Crucial role of phosphatase CD45 in determining signaling and proliferation of human myeloma cells

In multiple myeloma, a large number of growth factors (IL-6, IGF-1, FGF, HGF and HB-EGF) are involved in promoting myeloma cell growth. In the present study, a serum-free, cytokine-free, collagen-based assay, which does not allow the generation of spontaneous myeloma colonies, was used to identify the clonogenic growth factors for fourteen myeloma cell lines. IL-6 is the only clonogenic factor able to stimulate both CD45+ and CD45- myeloma cell lines, generating myeloma colonies from 10 out of 14 myeloma cell lines. Using a pharmacological Erk inhibitor, we show that the Erk/MAPK pathway is involved in IL-6-induced clonogenicity of CD45+, but not CD45- myeloma cell lines. In contrast to IL-6, the other growth factors (IGF-1, FGF, HGF and HB-EGF) stimulate only some myeloma cell lines, but always CD45-, and less effectively than IL-6. Among them, IGF-1 is the most potent, generating myeloma colonies from five out of eight CD45- myeloma cell lines. Finally, the capacity of IGF-1 and FGF to stimulate the clonogenicity of CD45- myeloma cells correlates with their ability to stimulate the Erk/MAPK pathway. We conclude that CD45 expression plays a crucial role in determining signaling and proliferation of human myeloma cell responses to IL-6, IGF-1 and other growth factors. The poor outcome of CD45- myeloma patients could be related to the capacity of CD45-myeloma cells to take advantage of multiple growth factors.     

IFN-alpha induces autocrine production of IL-6 in myeloma cell lines.

IL-6 is a major tumor growth factor in human multiple myeloma. Myeloma cell lines, which have the same phenotypic characteristics and Ig gene rearrangements as the original fresh myeloma cells and whose growth is strictly dependent on exogenous IL-6 similar to fresh myeloma cells, have been reproducibly established. We show here that IFN-alpha stimulated the growth of five of six of these human myeloma cell lines by inducing an autocrine production of IL-6 in myeloma cells. Indeed, IFN-alpha induced IL-6 mRNA accumulation and IL-6 production in myeloma cells and the IFN-alpha-induced growth of these cells was inhibited by anti-IL-6 mAb. Moreover, IFN-alpha made possible the rapid emergence of autonomously growing myeloma cell sublines, which produced IL-6 as an autocrine growth factor. As IFN-alpha has a potential therapeutical interest for multiple myeloma, the present study opens up new directions for studying its effects on the myeloma clone in vivo.     

Up-regulation of interleukin (IL)-6 receptor gene expression in vitro and in vivo in IL-6 deprived myeloma cells.

Myeloma cells absolutely require interleukin-6 (IL-6) for growing in vivo in patients with multiple myeloma and exogenous IL-6-dependent myeloma cell lines have been reproducibly obtained. In this study we show a dramatic up-regulation of the IL-6 receptor (gp80 chain) gene expression in myeloma cell lines following the removal of exogenous IL-6. Such a regulation was also known to occur in IL-6-deprived myeloma cells in vivo in three patients who were treated with optimal doses of anti-IL-6 monoclonal antibodies. The direct effect of IL-6 on IL-6 receptor gene expression in myeloma cells was further confirmed by adding IL-6 to an autonomously growing myeloma cell line.     

Development of new adherent mutant from human myeloma-derived cell line: in vitro model of anaplastic transformation of myeloma

Anaplastic myeloma is a rare but distinct, biologically aggressive variant of myeloma which usually results from dedifferentiation or anaplastic transformation of the myeloma cells. The molecular mechanisms that determine the biologic behavior of anaplastic myeloma and effective treatment modalities have not been well known due to lack of in vitro models. In the present study, we have developed an anaplastically transformed mutant from a human myeloma-derived cell line. In the process of long-term culture of the myeloma-derived IM-9 cell line in low serum and nutrient conditions, an adherent mutant line was developed and named IM-9/AD. This mutant cell line displayed several characteristics resembling anaplastic myeloma such as: 1, large cells with large vesicular nucleus and prominent nucleolus, multinuclearity and high mitotic figures; 2, loss of leukocyte-associated antigens; and 3, higher tumorigenecity in scid mice than its parental cell line. This newly developed mutant cell line may serve as a readily available in vitro model to investigate the biology of anaplastic myeloma.     

Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-alpha and insulin-like growth factor 1

As survival regulation is a key process in multiple myeloma biology, we have studied the Bcl-2 family proteins that can be regulated by three myeloma cell survival factors: interleukin-6 (IL-6), interferon-alpha (IFN-alpha) and insulin-like growth factor (IGF-1). Eleven myeloma cell lines, whose survival and proliferation are dependent on addition of IL-6, variably expressed 10 anti-apoptotic or pro-apoptotic proteins of the Bcl-2-family. When myeloma cells from four cell lines were IL-6 starved and activated with IL-6 or IFN-alpha, we observed that only Mcl-1 expression was up-regulated with myeloma cell survival induction. Nor was obvious regulation of these 10 pro-apoptotic or anti-apoptotic proteins found with IGF-1, another potent myeloma cell survival factor. Our results indicate that the myeloma cell survival activity of IL-6 linked to Bcl-xL regulation cannot be generalized and emphasize that Mcl-1 is the main target of IL-6 and IFN-alpha stimulation. However, other changes in the activity of the Bcl-2 protein family or other apoptosis regulators must be identified to elucidate the IGF-1 action mechanism. Cell Death and Differentiation (2000) 7, 1244 - 1252.     

The type III transforming growth factor-β receptor inhibits proliferation, migration, and adhesion in human myeloma cells

Transforming growth factor-β (TGF-β) plays an important role in regulating hematopoiesis, inhibiting proliferation while stimulating differentiation when appropriate. We previously demonstrated that the type III TGF-β receptor (TβRIII, or betaglycan) serves as a novel suppressor of cancer progression in epithelial tumors; however, its role in hematologic malignancies is unknown. Here we demonstrate that TβRIII protein expression is decreased or lost in the majority of human multiple myeloma specimens. Functionally, restoring TβRIII expression in myeloma cells significantly inhibited cell growth, proliferation, and motility, largely independent of its ligand presentation role. In a reciprocal fashion, shRNA-mediated silencing of endogenous TβRIII expression enhanced cell growth, proliferation, and motility. Although apoptosis was not affected, TβRIII inhibited proliferation through induction of the cyclin-dependent kinase inhibitors p21 and p27. TβRIII further regulated myeloma cell adhesion, increasing homotypic myeloma cell adhesion while decreasing myeloma heterotropic adhesion to bone marrow stromal cells. Mechanistically, live cell imaging of myeloma and stroma cell cocultures revealed that TβRIII-mediated inhibition of heterotropic adhesion was associated with decreased duration of myeloma/bone marrow stromal cell interaction. These results suggest that loss of TβRIII expression during multiple myeloma progression contributes to disease progression through its functional effects on increased cell growth, proliferation, motility, and adhesion.     

Targeted knockdown of DJ-1 induces multiple myeloma cell death via KLF6 upregulation

Multiple myeloma (MM) is an incurable plasma B cell malignancy. Despite recent advancements in anti-MM therapies, development of drug resistance remains a major clinical hurdle. DJ-1, a Parkinson’s disease-associated protein, is upregulated in many cancers and its knockdown suppresses tumor growth and overcomes chemoresistance. However, the role of DJ-1 in MM remains unknown. Using gene expression databases we found increased DJ-1 expression in MM patient cells, which correlated with shorter overall survival and poor prognosis in MM patients. Targeted DJ-1 knockdown using siRNAs induced necroptosis in myeloma cells. We found that Krüppel-like factor 6 (KLF6) is expressed at lower levels in myeloma cells compared to PBMCs, and DJ-1 knockdown increased KLF6 expression in myeloma cells. Targeted knockdown of KLF6 expression in DJ-1 knockdown myeloma cells rescued these cells from undergoing cell death. Higher DJ-1 levels were observed in bortezomib-resistant myeloma cells compared to parent cells, and siRNA-mediated DJ-1 knockdown reversed bortezomib resistance. DJ-1 knockdown increased KLF6 expression in bortezomib-resistant myeloma cells, and subsequent siRNA-mediated KLF6 knockdown rescued bortezomib-resistant myeloma cells from undergoing cell death. We also demonstrated that specific siRNA-mediated DJ-1 knockdown reduced myeloma cell growth under a hypoxic microenvironment. DJ-1 knockdown reduced the expression of HIF-1α and its target genes in hypoxic-myeloma cells, and overcame hypoxia-induced bortezomib resistance. Our findings demonstrate that elevated DJ-1 levels correlate with myeloma cell survival and acquisition of bortezomib resistance. Thus, we propose that inhibiting DJ-1 may be an effective therapeutic strategy to treat newly diagnosed as well as relapsed/refractory MM patients.     

多发性骨髓瘤中NF-κB作用机制研究

多发性骨髓瘤(multiple myeloma,MM)是浆细胞异常增生的恶性肿瘤,其发病机制比较复杂。研究表明,活化NF-κB具有多种生物学功能,它既能调节细胞因子;影响细胞周期,又与血管发生有关,并且与多发性骨髓瘤治疗密切相关。该文就NF-κB在多发性骨髓瘤中的作用机制做一综述。     

Karyotypic characteristics of a number of clonal lines of an interspecific mouse-mink hybridoma

Using G-banding method, a study was made of the karyotypes of mouse myeloma cell line P3-X63-Ag8.653 and some cell hybrid lines originated from a fusion of mink immunized spleen cells and this myeloma. Normal chromosomes 6, 12 and X were not detected in either examined cell of the parental myeloma. The mink chromosomes are preferentially lost from cells of the hybrid lines. These lines varied significantly from cell to cell and from each other in the retention of some mink chromosomes. The karyological study of the hybrid lines revealed some cytogenetical specificities of mouse chromosome composition that were general for cells of hybrid lines, but atypical for myeloma parental cells. This data suggest the cells of myeloma parental line to take part in the processes of somatic hybridization and/or selection for mink immunoglobulin production nonrandomly.     

In vitro inhibitory effect of interferon on colony formation of myeloma stem cells

Summary The inhibitory effect of interferon on colony formation of myeloma stem cells in two layer plasma clot-soft agar cultures was studied. Human lymphoblast interferon inhibited in therapeutically attainable concentrations myeloma stem cell proliferation in 50% and human fibroblast interferon in 23% of the 14 myeloma patients in whom in vitro colony formation could be achieved. In interferon-sensitive patients the numbers of myeloma stem cell clusters and colonies were decreased to 34.4%–54.9% of control cultures. In addition, maturation of myeloma stem cells in differentiated plasma cells was reduced by interferon in most of these cases.     

The magnitude of Akt/phosphatidylinositol 3'-kinase proliferating signaling is related to CD45 expression in human myeloma cells

In multiple myeloma, the Akt/PI3K pathway is involved in the proliferation of myeloma cells. In the current study, we have investigated the impact of the CD45 phosphatase in the control of Akt/PI3K activation. We show that Akt activation in response to insulin-like growth factor-1 (IGF-1) is highly variable from one human myeloma cell line to another one. Actually, Akt activation is highly related to whether CD45 is expressed or not. Indeed, both the magnitude and the duration of Akt phosphorylation in response to IGF-1 are more important in CD45- than in CD45+ myeloma cell lines. We next demonstrate a physical association between CD45 and IGF-1 receptor (IGF-1R) suggesting that CD45 could be involved in the dephosphorylation of the IGF-1R. Furthermore, the growth of CD45- myeloma cell lines is mainly or even totally controlled by the PI3K pathway whereas that of CD45+ myeloma cell lines is modestly controlled by it. Indeed, wortmannin, a specific PI3K inhibitor, induced a dramatic growth inhibition in the CD45- myeloma cell lines characterized by a G1 growth arrest, whereas it has almost no effect on CD45+ myeloma cell lines. Altogether, these results suggest that CD45 negatively regulates IGF-1-dependent activation of PI3K. Thus, strategies that block IGF-1R signaling and consequently the Akt/PI3K pathway could be a priority in the treatment of patients with multiple myeloma, especially those lacking CD45 expression that have a very poor clinical outcome.     

Delineation of the roles of paracrine and autocrine interleukin-6 (IL-6) in myeloma cell lines in survival versus cell cycle. A possible model for the cooperation of myeloma cell growth factors

Primary myeloma cells rapidly apoptose as soon as they are removed from their bone-marrow environment. A likely explanation is that the tumor environment produces survival factors that may counteract a spontaneous activation of pro-apoptotic program. Additional factors may trigger cell cycling in surviving myeloma cells. Interleukin-6 (IL-6) is a well recognized myeloma cell growth factor produced mainly by the tumor environment. However, myeloma cells themselves may produce low levels of autocrine IL-6. The respective roles of paracrine versus autocrine IL-6 are a matter of debate. We investigated these roles using the XG-6 myeloma cell line whose growth is dependent on addition of exogenous IL-6, despite its weak IL-6 mRNA and protein expression. The apoptosis induced by exogenous IL-6 deprivation was blocked by transferring the Mcl-1 gene coding for an anti-apoptotic protein in XG-6 cells. An XG-6Mcl-1 cell line which can survive and grow without adding IL-6 was obtained. We show that anti-IL-6 or anti-gp130 antibodies abrogated cell cycling whereas they did not affect cell survival. These data indicate that the weak autocrine IL-6 produced by myeloma cells is sufficient to trigger cell cycling whereas their survival requires large exogenous IL-6 concentrations. This important role of autocrine IL-6 has to be considered when evaluating the mechanism of action of myeloma cell growth factors. These factors may actually block an activated pro-apoptotic program, making possible a weak production of autocrine IL-6 to promote cell cycling.     

精氨酸缺乏对硼替佐米治疗多发性骨髓瘤的作用研究

摘要 目的：探讨精氨酸缺乏对硼替佐米(Bortezomib,BTZ) 治疗多发性骨髓瘤细胞的影响。方法：通过CCK8筛选BTZ对骨髓瘤细胞株(H929和RPMI 8226)治疗的最适药物浓度,比较在缺乏和富含精氨酸的两种培养基中的细胞增殖情况;通过使用PI染料标记细胞检测不同试验组细胞周期的分布,以及使用Annexin V/7AAD凋亡试剂盒检测BTZ对不同试验组细胞凋亡的影响。结果：BTZ降低了骨髓瘤细胞的存活率,并通过将细胞周期阻滞于G2/M、S期,抑制骨髓瘤细胞的增殖。缺乏精氨酸使细胞周期阻滞于S期,也抑制了骨髓瘤细胞的增殖。BTZ作用于缺乏精氨酸组的骨髓瘤细胞后,细胞凋亡百分比明显低于富含精氨酸组（H929细胞由约40%降至13.6%,RPMI8226凋亡百分比分别7.13%和19.27%）。结论：缺乏精氨酸和给予BTZ均阻滞细胞周期,抑制骨髓瘤细胞增殖;同时缺乏精氨酸降低了BTZ诱导骨髓瘤细胞的凋亡作用。     

Induced expression of B7-1 on myeloma cells following retroviral gene transfer results in tumor-specific recognition by cytotoxic T cells.

The aim of this study was to evaluate whether tumor cells from patients with multiple myeloma activate allogeneic and autologous T cells. Results showed that myeloma cells expressed few B7-2 and no B7-1 in six cell lines and primary cells from 11 patients. They expressed substantial levels of HLA class I, CD40, and a set of adhesion molecules. In accordance with the low density of B7 molecules on these cells, they were poor allogeneic CD8+ T cell stimulators. Neither IFN-gamma plus TNF-alpha nor CD40 stimulation significantly induced B7-1 or up-regulated B7-2 on human myeloma cell line or primary myeloma cells from six of seven patients. However, such induction was found on autologous bone-marrow nontumoral cells and on autologous dendritic cells following CD40 stimulation. High B7-1 expression was stably obtained on human myeloma cell line using transduction with a B7-1 retrovirus, enabling these cells to stimulate allogeneic CD8+, though not CD4+, T cell proliferation. For one patient with advanced disease, B7-1 gene transfer made it possible to amplify autologous cytotoxic T cells that killed autologous myeloma cells in an HLA class I-restricted manner, but not autologous PHA blasts. These results suggest that B7-1 gene transfer could be a promising immunotherapeutic approach in multiple myeloma.     

Myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones include double-positive CD8+CD4+ T cells: evidence from myeloma-bearing mouse model

The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122(+) cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8(+) T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8αα and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8(+) T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-γ and/or perforin compared with single-positive CD8(+) T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.     

CD45neg but not CD45pos human myeloma cells are sensitive to the inhibition of IGF-1 signaling by a murine anti-IGF-1R monoclonal antibody, mAVE1642

Insulin-like growth factor 1 (IGF-1) is a well-known growth factor for myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for multiple myeloma treatment. In this study, we investigated the effect of the antagonistic anti-IGF-1R murineAVE1642 Ab (mAVE1642). We show that mAVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines. Since we have previously shown the functional relevance of CD45 expression in the growth of myeloma cells and the association of CD45-negative (CD45neg) status with a less favorable clinical outcome, both CD45-positive (CD45pos) and CD45neg myeloma cell lines were selected for our study. We found that mAVE1642 strongly inhibits the growth of CD45neg myeloma cell lines, leading to a G1 growth arrest, whereas it has almost no effect on the growth of CD45pos myeloma cell lines. Furthermore, mAVE1642 binding induced a significant reduction of IGF-1R expression. We next demonstrated that the overexpression of IGF-1R in the CD45pos myeloma cell line increased Akt phosphorylation but was not sufficient to sensitize these cells to mAVE1642. In contrast, we generated a stable CD45-silencing XG-1 cell line and showed that it became sensitive to mAVE1642. Thus, for the first time, we provided direct evidence that the expression of CD45 renders cells resistant to mAVE1642. Taken together, these results support that therapy directed against IGF-1R can be beneficial in treating CD45neg patients.     

Mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6

Multiple myeloma is characterized by the malignant growth of immunoglobulin producing plasma cells, predominantly in the bone marrow. The effects of primary human mesenchymal stromal cells on the differentiation phenotype of multiple myeloma cells were studied by co-culture experiments. The incubation of multiple myeloma cells with bone marrow-derived mesenchymal stromal cells resulted in significant reduction of the expression of the predominant plasma cell differentiation markers CD38 and CD138, and cell surface immunoglobulin light chain. While the down-regulation of CD138 by stromal cells was completely dependent on their adhesive interactions with the multiple myeloma cells, interleukin-6 induced specific down-regulation of CD38. Mesenchymal stromal cells or their conditioned media inhibited the growth of multiple myeloma cell line, thereby reducing the overall amounts of secreted light chains. Analysis of primary multiple myeloma bone marrow samples reveled that the expression of CD38 on multiple myeloma cells was not affected by adhesive interactions. The ex vivo propagation of primary multiple myeloma cells resulted in significant increase in their differentiation markers. Overall, the data indicate that the bone marrow-derived mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.     

Inhibition of Fatty Acid Metabolism Reduces Human Myeloma Cells Proliferation

Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40–70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma.     

Unique biomechanical interactions between myeloma cells and bone marrow stroma cells

We observed that BMSCs (bone marrow stromal cells) from myeloma patients (myeloma BMSCs) were significantly stiffer than control BMSCs using a cytocompression device. The stiffness of myeloma BMSCs and control BMSCs was further increased upon priming by myeloma cells. Additionally, myeloma cells became stiffer when primed by myeloma BMSCs. The focal adhesion kinase activity of myeloma cells was increased when cells were on stiffer collagen gels and on myeloma BMSCs. This change in myeloma stiffness is associated with increased colony formation of myeloma cells and FAK activation when co-cultured with stiffer myeloma BMSCs or stiffer collagen. Additionally, stem cells of RPMI8226 cells became stiffer after priming by myeloma BMSCs, with concomitant increases of stem cell colony formation. These results suggest the presence of a mechanotransduction loop between myeloma cells and myeloma BMSCs to increase the stiffness of both types of cells via FAK activation. The increase of stiffness may in turn support the growth of myeloma cells and myeloma stem cells.