Hepatocyte growth factor promotes migration of human myeloma cells

Multiple myeloma is characterized by the accumulation and disseminationof malignant plasma cells in the bone marrow. Cell migrationis thought to be important for these events. We studied migrationin a Transwell two-chamber assay and tested the motogenic effectof various cytokines. In addition to insulin-like growth factor-1and stromal cell-derived growth factor-1 ${alpha}$ , previously known aschemoattractants for myeloma cells, we identified hepatocytegrowth factor as a potent attractant for myeloma cells. Hepatocytegrowth factor-mediated migration was dependent on phosphatidylinositol-3-kinase,involved the MAPK/Erk signaling cascade and VLA-4 integrins,but did not involve Akt, mTOR or G proteins.

Key words: myeloma, cell migration, hepatocyte growth factor, stromal cell-derived growth factor-1.

Introduction

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Introduction

Multiple myeloma (MM) is an incurable malignant clonal plasmacell disorder.¹ It is believed that MM precursor cells migratefrom blood vessels into the BM where they adhere to stromalcells and matrix proteins, receiving signals supporting thelong life of MM cells. As the disease progresses, the MM cellsdisseminate by migration through endothelial barriers.¹

Several cytokines that are involved in the pathogenesis of MMdisease, such as IGF-1, tumor necrosis factor (TNF)- ${alpha}$ , vascularendothelial growth factor (VEGF), fibroblast growth factor (FGF)-2and SDF-1 ${alpha}$ , promote MM cell migration.^2–6

The 68 kDa glycoprotein HGF, produced by stromal cells in theBM, is a pleiotropic cytokine with mitogenic, motogenic andmorphogenic properties.⁷ The HGF receptor c-Met is a tyrosinekinase type receptor. In hematologic cancer, HGF/c-Met seemto be of particular importance for MM.^8,9 The concentrationof HGF is significantly higher in serum¹⁰ and in the bone marrowplasma of myeloma patients than in control samples from healthysubjects. Besides, HGF is a negative prognostic factor for MMpatients.^11,12 We showed that HGF promoted adhesion of MM cellsto the matrix protein fibronectin, which increased cell proliferation.¹³HGF is also reported to facilitate invasiveness of MM cells,¹⁴and to inhibit bone formation in multiple myeloma.¹⁵

We studied chemotaxis of MM cells, and found myeloma cells tobe attracted to HGF in concentrations known to be present inthe BM of myeloma patients.

Design and Methods

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Design and Methods

Cytokines, antibodies and other reagents
HGF was purified in our own laboratory.¹⁶ NK1 and tumor necrosisfactor (TNF) were from Genentech Inc. (San Francisco, CA, USA).Recombinant human FGF2, IGF-1, interleukin (IL)-15 and VEGFwere from R&D Systems Inc. (Minneapolis, MN, USA), IL-6was from Biosource (Camarillo, CA, USA) and SDF-1 ${alpha}$ from Peprotech(London, UK). The neutralizing antibody against the VLA-4 integrinsubunit ${alpha}$ 4 and irrelevant IgGs came from Becton and Dickinson(San José, CA, USA). Antibodies against human HGF andc-Met were from R&D Systems Inc. (Minneapolis, MN, USA).The c-Met inhibitor PHA-665752 was a gift from Dr. James Christensen,Pfizer (San Diego, CA, USA). Pertussis toxin, rapamycin andwortmannin were from Sigma-Aldrich (St. Louis, MO, USA). LY294002,PD98059 and U0126 were from LC Laboratories (San Diego, CA,USA). The JAK2/STAT3 inhibitor AG 490 and the Akt inhibitorSH-5 were from Calbiochem (CNbiosciences, Inc., La Jolla, CA,USA). Compounds were diluted to final concentrations in RPMI-1640(Gibco, Paisley, UK) supplemented with l-glutamine (2 mM) andgentamicin (40 µg/mL).

Cell culture conditions
We used the IL-6-dependent human myeloma cell lines INA-6 andANBL-6, and myeloma cells from 11 randomly selected patientsadmitted to the hematology department of St. Olavs Hospital,Trondheim. Cells were harvested and cell lines were grown aspreviously described.¹³ The study was approved by the regionalethics commitee and patients gave their informed consent.

Migration assay
INA-6 and ANBL-6 cells were washed in Hanks’ balancedsalt solution (HBSS), and resuspended in RPMI-1640 with 0.1%bovine serum albumin (BSA) (Sigma-Aldrich) and IL-6 (0.1 ng/mL)(referred to as RPMI-medium). Cells were seeded (2x10⁵ cellsin 100 µL RPMI-medium) in the upper compartments of polycarbonateTranswell two-chamber migration plates (pore size: 5 µm)(Costar, Corning, NY, USA). The volume of RPMI-medium in thelower compartments was 600 µl. All experiments were performedin duplicates and repeated at least three times. After 22 h,at 37°C and 5% CO₂, the number of cells that had migratedthrough the membrane to the lower chamber was determined bya Coulter Counter Z1 (Beckman Coulter, Fullerton, CA, USA)

Statistics
Statistical significance was determined using a two-tailed,unpaired Student’s t-test. The minimal level of significancewas p=0.05.

Results and Discussion

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Results and Discussion

HGF promoted myeloma cell migration
The dissemination of malignant plasma cells throughout the bonemarrow is one of the hallmarks of multiple myeloma. In our studyselected cytokines known to induce biological responses in MMcells were assessed for their influence on MM cell migration.Compared with non-stimulated cell locomotion, migration wasincreased 5-fold by HGF or SDF-1 ${alpha}$ , and 2 to 3-fold by IGF-1.FGF2, IL-15, TNF or VEGF gave no significant increase in INA-6cell migration above control level (Figure 1A).

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Figure 1. INA-6 cells (A) or primary MM cells (B) were seeded in the upper compartment of a two-chamber Transwell migration assay. To the bottom compartment was added RPMI-medium with or without FGF2 (10 ng/mL), HGF (150 ng/mL), IGF-1 (100 ng/mL), IL-15 (20 ng/mL), SDF-1 ${alpha}$ (75 ng/mL), TNF (20 ng/mL) or VEGF (100 ng/mL). After 22 hrs. at 37°C, cells in the bottom compartments were counted and percentage of migrated cells calculated. Error bars represent +1 standard deviation (SD) of three repeated counts in two independent measurements. A is representative for three similar experiments. B represents 1 out of 5 primary cell samples. Migration increased significantly when cells were subjected to specific cytokines, according to an unpaired Student’s two-tailed test, *(p<0.01).

HGF and SDF-1 ${alpha}$ also induced migration of ANBL-6 cells significantlyabove the control level (Online Supplementary Figure S1A). Furthermore,HGF significantly promoted cell migration in 5 out of 11 patientMM cell samples tested (represented by Figure 1B), showing theclinical relevance of HGF-mediated migration. Cells migratedin response to HGF in a dose-dependent manner (Online SupplementaryFigure S1B). The 50% effective dose (ED₅₀) was calculated to38 ng/mL, which is equal to or lower than the concentrationof HGF found in the BM plasma of 30% of MM patients.¹² Migrationreached its maximum at 100–150 ng/mL and decreased atconcentrations above 200 ng/mL. In further experiments, 150ng/mL of HGF was used. Migration increased only as long as apositive HGF gradient was maintained (data not shown). We confirmedSDF-1 ${alpha}$ to be a strong promoter of myeloma cell migration. However,in cell lines, we found HGF to be an equally strong chemoattractantfor MM cells. Cell migration was an active process enablingcells to migrate through pores (5 µm) half the size ofa cell diameter (11 µm).

HGF stimulated cell migration through c-Met
HGF stimulated migration through its high-affinity receptorc-Met, since migration decreased almost to control level whencells were given antibodies against HGF or against c-Met, orwhen given the c-Met inhibitor PHA665752 at concentrations knownto fully inhibit c-Met phosphorylation. NK1, a natural splicevariant of HGF containing the HGF receptor-binding sites,⁷ waspreviously found to have varying effects on cell behaviour.¹⁷In our study, NK1 did not promote cell migration. On the contrary,it partly (42%) inhibited HGF-induced migration (Figure 2A).

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Figure 2. (A) Neutralizing antibodies against HGF (10 µg/mL) or c-Met (2 µg/mL) inhibited INA-6 cell migration to control levels. Isotype control antibody (10 µg/mL) was used. The c-Met inhibitor PHA 665752 (50 nM) and the isotype variant of HGF, NK1 (100 ng/mL), reduced HGF-mediated cell migration. (B) Neutralizing antibody against the VLA4-integrin ${alpha}$ 4 sub-unit (0.1 µg/mL) reduced HGF-mediated cell migration by 40%. The PI3K inhibitors Ly2940002 (3 µM) and wortmannin (50 nM) reduced migration to control levels. The Akt inhibitor SH-5 (10 µM) did not influence migration. (C) The inhibitors of MAPK, PD98059 (5 µM) and U0126 (5 µM) reduced HGF-mediated migration more than 40%, while HGF-mediated migration was not influenced by rapamycin (5 ng/mL) an inhibitor of mTOR, AG490 (5 µM) an inhibitor of JAK/STAT, or an isotype control IgG (0.1 µg/mL). The error bars represent ± 1 SD of three repeated counts of two independent measurements. The figures show one representative out of three similar experiments.

HGF-mediated cell migration was dependent on the intracellular signaling molecules PI-3K and involved the MAPK/Erk signaling pathway and the VLA-4 integrin
In order to delineate signaling pathways involved in HGF-stimulatedmigration, we subjected INA-6 cells to selected inhibitors ofintracellular signaling molecules and to antibodies againstthe VLA-4 integrin. Lipid products of PI3K are known to activatethe serin/threonine kinase Akt,¹⁸ and mTOR is a signaling mediatordownstream of Akt. We have previously shown that HGF activatesAkt, and have thereby indirectly shown PI3K activation in INA-6cells.¹³ The PI3K inhibitors wortmannin and LY2940002 blockedHGF-mediated migration completely (Figure 2B) showing that HGF-mediatedmigration was fully dependent on PI3K. HGF has previously beenshown to activate the MAPK/Erk pathway in INA-6 cells.¹³ PD98059and U0126, in concentrations that fully inhibit phosphorylationof p44/p42 MAPK in INA-6 cells,¹³ decreased HGF-mediated migrationby 42% and 47%, respectively (Figure 2C). This suggests thatcytokine-induced migration, in contrast to HGF-induced celladhesion,¹³ does involve the MAPK/Erk signaling pathway in MMcells. Here MM cells could be similar to carcinoma cells, inwhich MAP kinases are known to increase the activation of actin/myosinmotors by enhancing the activity of myosin light chain kinasesand thereby promoting motility.¹⁹

We have previously found VLA-4 to be an abundant integrin onMM cells and shown that HGF increases VLA-4-dependent adhesionof MM cells to fibronectin.¹³ Use of neutralizing antibodiesagainst the integrin subunit ${alpha}$ 4 decreased migration by 47% (Figure 2B).These data indicate that adhesion was an important part of migrationand that cell migration, not surprisingly, is a complex eventin which additional adhesion molecules, besides VLA-4, may participate.

We found that the Akt inhibitor SH-5, the mTOR inhibitor rapamycin,as well as the Jak2/STAT3 inhibitor AG490 did not influenceHGF-mediated migration (Figure 2B and C). The efficacy of SH-5,AG490 and rapamycin was proven in a methyl-3H-thymidine proliferationassay (Online Supplementary text) where all inhibitors, at concentrationsused in our experiments, decreased INA-6 cell proliferation(Online Supplementary Figure S2). The efficacy of PD98059 andU0126 has previously been tested elsewhere.¹³

All inhibitors used in our experiments were tested for theirinfluence on cell viability after 22 h using an Apoptest-FITCkit (Online Supplementary text). No significant difference inviability was seen in treated compared with non-treated cellsat concentrations of inhibitors that were effective in the migrationassay (data not shown).

In some carcinoma cell lines, c-Met can be transactivated byligand binding to G-protein-coupled receptors (GPCR).²⁰ Pertussistoxin, an inhibitor of GPCR, did not influence HGF-stimulatedINA-6 cell migration, while it decreased SDF-1 ${alpha}$ -stimulated migration(Online Supplementary Figure S3). This indicates that HGF-mediatedmigration does not signal through GPCRs in our cells.

Taken together, our study shows that HGF-mediated migrationwas dependent on PI3K, it involved the MAPK/Erk-pathway andthe integrin VLA-4, but HGF-mediated migration did not involveJAK/STAT, Akt or its downstream effector mTOR.

In conclusion, HGF, which is a cytokine known to play a rolein MM biology, is here shown to be a promoter of MM cell migrationwith potency similar to that of SDF-1 ${alpha}$ . HGF may contribute tothe dissemination of myeloma cells within the patient’sbone marrow and as such could be an attractive target for therapy.

Acknowledgments

we thank Berit Størdal, Hanne Hella and Toril Holienfor their excellent technical help

Footnotes

Funding: this work is supported by grants from The NorwegianCancer Society, The Cancer Fund of St.Olavs Hospital, FamilienBlix’ fond, Rakel og Otto Kr. Bruuns legat and The ResearchCouncil of Norway.

The online version of this article contains a supplemental appendix.

Authorship and Disclosures

RUH: designed research, performed experiments and wrote thepaper. UMF: performed experiments and contributed to the writingof the paper. VB, TBR and HH: performed experiments. AW: collectedpatient samples. AS: advised throughout the work and discussions.MB: contributed in design of the research and wrote the finalversion of the manuscript

The authors reported no potential conflicts of interest.

Received for publication June 21, 2007. Revision received October 30, 2007. Accepted for publication December 21, 2007.

References

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