ABSTRACT
Multiple myeloma (MM) is an incurable neoplasm caused by proliferation of malignant plasma cells in the bone marrow (BM). MM is characterized frequently by a complete or partial deletion of chromosome 13q14, seen in more than 50% of patients at diagnosis. Within this deleted region the tripartite motif containing 13 (TRIM13, also termed RFP2) gene product has been proposed to be a tumour suppressor gene (TSG). Here, we show that low expression levels of TRIM13 in MM are associated with chromosome 13q deletion and poor clinical outcome. We present a functional analysis of TRIM13 using a loss-of-function approach, and demonstrate that TRIM13 downregulation decreases tumour cell survival as well as cell cycle progression and proliferation of MM cells. In addition, we provide evidence for the involvement of TRIM13 downregulation in inhibiting the NF kappa B pathway and the activity of the 20S proteasome. Although this data does not support a role of TRIM13 as a TSG, it substantiates important roles of TRIM13 in MM tumour survival and proliferation, underscoring its potential role as a novel target for therapeutic intervention.
Subject(s)
DNA-Binding Proteins/biosynthesis , Multiple Myeloma/genetics , Multiple Myeloma/pathology , NF-kappa B/antagonists & inhibitors , Proteasome Endopeptidase Complex/metabolism , Tumor Suppressor Proteins/biosynthesis , Apoptosis/genetics , Cell Cycle/genetics , Cell Division/genetics , Cell Line, Tumor , Cell Survival/genetics , Chromosome Deletion , Chromosomes, Human, Pair 13 , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Down-Regulation , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Multiple Myeloma/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Proteasome Endopeptidase Complex/genetics , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Deregulated Wnt/ß-catenin signaling underlies the pathogenesis of a broad range of human cancers, yet the development of targeted therapies to disrupt the resulting aberrant transcription has proved difficult because the pathway comprises large protein interaction surfaces and regulates many homeostatic functions. Therefore, we have directed our efforts toward blocking the interaction of ß-catenin with B cell lymphoma 9 (BCL9), a co-activator for ß-catenin-mediated transcription that is highly expressed in tumors but not in the cells of origin. BCL9 drives ß-catenin signaling through direct binding mediated by its α-helical homology domain 2. We developed a stabilized α helix of BCL9 (SAH-BCL9), which we show targets ß-catenin, dissociates native ß-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and exhibits mechanism-based antitumor effects. SAH-BCL9 also suppresses tumor growth, angiogenesis, invasion, and metastasis in mouse xenograft models of Colo320 colorectal carcinoma and INA-6 multiple myeloma. By inhibiting the BCL9-ß-catenin interaction and selectively suppressing oncogenic Wnt transcription, SAH-BCL9 may serve as a prototype therapeutic agent for cancers driven by deregulated Wnt signaling.
Subject(s)
Gene Targeting , Neoplasm Proteins/metabolism , Oncogenes/genetics , Wnt Signaling Pathway/genetics , beta Catenin/metabolism , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation , Colorectal Neoplasms/blood supply , Colorectal Neoplasms/pathology , Humans , Intestinal Mucosa/metabolism , Mice , Molecular Sequence Data , Neoplasm Proteins/chemistry , Neovascularization, Pathologic/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Stability , Protein Structure, Secondary , TCF Transcription Factors/metabolism , Transcription Factors , Transcription, Genetic , Xenograft Model Antitumor AssaysABSTRACT
Multiple myeloma is characterized by frequent chromosomal alterations. Deletion of chr 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. Here, we functionally validate the role of the microRNAs-15a/16-1 cluster, centered at the deleted region, as TSGs and delineate their downstream target genes in MM. Using "sponge" lentiviral vectors to competitive stably inhibit mature microRNAs in vitro and in vivo, we have documented enhanced proliferative and invasive capacity of cells with stably inhibition of miR-16. Importantly, miR-16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load and host angiogenesis. Expression profiling analysis of miR-16-deficient cells identified a large number of downstream target genes including FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. We validated designated genes showing binding sites within the conserved 3'-UTR and also within the mRNA coding region as direct miR-16 targets, thus indicating that the miRNAs may have many more targets than anticipated by conventional prediction methods. This loss-of-function system, which mimics the 13q chromosomal deletion, provides a valuable tool to investigate their function in MM pathogenesis and their potential use as therapeutic targets.
ABSTRACT
Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as in vitro and in vivo functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances beta-catenin-mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling.
Subject(s)
Colonic Neoplasms/metabolism , Multiple Myeloma/metabolism , Neoplasm Proteins/biosynthesis , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Movement/physiology , Colonic Neoplasms/blood supply , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Disease Progression , Humans , Hyaluronan Receptors/biosynthesis , Hyaluronan Receptors/genetics , Multiple Myeloma/blood supply , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Metastasis , Neoplasm Proteins/genetics , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/genetics , Wnt Proteins/metabolismABSTRACT
Multiple myeloma (MM) is a cancer of plasma cells with complex molecular characteristics that evolves from monoclonal gammopathy of undetermined significance, a highly prevalent premalignant condition. MM is the second most frequent hematologic cancer in the United States, and it remains incurable, thereby highlighting the need for new therapeutic approaches, particularly those targeting common molecular pathways involved in disease progression and maintenance, shared across different MM subtypes. Here we report that Wnt/beta-catenin is one such pathway. We document the involvement of beta-catenin in cell-cycle regulation, proliferation, and invasion contributing to enhanced proliferative and metastatic properties of MM. The pleiotropic effects of beta-catenin in MM correlate with its transcriptional function, and we demonstrate regulation of a novel target gene, Aurora kinase A, implicating beta-catenin in G2/M regulation. beta-catenin and Aurora kinase A are present in most MM but not in normal plasma cells and are expressed in a pattern that parallels progression from monoclonal gammopathy of undetermined significance to MM. Our data provide evidence for a novel functional link between beta-catenin and Aurora kinase A, underscoring a critical role of these pathways in MM disease progression.
Subject(s)
Multiple Myeloma/genetics , Protein Serine-Threonine Kinases/genetics , Wnt Proteins/physiology , beta Catenin/physiology , Animals , Aurora Kinase A , Aurora Kinases , Cell Cycle/genetics , Cell Proliferation , Disease Progression , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Multiple Myeloma/pathology , Protein Serine-Threonine Kinases/physiology , Transplantation, Heterologous , Tumor Cells, Cultured , Wnt Proteins/genetics , beta Catenin/geneticsABSTRACT
Multiple myeloma (MM) evolves from a highly prevalent premalignant condition termed MGUS. The factors underlying the malignant transformation of MGUS are unknown. We report a MGUS/MM phenotype in transgenic mice with Emu-directed expression of the XBP-1 spliced isoform (XBP-1s), a factor governing unfolded protein/ER stress response and plasma-cell development. Emu-XBP-1s elicited elevated serum Ig and skin alterations. With age, Emu-xbp-1s transgenics develop features diagnostic of human MM, including bone lytic lesions and subendothelial Ig deposition. Furthermore, transcriptional profiles of Emu-xbp-1s lymphoid and MM cells show aberrant expression of known human MM dysregulated genes. The similarities of this model with the human disease, coupled with documented frequent XBP-1s overexpression in human MM, serve to implicate XBP-1s dysregulation in MM pathogenesis.
Subject(s)
Cell Differentiation , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/pathology , Multiple Myeloma/pathology , Nuclear Proteins/metabolism , Plasma Cells/cytology , Aging/pathology , Animals , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Bone Diseases/pathology , Cells, Cultured , DNA-Binding Proteins/genetics , Dromaiidae/genetics , Electrophoretic Mobility Shift Assay , Endoplasmic Reticulum/metabolism , Female , Humans , Hypergammaglobulinemia/pathology , Kidney Diseases/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multiple Myeloma/metabolism , Nuclear Proteins/genetics , Plasma Cells/immunology , Plasma Cells/metabolism , RNA Splicing , Regulatory Factor X Transcription Factors , Skin Diseases/pathology , Transcription Factors , Transcription, Genetic , X-Box Binding Protein 1ABSTRACT
Multiple myeloma (MM) is an invariably fatal form of cancer characterized by clonal proliferation of malignant plasma cells in the bone marrow. The canonical Wnt signaling pathway is activated in MM cells through constitutively active beta-catenin, a messenger molecule relevant to growth, survival, and migration of MM cells. The identification of a number of small molecular compounds, such as PKF115-584, which disrupt the interaction of the transcriptionally active beta-catenin/TCF protein complex, provides valuable new therapeutic tools to target an alternative pathway in MM independent of the proteasome. Here we evaluated the transcriptional, proteomic, signaling changes, and biological sequelae associated with the inhibition of Wnt signaling in MM by PKF115-584. The compound blocks expression of Wnt target genes and induces cytotoxicity in both patient MM cells and MM cell lines without a significant effect in normal plasma cells. In xenograft models of human MM, PKF115-584 inhibits tumor growth and prolongs survival. Taken together, these data demonstrate the efficacy of disrupting the beta-catenin/TCF transcriptional complex to exploit tumor dependence on Wnt signaling as a therapeutic approach in the treatment of MM.