Randi Utne Holt

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.

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 (Tyr1003, Tyr1230/1234/1235, and Tyr1349), 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.

Serum/glucocorticoid-regulated kinase 1 (SGK1) is a prominent target gene of the transcriptional response to cytokines in multiple myeloma and supports the growth of myeloma cells.

Multiple myeloma (MM) is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We determined the changes in gene expression induced by interleukin (IL)-6, tumor necrosis factor-α, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase serum/glucocorticoid-regulated kinase 1 (SGK1), which is a down-stream effector of PI3-kinase. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, small hairpin RNA (shRNA)-mediated knock-down of STAT3 reduced basal and induced SGK1 levels. Furthermore, downregulation of SGK1 by shRNAs resulted in decreased proliferation of myeloma cell lines and reduced cell numbers. On the molecular level, this was reflected by the induction of cell cycle inhibitory genes, for example, CDKNA1/p21, whereas positively acting factors such as CDK6 and RBL2/p130 were downregulated. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their malignant growth.

Hepatocyte growth factor promotes migration of human myeloma cells

This study demonstrates that myeloma cells can be attracted to hepatocyte growth factor in concentrations known to be present in the bone marrow of patients with 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-1α, 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.

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.

Elevated serum concentrations of activated hepatocyte growth factor activator in patients with multiple myeloma

Objectives:  Hepatocyte growth factor (HGF) is a potential key factor in multiple myeloma. Conversion of pro‐HGF to its active form is a critical limiting step for its biological effects. We aimed to examine the levels of the most potent activator, the hepatocyte growth factor activator (HGFA), in serum and bone marrow plasma of patients with multiple myeloma.

Soluble c-Met in serum of patients with multiple myeloma: correlation with clinical parameters

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.

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 β1, α2, α3 and α5 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 β1 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 α‐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.

Mn2+ regulates myeloma cell adhesion differently than the proadhesive cytokines HGF, IGF‐1, and SDF‐1α

Adhesion of multiple myeloma (MM) cells in the bone marrow (BM) is important for the growth and survival of the myeloma cells. Very late antigen‐4 (VLA‐4) is one of the main adhesion receptors that mediate MM cell binding to fibronectin (FN). In this study we have examined the effect of divalent cations on adhesion of MM cells to FN, and compared this type of adhesion with the adhesion induced by the cytokines HGF, IGF‐1 and SDF‐1α. Mn2+ induced adhesion in all cell lines tested. Cytokine‐ and Mn2+‐induced VLA‐4‐mediated adhesion were different in many respects, including binding specificity, adhesion kinetics and the activation state of VLA‐4. To study a potential role of divalent cations in vivo, we measured the concentrations of divalent cations in BM plasma from 14 MM patients. We also found that Mn2+‐mediated adhesion to FN activated the MAPK pathway, indicating that the interaction of MM‐cells with FN mediated by Mn2+ could play a critical role for growth and proliferation. In conclusion, this study shows a potential important role of divalent cations in MM cell biology and supports earlier studies pointing to activated VLA‐4 as a key for homing of MM cells to the BM.