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Nicholas Mitsiades

Publications and source records attributed to Nicholas Mitsiades.

At least 37 records · Page 2Linked to original sources
PubMed2003

Proteasome inhibitor PS-341 abrogates IL-6 triggered signaling cascades via caspase-dependent downregulation of gp130 in multiple myeloma.

Proteasome inhibitor PS-341 is one of the most promising novel agents against multiple myeloma (MM). We have previously shown that PS-341 inhibits IL-6 triggered phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 (also known as p42/44 mitogen-activated protein kinases) in MM cells. In this study, we further examined whether clinically achievable concentrations of PS-341 could inhibit IL-6 triggered signaling cascades in MM. We found that PS-341 inhibited not only ERK, but also signal transducers and activators of transcription (STAT) 3 as well as Akt phosphorylation. Since gp130 (CD130) dimerizes and is phosphorylated after IL-6 binding to gp80 (IL-6 receptor), we hypothesized that gp130 could be involved in PS-341-induced blockade of signaling cascades mediating MM cell growth, survival, and drug resistance in the bone marrow (BM) microenvironment. In this study, we first demonstrate that PS-341 induces downregulation of gp130 in a time- and dose-dependent manner in vitro, prior to MM cell death. Conversely, downregulation of gp130 is completely abrogated by the pan-caspase inhibitor Z-VAD-FMK, suggesting that downregulation of gp130 is mediated via caspase activation. Z-VAD-FMK also abrogates the inhibitory effect of PS-341 on IL-6-triggered signaling cascades. Importantly, we demonstrate that phosphorylation of ERK, STAT3, and Akt in MM.1S cells induced by either exogenous IL-6 or by binding of MM cells to BM stromal cells is abrogated by PS-341. These studies, therefore, define another novel mechanism whereby PS-341 can overcome the growth and survival advantage in MM cells conferred by the BM milieu. Importantly, this effect on cytokine-induced gp130 signaling cascades may account, at least in part, for the remarkable preclinical sensitivity and clinical responses achieved in MM with PS-341 treatment.

Boronic Acids↗
PubMed2003

Immunomodulatory drug costimulates T cells via the B7-CD28 pathway.

Although thalidomide (Thal) does not directly induce T-cell activation, it increases proliferation of T cells following CD3 activation. In this study, we examined the immunomodulatory effects of a more potent analog of Thal, immunomodulatory drug (IMiD), on T cells. Although IMiD3 does not directly stimulate proliferation of normal donor CD3+ T cells, it significantly costimulates proliferation of CD3+ T cells induced by CD3 ligation (stimulation index [SI], 2.4), immature dendritic cells (DCs; SI, 2.1), and mature DCs (SI, 2.6). T-cell proliferation triggered by DCs was abrogated by cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA-4-Ig), and IMiD3 partially overcomes this inhibitory effect. IMiD3 also overcomes the inhibitory effects of CTLA-4-Ig on Epstein-Barr virus (EBV) and influenza (Flu)-specific CD4 and CD8 T-cell responses, as measured by cytokine capture and enzyme-linked immunosorbent spot (ELISPOT) assay. IMiD3 did not induce up-regulation of CD28 expression on T cells, or of CD80-CD86 expression on dendritic cells. Importantly, IMiD3 triggers tyrosine phosphorylation of CD28 on T cells, followed by activation of nuclear factor kappaB (NF-kappaB), a known downstream target of CD28 signaling. These results therefore define the costimulatory mechanism whereby IMiD3 induces T-cell activation and provide the cellular and molecular basis for use of IMiD3 as an adjuvant in immunotherapeutic treatment strategies for multiple myeloma.

Antigens, CD↗
PubMed2003

Superoxide-dependent and -independent mitochondrial signaling during apoptosis in multiple myeloma cells.

Superoxide (O2-) radicals have been linked to apoptosis. Here, we show that 2-methoxyestradiol (2ME2)-induced apoptosis in multiple myeloma (MM) cells is associated with O2- generation, whereas dexamethasone (Dex)-induced apoptosis occurs without concurrent increase in O2-. In contrast, both these agents decrease mitochondrial transmembrane potential (Deltapsi(m)). Treatment of MM cells with an antioxidant N-acetyl-L-cysteine blocks 2ME2, but not Dex-induced apoptosis as well as release of mitochondrial proteins cytochrome c (cyto c) and Smac. Taken together, these results demonstrate that there are at least two distinct apoptotic pathways: one dependent on O2-, which is induced by 2ME2 and is associated with release of cyto c and Smac; and the other an independent of O2-, which is triggered by Dex and associated with Smac release.

2-Methoxyestradiol↗
PubMed2003

Insulin-like growth factor-1 induces adhesion and migration in human multiple myeloma cells via activation of beta1-integrin and phosphatidylinositol 3'-kinase/AKT signaling.

Insulin-like growth factor-1 (IGF-I) is a growth and survival factor in human multiple myeloma (MM) cells. Here we examine the effect of IGF-I on MM cell adhesion and migration, and define the role of beta1 integrin in these processes. IGF-I increases adhesion of MM.1S and OPM6 MM cells to fibronectin (FN) in a time- and dose-dependent manner, as a consequence of IGF-IR activation. Conversely, blocking anti-beta1 integrin monoclonal antibody, RGD peptide, and cytochalasin D inhibit IGF-I-induced cell adhesion to FN. IGF-I rapidly and transiently induces association of IGF-IR and beta1 integrin, with phosphorylation of IGF-IR, IRS-1, and p85(PI3-K). IGF-I also triggers phosphorylation of AKT and ERK significantly. Both IGF-IR and beta1 integrin colocalize to lipid rafts on the plasma membrane after IGF-I stimulation. In addition, IGF-I triggers polymerization of F-actin, induces phosphorylation of p125(FAK) and paxillin, and enhances beta1 integrin interaction with these focal adhesion proteins. Importantly, using pharmacological inhibitors of phosphatidylinositol 3'-kinase (PI3-K) (LY294002 and wortmannin) and extracellular signal-regulated kinase (PD98059), we demonstrate that IGF-I-induced MM cell adhesion to FN is achieved only when PI3-K/AKT is activated. IGF-I induces a 1.7-2.2 (MM.1S) and 2-2.5-fold (OPM6) increase in migration, whereas blocking anti-IGF-I and anti-beta1 integrin monoclonal antibodies, PI3-K inhibitors, as well as cytochalasin D abrogate IGF-I-induced MM cell transmigration. Finally, IGF-I induces adhesion of CD138+ patient MM cells. Therefore, these studies suggest a role for IGF-I in trafficking and localization of MM cells in the bone marrow microenvironment. Moreover, they define the functional association of IGF-IR and beta1 integrin in mediating MM cell homing, providing the preclinical rationale for novel treatment strategies targeting IGF-I/IGF-IR in MM.

Antibodies, Monoclonal↗
PubMed2003

Identification of genes modulated in multiple myeloma using genetically identical twin samples.

Genetic heterogeneity between individuals confounds the comparison of gene profiling of multiple myeloma (MM) cells versus normal plasma cells (PCs). To overcome this barrier, we compared the gene expression profile of CD138+ MM cells from a patient bone marrow (BM) sample with CD138+ PCs from a genetically identical twin BM sample using microarray profiling. Two hundred and ninety-six genes were up-regulated and 103 genes were down-regulated at least 2-fold in MM cells versus normal twin PCs. Highly expressed genes in MM cells included cell survival pathway genes such as mcl-1, dad-1, caspase 8, and FADD-like apoptosis regulator (FLIP); oncogenes/transcriptional factors such as Jun-D, Xbp-1, calmodulin, Calnexin, and FGFR-3; stress response and ubiquitin/proteasome pathway-related genes and various ribosomal genes reflecting increased metabolic and translational activity. Genes that were down-regulated in MM cells versus healthy twin PCs included RAD51, killer cell immunoglobulin-like receptor protein, and apoptotic protease activating factor. Microarray results were further confirmed by Western blot analyses, immunohistochemistry, fluorescent in situ hybridization (FISH), and functional assays of telomerase activity and bone marrow angiogenesis. This molecular profiling provides potential insights into mechanisms of malignant transformation in MM. For example, FGFR3, xbp-1, and both mcl-1 and dad-1 may mediate transformation, differentiation, and survival, respectively, and may have clinical implications. By identifying genes uniquely altered in MM cells compared with normal PCs in an identical genotypic background, the current study provides the framework to identify novel therapeutic targets.

Adult↗
PubMed2003

Insights into the multistep transformation of MGUS to myeloma using microarray expression analysis.

To define specific pathways important in the multistep transformation process of normal plasma cells (PCs) to monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM), we have applied microarray analysis to PCs from 5 healthy donors (N), 7 patients with MGUS, and 24 patients with newly diagnosed MM. Unsupervised hierarchical clustering using 125 genes with a large variation across all samples defined 2 groups: N and MGUS/MM. Supervised analysis identified 263 genes differentially expressed between N and MGUS and 380 genes differentially expressed between N and MM, 197 of which were also differentially regulated between N and MGUS. Only 74 genes were differentially expressed between MGUS and MM samples, indicating that the differences between MGUS and MM are smaller than those between N and MM or N and MGUS. Differentially expressed genes included oncogenes/tumor-suppressor genes (LAF4, RB1, and disabled homolog 2), cell-signaling genes (RAS family members, B-cell signaling and NF-kappaB genes), DNA-binding and transcription-factor genes (XBP1, zinc finger proteins, forkhead box, and ring finger proteins), and developmental genes (WNT and SHH pathways). Understanding the molecular pathogenesis of MM by gene expression profiling has demonstrated sequential genetic changes from N to malignant PCs and highlighted important pathways involved in the transformation of MGUS to MM.

Cell Transformation, Neoplastic↗
PubMed2003

Hsp27 inhibits release of mitochondrial protein Smac in multiple myeloma cells and confers dexamethasone resistance.

Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Heat shock protein 27 (Hsp27) negatively regulates another mitochondrial protein, cytochrome c, during apoptosis; however, the role of Hsp27 in modulating Smac release is unknown. Here we show that Hsp27 is overexpressed in both dexamethasone (Dex)-resistant multiple myeloma (MM) cell lines (MM.1R, U266, RPMI-8226) and primary patient cells. Blocking Hsp27 by an antisense (AS) strategy restores the apoptotic response to Dex in Dex-resistant MM cells by triggering the release of mitochondrial protein Smac, followed by activation of caspase-9 and caspase-3. Moreover, AS-Hsp27 overcomes interleukin-6 (IL-6)-mediated protection against Dex-induced apoptosis. These data demonstrate that Hsp27 inhibits the release of Smac, and thereby confers Dex resistance in MM cells.

Apoptosis↗
PubMed2003

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

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.

Acetylation↗
PubMed2003

JNK-dependent release of mitochondrial protein, Smac, during apoptosis in multiple myeloma (MM) cells.

Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Previous studies have shown that c-Jun NH(2)-terminal kinase (JNK) is involved in regulating another mitochondrial protein, cytochrome c during apoptosis; however, the role of JNK in the release of mitochondrial Smac is unknown. Here we show that induction of apoptosis in multiple myeloma (MM) cells is associated with activation of JNK, translocation of JNK from cytosol to mitochondria, and release of Smac from mitochondria to cytosol. Blocking JNK either by dominant-negative mutant (DN-JNK) or cotreatment with a specific JNK inhibitor, SP600125, abrogates both stress-induced release of Smac and induction of apoptosis. These findings demonstrate that activation of JNK is an obligatory event for the release of Smac during stress-induced apoptosis in MM cells.

Anthracenes↗
PubMed2003

Molecular sequelae of histone deacetylase inhibition in human malignant B cells.

Histone acetylation modulates gene expression, cellular differentiation, and survival and is regulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibition results in accumulation of acetylated nucleosomal histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of suberoylanilide hydroxamic acid (SAHA), the prototype of a series of hydroxamic acid-based HDAC inhibitors, in cell lines and patient cells from B-cell malignancies, including multiple myeloma (MM) and related disorders. SAHA induced apoptosis in all tumor cells tested, with increased p21 and p53 protein levels and dephosphorylation of Rb. We also detected cleavage of Bid, suggesting a role for Bcl-2 family members in regulation of SAHA-induced cell death. Transfection of Bcl-2 cDNA into MM.1S cells completely abrogated SAHA-induced apoptosis, confirming its protective role. SAHA did not induce cleavage of caspase-8, -9, or -3 in MM.1S cells during the early phase of apoptosis, and the pan-caspase inhibitor ZVAD-FMK did not protect against SAHA. Conversely, poly(ADP)ribose polymerase (PARP) was cleaved in a pattern indicative of calpain activation, and the calpain inhibitor calpeptin abrogated SAHA-induced cell death. Importantly, SAHA sensitized MM.1S cells to death receptor-mediated apoptosis and inhibited the secretion of interleukin 6 (IL-6) induced in bone marrow stromal cells (BMSCs) by binding of MM cells, suggesting that it can overcome cell adhesion-mediated drug resistance. Our studies delineate the mechanisms whereby HDAC inhibitors mediate anti-MM activity and overcome drug resistance in the BM milieu and provide the framework for clinical evaluation of SAHA, which is bioavailable, well tolerated, and bioactive after oral administration, to improve patient outcome.

Acetylation↗
PubMed2003

Nuclear factor-kappaB p65 mediates tumor necrosis factor alpha-induced nuclear translocation of telomerase reverse transcriptase protein.

Sustained proliferation of cancer cells requires telomerase to maintain telomeres that regulate chromosomal stability and cellular mitosis. Expression of human telomerase reverse transcriptase (hTERT) catalytic subunit, which modulates telomerase activity, is regulated at both the transcriptional level and via phosphorylation by Akt kinase. Moreover, nuclear localization of hTERT is required to promote elongation of telomere sequences. In this study, we show for the first time that hTERT protein interacts directly with nuclear factor (NF)-kappaB p65 in MM.1S cells. Importantly, tumor necrosis factor alpha (TNFalpha) modulates telomerase activity by inducing translocation from the cytoplasm to the nucleus of hTERT protein bound to NF-kappaB p65. Conversely, a specific IkappaB kinase (IKK) inhibitor PS-1145, and a specific NF-kappaB nuclear translocation inhibitor SN-50, both block TNFalpha-induced hTERT nuclear translocation. These studies suggest that NF-kappaB p65 plays a pivotal role in regulating telomerase by modulating its nuclear translocation.

Cell Nucleus↗
PubMed2003

Expression of serotherapy target antigens in Waldenstrom's macroglobulinemia: therapeutic applications and considerations.

Monoclonal antibody (mAb) therapy (serotherapy) has been successfully used in the treatment of many B-cell malignancies, among them lymphoplasmacytic lymphoma, an uncommon disorder that includes patients with the clinicopathological diagnosis of Waldenstrom's macroglobulinemia (WM). Rituximab, a mAb directed at CD20, was recently demonstrated by us and others to induce remissions and facilitate hematological recovery in patients with WM. The expression of CD20, along with targets of other mAbs which are commercially available, currently in clinical trials, or in preclinical development, have not been extensively studied or well documented in lymphoplasmacytic lymphoma. As such, we examined by flow cytometric analysis tumor cells from a large series of patients with the histopathlogical diagnosis of lymphoplasmacytic lymphoma and the clinicopathological diagnosis of WM for expression of the serotherapy target antigens CD20, CD22, CD40, CD52, IgM, MUC1 core protein, and 1D10. These studies demonstrated antigen expression on >or=50% of bone marrow tumor cells (CD19(+), kappa/lambda light chain-restricted), respectively, from patients as follows: CD20 (98.3%), CD22 (88.3%), CD40 (83.3%), CD52 (77.4%), IgM (83.3%), MUC1 core protein (57.8%), and 1D10 (50%). Both interpatient and intrapatient tumor clone antigen expression was heterogeneous. Combined mAb therapy might be a useful approach to cope with this variation, and could be tailored to target all members of the tumor clone for an individual patient.

Antibodies, Monoclonal↗
PubMed2003

Novel biologically based therapies for Waldenstrom's macroglobulinemia.

Waldenstrom's macroglobulinemia (WM) remains an incurable B-cell malignancy, necessitating urgent development of novel treatment strategies. Building on our experience on bed-to-bedside translational studies for multiple myeloma (mm), we identified a constellation of novel classes of anti-WM agents, including the proteasome inhibitor PS-341; the ansamycin family of inhibitors (eg, geldanamycin and its analogues) of the heat-shock protein 90 (hsp90) molecular chaperone; histone deacetylase inhibitors, such as suberoylanilide hydroxamic acid (SAHA); and the thiazolidinedione group of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists (eg, ciglitazone or rosiglitazone). Our preclinical data show that these classes of agents induce growth arrest and apoptosis of WM cells, at concentrations relevant to those achieved in previous clinical uses of these drugs, and suggest that novel therapeutic strategies for WM can be designed to include combinations of these agents, to simultaneously target multiple levels of diverse pathways important for tumor cell growth and survival, and thus maximize the pro-apoptotic activities of these agents and/or neutralize protective responses of WM against their effects. These molecular studies provide a framework for rational design of the next generation of combination therapies for WM.

Adjuvants, Immunologic↗
PubMed2003

Proteomic analyses in Waldenstrom's macroglobulinemia and other plasma cell dyscrasias.

The proteomic analysis of tumor cells emerges as a key complement to gene expression profiling, primarily because regulation of protein expression (at the translational and post-translational levels) can buffer the magnitude of changes occurring at the gene transcription level, in order to fine tune cellular functions. Herein we describe the concept of proteomic analysis of the signaling state of tumor cells, as well as its application in the study of signaling pathways in plasma cell dyscrasias, such as Waldenstrom's macroglobulinemia (WM) and multiple myeloma (MM). Comparative studies of WM versus MM cells at baseline and in the setting of drug treatment reveals proteomic profiles of the signaling state with significant overlap (that could reflect a putative B-cell lineage-related proteomic signature), but also distinct differences, possibly associated with differential features in the biologic behavior and drug sensitivity of these diseases. These proteomic studies pave the way for a more comprehensive insight into the molecular basis of WM versus other B-cell malignancies.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

VEGF-dependent conjunctivalization of the corneal surface.

PURPOSE: To investigate the mechanisms governing corneal neovascularization and the appearance of goblet cells in a murine model of limbal insufficiency. METHODS: The spatial and time-dependent relationship between corneal neovascularization and goblet cell density was analyzed in corneal flatmounts. Immunohistochemical detection of the vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR1) was performed in paraffin-embedded sections. A transgenic mouse that expresses the reporter gene lacZ targeted to the Flt-1 locus through homologous recombination was used to analyze corneal expression of Flt-1. The presence of soluble and membranous goblet cell Flt-1 mRNA and protein content was assessed with Northern and Western blot analyses, respectively. Finally, systemic adenoviral expression of a soluble Flt-1/Fc construct was used to study the effect of inhibition of VEGF bioactivity on the appearance of goblet cells and neovascularization. RESULTS: Corneal neovascularization preceded the appearance of goblet cells, although both processes overlapped temporally. Flt-1 was abundant in the conjunctiva-like epithelium covering the cornea, as well as in the goblet cells, invading leukocytes, and vasculature. A similar expression pattern was observed in the transgenic mice expressing the lacZ gene downstream from the Flt-1 promoter. Isolated human and rat goblet cells in culture expressed Flt-1 mRNA and protein, as did freshly isolated human conjunctiva. The systemic inhibition of VEGF bioactivity potently suppressed both corneal neovascularization (8.3% +/- 8.1% vs. 41.1% +/- 15.3% corneal area; P < 0.001) and corneal goblet cell density (1.6% +/- 2.5% vs. 12.2% +/- 2.4% corneal area; P < 0.001). CONCLUSIONS: Two important features of corneal conjunctivalization, the appearance of goblet cells and neovascularization, are regulated by VEGF. Both processes are probably mediated, in part, through the Flt-1 receptor. Taken together, these data indicate that an anti-VEGF therapeutic approach may limit the visual loss associated with conjunctivalization of the corneal surface.

Animals↗
PubMed2003

Regulation of vascular endothelial growth factor expression by insulin-like growth factor I in thyroid carcinomas.

Vascular endothelial growth factor (VEGF) produced by tumor cells potently stimulates endothelial cell proliferation and angiogenesis and plays a key role in the pathophysiology of several neoplasias. Hypoxia activates the VEGF promoter via response elements that bind the transcription factors hypoxia-inducible factor-1 alpha (HIF-1 alpha) and activator protein-1 (AP-1). Yet, the paracrine signaling pathways regulating VEGF production and angiogenesis in thyroid cancer have not been fully elucidated. In this study, we, therefore, investigated the regulation of VEGF production by the thyroid carcinoma cell line SW579. We found that IGF-I up-regulated VEGF mRNA expression and protein secretion. Furthermore, transfection of SW579 cells with vector expressing a constitutively active form of Akt, a major mediator of IGF-I signaling, also stimulated VEGF expression. The IGF-I-induced up-regulation of VEGF production was associated with activation of AP-1 and HIF-1 alpha and was abrogated by phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002); Jun kinase inhibitor (SP600125); HIF-1 alpha antisense oligonucleotide; or geldanamycin, an inhibitor of the heat shock protein 90 molecular chaperone, which regulates the three-dimensional conformation and function of IGF-I-receptor and Akt. These data indicate that IGF-I stimulates VEGF synthesis in thyroid carcinomas in an Akt-dependent pathway via AP-1 and HIF-1 alpha and provide the framework for clinical use of small-molecule inhibitors, including geldanamycin analogs, to abrogate proangiogenic cascades in thyroid cancer.

Adenocarcinoma↗