Masanori Obata

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.

Dehydroepiandrosterone can inhibit the proliferation of myeloma cells and the interleukin-6 production of bone marrow mononuclear cells from patients with myeloma.

The serum levels of an adrenal sex hormone, dehydroepiandrosterone sulfate (DHEA-S), are significantly more decreased in human myelomas compared with the reduction brought by physiologic decline with age. In order to clarify the effect of DHEA on myeloma cells, we investigated whether DHEA and DHEA-S could inhibit interleukin-6 (IL-6) production of bone marrow mononuclear cells and the proliferation of myeloma cells from patients with myeloma. DHEA-S and DHEA suppressed IL-6 production from a bone marrow stromal cell line, KM-102, as well as in bone marrow mononuclear cells from patients with myeloma. Furthermore, DHEA inhibited in vitro growth of the U-266 cell line and primary myeloma cells from the patients, as well as the in vivo growth of U-266 cells implanted i.p. in severe combined immunodeficiency-hIL6 transgenic mice. DHEA up-regulated the expression of peroxisome proliferator-activated receptor (PPAR), PPAR beta, but not PPARgamma or PPARalpha, and the expression of IkappaBalpha gene in myeloma cells and bone marrow stromal cells, which could explain the suppressive effect of DHEA on IL-6 production through the down-regulation of NF-kappaB activity. Therefore, these data revealed that DHEA-S, as well as DHEA, had a direct effect on myeloma and bone marrow stromal cells to inhibit their proliferation and IL-6 production, respectively.

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.

The regulatory mechanism of IL-6-dependent proliferation of human myeloma cells.

Abstract Multiple myeloma (MM) is a malignant tumor of plasma cells in the bone marrow. Interleukin 6 (IL-6) is an indispensable growth factor for myeloma cells. The heterogeneity of myeloma cells are the characteristics of MM, categorized into five sub-populations, two immature cells, MPC-1<PRE>-</PRE> CD49e<PRE>-</PRE> CD45<PRE>+/-</PRE>, intermediate cells, MPC-1<PRE>+</PRE> CD49e<PRE>-</PRE> CD45<PRE>+/-</PRE>, and mature cells, MPC-1<PRE>+</PRE> CD49e<PRE>+</PRE> CD45<PRE>+</PRE>. Only MPC-1<PRE>-</PRE> CD49e<PRE>-</PRE> CD45<PRE>+</PRE> immature cells (∼2% of total myeloma cells) respond to IL-6 to proliferate. CD45 protein tyrosine phosphatase is the determinant of IL-6 dependent cell growth of myeloma cells, although well studied IL-6 signal transducing factors, such as, IL-6Ra, gp130, Jak2, STAT3, and MAPK, are activated and involved in the process. Immature CD45<PRE>-</PRE> cells converted to CD45<PRE>+</PRE> cells after IL-6 stimulation both in U266 cells and sorted myeloma cells from the bone marrow aspirates of MM patients. CD45<PRE>-</PRE> cells are relatively resistant to serum starvation compared to CD45<PRE>+</PRE> cells. Because IL-6 level in the bone marrow is low even in MM patients, the CD45<PRE>-</PRE> phenotype of myeloma cells may protect the cells from apoptosis. These findings of a tuning effect of CD45 on myeloma cell proliferation may aid the study of IL-6 dependent proliferation of myeloma cells and lead to the development of new therapies for MM patients.