CS1 Promotes Multiple Myeloma Cell Adhesion, Clonogenic Growth, and Tumorigenicity Via C-Maf-Mediated Interactions with Bone Marrow Stromal Cells (BMSCs)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 840-840
Author(s):  
Yu-Tzu Tai ◽  
Ender Soydan ◽  
Mariateresa Fulciniti ◽  
Weihua Song ◽  
Xian-Feng Li ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Adhesion ◽  
Membrane Protein ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Lentiviral Vector ◽  
Myeloma Cell ◽  
Differentially Expressed ◽  
Parp Cleavage ◽  
Cyclin D2

Abstract Cell membrane protein CS1 is highly expressed by tumor cells from the majority of multiple myeloma (MM) patients (>95%) regardless of cytogenetic abnormalities and response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active MM. However, its role in MM pathophysiology is undefined. In the present study, we first generated CS1 null OPM2 MM cells using lentiviral CS1 short interfering RNA. Specific CS1 knockdown was confirmed by depletion of CS1 mRNA and membrane protein, whereas CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cntOPM2). Immunoblotting of phopho-site of multiple kinase screen analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in CS1null OPM2 cells vs. cntOPM2 cells. Serum deprivation markedly blocked survival at earlier time points in CS1null OPM2 cells vs. cntOPM2 cells. Earlier apoptosis in CS1null OPM2 cells correlated with earlier activation of caspases, PARP cleavage, and increased proapoptotic proteins BNIP3, BIK. CS1 knockdown further delayed development of OPM2 tumor and prolonged survival in mice. CS1null OPM2 cells failed to grow tumors in the majority of mice (n=8) at 5 weeks after cell inoculation, whereas cntOPM2 cells formed tumors within 1.5 weeks in all animals (n=8). Interestingly, CS1 was expressed in tumors that developed late in mice injected with CS1null OPM2 cells. Concomitantly, we overexpressed CS1 in CS1-low expressing U266 cells by transfecting an expressing plasmid pflagCS1 or control vector. Enforced CS1 expression enhanced U266 cell growth and survival. In contrast to the majority of U266 cells (>95%) that grow in suspension in standard tissue culture flasks, all U266CS1 cells exhibited adherent growth and homotypic adhesion. Importantly, overexpressed CS1 increased adhesion of U266 and MM1S cells to BMSCs. Furthermore, U266CS1 cells formed more and larger colonies in methylcellulose than U266 cells. Interestingly, tumors that developed in mice injected with U266 cells expressed significantly higher levels of CS1 than injected U266 cells; moreover, exercised tumors grew in an adherent manner in vitro. Overlapping differentially expressed genes in U266CS1 vs. U266 and CS1null OPM2 vs. cntOPM2 was next analyzed by gene expression profiling. Importantly, c-maf pathway was significantly upregulated in U266CS1 vs. U266 cells and downregulated in CS1null OPM2 vs. cntOPM2 cells, as evidenced by differentially expressed c-maf and its target genes, i.e., cyclin D2, integrin αE/β7 at both mRNA and protein levels. Myeloma cell adhesion-induced VEGF secretion by BMSCs was greater with U266CS1 than U266 cells. Finally, immunoblotting showed upregulation of c-maf and cyclin D2 in U266 tumors overexpressing CS1. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

Drugs used to treat bipolar disorder act via microRNAs to regulate expression of genes involved in neurite outgrowth

2020 ◽  
Vol 34 (3) ◽  
pp. 370-379 ◽  
Author(s):  
Srisaiyini Kidnapillai ◽  
Ben Wade ◽  
Chiara C Bortolasci ◽  
Bruna Panizzutti ◽  
Briana Spolding ◽  
...  
Keyword(s):  
Bipolar Disorder ◽  
Neurite Outgrowth ◽  
Neural Plasticity ◽  
Drug Combination ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Drug Effects ◽  
Differentially Expressed ◽  
Transcriptional Level ◽  
Expression Of Genes

Background: The drugs commonly used to treat bipolar disorder have limited efficacy and drug discovery is hampered by the paucity of knowledge of the pathophysiology of this disease. This study aims to explore the role of microRNAs in bipolar disorder and understand the molecular mechanisms of action of commonly used bipolar disorder drugs. Methods: The transcriptional effects of bipolar disorder drug combination (lithium, valproate, lamotrigine and quetiapine) in cultured human neuronal cells were studied using next generation sequencing. Differential expression of genes ( n=20) and microRNAs ( n=6) was assessed and the differentially expressed microRNAs were confirmed with TaqMan MicroRNA Assays. The expression of the differentially expressed microRNAs were inhibited to determine bipolar disorder drug effects on their target genes ( n=8). Independent samples t-test was used for normally distributed data and Kruskal-Wallis/Mann-Whitney U test was used for data not distributed normally. Significance levels were set at p<0.05. Results: We found that bipolar disorder drugs tended to increase the expression of miR-128 and miR-378 ( p<0.05). Putative target genes of these microRNAs targeted pathways including those identified as “neuron projection development” and “axonogenesis”. Many of the target genes are inhibitors of neurite outgrowth and neurogenesis and were downregulated following bipolar disorder drug combination treatment (all p<0.05). The bipolar disorder drug combination tended to decrease the expression of the target genes ( NOVA1, GRIN3A, and VIM), however this effect could be reversed by the application of microRNA inhibitors. Conclusions: We conclude that at a transcriptional level, bipolar disorder drugs affect several genes in concert that would increase neurite outgrowth and neurogenesis and hence neural plasticity, and that this effect is mediated (at least in part) by modulation of the expression of these two key microRNAs.

scholarly journals Elucidating the expression and function of Numbl during cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma (MM)

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Yuejiao Huang ◽  
Xianting Huang ◽  
Chun Cheng ◽  
Xiaohong Xu ◽  
Hong Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Clinical Problem ◽  
Integrin Β1 ◽  
Cell Viability Assay ◽  
Adhesion Model

Abstract Background Cell adhesion-mediated drug resistance (CAM-DR) is a major clinical problem that prevents successful treatment of multiple myeloma (MM). In particular, the expression levels of integrin β1 and its sub-cellular distribution (internalization and trafficking) are strongly associated with CAM-DR development. Methods Development of an adhesion model of established MM cell lines and detection of Numbl and Integrinβ1 expression by Western Blot analysis. The interaction between Numbl and Integrinβ1 was assessed by a co-immunoprecipitation (CO-IP) method. Calcein AM assay was performed to investigate the levels of cell adhesion. Finally, the extent of CAM-DR in myeloma cells was measured using cell viability assay and flow cytometry analysis. Results Our preliminary date suggest that Numbl is differentially expressed in a cell adhesion model of MM cell lines. In addition to binding to the phosphotyrosine-binding (PTB) domain, the carboxyl terminal of Numbl can also interact with integrin β1 to regulate the cell cycle by activating the pro-survival PI3K/AKT signaling pathway. This study intends to verify and elucidate the interaction between Numbl and integrin β1 and its functional outcome on CAM-DR. We have designed and developed a CAM-DR model using MM cells coated with either fibronectin or bone marrow stromal cells. We assessed whether Numbl influences cell-cycle progression and whether it, in turn, contributes to activation of PI3K/AKT signal pathway through the adjustment of its carboxyl end. Finally, we showed that the interaction of Numbl with integrin β1 promotes the formation of CAM-DR in MM cells. Conclusions Our findings elucidated the specific molecular mechanisms of CAM-DR induction and confirmed that Numbl is crucial for the development of CAM-DR in MM cells.

scholarly journals Identification of lncRNAs Involved in PCV2 Infection of PK-15 Cells

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 479
Author(s):  
Jin He ◽  
Chaoliang Leng ◽  
Jiazhen Pan ◽  
Aoqi Li ◽  
Hua Zhang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Severe Disease ◽  
Enrichment Analysis ◽  
Economic Loss ◽  
Porcine Circovirus Type ◽  
Pcv2 Infection ◽  
Differentially Expressed ◽  
Porcine Circovirus ◽  
Swine Industry

Porcine circovirus type 2 (PCV2) can cause severe disease in infected pigs, resulting in massive economic loss for the swine industry. Transcriptomic and proteomic approaches have been widely employed to identify the underlying molecular mechanisms of the PCV2 infection. Numerous differentially expressed mRNAs, miRNAs, and proteins, together with their associated signaling pathways, have been identified during PCV2 infection, paving the way for analysis of their biological functions. Long noncoding RNAs (lncRNAs) are important regulators of multiple biological processes. However, little is known regarding their role in the PCV2 infection. Hence, in our study, RNA-seq was performed by infecting PK-15 cells with PCV2. Analysis of the differentially expressed genes (DEGs) suggested that the cytoskeleton, apoptosis, cell division, and protein phosphorylation were significantly disturbed. Then, using stringent parameters, six lncRNAs were identified. Additionally, potential targets of the lncRNAs were predicted using both cis- and trans-prediction methods. Interestingly, we found that the HOXB (Homeobox B) gene cluster was probably the target of the lncRNA LOC106505099. Enrichment analysis of the target genes showed that numerous developmental processes were altered during PCV2 infection. Therefore, our study revealed that lncRNAs might affect porcine embryonic development through the regulation of the HOXB genes.

Inhibition of Multiple Myeloma Cell Adhesion to Fibronectin by Ephrin Ligation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2360-2360
Author(s):  
Stuart Ratner ◽  
Charles A. Schiffer ◽  
Jeffrey A. Zonder
Keyword(s):  
Multiple Myeloma ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Myeloma Cell ◽  
Separate Experiment ◽  
Partial Recovery ◽  
Multiple Myeloma Cell ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) cell adhesion to fibronectin (FN), mediated via VLA-4 and VLA-5, has been shown to induce resistance to several chemotherapeutic drugs. Disruption of MM cell adhesion to FN and other marrow microenvironment elements might therefore enhance the effects of therapy. We now present the first evidence that Eph-ephrin signaling may be exploited to inhibit MM cell binding to fibronectin. Ephs are transmembrane tyrosine kinases and ephrins are their cell-surface ligands. There are two classes of Ephs and ephrins, A and B. Both Ephs and ephrins can transduce repulsive signals that cause interacting cells to lose contact with each other and with extracellular matrix. We are not aware of any previous systematic study of Eph and ephrin expression or function in MM cells. We have found MM cell lines H929, U266, and RPMI 8226 express members of the A classes of both Ephs and ephrins, but not the B classes. First, we demonstrated ligation with commercially available anti-ephrin A3 antibody was followed by ephrin capping and shedding from the cell surface. We next explored whether ephrin ligation affects MM cell adhesiveness in culture. Whereas H929, U266, and RPMI 8226 cells adhered rapidly to fibronectin-coated plastic surfaces, all three cell lines failed completely to adhere to a mixed coating of FN and rabbit anti-ephrin A3 antibody for a period of 2 hrs. This effect was not seen with FN + normal rabbit Ig. This suggests binding of ephrin A3 (or another cross-reacting A-class ephrin) by solid-state antibody triggers intracellular signals that interfere with initial steps of integrin-mediated adhesion. After 2 hr, spontaneous partial recovery of adhesion occurred, reaching a plateau of approximately 30% of control values by 24 hr. We postulate this recovery occurs via clipping of the extracellular ephrin domain by transmembrane metalloproteases, since recovery of FN adhesion was partially prevented by the metalloprotease inhibitor GM6001 (25 uM). Also consistent with this theory, we found in a separate experiment that GM6001 reduced the shedding of cross-linked A-class ephrins from MM cell lines. In summary, we have demonstrated that manipulation of EPH-ephrin signaling can impair MM-cell adhesion to FN, and that this effect is enhanced by simultaneous inhibition of metalloprotease activity. We are currently studying the effect of A-class ephrin ligation on adhesion-mediated drug resistance in MM cell lines. We also intend to evaluate EPH-ephrin expression in marrow specimens from patients with MM.

A High Throughput Seeking Myeloma Therapeutics Identifies Glucocorticoids as Inhibitors of c-Maf-Dependent Transactivation of Cyclin D2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3384-3384
Author(s):  
Xinliang Mao ◽  
A. Keith Stewart ◽  
Alessandro Datti ◽  
Aaron D. Schimmer
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
High Throughput ◽  
Myeloma Cell ◽  
Final Concentration ◽  
Biologically Active ◽  
Luciferase Assay ◽  
Myeloma Cell Line ◽  
Cyclin D2 ◽  
Nih3t3 Cells

Abstract c-Maf is a transcription factor that regulates expression of several genes including cyclin D2. c-Maf and cyclin D2 are frequently over expressed in multiple myeloma and associated with chemoresistance and poor clinical outcome. Therefore, molecules that inhibit c-Maf-dependent transactivation of cyclin D2 may be important biological tools to understand the pathogenesis of myeloma and could be therapeutically useful. To identify such compounds, we devised a high throughput screen in NIH3T3 cells. NIH3T3 cells over expressing c-Maf and the cyclin D2 promoter-driving luciferase were treated with aliquots at a final concentration of 5 μM from the LOPAC and Prestwick libraries of totally 2400 biologically active chemicals and off-patent drugs. Cyclin D2 transactivation (luciferase assay) and cell viability (MTS-based assay) were measured 24hr later. From this screen, compounds that preferentially reduced cyclin D2 transactivation over viability were identified. Hits were defined mathematically as (sample luciferase/control luciferase)/(sample MTS/control MTS) &lt; 0.5. The Z score of the screening assay was 0.58, and the coefficient of variance was &lt;3%, denoting a robust screen. Forty compounds that reproducibly repressed cyclin D2 transactivation were identified through this screen. To classify groups of drugs that influenced cyclin D2 transactivation, microarray software Treeview and Cluster were used to cluster the drugs into families. Thirty-one of the 40 hits from the screen were members of the glucocorticoid family and the screen identified all of the glucocorticoids found in the two libraries. To test the specificity of these hits, NIH3T3 cells over-expressing the cyclin D2 promoter-driving luciferase with or without c-Maf expression, and NIH3T3 cells expressing the RSV promoter-driving luciferase were treated with the compounds. Luciferase expression driven by the RSV promoter was reduced by one compound. Eight compounds repressed cyclin D2 transactivation independent of c-Maf expression. The 31 glucocorticoids identified in this screen preferentially reduced cyclin D2 transactivation in the presence of c-Maf. For example, the EC50 was &lt; 1 μM for betamethasone, budesonide and dexamethasone in NIH3T3 cells over-expressing c-Maf and was &gt; 50 μM for these glucocorticoids in the NIH3T3 cells without c-Maf expression, where the EC50 represents the concentration of the compound required to reduce luciferase activity by 50%. Glucocorticoids significantly reduced c-Maf expression by immunoblotting at 5 μM, consistent with a c-Maf-dependent inhibition of cyclin D2 transactivation. Given their effects on c-Maf and cyclin D2, we tested the selected glucocorticoids in the c-Maf over-expressing myeloma cell line 8226. At a final concentration of 5 μM, glucocorticoids betamethasone, budesonide and dexamethasone induced 30, 31, and 32% apoptosis, respectively 24 hours after treatment, but induced &lt; 5% apoptosis in the myeloma cell line KMS12 that lacks c-maf expression. In conclusion, glucocorticoids repress c-Maf dependent transactivation of cyclin D2. These findings demonstrate a new mechanism for glucocorticoid-induced apoptosis and help explain their activity in multiple myeloma.

The Oral PKC-β Inhibitor Enzastaurin (LY317615) Suppress Phosphorylation and Induces Apoptosis in Multiple Myeloma Cell Lines by Inhibition of AKT Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1371-1371
Author(s):  
Antonino Neri ◽  
Sandra Marmiroli ◽  
Pierfrancesco Tassone ◽  
Luigia Lombardi ◽  
Lucia Nobili ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Time Dependent ◽  
Parp Cleavage ◽  
Akt Phosphorylation ◽  
Downstream Target ◽  
Pkc Β

Abstract The PKC pathway has been shown to play a role in the regulation of cell proliferation in several hematologic malignancies. In this study we tested the oral PKC-β inhibitor, Enzastaurin (LY317615 - Eli Lilly) for its therapeutic efficacy in Multiple Myeloma (MM). We first analyzed PKC-β I and II expression by Western blot in a panel of 19 human MM cell lines, showing that 9 cell lines express either 1 or both isoforms. We next examined the growth inhibition effect of Enzastaurin in the same panel of MM cell lines using either WST-1 or MTT assay and cell viability assessment by Tripan Blue exclusion. Eighteen cell lines have IC50 value ranging from 1,2 μM to 12,5 μM. To examine molecular mechanisms whereby Enzastaurin induces cytotoxicity, we performed cell cycle profiling using PI and observed a significant increase of the percentage of cells in the sub G0–G1 fraction. To determine whether Enzastaurin-induced cell death is mediated by apoptosis, we studied by ELISA and Western blot caspase 3 and PARP cleavage. We observed induction of caspase 3 and PARP cleavage in a dose and time dependent fashion. Notably, the broad caspase (Z-VAD-FMK) inhibitor reduced Enzastaurin-induced cytotoxicity. We next determined whether Enzastaurin could inhibit AKT phosphorylation in MM cell lines with constitutive phosphorylation of AKT. Enzastaurin decreased AKT phosphorylation in a dose and time dependent fashion. Phosphorylation of GSK3β, a downstream target protein of AKT, was also markedly inhibited. Phosphorylation of PDK-1, a known upstream activator of AKT, was not affected by Enzastaurin. In conclusion, our results indicate that Enzastaurin-induced cytotoxicity is mediated via activation of caspase. This effect is associated with significant inhibition of AKT activity and its downstream target GSK3 β. Enzastaurin does not alter the phosphorylation of the upstream AKT activator PDK-1. These data suggest that Enzastaurin inhibit AKT signalling pathway and support its evaluation in a murine model of human MM.

NVP-LAQ824 is a potent novel histone deacetylase inhibitor with significant activity against multiple myeloma

Blood ◽  
2003 ◽  
Vol 102 (7) ◽  
pp. 2615-2622 ◽  
Author(s):  
Laurence Catley ◽  
Ellen Weisberg ◽  
Yu-Tzu Tai ◽  
Peter Atadja ◽  
Stacy Remiszewski ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Histone Deacetylase ◽  
Hdac Inhibitors ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Significant Activity ◽  
Dependent Manner

Abstract Histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in hematologic malignancies. Here we show that NVP-LAQ824, a novel hydroxamic acid derivative, induces apoptosis at physiologically achievable concentrations (median inhibitory concentration [IC50] of 100 nM at 24 hours) in multiple myeloma (MM) cell lines resistant to conventional therapies. MM.1S myeloma cell proliferation was also inhibited when cocultured with bone marrow stromal cells, demonstrating ability to overcome the stimulatory effects of the bone marrow microenvironment. Importantly, NVP-LAQ824 also inhibited patient MM cell growth in a dose- and time-dependent manner. NVP-LAQ824-induced apoptotic signaling includes up-regulation of p21, caspase cascade activation, and poly (adenosine diphosphate [ADP]) ribose (PARP) cleavage. Apoptosis was confirmed with cell cycle analysis and annexin-propidium iodide staining. Interestingly, treatment of MM cells with NVPLAQ824 also led to proteasome inhibition, as determined by reduced proteasome chymotrypsin-like activity and increased levels of cellular polyubiquitin conjugates. Finally, a study using NVP-LAQ824 in a preclinical murine myeloma model provides in vivo relevance to our in vitro studies. Taken together, these findings provide the framework for NVP-LAQ824 as a novel therapeutic in MM. (Blood. 2003;102:2615-2622)

B026 CS1 Promotes Multiple Myeloma Cell Adhesion, Clonogenic Growth, and Tumorigenicity via c-Maf Mediation

2009 ◽  
Vol 9 ◽  
pp. S96-S97
Author(s):  
YT Tai ◽  
E Soydan ◽  
M Fulciniti ◽  
W Song ◽  
K Kim ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Adhesion ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Clonogenic Growth

scholarly journals CS1 promotes multiple myeloma cell adhesion, clonogenic growth, and tumorigenicity via c-maf–mediated interactions with bone marrow stromal cells

Blood ◽  
2009 ◽  
Vol 113 (18) ◽  
pp. 4309-4318 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Ender Soydan ◽  
Weihua Song ◽  
Mariateresa Fulciniti ◽  
Kihyun Kim ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Molecular Mechanisms ◽  
Myeloma Cell ◽  
Marrow Stromal Cells ◽  
Growth And Survival ◽  
Multiple Myeloma Cell

Abstract CS1 is highly expressed on tumor cells from the majority of multiple myeloma (MM) patients regardless of cytogenetic abnormalities or response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active disease. However, its contribution to MM pathophysiology is undefined. We here show that CS1 knockdown using lentiviral short-interfering RNA decreased phosphorylation of ERK1/2, AKT, and STAT3, suggesting that CS1 induces central growth and survival signaling pathways in MM cells. Serum deprivation markedly blocked survival at earlier time points in CS1 knockdown compared with control MM cells, associated with earlier activation of caspases, poly(ADP-ribose) polymerase, and proapoptotic proteins BNIP3 and BIK. CS1 knockdown further delayed development of MM tumor and prolonged survival in mice. Conversely, CS1 overexpression promoted myeloma cell growth and survival by significantly increasing myeloma adhesion to bone marrow stromal cells (BMSCs) and enhancing myeloma colony formation in semisolid culture. Moreover, CS1 increased c-maf–targeted cyclin D2-dependent proliferation, -integrin β7/αE-mediated myeloma adhesion to BMSCs, and -vascular endothelial growth factor-induced bone marrow angiogenesis in vivo. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

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