HGF and IGF-1 synergize with SDF-1α in promoting migration of myeloma cells by cooperative activation of p21-activated kinase.

Stromal-derived factor (SDF)-1α, insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF) are potent mediators of cell migration. We studied the effect of combinations of these cytokines on the migration of myeloma cells. When SDF-1α was combined with either HGF or IGF-1, we found a striking synergy in the cytokines' ability to guide cells across a transwell membrane. Between HGF and IGF-1 there was no cooperativity. However, the effects of HGF and IGF-1 were not redundant. HGF and SDF-1 caused concentration gradient-directed migration, as opposed to IGF-1, which apparently caused randomly directed cell movement. The SDF-1α-driven migration of JJN-3 cells, a myeloma cell line secreting large amounts of HGF, was reduced when JJN-3 cells were given an inhibitor of the HGF receptor, demonstrating a cooperative activity between autocrine HGF and exogenous SDF-1α. There was a clear positive correlation between the degree of cytokine-induced migration and phosphorylation of p21-activated kinase (PAK) both in primary myeloma cells and in cell lines including INA-6 and IH-1. Downregulation of PAK with small interfering RNA in INA-6 cells resulted in decreased cytokine-driven migration. This study shows synergy between SDF-1α and HGF/IGF-1 in inducing migration of myeloma cells, yet each cytokine has distinct properties in the way it regulates cell migration. These findings are likely to be of clinical relevance because multiple myeloma cells are located in an environment containing HGF and IGF-1 and are exposed to an SDF-1α gradient between the bone marrow and peripheral blood.

c-Met signaling promotes IL-6-induced myeloma cell proliferation.

OBJECTIVES: Hepatocyte growth factor (HGF) is a constituent of the myeloma microenvironment and is elevated in sera from myeloma patients compared to healthy individuals. Increased levels of serum HGF predict a poor prognosis. It has previously been shown by us and others HGF can act as a growth factor to myeloma cells in vitro although these effects have been moderate. We therefore wanted to investigate if HGF could influence the effects of interleukin (IL)-6. METHODS: Myeloma cell lines and primary samples were tested for the combined effects of IL-6 and HGF in inducing DNA synthesis and migration. Expression levels of c-Met protein were analysed by Western blotting and flow cytometry. Signaling pathways were examined by Western blotting using phosphospecific antibodies and a Ras-GTP pull down assay. RESULTS: HGF potentiated IL-6-induced growth in human myeloma cell lines and in purified primary myeloma cells. There was also cooperation between HGF and IL-6 in induction of migration. There seemed to be two explanations for this synergy. IL-6-treatment increased the expression of c-Met making cells HGF responsive, and IL-6 was dependent on c-Met signaling in activating both Ras and p44/42 MAPK by a mechanism involving the tyrosine phosphatase Shp2. CONCLUSIONS: The results indicate that besides from being a myeloma growth factor alone, HGF can also potentiate the effects of IL-6 in myeloma proliferation and migration. Thus, c-Met signaling could be a target for therapy of multiple myeloma.

Hepatocyte growth factor promotes migration of human myeloma cells.

Multiple myeloma is characterized by the accumulation and dissemination of malignant plasma cells in the bone marrow. Cell migration is thought to be important for these events. We studied migration in a Transwell two-chamber assay and tested the motogenic effect of various cytokines. In addition to insulin-like growth factor-1 and stromal cell-derived growth factor-1alpha, previously known as chemoattractants for myeloma cells, we identified hepatocyte growth factor as a potent attractant for myeloma cells. Hepatocyte growth 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.

Tyrosine kinase inhibitors alter adhesivity of prostatic cancer cells to extracellular matrix components.

Tyrosine kinase inhibitors (TKIs) are thought to have potential as a new generation of anti-cancer drugs. Since invasiveness, the main characteristic of malignant behaviour, is believed to depend on altered cell-matrix interactions, we investigated the effect of two potent TKIs, genistein and tyrphostin AG-1478, on the interaction of prostate cancer cells with extracellular matrix components. PC-3 and DU-145 cells were treated with various concentrations of genistein and tyrphostin AG-1478. Adhesion to extracellular matrix was assayed using fluorescence-labelled cells seeded on collagen type I, collagen type IV, fibronectin, laminin and vitronectin. The expression levels of integrin beta1, alpha2, alpha3 and alpha5 subunits were measured using flow cytometry of cells labelled with monoclonal murine antibodies. Genistein treatment reduced the ability of both cell lines to adhere to the matrix proteins tested. This effect was more pronounced for PC-3 cells than for DU-145 cells. Genistein treatment decreased the expression of beta1 integrins by 40% in PC-3 cells and 22% in DU-145. AG-1478 treatment slightly reduced the ability of DU-145 cells to adhere, but did not decrease PC-3 cell adhesion. Nevertheless, expression levels were reduced for most integrins tested, except the expression of alpha-5, for which no significant effect was measured. Our results point to a possible role of TKIs as suppressors of prostate carcinoma cell adhesion to extracellular matrix components, by acting as inhibitors of integrin expression.

Human myeloma cells adhere to fibronectin in response to hepatocyte growth factor.

BACKGROUND AND OBJECTIVES: Multiple myeloma is characterized by an accumulation of malignant plasma cells in the bone marrow. Inside the bone marrow, adhesion of myeloma cells to extracellular matrix proteins such as fibronectin may promote cell survival and induce drug resistance. In this work we examined the effect of hepatocyte growth factor (HGF) on the adhesion of myeloma cells and the signaling pathways involved. DESIGN AND METHODS: Cell adhesion experiments were performed with the human myeloma cell line INA-6 and primary myeloma cells. The HGF signaling pathway was studied in INA-6 cells with the use of antibodies against VLA-4 integrin, and with inhibitors of various intracellular signaling molecules. RESULTS: We found that HGF stimulated adhesion of myeloma cells to fibronectin. This event was dependent on the alpha4 and beta1 integrin subunits (VLA-4), but HGF did not increase the expression of integrins on the cell surface. Our findings suggest that HGF promotes myeloma cells to adhere via activation of the phosphatidylinositol 3-kinase (PI3K) pathway independently of AKT, but possibly through the involvement of nuclear factor kappa B (NF-kappaB). INA-6 cells adhered to fibronectin after stimulation by insulin-like growth factor or stromal cell-derived factor 1alpha, but this adhesion was less dependent on PI3K than HGF-mediated adhesion. INTERPRETATION AND CONCLUSIONS: his work points to HGF as a pro-adhesive factor in cell adherence to the bone marrow matrix protein fibronectin, an event known to promote cancer cell survival and drug resistance. Inhibiting HGF, its receptor c-Met or the VLA-4 integrin may be beneficial to the myeloma patient.

A selective c-met inhibitor blocks an autocrine hepatocyte growth factor growth loop in ANBL-6 cells and prevents migration and adhesion of myeloma cells.

PURPOSE: We wanted to examine the role of the hepatocyte growth factor (HGF) receptor c-Met in multiple myeloma by applying a novel selective small molecule tyrosine kinase inhibitor, PHA-665752, directed against the receptor. EXPERIMENTAL DESIGN: Four biological sequels of HGF related to multiple myeloma were studied: (1) proliferation of myeloma cells, (2) secretion of interleukin-11 from osteogenic cells, (3) migration of myeloma cells, and (4) adhesion of myeloma cells to fibronectin. We also examined effects of the c-Met inhibitor on intracellular signaling pathways in myeloma cells. RESULTS: PHA-665752 effectively blocked the biological responses to HGF in all assays, with 50% inhibition at 5 to 15 nmol/L concentration and complete inhibition at around 100 nmol/L. PHA-665752 inhibited phosphorylation of several tyrosine residues in c-Met (Tyr(1003), Tyr(1230/1234/1235), and Tyr(1349)), blocked HGF-mediated activation of Akt and p44/42 mitogen-activated protein kinase, and prevented the adaptor molecule Gab1 from complexing with c-Met. In the HGF-producing myeloma cell line ANBL-6, PHA-665752 revealed an autocrine HGF-c-Met-mediated growth loop. The inhibitor also blocked proliferation of purified primary myeloma cells, suggesting that autocrine HGF-c-Met-driven growth loops are important for progression of multiple myeloma. CONCLUSIONS: Collectively, these findings support the role of c-Met and HGF in the proliferation, migration, and adhesion of myeloma cells and identify c-Met kinase as a therapeutic target for treatment of patients with multiple myeloma.

Bone morphogenetic protein-5, -6 and -7 inhibit growth and induce apoptosis in human myeloma cells.

Previously, bone morphogenetic protein (BMP)-2 and -4 have been shown to inhibit proliferation and induce apoptosis in human myeloma cells. BMP-2 and -4 belong to a subgroup of BMPs using the BMP receptors Alk-3 or -6. In this study, we examined the effects on human myeloma cells of BMP-6 and -7, members of a different BMP subgroup, which mainly utilize Alk-2 as their receptor. All cell lines examined expressed mRNA for the BMP-6 and -7 receptor Alk-2. We did not detect transcripts for the BMP-2 and -4 receptors Alk-3 or Alk-6 in INA-6 and RPMI-8226 cells by RT-PCR. Accordingly, the intracellular signalling molecules Smad-1, -5 and -8 were not phosphorylated by BMP-4 in INA-6 and RPMI-8226 cells. The expression patterns of various BMP receptors in the myeloma cell lines explained the differences in responses to the various BMPs. Alk-2-expressing cell lines responded with growth inhibition and apoptosis to BMP-6 and -7, whereas cell lines lacking both Alk-3 and -6 were resistant to BMP-4. Soluble Alk-3 and -6 were able to neutralize the BMP-4 effects in BMP-4-responsive cell lines. All BMPs reduced viability in more than 70% of purified primary myeloma cell samples. BMPs have intriguing antitumor effects in vitro. Importantly, myeloma cells not responsive to BMP-2 and -4 may still be sensitive to BMP-6 or -7. It is possible that therapeutic use of BMP or BMP analogues could have an impact on both myeloma bone disease and myeloma cell growth.