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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., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Aims: Interaction with the bone marrow microenvironment is important for homing and survival of myeloma cells. One cytokine involved in this process is hepatocyte growth factor (HGF). HGF, by binding to the receptor tyrosine kinase c-Met, mediates a broad range of tumour progression activities. Our aims were to investigate whether HGF and c-Met are present in bone marrow and extramedullary tumours from patients with monoclonal plasma cell disease, and whether c-Met is activated., Methods and Results: Expression of HGF, c-Met and phospho-c-Met was studied by immunohistochemistry in biopsies from 80 patients with monoclonal plasma cell disease. Cytoplasmic staining for HGF in plasma cells was demonstrated in 58 of 68 biopsies from multiple myeloma patients (85%), but also in biopsies from nine of 10 healthy individuals. Membranous staining for c-Met was found in 25 of 63 multiple myeloma patients (40%) and in none of 10 healthy individuals. Membranous staining for phospho-c-Met was found in biopsies from 15 of 21 c-Met-positive myeloma patients (71%)., Conclusions: Our data point to c-Met expression as one of the factors that distinguishes normal from malignant plasma cells, and indicate that the HGF/c-Met system is activated in multiple myeloma patients., (© 2012 Blackwell Publishing Limited.)

Objectives: The receptor tyrosine kinase c-Met and its ligand, hepatocyte growth factor (HGF), play key roles in tumour genesis and metastasis and contribute in multiple myeloma pathogenesis. Substantial data support that a soluble extracellular fragment of c-Met may function as a decoy receptor that downregulates the biological effects of HGF and c-Met. We examined serum levels of soluble c-Met in patients with myeloma and healthy individuals and investigated a possible relationship with clinical disease parameters and survival., Methods: The concentration of c-Met and HGF was measured by enzyme-linked immunosorbent assay in serum (n=49) and bone marrow plasma (n=16) from patients with multiple myeloma and in serum from healthy controls (n=26)., Results: The median serum concentration of soluble c-Met was 186 ng/mL (range 22-562) in patients with multiple myeloma and 189 ng/mL (range 124-397) in healthy individuals. There was a significant negative correlation between serum c-Met levels and disease stage, bone marrow plasma cell percentage and serum concentration of M-protein., Conclusion: We have for the first time examined the concentration of soluble c-Met in serum from patients with myeloma and found equal median levels in patients with myeloma as a group and healthy individuals. Still, serum levels of soluble c-Met correlated negatively with parameters of disease burden in patients with myeloma. We suggest that a possible role for the c-Met ectodomain as a negative regulator of HGF/c-Met activity should be examined in multiple myeloma., (© 2011 John Wiley & Sons A/S.)

Background: BCL3 is a putative oncogene encoding for a protein belonging to the inhibitory kappaB-family. We experienced that this putative oncogene was a common target gene for growth-promoting cytokines in myeloma cell lines., Methods: Gene expression of BCL3 was studied in 351 newly diagnosed myeloma patients, 12 patients with smouldering myeloma, 44 patients with monoclonal gammopathy of undetermined significance and 22 healthy individuals. Smaller material of samples was included for mRNA detection by RT-PCR, protein detection by Western blot and immunohistochemistry, and for cytogenetic studies. A total of eight different myeloma cell lines were studied., Results: Bcl-3 was induced in myeloma cell lines by interleukin (IL)-6, IL-21, IL-15, tumor necrosis factor-alpha and IGF-1, and its upregulation was associated with increased proliferation of the cells. In a population of 351 patients, expression levels of BCL3 above 75th percentile were associated with shorter 5-yr survival. When this patient population was divided into subgroups based on molecular classification, BCL3 was significantly increased in a poor risk subgroup characterized by overexpression of cell cycle and proliferation related genes. Intracellular localization of Bcl-3 was dependent on type of stimulus given to the cell., Conclusion: BCL3 is a common target gene for several growth-promoting cytokines in myeloma cells and high expression of BCL3 at the time of diagnosis is associated with poor prognosis of patients with multiple myeloma (MM). These data may indicate a potential oncogenic role for Bcl-3 in MM.

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.

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.

Multiple myeloma (MM) is characterized by accumulation and dissemination of malignant plasma cells (PCs) in the bone marrow (BM). Gene expression profiling of 2 MM cell lines (OH-2 and IH-1) indicated that expression of PRL-3, a metastasis-associated tyrosine phosphatase, was induced by several mitogenic cytokines. Cytokine-driven PRL-3 expression could be shown in several myeloma cell lines at both the mRNA and protein levels. There was significantly higher expression of the PRL-3 gene in PCs from patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), and myeloma than in PCs from healthy persons. Among 7 MM subgroups identified by unsupervised hierarchical cluster analysis, PRL-3 gene expression was significantly higher in the 3 groups denoted as "proliferation," "low bone disease," and "MMSET/FGFR3." PRL-3 protein was detected in 18 of 20 BM biopsies from patients with MM. Silencing of the PRL-3 gene by siRNA reduced cell migration in the MM cell line INA-6, but had no detectable effect on proliferation and cell-cycle phase distribution of the cells. In conclusion, PRL-3 is a gene product specifically expressed in malignant plasma cells and may have a role in migration of these cells.

The bone disease in multiple myeloma is caused by an uncoupling of bone formation from bone resorption. A key difference between patients with and patients without osteolytic lesion is that the latter have fewer and less active osteoblasts. Hepatocyte growth factor (HGF) is often produced by myeloma cells and is found at high concentrations in the bone marrow of patients with multiple myeloma. Here we show that HGF inhibited bone morphogenetic protein (BMP)-induced in vitro osteoblastogenesis. Thus, HGF inhibited BMP-induced expression of alkaline phosphatase in human mesenchymal stem cells (hMSCs) and the murine myoid cell line C2C12, as well as mineralization by hMSCs. Furthermore, the expression of the osteoblast-specific transcription factors Runx2 and Osterix was reduced by HGF treatment. HGF promoted proliferation of hMSCs, and the BMP-induced halt in proliferation was overridden by HGF, keeping the cells in a proliferative, undifferentiating state. BMP-induced nuclear translocation of receptor-activated Smads was inhibited by HGF, providing a possible explanation of how HGF inhibits BMP signaling. The in vitro data were supported by the observation of a negative correlation between HGF and a marker of osteoblast activity, bone-specific alkaline phosphatase (rho = -0.45, P = .008), in sera from 34 patients with myeloma. These observations suggest that HGF inhibits bone formation in multiple myeloma.