Toll-Like Receptor (TLR)-1/2 Triggering of Multiple Myeloma Cells Modulates Their Adhesion to Bone Marrow Stromal Cells and Enhances Bortezomib-Induced Apoptosis

AbstractThe interleukin 6/glycoprotein 130/signal transducer and activator of transcription 3 (IL-6/gp130/STAT3) pathway has been reported to play an important role in the pathogenesis of multiple myeloma (MM) and for survival of MM cells. However, most data concerning the role of IL-6 and IL-6–triggered signaling pathways were obtained from experiments performed with MM cell lines and without considering the bone marrow microenvironment. Thus, the precise role of IL-6 and its intracellular signaling pathways for survival of human MM cells is still unclear. Here we show that treatment of human MM cells (IL-6–dependent MM cell line INA-6 and primary MM cells) with the IL-6 receptor antagonist Sant7 or with an anti-gp130 monoclonal antibody (mAb) induced apoptosis if the cells were cultured in the absence of bone marrow stromal cells (BMSCs). In contrast, apoptosis could not be observed if the MM cells were cocultured with BMSCs. The analysis of intracellular pathways revealed that Sant7 and anti-gp130 mAb were effectively inhibiting the phosphorylation of gp130 and STAT3 in the absence and presence of BMSCs, whereas ERK1 and ERK2 (ERK1,2) phosphorylation was only slightly affected. In contrast, treatment with the farnesyl transferase inhibitor, FPT III, induced apoptosis in MM cells in the absence or presence of BMSCs and led to a complete inhibition of the Ras/mitogen-activated protein kinase pathway. These observations indicate that the IL-6/gp130/STAT3 pathway is not essential for survival of human myeloma cells if they are grown in the presence of cells from the bone marrow microenvironment. Furthermore, we provide evidence that farnesyl transferase inhibitors might be useful for the development of novel therapeutic strategies for the treatment of MM.

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Dexmethasone Down-Regulates Stromal Cell-Derived Factor-1 Production in Bone Marrow Stromal Cells: Implications in Myeloma Cell Biology.

Blood ◽

10.1182/blood.v106.11.2510.2510 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2510-2510

Author(s):

Seong-Woo Kim ◽

Jin-Hee Hwang ◽

Hwan-Jung Yun ◽

Samyong Kim ◽

Deog-Yeon Jo

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Myeloma Cell ◽

Cxcr4 Expression ◽

Marrow Stromal Cells ◽

Myeloma Cells ◽

Regulatory Effects ◽

Stromal Cell Derived Factor ◽

Induced Apoptosis

Abstract Stromal cell-derived factor-1 (SDF-1) plays a role in the homing of myeloma cells to bone marrow. In addition, SDF-1 modestly enhances the proliferation of myeloma cells and inhibits Dexmethasone (Dex)-induced apoptosis of the cells. Dex is currently used to treat multiple myeloma, based on its apoptic effects. In this study, we investigated the regulatory effects of Dex on SDF-1 production in bone marrow stromal cells (BMSCs) and on CXCR4 expression in myeloma cells. As previously reported, it was evident that primary myeloma cells (CD138+ cells obtained from patients with multiple myeloma) and Dex-resistant myeloma cell line RPMI8226 expressed CXCR4 and responded to SDF-1, resulting in chemotaxis. SDF-1 modestly stimulated the proliferation of primary myeloma cells and RPMI8226 cells and protected the cells from Dex-induced apoptosis. Human umbilical vein endothelial cells transduced with the SDF-1 gene using adenoviral vectors better supported the formation of cobblestone areas of primary myeloma cells and RPMI8226 cells in co-culture, similar to hematopoietic progenitor cells; this was blocked by pretreating the myeloma cells with pertussis toxin, indicating that SDF-1 plays a critical role not only in migration of the cells underneath the SDF-1-producing stromal cells but also in proliferation of the cells in contact. Dex up-regulated CXCR4 expression in RPMI8226 cells; however, its regulatory effects on CXCR4 in primary myeloma cells differed among patients. RT-PCR and Northern blot analyses revealed that Dex down-regulated SDF-1 mRNA expression in both primary BMSCs and murine stromal MS-5 cells in a dose-dependent manner. Western blot analysis and ELISA assay confirmed that Dex inhibited SDF-1 production in BMSCs. Furthermore, Dex inhibited cobblestone area formation of RPMI8226 cells in co-culture with MS-5. Interestingly, Dex up-regulated CXCR4 mRNA expression and cytoplasmic CXCR4 in BMSCs. These results indicate that Dexamethasone induces the down-regulation of SDF-1 production in BMSCs, which might mediate, at least in part, its anti-myeloma effects in vivo.

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CD56 Expression Is the Potent Prognostic Marker of the Thalidomide Efficacy in the Patients with Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.5142.5142 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5142-5142

Author(s):

Akio Mori ◽

Yutaka Tsutsumi ◽

Satoshi Hashino ◽

Hiroe Kanamori ◽

Makoto Ibata ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Adhesion ◽

Adhesion Molecules ◽

Prognostic Marker ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Myeloma Cells ◽

Cd56 Expression

Abstract Thalidomide (Thal) alone or in combination with steroids achieves responses even in the setting of refractory multiple myeloma (MM), however, responses are still limited. The precise mechanism of Thal action is unknown, further, no distinct marker, which could prognosticate the efficacy of Thal, is known. Therefore, we evaluated the correlation between the efficacy of Thal and the potent prognostic factors in patients with refractory MM. Ten patients with refractory MM received Thal at doses of 50 or 100 mg per day and steroids, either dexamethasone (Dex) or prednisolone (PSL). Dex was administrated 20 mg per day, 4 days every 28 days, and PSL was administrated 10 mg per day. The median age was 71.5 years (range, 62–79 years) and 20 % were man, and all patients were diagnosed as clinical stage IIIA based on the Durie and Salmon classification. The therapeutic response was assessed according to the modified criteria of Southwest Oncology Group (SWOG). Among 10 patients, 7 patients were the responders; 2 had complete remission, 3 had partial remission, and 2 had minimal remission. There were no differences in the pretreatment characteristics of responders and nonresponders (age, sex, type and concentration of serum and/or urine monoclonal component, international prognostic index, presence of bone lesion, and chromosomal abnormalities). However, flow cytometric evaluation of the myeloma cells revealed that CD56, which is one of the adhesion molecules N-CAM, expressed more than 45 % in all responders, while those expressed less than 5 % in all nonresponders (84 ± 19 (±SD) % v/s 4 ± 2 %, P=0.017). Furthermore, CD56 expression of the myeloma cells was reduced from 84% to 70 ± 32 % after Thal therapy in all evaluated responders (P =0.048). These results suggest that CD56 expression of the myeloma cells could be the potent prognostic marker of the Thal efficacy. Moreover, it was reported that Thal reduced the expression of cell adhesion molecules, such as LFA-1 and ICAM-1, and abrogated the binding of MM cells to bone marrow stromal cells, that triggered the secretion of interleukin-6 and vascular endothelial growth factor. Taken together, it was suggested that Thal reduced the expression of CD56 and altered the MM cell adhesion to bone marrow stromal cells, and that could be one of the pathogenesis of anti-MM activity of Thal.

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Nifuroxazide Inhibits STAT3 Function and Shows Potent Anti-Tumor Activity Against Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.3450.3450 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3450-3450

Author(s):

Erik A. Nelson ◽

Teru Hideshima ◽

Laurie Gashin ◽

Sarah R. Walker ◽

Rebecca A. Lynch ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Transcriptional Activity ◽

Marrow Stromal Cells ◽

Normal Peripheral Blood ◽

Myeloma Cells ◽

Stat3 Signaling ◽

Nf Kappab

Abstract Activation of the transcription factor STAT3 is essential for the pathogenesis of many cancers, including multiple myeloma. While normal cells can tolerate a reduction in STAT3 function, tumors often require constitutive STAT3 signaling for survival. Thus, identifying drugs that inhibit STAT3 activity may provide new therapeutic agents useful for cancer treatment. We have developed a high throughput cell-based screen to identify drugs that inhibit STAT3-dependent transcriptional activity. To assure the specificity of these drugs for STAT3 function, we performed a counter screen assessing NF-kappaB-dependent transcriptional activity. To bypass the difficulties inherent in the development of novel small molecules for clinical use, we analyzed a library of 1120 drugs that are either FDA approved, or are otherwise known to be safe in humans. From this screen, we identified nifuroxazide, a drug used to treat dehydration associated with diarrheal illness, as a potent inhibitor of STAT3 transcriptional activity. By contrast, nifuroxazide has no effect on NF-kappaB-dependent transcription. Myeloma cells containing constitutive STAT3 activation show decreased STAT3 tyrosine phosphorylation when incubated with 10 uM nifuroxazide. In addition, expression of STAT3 target genes necessary for myeloma survival, including bcl-x, mcl-1, and cyclin D1, is markedly reduced by 10 uM nifuroxazide. To determine whether these effects of nifuroxazide on STAT3 signaling alter cell viability, we utilized U266 myeloma cells, which depend on STAT3 activation for survival. U266 viability is inhibited by nifuroxazide at an EC50 of approximately 3 uM. Notably, RPMI 8226 myeloma cells, which do not contain activated STAT3, are not affected by comparable concentrations of nifuroxazide. In addition, this dose has no effect on normal peripheral blood mononuclear cells. Given that myeloma cells receive survival signals from bone marrow stromal cells, we determined if nifuroxazide affects myeloma survival in stromal cell co-cultures. Nifuroxazide is effective at reducing U266 viability in the presence of bone marrow stromal cells at an EC50 of approximately 3 uM. Thus, screening for compounds that inhibit STAT3 transcriptional activity is useful in identifying potential drugs for myeloma therapy. Through this approach, we have identified a novel STAT3 inhibitory function for nifuroxazide. Nifuroxazide inhibits STAT3 mediated survival of myeloma cells and may be useful, either alone or in combination with other drugs, for the treatment of patients with multiple myeloma.

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The Relationship of Myeloma Cells, Stromal Cells and Monocytes Under Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v120.21.4987.4987 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4987-4987

Author(s):

Hiroshi Ikeda ◽

Yuka Aoki ◽

Nasanori Nojima ◽

Hiroshi Yasui ◽

Toshiaki Hayashi ◽

...

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Bone Marrow ◽

Cell Proliferation ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Myeloma Cells ◽

Growth And Survival

Abstract Abstract 4987 The Bone marrow (BM) microenvironment plays crucial role in pathogenesis of Multiple myeloma(MM). Myeloma cells contacts with bone marrow stromal cells (BMSCs), which secrete factors/cytokines, promoting tumor cell growth and survival. Paracrine secretion of cytokines(i. e., interleukin-6 (IL-6) insulin-like growth factor-1, inflammatory protein-1a) in BM stromal cells promotes multiple myeloma cell proliferation and protects against drug-induced cytotoxicity. These cytokines provide stimulatory signals for multiple myeloma growth and survival. Bone involvement is a common feature in MM patient, solid and hematologic cancers. MM localizes to the bone in nearly all patients ranges between 40% and 75%. Disease-related skeletal complications result in significant morbidity due to pain, pathologic fractures and spinal cord compression. The bone microenvironment creates a supportive niche for tumor growth. Osteoclasts and bone marrow stromal cells, along with extracellular matrix and cytokines stimulate tumor cell proliferation and confer chemoresistance. Therefore, the reciprocal interactions between tumor cells, osteoclasts, osteoblasts, and bone marrow stromal cells present an important. In current study, monocyte can directly promote mesenchymal stem cells osteogenic differentiation through cell contact interactions, thus resulting in the production of osteogenic factors by the monocytes. This mechanism is mediated by the activation of STAT3 signaling pathway in the mesechymal stem cells that leads to the upregulation of Osteoblasts-associated genes such as Runx2 and alkaline phosphatase (ALP), and the down-regulation of inhibitors such as DKK1 to drive the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocyte, component of BM cells, as a potential niche component that supports myeloma cells. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BM stromal cells, monocytes, or a combination of BM stromal cells and monocytes. Consistently, we observed increased proliferation of MM cell lines in the presence of either BM stromal cells or monocytes compared to cell line-only control. Furthermore, the co-culture of BM stromal cells plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin

Blood ◽

10.1182/blood.v126.23.1772.1772 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1772-1772 ◽

Cited By ~ 3

Author(s):

Jahangir Abdi ◽

Yijun Yang ◽

Patrick Meyer-Erlach ◽

Hong Chang

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Drug Resistance ◽

Cell Lines ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Molecular Mechanisms ◽

Marrow Stromal Cells ◽

Survivin Gene ◽

Induced Apoptosis

Abstract INTRODUCTION It is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known. METHODS Human myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia). RESULTS BMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05). CONCLUSION Our results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

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Bone marrow stromal cells protect myeloma cells from bortezomib induced apoptosis by suppressing microRNA-15a expression

Leukemia & Lymphoma ◽

10.3109/10428194.2011.576791 ◽

2011 ◽

Vol 52 (9) ◽

pp. 1787-1794 ◽

Cited By ~ 75

Author(s):

Mu Hao ◽

Li Zhang ◽

Gang An ◽

Hengxing Meng ◽

Youjin Han ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Myeloma Cells ◽

Induced Apoptosis

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Etodolac Induces Apoptosis and Inhibits Cell Adhesion to Bone Marrow Stromal Cells in Human Myeloma Cells.

Blood ◽

10.1182/blood.v104.11.4836.4836 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4836-4836

Author(s):

Satoki Nakamura ◽

Miki Kobayashi ◽

Kiyoshi Shibata ◽

Naohi Sahara ◽

Kazuyuki Shigeno ◽

...

Keyword(s):

Bone Marrow ◽

Cell Adhesion ◽

Adhesion Molecules ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Myeloma Cell ◽

Marrow Stromal Cells ◽

Myeloma Cells ◽

Cox 2 ◽

Induced Apoptosis

Abstract Cyclooxygenase-2 (COX-2) is reported to regulate apoptosis and to be an important cellular target for therapy. In this study, we demonstrated that etodolac, a COX-2 inhibitor, inhibited proliferation and induced apoptosis in myeloma cell lines (RPMI 8226 and MC/CAR cells), expressing the COX-2 enzyme. In both cell lines, etodolac more strongly induced apoptosis compared with thalidomide or meloxicam. Etodolac induced down-regulation of bcl-2 protein and mRNA, activation of caspase-9, -7 and -3, down-regulation of caspase inhibitors, cIAP-1 and survivin, and loss of mitochondrial membrane potential in a dose-dependent manner. In addition, our data demonstrated that when myeloma cells were coincubated with 50 mM etodolac on bone marrow stromal cells (BMSC), myeloma cell adhesion to BMSC was significantly inhibited compared with thalidomide or meloxicam coincubation, and the adhesion molecules VLA-4, LFA-1 (CD11a), CXCX4, and CD44 were suppressed on myeloma cells treated with etodolac. Moreover, we found that 100 mM R-etodolac, S-etodolac, and the combination of R- and S-etodolac, which are the stereoisomers of etodolac, slightly inhibited the proliferation of myeloma cells, while 50 to 100 mM etodolac significantly inhibited the proliferation of myeloma cells. In conclusion, our findings indicate that etodolac induced apoptosis via a bcl-2 dependent pathway, suppressed the expression of adhesion molecules, and inhibited myeloma cell adhesion to BMSC compared with thalidomide or meloxicam. Thus, the activities of etodolac potentially extend to the treatment of patients with myeloma resistant to standard chemotherapy, including thalidomide.

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