

Hepatocyte growth factor (HGF)/scatter factor (SF) is a potent angiogenic cytokine. Bussolino et al. 22

first showed that HGF vividly induces endothelial cell (ECs) proliferation and migration in vitro by 23

triggering their specific tirosine kinase receptor mesenchymal-epithelial transition factor (cMET), 24

and angiogenesis in vivo using the rodent cornea assay (6). It also enhances the expression of other 25

angiogenic factors, including vascular endothelial growth factor (VEGF) and its receptors, and 26




5

suppresses the expression of thrombospondin 1, an endogenous angiogenesis inhibitor (7). Aberrant 1

HGF/cMET pathway activation has been described in both solid (8) and blood tumors (9) in which 2

it triggers several signaling pathways, including Src/FAK, p120/STAT3, PI3K/Akt and Ras/MEK, 3

which results in cell proliferation, migration, invasion, and resistance to apoptosis. The HGF/cMET 4

pathway is thus important for tumor growth, angiogenesis, and metastatic spread. 5

The HGF/cMET pathway is involved in the MM pathogenesis. Coexpression of HGF and cMET 6

has been observed in MM PCs, implying the existence of an autocrine loop (9, 10). Since BM 7

stromal cells (BMSCs) produce HGF (11), a paracrine stimulation of PCs within their 8

microenvironment may occur, and provide mitogenic, migratory and morphogenic effects (9). HGF 9

levels are significantly increased in peripheral and BM blood of MM patients at diagnosis compared 10

with healthy controls, and represent a negative prognostic factor (12). We have shown that the 11

HGF/cMET pathway is constitutively activated in PCs from relapsed and resistant patients, and that 12

it mediates the multidrug resistance (10). We have also shown a therapeutic activity of SU11274, a 13

specific adenosine triphosphate-competitive small-molecule cMET inhibitor in a MM xenograft 14

model (10). Furthermore, SU11274 is able to inhibit cMET phosphorylation and cMET-dependent 15

motility, invasion and proliferation in preclinical models of lung (13) and ovarian (14) carcinomas. 16

Data to be presented show the role of HGF/cMET pathway in MM-associated angiogenesis, and the 17

effects of cMET inhibition by SU11274 on ECs of MM patients (MMECs) both in vitro and in vivo. 18

19

MATERIAL AND METHODS 20

Patients and ECs 21

Patients fulfilling the International Myeloma Working Group diagnostic criteria (15) for MM 22

(n=32) and MGUS (n=24) were studied. MM patients (18 men/14 women), aged 41–82 (median 23

61.5) years, were at first diagnosis (n=9), on refractory phase to bortezomib- or lenalidomide-based 24

chemotherapies (n=12), or at relapse after these therapies (n=11). The M-component was IgG 25

(n=18), IgA (n=8), and κ or λ (n=6). MGUS patients (15 men/9 women), aged 42–79 (median 60.5) 26




6

years, were IgG (n=13), IgA (n=7), or κ or λ (n=4). Control BM ECs were harvested from 10 1

subjects with anemia due to iron or vitamin B12 deficiency (2). The study was approved by the 2

Ethics Committee of the University of Bari Medical School, and all patients provided their 3

informed consent according to the Declaration of Helsinki. MMECs, MGUS derived ECs (MGECs) 4

and control ECs were harvested and cultured as described (16). 5

ELISA, immunoprecipitation, western blot, and real time reverse transcriptase (RT)-PCR 6

MMECs, MGECs and control ECs (1×106 cells/mL) were cultured for 24 h in serum-free medium 7

(SFM) 1% glutamine, and their conditioned media (CM) were prepared (16). HGF was quantified in 8

the CM by an ELISA (Quantikine® Human HGF, R&D Systems, Inc., Minneapolis, MN, USA). 9

Protein lysates from all the ECs types (7×105 cells/sample) were incubated with an anti-cMET 10

antibody (Ab) (Cell Signaling Technology, Danvers, MA, USA), then antigen-Ab complexes 11

immunoprecipitated by Protein G/agarose (Sepharose®, Sigma-Aldrich, St Louis, MO, USA). 12

Protein aliquots (50 μg) were immunoblotted with anti-cMET and anti-phospho(p)-cMET 13

(Tyr1349) Abs (Cell Signaling Technology). Immunoreactive bands were detected using enhanced 14

chemiluminescence (LiteAblot, Euroclone, Milan, Italy) and the Gel-Logic1500 system (Eastman 15

Kodak Co., Rochester, NY, USA), quantified by the Kodak Imaging software, and expressed as 16

optical density (OD) units (17). 17

Real time RT-PCR was performed using primers (Invitrogen, Paisley, UK) shown in 18

Supplementary Table 1, and the Applied Biosystems methodology (18). Relative quantification of 19

the mRNA was performed using the comparative threshold cycle (Ct) method with GAPDH as the 20

reference gene and with the 2-ΔΔCT formula (19). 21

Fluorescence-activated cell sorting (FACS) 22

Three×105 MMECs, MGECs or normal ECs/tube were incubated with fluorescein isothiocyanate 23

(FITC)-conjugated anti-cMET and phycoerythrin (PE)-labeled anti-p-cMET (Y1234/1235) Abs 24

(R&D Systems). At least 100,000 events/sample were acquired and analyzed using FACScantoII 25




7

cytofluorimeter and the FACSDiva software (Becton Dickinson-BD, San Jose, CA, USA). Negative 1

controls were stained with isotype-matched irrelevant antibodies (BD). 2

Immunofluorescence 3

Five×103 MMECs, MGECs and control ECs/chamber were cultured on fibronectin-coated chamber 4

slides (LabTek, Nalge Nunc International, Naperville, IL, USA), fixed (paraformaldehyde), 5

permeabilized (Triton X-100), and incubated with an anti-cMET mouse monoclonal Ab and with an 6

anti-p-cMET rabbit Ab (both from Abcam, Cambridge, UK), then with the anti-mouse IgG-FITC 7

and with the anti-rabbit IgG-tetramethylrhodamine isothiocyanate (TRITC) Abs (both from Sigma-8

Aldrich). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, in Vectashield® 9

Hard_Set™ mounting medium, Vector, Burlingame, CA, USA). Pictures were acquired by an 10

Axioplan-2 microscope (Carl Zeiss, Jena, Germany), and analyzed by the Leica Application Suite 11

Advanced Fluorescence software (Leica Microsystems, Wetzlar, Germany). 12

Functional studies 13

Treatment of MMECs with HGF and cMET inhibitors and anti-MM drugs. The anti-HGF and 14

anti-cMET neutralizing Abs (R&D Systems) were titrated and used at final doses of 80 ng/µL and 15

40 ng/µL respectively, which were capable to significantly decrease the p-cMET expression in 16

MMECs. The cMET inhibitor SU11274 (Selleck Chemicals, Houston, TX, USA) was used at 0.5 17

and 1 µmol/L (14). Bortezomib (Velcade®, Millennium Pharmaceuticals Inc., Cambridge, MA, 18

USA) and lenalidomide (Revlimid®, Celgene Corporation, Summit, NJ, USA) were dissolved in 19

dimethylsulfoxide (Sigma-Aldrich) at final doses of 7.5 nmol/L in vitro/20 nmol/L in vivo (20), and 20

1.75 µmol/L (17), respectively. 21

Cell viability. Five×103 cells/100 µL/well were plated in triplicate in 96-well plates in serum-free 22

Dulbecco’s modified eagle’s medium (DMEM, Euroclone) alone (negative control), or 23

supplemented with 20% fetal calf serum alone (positive control), or added with the neutralizing Abs 24

(singularly and in combination), or with SU11274 for 24 h, then tested with the MTT assay (Cell 25

Growth Determination kit, Sigma-Aldrich) on the last 4 h, and measured at 570 nm absorbance 26




8

referenced to 655 nm (17). 1

Angiogenesis on Matrigel. Two×104 MMECs/well were seeded in triplicate on Matrigel-coated 2

(BD) 48-well plates in SFM alone (positive control), or added with the neutralizing Abs, or with 3

SU11274 and/or bortezomib or lenalidomide. After 18 h the skeletonization of the mesh was 4

followed by measurement of mesh areas and vessel length in three randomly-chosen ×200 fields on 5

an EVOS digital inverted microscope (Euroclone) (18). Three different doses of SU11274 (0.1, 0.5, 6

and 1 µmol/L), bortezomib (7.5, 10, and 20 nmol/L) and lenalidomide (0.5, 0.25, and 1.75 µmol/L) 7

were singularly tested in the Matrigel assay. The antiangiogenic potency of the drug associations 8

was assessed by combining the highest dose of SU11274 with the highest dose of bortezomib or 9

lenalidomide. 10

“Wound” healing. Confluent MMECs on fibronectin (10 μg/mL)-coated (Sigma-Aldrich) 6 cm2 11

dishes were scraped as a “wound” with a pipette tip, and left to move into the wound for 24 h in 12

SFM alone (control), or added with the neutralizing Abs or with SU11274. Cells were fixed, and 13

counted in at least three randomly chosen ×10 wound fields on the EVOS microscope (17). 14

Chemotaxis. By using the Boyden microchamber assay (16), 1×105 MMECs/well were seeded in 15

triplicate on the upper compartment of the chamber, exposed to the neutralizing Abs or SU11274, 16

and left to migrate towards DMEM with 1.5% fetal calf serum (negative control) or added with 17

VEGF (10 ng/mL, Sigma Chemical Co.) and FGF-2 (10 ng/mL, Peprotech Inc., Rocky Hill, NJ, 18

USA) (positive control) in the lower compartment. After 8 h at 37°C, the migrated cells were fixed, 19

stained (Snabb-Diff Kit, Labex AB, Helsingborg, Sweden), and counted on 3-4 ×400 20

fields/membrane using the EVOS microscope. 21

Adhesion to and spreading on fibronectin. Two×103 MMECs/well were plated in triplicate on 22

fibronectin-coated 96-well plates in DMEM alone (control) or added with the neutralizing Abs or 23

SU11274 for 30 min (to assess adhesion) or 90 min (to assess spreading), fixed (4% 24

paraformaldehyde), and quantified by the crystal violet assay at 595 nm in a Microplate Reader 25

(Molecular Devices Corp., Sunnyvale, CA, USA) (18). 26




9

Apoptosis. Five×105 MMECs untreated or treated with the neutralizing Abs or SU11274 were 1

washed with ice-cold PBS without Ca2+ and Mg2+, incubated with aminoactinomycin D (7-ADD) 2

and FITC Annexin V (Becton Dickinson-BD Biosciences™, San Jose, CA), and analyzed on 3

FACScantoII with the FACSDiva software (BD). 4

Proliferation assay with carboxyfluorescein succinimidyl ester (CFSE) staining. Five×105 5

MMECs untreated or treated with the neutralizing Abs or SU11274 were labeled with CFSE at 24 6

and 72 h by using CellTrace™ cell proliferation kit (Molecular Probes Inc., Eugene, OR, USA), 7

acquired by the FACScantoII with the FlowJo software (Tree Star, Inc., Ashland, OR, USA), and 8

shown as proliferation index (PI). 9

Angiogenesis assay 10

CM of MMECs untreated or treated with SU11274 was tested for the expression of 55 human 11

angiogenesis-related proteins by using the Human Angiogenesis Western blot Array (R&D 12

Systems). Spots were detected and quantified as for western blot. 13

Two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) protein identification 14

The 2-DE was done in duplicate by isoelectric focusing (IEF), followed by SDS-PAGE. Gels were 15

silver stained, and analyzed on PD-Quest software (Bio-Rad, Hercules, CA, USA) (21). Spots of 16

interest were excised, destained, dehydrated in acetonitrile and digested with trypsin. Proteins were 17

identified by peptide mass fingerprinting (PMF) and tandem MS/MS analysis with a matrix-assisted 18

laser desorption/ionization (MALDI)-time of flight (TOF)/TOF Ultraflextreme (Bruker Daltonics, 19

Billerica, MA, USA) (22). Data were exported by the BioTools software (version 3.2, Bruker 20

Daltonics), subjected to database search by using the Matrix Science system 21

(www.matrixscience.com), and referred for mass to the SwissProt human protein database (release 22

2014_03 of 19-Mar-2014 of UniProtKB/TrEMBL, 54247468 sequence entries). 23

In vivo chorioallantoic membrane (CAM) assay 24

Fertilized white Leghorn chicken eggs were incubated at 37°C at constant humidity (23). On day 3, 25

the shell was opened and 2-to-3 mL of albumen removed to detach the CAM. On day 8, the CAMs 26




10

were implanted with 1 mm3 sterilized gelatin sponges (Gelfoam Upjohn Co., Kalamazoo, MI, USA) 1

loaded with SFM alone (negative control) or with the CM of MMECs untreated (positive control) or 2

treated with SU11274 alone or added with bortezomib and/or lenalidomide. On day 12, the 3

angiogenic response was evaluated as the number of vessels converging toward the sponge at ×50 4

and photographed in ovo by a stereomicroscope (Olympus Italia Srl, Segrate, Milan, Italy). 5

6

RESULTS 7

Expression of HGF and cMET in the ECs types 8

MMECs (at diagnosis, at relapse, and in refractory disease), MGECs and control ECs were 9

evaluated. MMECs expressed (as average) ~2-fold more HGF and cMET mRNA vs MGECs; and 10

~4-fold more vs control ECs (P<0.01; Fig. 1A). Accordingly, MMECs secreted ~2.5-fold more 11

HGF than MGECs (P<0.03) and ~12-fold more than control ECs (P<0.01) in a 24 h serum-free 12

medium (SFM) culture (Fig. 1B, left panel). Similarly, immunoprecipitated total cMET protein was 13

~3-fold more in MMECs vs MGECs and control ECs (P<0.01; Fig. 1B, right panel). The activation 14

of cMET in MMECs was assessed by western blot and optical density (OD) of p-cMET: it was ~4-15

fold more vs MGECs and control ECs (P<0.01; Fig. 1B, right panel). FACS analysis (Fig. 1C) 16

showed high variability in cMET and p-cMET expression among 1st diagnosed MM patients (20 ± 17

10% double positive MMECs), while refractory and relapsed patients gave substantially higher 18

expression (60 ± 15% and 80 ± 20% vs 9 ± 7% MGECs and 2 ± 1% control ECs; P<0.001; Fig. 19

1C). Immunofluorescence studies (Fig. 1D) confirmed these data, and showed a cytoplasmatic 20

colocalization of both molecules in MMECs, but not in MGECs and control ECs. 21

Overall data suggest that MMECs, but not MGECs and control ECs, have an activated HGF/cMET 22

pathway. 23

24

Existence of a HGF/cMET autocrine loop in MMECs but not in MGECs and control ECs 25




11

The simultaneous expression of both HGF and cMET, and the evidence of a constitutive p-cMET 1

expression in MMECs suggest that a HGF/cMET autocrine loop is operative in these cells. To 2

validate this hypothesis, p-cMET was evaluated by flow cytometry in all ECs types starved in SFM 3

for 24 h (Fig. 2A): while the p-cMET/cMET double positive cells decreased in MGECs and control 4

ECs, they persisted in MMECs (18% vs 20% in a 1st diagnosed, 72% vs 80% in a refractory, and 5

70% vs 79% in a relapsed representative patients; Fig. 2A). Treatment of MMECs from refractory 6

and relapsed patients with anti-HGF (80 ng/µL) or anti-cMET (40 ng/µL) neutralizing Abs reduced 7

sizeably the p-cMET expression (Fig. 2B). Similar data were obtained in 1st diagnosed patients 8

(data not shown). 9

The evidence that MMECs unchange the p-cMET expression on starvation (i.e., in absence of 10

external stimulation), together with its inhibition by anti-HGF and anti-cMET Abs suggest that an 11

autocrine HGF/cMET pathway is operative in MMECs of active patients. 12

13

The HGF/cMET autocrine loop mediates MMECs migration and angiogenesis 14

We next investigated whether the HGF/cMET pathway regulates the MMECs angiogenic activities. 15

A 24 h treatment with anti-HGF and anti-cMET Abs in SFM did not modify neither MMECs 16

viability (Fig. 3A), nor apoptosis, nor proliferation (Supplementary Fig. S1A; PI=1). Data were 17

confirmed at 72 h (not shown). However, the spontaneous MMECs migration into the "wound" 18

lowered by 78% and 80% (as average) with anti-HGF or anti-cMET Abs, and by 82% with both 19

(migrated cells: 20 ± 3, 18 ± 2, and 16 ± 2 vs 88 ± 15 in untreated MMECs; P<0.001; Fig. 3B). 20

Similarly, in the Boyden microchambers, the MMECs migratory activity towards VEGF+FGF-2 21

(both 10 ng/mL) as chemoattractants was inhibited by 40%, 37%, and 45% (P<0.01; Fig. 3C, left 22

panel). The lack of differences between each single and combined treatments in absence of external 23

stimulation further confirms the existence of an autocrine HGF/cMET loop in MMECs. 24

MMECs adhesion and spreading were inhibited by 30% with the anti-cMET Ab (P<0.03; Fig. 3C, 25

middle and right panels) but not with anti-HGF Ab, perhaps because an intracrine secretion of HGF 26




12

stimulating the intracellular moiety of cMET (24) takes place in MMECs, and contributes to these 1

cell functions. 2

MMECs are able to spontaneously spread and form a closely knit capillary network when seeded on 3

Matrigel surface due to their overangiogenic phenotype (Fig. 3D) (16). The anti-HGF and anti-4

cMET Abs significantly inhibited the angiogenic network, since they reduced the mesh areas by 5

48% and 55%, respectively, and the vessel length by 47% and 57%, respectively, vs untreated cells. 6

When Abs were combined both areas and length were inhibited approximately at the same extent, 7

which further corroborates the existence of an autocrine HGF/cMET loop in MMECs. 8

Overall data suggest that HGF/cMET pathway regulates MMECs migratory activities, and that it is 9

implicated in the whole MM angiogenesis. 10

11

Effects of cMET inhibition by SU11274 in vitro 12

MMECs were treated with SU11274 0.1-10 µmol/L: no effect on cell viability, except for the 13

highest (toxic) dose (Fig. 4A), nor on cell apoptosis, nor proliferation (Supplementary Fig. S1B; 14

PI=1) was observed at 24 h. Similar data were obtained at 72 h (not shown). Of note, 0.5 and 1 15

µmol/L were able to decrease the p-cMET expression respectively by 23% and 40% 16

(Supplementary Fig. S2A). 17

Much in the same way as the Abs, SU11274 reduced dose dependently both spontaneous and 18

chemotactic migration: at 0.5 and 1 µmol/L, the former ("wound" assay; Fig. 4B) lowered 19

respectively by 60% and 87% (as average) vs untreated cells (migrated cells: 40 ± 7 and 14 ± 4 vs 20

90 ± 11; P<0.001); the latter by 36% and 70% (P<0.01; Fig. 4C, left panel). SU11274 impacted 21

dose dependently with MMECs attachment to and spread on fibronectin: at 1 µmol/L, these cell 22

activities were reduced respectively by 42% and 30% (P<0.01 and P<0.03; Fig. 4C, middle and 23

right panels). SU11274 was able to inhibit dose dependently the whole MMECs angiogenesis: 0.5 24

and 1 µmol/L reduced the mesh areas respectively by 58% and 80%; the vessel length by 61% and 25

83% vs untreatred cells (Supplementary Fig. S2B). 26




13

SU11274 enhances the antiangiogenic power of bortezomib and lenalidomide. In fact, SU11274 (1 1

µmol/L), bortezomib (7.5 nmol/L [20]), and lenalidomide (1.75 µmol/L [17]) gave an overlapping 2

antiangiogenic effect since inhibited the mesh areas by 48%, 42% and 45%, respectively; the vessel 3

length by 53%, 41% and 43% (Fig. 4D). When SU11274 was combined with bortezomib or 4

lenalidomide, it gave statistically significant antiangiogenesis: inhibition of mesh areas by 92% and 5

95%, respectively; of vessel length by 96% and 98% vs SU11274-treated cells (P<0.001; Fig. 4D). 6

7

cMET inhibition leads to a particular modulation of angiogenesis-related cytokines in 8

MMECs 9

To further study the activity of HGF/cMET pathway on MM angiogenesis, MMECs CM were 10

tested for 55 angiogenesis-related cytokines upon the SU11274 treatment (1 µmol/L for 24 h). The 11

drug was able to significantly modulate four cytokines (P<0.03 or better; Fig. 5A): three were 12

angiogenic, i.e., SERPIN E1 [serpin peptidase inhibitor, clade E (nexin, plasminogen activator 13

inhibitor type 1), member 1] (25), CXCL16 (chemokine ligand 16) (26), and MCP-1 (monocyte 14

chemotactic protein-1, or CCL2) (27), and were reduced respectively by 42%, 38%, and 32% (as 15

average); one was antiangiogenic, i.e., SERPIN F1 [serpin peptidase inhibitor, clade F (α2 16

antiplasmin, pigment epithelium derived factor), member 1] (28), and was increased by 30%. The 17

drug-modulation of these cytokines was also confirmed by real time RT-PCR analysis of SU11274-18

treated vs untreated MMECs (Supplementary Fig. S4A). 19

Data suggest that the HGF/cMET pathway is implicated in regulating the overangiogenic MMECs 20

profile. 21

22

SU11274 alone and in combination with anti-MM drugs changes the MMECs proteome 23

The MMECs proteome was studied following treatment with SU11274 alone or combined with 24

bortezomib or lenalidomide vs untreated cells (control). At least three 2-DE-gels were run per 25

sample, and analyzed by computer assisted spot matching (to identify spot variations), peptide 26




14

sequencing, and MS/MS followed by database searching. Fourteen proteins varied significantly (2-1

fold changes vs control) upon treatment with SU11274 alone (Fig. 5B) and in combination 2

(Supplementary Fig. S3): four angiogenic proteins (Supplementary Table S2), i.e., annexin A4 3

(ANXA4, #2), prohibitin (PHB, #3), peroxiredoxin-6 (PRDX6, #10), and annexin A2 (ANXA2, 4

#12), which govern cell shape and migration, were downregulated; while one protein, i.e., calpain 5

small subunit 1 (CPNS1, #1), which regulates cell senescence, was upregulated. A real time RT-6

PCR for these last five proteins showed a symmetric gene modulation following the cMET 7

inhibition (Supplementary Fig. S4B). 8

9

Antiangiogenic effect on MMECs in vivo 10

When CAMs were implanted with a gelatine sponge soaked with the MMECs CM, many newly-11

formed capillaries converging radially toward the sponge in a “spoked-wheel” pattern were seen 12

(vessel count = 27 ± 4 vs 8 ± 3 of physiological angiogenesis in serum-free medium; P<0.001; Fig. 13

6, panels a and b). In contrast, MMECs treated with SU11274 were poorly angiogenic (16 ± 2; 14

panel c), and even less with SU11274 plus bortezomib or lenalidomide in a statistically significant 15

fashion (7 ± 2 and 8 ± 3, respectively; P<0.001; Fig. 6, panels d and e). 16

17

DISCUSSION 18

Tumor angiogenesis is driven by several growth factors that activate receptor tyrosine kinases and 19

different signaling pathways in ECs (29). A key pathway is vascular endothelial growth factor 20

(VEGF)/VEGF receptors (VEGFRs), whose VEGF-A/VEGFR2 autocrine loop has been found in 21

ECs derived from BM of patients with active MM (MMECs) (30). Angiogenesis inhibitors 22

targeting this pathway, both alone and combined with chemotherapeutics, are a well defined 23

therapeutic option for cancer patients (31). So far, however, clinical trials have failed to show a 24

therapeutic power of VEGF-A/VEGFR2 inhibitors in MM (32). 25




15

Tentatively, we suggest that the HGF/cMET pathway may be one promising new target for 1

antiangiogenic management of MM patients. This pathway governes the tumor cell scattering, 2

hence it regulates cell motility and metastatic spread (33). HGF is released by mesenchyma-derived 3

cells, whereas cMET is expressed by several cell types, including vascular endothelial cells (34) and 4

accessory cells (pericytes) (35). Activation of cMET in tumors most often occurs through ligand 5

dependent autocrine or paracrine mechanisms. Either HGF is released from the surrounding stromal 6

cells, resulting in a constitutive paracrine cMET activation (11); or coexpression of HGF and cMET 7

leads to autocrine activation, as found in carcinomas, sarcomas, gliomas and B cell tumors (8, 9, 8

36). The HGF/cMET pathway is also involved in the MM pathogenesis: its paracrine and autocrine 9

activation have been found in both PCs and microenvironment cells (9, 10). 10

The HGF/cMET pathway plays a key role in angiogenesis, since it stimulates ECs both directly and 11

indirectly by enhancing the expression of the VEGF-A/VEGFR2 pathway and downregulating 12

thrombospondin 1, an angiogenesis inhibitor (7). Here, we show that the HGF/cMET pathway is 13

activated in MMECs of patients with active disease (at first diagnosis, and on refractory and relapse 14

phase), but not in ECs from MGUS patients (MGECs) and control (benign anemia) patients. The 15

significant increase of cMET phosphorylation in MMECs vs MGECs/control ECs suggests that this 16

pathway is constitutively activated in MM. Thus, parallel to the autocrine HGF/cMET loop found in 17

PCs (9, 10) an autocrine loop occurs in MMECs too, as confirmed by experiments with anti-HGF 18

and anti-cMET neutralizing Abs. Therefore much in the same way as the VEGF-A/VEGFR2 19

autocrine loop entails the overangiogenic phenotype of active MMECs vs norm-angiogenic MGECs 20

(30), the HGF/cMET autocrine loop may act as an additional angiogenesis amplifier for MMECs. 21

Worth of note is that VEGF synergizes with HGF in inducing angiogenesis (37). Treatment with the 22

neutralizing Abs to HGF and cMET interferes with MMECs spontaneous and chemo-induced 23

migration, while MMECs adhesion and spreading are not impacted by external blockade of HGF. 24

Perhaps an intracrine HGF secretion and stimulation of the cMET cytoplasmic moiety (24) may 25

occur in MMECs. An intracrine VEGF-A/VEGFR1 signaling has been shown in human primary 26




16

PCs (38). Plausibly, autocrine and intracrine HGF/cMET loops may be operative in MMECs, hence 1

the intracrine loop is sufficient to mediate cell adhesion and spreading. On the other hand, treatment 2

of cells with the anti-cMET Ab (Fig. 3C) or with SU11274 (Fig. 4C) is able to inhibit both 3

autocrine and intracrine loops, hence MMECs adhesion and spreading, which supports a role of 4

HGF/cMET pathway also in these cell functions. 5

We found that the cMET pathway is overactivated in drug-resistant MM cell lines as well as in PCs 6

from MM patients at relapse or on refractory phase (10). Rocci et al. (39) reported that the 7

overexpression of the cMET oncogene in PCs indicates poor prognosis. Overall, an altered 8

HGF/cMET pathway activation in MM PCs implies a less responsive disease. Accordingly, 9

targeting of the HGF/cMET pathway may disrupt the interactions between tumor PCs and their 10

microenvironment, which may significantly increase treatment efficacy in refractory disease (39). 11

Several compounds targeting cMET have been developed, and are now being tested in clinical 12

trials. Results of a phase I trial using a selective oral cMET inhibitor (ARQ197) have been reported 13

(40). Also, an anti-cMET antiboby (METMab) significantly improves progression-free survival in 14

patients with non-small-cell lung cancer with high cMET expression and receiving erlotinib, an 15

epidermal growth factor receptor inhibitor (41). Here, we tested SU11274, a novel cMET tyrosine 16

kinase inhibitor, and demonstrated its antiangiogenic activity on MMECs both singularly and in 17

combination with other two antiangiogenic/anti-MM drugs, i.e., bortezomib (20) and lenalidomide 18

(17), in a statistically significant fashion, as demonstrated in vitro (Matrigel assay) and in vivo 19

(CAM assay). SU11274 interferes with the MMECs angiogenic activities, such as spontaneous and 20

chemo-induced migration, adhesion, spreading, and whole angiogenesis, which are all partly 21

mediated by the HGF promigratory effects (9). It inhibits three angiogenic cytokines, i.e., SERPIN 22

E1 (25), CXCL16 (26) and MCP-1 (27) which are involved in adhesion, migration, chemotaxis and 23

homing of MM cells, and upregulates the antiangiogenic cytokine SERPIN F1 (28), which is 24

expressed by MMECs but not MGECs (42). 25




17

cMET inhibitor also downregulates angiogenic proteins, such as ANXA2 (43), ANXA4 (44), 1

PRDX6 (45) and PHB (46), and upregulates CPNS1, a protein favouring cell damage and 2

senescence (47). 3

In sum, SU11274 is able to exert a direct inhibitory effect on MMECs of patients on the active 4

phase, in whom the endothelial HGF/cMET autocrine loop is operative. This drug may be thus 5

envisaged as a possible antiangiogenic option for MM patients to be further tested in clinical trials 6

(48). 7

8

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14

LEGENDS 15

Figure 1 16

Expression of HGF and cMET in the ECs types. 17

A) HGF (left panel) and cMET (right panel) mRNA levels were analyzed by real time RT-PCR and 18

normalized to GAPDH. B) HGF levels were assessed by ELISA (left panel); p-cMET and cMET by 19

western blot and optical density (OD) of bands (right panel). Gene and protein expression fold 20

change levels in control ECs were arbitrarily set as 1. Data are means ± SD of MMECs from 21

patients at first diagnosis (n=9), on refractory phase to bortezomib- or lenalidomide-based therapies 22

(n=12) or at relapse after these therapies (n=11); of MGECs (n=24) and of control ECs (n=10). 23

*P<0.03; and **P<0.01 by Wilcoxon signed-rank test. C) FACS analysis shows percentages of p-24

cMET/cMET double positive ECs in the patients’ groups. D) Immunofluorescence for cMET (green 25

signal), p-cMET (red signal) and nuclei (blue signal) in ECs from representative MM, MGUS 26




23

patients, and control subjects. Merge (yellow signal) shows cytoplasmatic colocalization of cMET 1

and p-cMET. Left panels: merged pictures of cMET, p-cMET and nuclei; middle panels: merged 2

pictures of cMET and nuclei; right panels: merged pictures of p-cMET and nuclei, by an Axioplan-3

2 microscope. Original magnification ×63; Scale bar: 30 μm. 4

5

Figure 2 6

HGF/cMET autocrine loop in MMECs vs MGECs and control ECs. 7

A) FACS analysis on MMECs, MGECs and control ECs: percentages of double positive p-8

cMET/cMET cells at 24 h culture in complete or serum-free medium. B) Time and dose finding 9

effects of anti-HGF and anti-cMET neutralizing Abs on MMECs. A representative relapsed patient 10

is shown. 11

12

Figure 3 13

HGF/cMET autocrine loop mediates migration and angiogenesis of MMECs. 14

A) No effect of anti-HGF (80 ng/μL) and anti-cMET (40 ng/μL) neutralizing Ab on cell viability, 15

but B) inhibitory effects on the cell spontaneous migration into the “wound”. C) Inhibition of 16

chemotaxis (left panel) by the Abs. No effect with anti-HGF, but inhibition with anti-cMET on cell 17

adhesion (middle panel) and spreading (right panel). D) Matrigel angiogenesis assay: inhibition of 18

the whole angiogenesis by the Abs. Original magnification ×200; scale bar: 50 μm. Measurement of 19

mesh areas and vessel length by the EVOS image software: histograms are means ± SD of MMECs 20

from relapsed (n=8) or refractory (n=9) patients. *P<0.03; and **P<0.01 by Wilcoxon signed-rank 21

test. 22

23

Figure 4 24

Effects of the cMET inhibition by SU11274 and drug combinations on MMECs in vitro. 25




24

A) No effects of SU11274 increasing doses on cell viability except for the highest dose due to 1

toxicity. B) Dose dependent inhibition by SU11274 of the cell spontaneous migration into the 2

MMECs “wound”; and C) on the cell chemotaxis (left panel), adhesion (middle panel) and 3

spreading (right panel). D) Matrigel angiogenesis assay showing inhibitory effects of SU11274 and 4

even more by its combination with bortezomib or lenalidomide. Original magnification ×200; scale 5

bar: 50 μm. Measurement of mesh areas and vessel length by the EVOS image software showing 6

statistically significant decrease by the drug combinations: histograms are means ± SD of MMECs 7

from relapsed (n=8) or refractory (n=9) patients. *P<0.03; and **P<0.01 by Wilcoxon signed-rank 8

test. 9

10

Figure 5 11

SU11274 modifies MMECs angiogenesis-related cytokines and proteins. 12

A) The CM of SU11274-treated vs -untreated MMECs was tested by a cytokine angiogenesis assay. 13

Values from untreated cells were arbitrarily set as 0. Data are given as average of 3 independent 14

experiments. Significant downregulation of three angiogenic cytokines (SERPIN E1, CXCL16, 15

MCP-1), and upregulation of an antiangiogenic cytokine (SERPIN F1) are shown. *P<0.03 or 16

better; Wilcoxon signed-rank test. B) Proteome analysis of SU11274-treated vs untreated MMECs. 17

Silver-stained 2-DE gels of whole protein lysates from a representative refractory patient out of 2 18

refractory and 3 relapsed patients. Black and red squares indicate downregulated or upregulated 19

proteins, respectively. 20

21

Figure 6 22

SU11274 antiangiogenic effect in vivo. 23

CAMs were incubated with gelatine sponges loaded with serum-free medium (physiological 24

angiogenesis, panel a), with the CM of untreated MMECs (positive control, panel b) or treated with 25

SU11274 alone (panel c) or combined with bortezomib (panel d) or lenalidomide (panel e). A 26




25

statistically significant antiangiogenic effect by the drug combinations is shown (P<0.001). A 1

representative patient at relapse is shown. Original magnification ×50 on a stereomicroscope. 2




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