Mohd S. Iqbal

Accelerated proliferation of myeloma cells by interleukin-6 cooperating with fibroblast growth factor receptor 3-mediated signals.

Interleukin-6 (IL-6) is a cytokine that regulates the proliferation of some tumor cells including multiple myeloma (MM). Ectopic expression of fibroblast growth factor receptor 3 (FGFR3) associated with the chromosomal translocation, t(4;14)(p16.3;q32), is frequently found in MM, and therefore, has been implicated in the neoplastic transformation of this disease. Here, we show that IL-6 together with FGF enhanced proliferation of a myeloma cell line, KMS-11 carrying t(4;14)(p16.3;q32) and the FGFR3-transfected U266 myeloma cell line which ectopically expressed FGFR3 but responded to neither IL-6 nor FGF alone. In KMS-11, IL-6 activated signal transducer and activator of transcription 3 (STAT3) while FGF activated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol (PI)-3 kinase. As both MEK inhibitors and a PI 3-kinase inhibitor abolished the effect of IL-6 and FGF, the activation of both the ERK1/2 and PI 3-kinase signaling cascades is essential for the proliferation of KMS-11 enhanced by IL-6 and FGF. Furthermore, the FGF-induced activation of ERK1/2 contributed to the serine phosphorylation of STAT3, suggesting that the signaling crosstalk between the cytokine receptor, IL-6 receptor α/gp130 and the growth factor receptor tyrosine kinase, FGFR3. These results indicate that FGFR3 plays a crucial role in the accelerated proliferation of MM carrying t(4;14)(p16.3;q32).

Increased susceptibility to apoptosis in CD45(+) myeloma cells accompanied by the increased expression of VDAC1.

Expression of CD45 is quite variable in human myeloma cells and cell lines, such as U266, and CD45+ U266 proliferates in response to a growth factor, interleukin-6. Here, we show that CD45+ myeloma cell lines were more sensitive to various apoptotic stimuli, such as oxidative stress and endoplasmic reticulum (ER)-stress, than CD45− cells. Reactive oxygen species and calcium ion seemed to be involved in the susceptibility to apoptosis of CD45+ U266. The activation of the src family kinases associated with CD45 phosphatase played an important role in the augmented apoptosis in CD45+ U266 by oxidative stress. These results indicate that the CD45-expression renders myeloma cells competent for not only mitogenic but also apoptotic stimuli, resulting in either proliferation or apoptosis of CD45+ myeloma cells dependently upon the circumstantial stimuli. Furthermore, voltage-dependent anion channel (VDAC) 1 was identified as a gene highly expressed in CD45+ U266 by cDNA subtraction. The increased expression of VDAC1 seemed to augment the sensitivity to the ER-stress because the VDAC1-transfected U266 was more susceptible to the thapsigargin-induced apoptosis. Thus, CD45 expression accompanied by the increased VDAC1 expression sensitizes myeloma cells to the various extracellular stimuli that trigger apoptosis via the mitochondrial pathways.

Galectin-1 supports the survival of CD45RA(-) primary myeloma cells in vitro.

The survival and proliferation of human myeloma cells are considered to be heavily dependent on the microenvironment of bone marrow (BM). This study confirmed that galectin‐1 (Gal‐1) and SDF‐1α were produced by bone marrow mononuclear cells of myeloma patients. The addition of Gal‐1 and SDF‐1α to a serum‐free synthetic medium, maintained the viability of primary myeloma cells for 2 weeks similar to that before culture. While Gal‐1 reduced the viable cell number in CD45RA(+) B cell lines, it maintained the viability of CD45(−) U266 and CD45RA(−)RO(+) ILKM3 myeloma cell lines in the synthetic medium. This was confirmed with the transfection of the PTPRC (CD45) RA, ‐RB, or ‐RO gene into CD45(−) U266 cells. The combination of Gal‐1 and SDF‐1α significantly induced phosphorylation of Akt and IkB, while the phosphorylation of ERK1/2 was significantly reduced in CD45RA(+) U266 and Raji cells but not CD45(−) or CD45RA(−) U266 cells. Furthermore, we confirmed that Gal‐1 bound to CD45RA in CD45RA(+) Raji cells, and also physically interacted with β1‐integrin by immunoprecipitation followed by Western blotting and confocal microscopy. The results suggest that Gal‐1 has two different actions depending on its binding partner, and supports the survival of CD45RA(−) myeloma cells.

Constitutively lower expressions of CD54 on primary myeloma cells and their different localizations in bone marrow

To evaluate nuclear factor‐κB (NF‐κB) activity in primary myeloma cells from myeloma patients, we confirmed that the expression levels of CD54 showed a good correlation with the levels of DNA binding activity for NF‐κB in human myeloma cell lines, and thus analyzed the expression levels of CD54 on CD38(++) plasma cell fractions as one of NF‐κB activity. Primary myeloma cells unexpectedly showed constitutively lower expressions of CD54 than normal bone marrow (BM) plasma cells. Furthermore, the expression levels of CD54 on these plasma cells showed a significant correlation with the plasma levels of CXCL12 stromal cell‐derived factor‐1α (SDF‐1α) in their BM aspirates, and the expressions of CXCR4, the receptor for CXCL12, decreased on primary myeloma cells compared with normal BM plasma cells. It was also confirmed that the addition of CXCL12 to the in vitro culture significantly induced the up‐regulation of CD54 expression in primary myeloma cells. In addition, myeloma cells with lower expressions of CD54 were more unstable in the in vitro culture, resulting in a marked reduction of the viable cell number. In the immunohistochemical analysis of BM aspirates, myeloma cells with lower CD54 expression resided in the perivascular regions. Therefore, these data suggest that primary myeloma cells exhibit constitutively lower CD54 that might be partially regulated by CXCL12, and their localizations in the BM may be associated with the expression levels of CD54.

IL-6-induced activation of MYC is responsible for the down-regulation of CD33 expression in CD33(+) myeloma cells

Human myeloma cells from about 10% of cases with multiple myeloma expressed CD33 and had monocytoid morphology with convoluted nuclei, and all these patients had no increase in serum CRP values. In CD33(+) myeloma cells as well as myeloma cell lines, CD33 expression levels were correlated with the increased expression levels of CEBPA (C/EBPα). This correlation was confirmed by the finding that transfection with the CEBPA gene induced CD33 expression in a CD33(−) myeloma cell line. As suggested by the lack of an increase in serum CRP values in CD33(+) myelomas, IL-6 down-regulated the expression of CD33 in CD33(+) myeloma cell lines along with the down-regulation of CEBPA gene expression. Cucurbitacin I (STAT3 inhibitor), but not U0126 (MAPK inhibitor), could abolish the effect of IL-6. Furthermore, IL-6 up-regulated the expression of MYC via STAT3 phosphorylation and MYC bound to the promoter region of the CEBPA gene followed by the down-regulation of CEBPA expression. It was confirmed that introduction of shRNA for MYC into a CD33(+) myeloma cell line blocked the IL-6-induced down-regulation of CD33 and CEBPA expression. Therefore, these results indicate that IL-6 can reverse the expression level of CD33 by up-regulating MYC followed by the down-regulation of CEBPA expression.

CD56 expression in human myeloma cells derived from the neurogenic gene expression: possible role of the SRY-HMG box gene, SOX4

CD56 is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. In order to clarify the possible mechanisms of CD56 expression in human myeloma, we underwent screening for potential targets by microarray analysis, where the CD56(+) myeloma cell lines showed markedly increased expressions of transcription factors involved in the neuronal cell lineage compared to the CD56(−) myeloma cell lines. Here, we show that among the SOX family of transcription factors, SOX4 was highly up-regulated and SOX1 was down-regulated in the CD56(+) myeloma cell lines as well as in primary myeloma cases as confirmed by the RT-PCR. ChIP analysis of the CD56 promoter region showed specific bindings of SOX4 in the CD56(+) and SOX1 in the CD56(−) myeloma cell lines, respectively. shRNA against SOX1 failed to induce CD56 expression in CD56(−) myeloma cell line, U266. On the contrary, over-expression of SOX4 in the CD56(−) myeloma cell line could induce the CD56 expression. Silencing of SOX4 by shRNA transfection down-regulated CD56 expression and induced apoptosis to CD56(+) human myeloma cell line, AMO1. Thus, induction of SOX4 gene expression might be responsible for the CD56 expression in human myeloma cells.

A novel signaling pathway associated with Lyn, PI 3-kinase and Akt supports the proliferation of myeloma cells

Interleukin-6 (IL-6) is a growth factor for human myeloma cells. We have recently found that in myeloma cells the activation of both signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase (ERK) 1/2 is not sufficient for the IL-6-induced proliferation, which further requires the activation of the src family kinases, such as Lyn. Here we showed that the Lyn-overexpressed myeloma cell lines had the higher proliferative rate with IL-6 and the enhanced activation of the phosphatidylinositol (PI) 3-kinase and Akt. The IL-6-induced phosphorylation of STAT3 and ERK1/2 was not up-regulated in the Lyn-overexpressed cells, indicating that the Lyn-PI 3-kinase-Akt pathway is independent of these pathways. The PI 3-kinase was co-precipitated with Lyn in the Lyn-overexpressed cells of which proliferation with IL-6 was abrogated by the specific inhibitors for PI 3-kinase or Akt, suggesting that the activation of the PI 3-kinase-Akt pathway associated with Lyn is indeed related to the concomitant augmentation of myeloma cell growth. Furthermore, the decreased expression of p53 and p21(Cip1) proteins was observed in the Lyn-overexpressed cells, implicating a possible downstream target of Akt. This study identifies a novel IL-6-mediated signaling pathway that certainly plays a role in the proliferation of myeloma cells and this novel mechanism of MM tumor cell growth associated with Lyn would eventually contribute to the development of MM treatment.