ABSTRACT
Obesity contributes to many cancers, including breast cancer and multiple myeloma, two cancers that often colonize the bone marrow (BM). Obesity often causes metabolic disease, but at the cellular level, there is uncertainty regarding how these shifts affect cellular phenotypes. Evidence is building that different types of fuel affect tumor cell metabolism, mitochondrial function, and signaling pathways differently, but tumor cells are also flexible and adapt to less-than ideal metabolic conditions, suggesting that single-pronged attacks on tumor metabolism may not be efficacious enough to be effective clinically. In this review, we describe the newest research at the pre-clinical level on how tumor metabolic pathways and energy sources affect cancer cells, with a special focus on multiple myeloma (MM). We also describe the known forward-feedback loops between bone marrow adipocytes (BMAds) and local tumor cells that support tumor growth. We describe how metabolic targets and transcription factors related to fatty acid (FA) oxidation, FA biosynthesis, glycolysis, oxidative phosphorylation (OXPHOS), and other pathways hold great promise as new vulnerabilities in myeloma cells. Specifically, we describe the importance of the acetyl-CoA synthetase (ACSS) and the acyl-CoA synthetase long chain (ACSL) families, which are both involved in FA metabolism. We also describe new data on the importance of lactate metabolism and lactate transporters in supporting the growth of tumor cells in a hypoxic BM microenvironment. We highlight new data showing the dependency of myeloma cells on the mitochondrial pyruvate carrier (MPC), which transports pyruvate to the mitochondria to fuel the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), boosting OXPHOS. Inhibiting the MPC affects myeloma cell mitochondrial metabolism and growth, and synergizes with proteosome inhibitors in killing myeloma cells. We also describe how metabolic signaling pathways intersect established survival and proliferation pathways; for example, the fatty acid binding proteins (FABPs) affect MYC signaling and support growth, survival, and metabolism of myeloma cells. Our goal is to review the current the field so that novel, metabolic-focused therapeutic interventions and treatments can be imagined, developed and tested to decrease the burden of MM and related cancers.
ABSTRACT
Multiple myeloma (MM) is an incurable cancer of plasma cells with a 5-year survival rate of 59%. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression; however, the underlying mechanisms remain unclear. Acyl-CoA synthetase long-chain family members (ACSLs) convert free long-chain fatty acids into fatty acyl-CoA esters and play key roles in catabolic and anabolic fatty acid metabolism. The Cancer Dependency Map data suggested that ACSL3 and ACSL4 were among the top 25% Hallmark Fatty Acid Metabolism genes that support MM fitness. Here, we show that inhibition of ACSLs in human myeloma cell lines using the pharmacological inhibitor Triascin C (TriC) causes apoptosis and decreases proliferation in a dose- and time-dependent manner. RNA-seq of MM.1S cells treated with TriC for 24 h showed a significant enrichment in apoptosis, ferroptosis, and ER stress. Proteomics of MM.1S cells treated with TriC for 48 h revealed that mitochondrial dysfunction and oxidative phosphorylation were significantly enriched pathways of interest, consistent with our observations of decreased mitochondrial membrane potential and increased mitochondrial superoxide levels. Interestingly, MM.1S cells treated with TriC for 24 h also showed decreased mitochondrial ATP production rates and overall lower cellular respiration.
ABSTRACT
BACKGROUND: Multiple myeloma (MM) patients with t(14;20) have a poor prognosis and their outcome has not improved following the introduction of bortezomib (Bzb). The mechanism underlying the resistance to proteasome inhibitors (PIs) for this subset of patients is unknown. METHODS: IC50 of Bzb and carfilzomib (CFZ) in human myeloma cell lines (HMCLs) were established by MTT assay. Gene Expression profile (GEP) analysis was used to determine gene expression in primary myeloma cells. Immunoblotting analysis was performed for MAFb and caspase family proteins. Immunofluorescence staining was used to detect the location of MAFb protein in MM cells. Lentiviral infections were used to knock-down MAFb expression in two lines. Apoptosis detection by flow cytometry and western blot analysis was performed to determine the molecular mechanism MAFb confers resistance to proteasome inhibitors. RESULTS: We found high levels of MAFb protein in cell lines with t(14;20), in one line with t(6;20), in one with Igλ insertion into MAFb locus, and in primary plasma cells from MM patients with t(14;20). High MAFb protein levels correlated with higher IC50s of PIs in MM cells. Inhibition of GSK3ß activity or treatment with Bzb or CFZ prevented MAFb protein degradation without affecting the corresponding mRNA level indicating a role for GSK3 and proteasome inhibitors in regulation of MAFb stability. Silencing MAFb restored sensitivity to Bzb and CFZ, and enhanced PIs-induced apoptosis and activation of caspase-3, - 8, - 9, PARP and lamin A/C suggesting that high expression of MAFb protein leads to insensitivity to proteasome inhibitors. CONCLUSION: These results highlight the role of post-translational modification of MAFb in maintaining its protein level, and identify a mechanism by which proteasome inhibitors induced stabilization of MAFb confers resistance to proteasome inhibitors, and provide a rationale for the development of targeted therapeutic strategies for this subset of patients.
Subject(s)
MafB Transcription Factor/physiology , Multiple Myeloma/drug therapy , Proteasome Inhibitors/therapeutic use , Apoptosis/drug effects , Caspases/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm , Glycogen Synthase Kinase 3/antagonists & inhibitors , Humans , MafB Transcription Factor/analysis , MafB Transcription Factor/genetics , Multiple Myeloma/pathologyABSTRACT
The anabolic action of PTH in bone is mostly mediated by cAMP/PKA and Wnt-independent activation of ß-catenin/T-cell factor (TCF) signaling. ß-Catenin switches the PTH receptor (PTHR) signaling from cAMP/PKA to PLC/PKC activation by binding to the PTHR. Ixazomib (Izb) was recently approved as the first orally administered proteasome inhibitor for the treatment of multiple myeloma; it acts in part by inhibition of pathological bone destruction. Proteasome inhibitors were reported to stabilize ß-catenin by the ubiquitin-proteasome pathway. However, how Izb affects PTHR activation to regulate ß-catenin/TCF signaling is poorly understood. In the present study, using CRISPR/Cas9 genome-editing technology, we show that Izb reverses ß-catenin-mediated PTHR signaling switch and enhances PTH-induced cAMP generation and cAMP response element-luciferase activity in osteoblasts. Izb increases active forms of ß-catenin and promotes ß-catenin translocation, thereby dissociating ß-catenin from the PTHR at the plasma membrane. Furthermore, Izb facilitates PTH-stimulated GSK3ß phosphorylation and ß-catenin phosphorylation. Thus Izb enhances PTH stimulation of ß-catenin/TCF signaling via cAMP-dependent activation, and this effect is due to its separating ß-catenin from the PTHR. These findings provide evidence that Izb may be used to improve the therapeutic efficacy of PTH for the treatment of osteoporosis and other resorptive bone diseases.
Subject(s)
Boron Compounds/metabolism , Glycine/analogs & derivatives , Parathyroid Hormone/metabolism , beta Catenin/drug effects , Animals , Bone and Bones/metabolism , Boron Compounds/pharmacology , Cell Culture Techniques , Cyclic AMP/metabolism , Glycine/genetics , Glycine/metabolism , Glycine/pharmacology , Humans , Mice , Mice, Inbred C57BL , Osteoblasts , Phosphorylation , Proteasome Inhibitors/pharmacology , Receptor, Parathyroid Hormone, Type 1/drug effects , Receptor, Parathyroid Hormone, Type 1/metabolism , Signal Transduction/drug effects , T-Lymphocytes/metabolism , TCF Transcription Factors/metabolism , beta Catenin/metabolismABSTRACT
Objective To study the clinical efficacy of Qingfei Sanjie Pills combined with targeted cryoablation therapy on elderly patients with advanced non-small cell lung cancer (NSCLC) and its influence on the levels of immune function in patients. Methods Totally 94 elderly patients with NSCLC were divided into observation group and control group, with 47 cases in each group. Both groups were given targeted cryoablation therapy and under CT review after surgery to observe lesions condition. Observation group was given Qingfei Sanjie Pills, once a bottle, twice a day, orally, for three months. Recent efficacy of the two groups were evaluated. The levels of IgG, IgA, CD3+, CD4+, CD8+, and complications in the two groups were detected. The survival rate of 1 year, 2 years and 3 years in the two groups were observed. Results Two cases in the observation group and three cases in the control group were invalid. The total effective rate was 84.4% (38/45) in observation group and 65.9% (29/44) in the control group, with statistical significance (P<0.05). After the treatment, the levels of IgG, IgA, CD3+, CD4+and CD8+in the observation group were significantly higher than the control group (P<0.05). After treatment, the incidence rates of increased cough, postoperative pain, bloody sputum, fever and nausea and vomiting in the observation group were lower than the control group. The 1 year, 2 years and 3 years survival rates were 91.1%, 75.6% and 64.4% in observation group, and 79.5%,63.6% and 52.3% in the control group,with statistical significance(P<0.05).Conclusion Qingfei Sanjie Pills combined with targeted cryoablation therapy on elderly patients with advanced NSCLC has good clinical efficacy, which can improve immune system of patients and increase survival rate.
ABSTRACT
Objective To study the clinical efficacy of Qingfei Sanjie Pills combined with targeted cryoablation therapy on elderly patients with advanced non-small cell lung cancer (NSCLC) and its influence on the levels of immune function in patients. Methods Totally 94 elderly patients with NSCLC were divided into observation group and control group, with 47 cases in each group. Both groups were given targeted cryoablation therapy and under CT review after surgery to observe lesions condition. Observation group was given Qingfei Sanjie Pills, once a bottle, twice a day, orally, for three months. Recent efficacy of the two groups were evaluated. The levels of IgG, IgA, CD3+, CD4+, CD8+, and complications in the two groups were detected. The survival rate of 1 year, 2 years and 3 years in the two groups were observed. Results Two cases in the observation group and three cases in the control group were invalid. The total effective rate was 84.4% (38/45) in observation group and 65.9% (29/44) in the control group, with statistical significance (P<0.05). After the treatment, the levels of IgG, IgA, CD3+, CD4+and CD8+in the observation group were significantly higher than the control group (P<0.05). After treatment, the incidence rates of increased cough, postoperative pain, bloody sputum, fever and nausea and vomiting in the observation group were lower than the control group. The 1 year, 2 years and 3 years survival rates were 91.1%, 75.6% and 64.4% in observation group, and 79.5%,63.6% and 52.3% in the control group,with statistical significance(P<0.05).Conclusion Qingfei Sanjie Pills combined with targeted cryoablation therapy on elderly patients with advanced NSCLC has good clinical efficacy, which can improve immune system of patients and increase survival rate.
ABSTRACT
Multiple myeloma (MM) patients with the t(14;16) translocation have a poor prognosis, and unlike other molecular subgroups, their outcome has not improved with the introduction of bortezomib (Bzb). The mechanism underlying innate resistance of MM to Bzb is unknown. In the present study, we have investigated how MAF overexpression impacts resistance to proteasome inhibitor (PI) therapy (Bzb and carfilzomib). High levels of MAF protein were found in t(14;16) cell lines; cell lines from the t(4;14) subgroup had intermediate levels, whereas cell lines from the other subgroups had low levels. High expression of MAF protein in t(14;16) was associated with significantly higher PI half-maximum inhibitory concentration values compared with other molecular subgroups. PI exposure abrogated glycogen synthase kinase 3ß (GSK3ß)-mediated degradation of MAF protein, resulting in increased MAF protein stability and PI resistance. Subsequent studies using loss-of-function and gain-of-function models showed that silencing MAF led to increased sensitivity to PIs, enhanced apoptosis, and activation of caspase-3, -7, -8, -9, poly (ADP-ribose) polymerase, and lamin A/C. In contrast, overexpression of MAF resulted in increased resistance to PIs and reduced apoptosis. These results define the role of MAF and GSK3 in the resistance of t(14;16) MM to PIs and identifies a novel mechanism by which MAF protein levels are regulated by PIs, which in turn confers resistance to PIs.
Subject(s)
Drug Resistance, Neoplasm , Immunity, Innate , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Proteasome Inhibitors/therapeutic use , Proto-Oncogene Proteins c-maf/metabolism , Apoptosis/drug effects , Caspases/metabolism , Cell Line, Tumor , Chromosomes, Human, Pair 14/genetics , Chromosomes, Human, Pair 16/genetics , Drug Resistance, Neoplasm/drug effects , Enzyme Activation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Gene Silencing/drug effects , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Immunity, Innate/drug effects , Lamins/metabolism , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Phosphorylation/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Prognosis , Proteasome Inhibitors/pharmacology , Proteolysis/drug effects , Proto-Oncogene Proteins c-maf/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Translocation, GeneticABSTRACT
We have sequenced 463 presenting cases of myeloma entered into the UK Myeloma XI study using whole exome sequencing. Here we identify mutations induced as a consequence of misdirected AID in the partner oncogenes of IGH translocations, which are activating and associated with impaired clinical outcome. An APOBEC mutational signature is seen in 3.8% of cases and is linked to the translocation-mediated deregulation of MAF and MAFB, a known poor prognostic factor. Patients with this signature have an increased mutational load and a poor prognosis. Loss of MAF or MAFB expression results in decreased APOBEC3B and APOBEC4 expression, indicating a transcriptional control mechanism. Kataegis, a further mutational pattern associated with APOBEC deregulation, is seen at the sites of the MYC translocation. The APOBEC mutational signature seen in myeloma is, therefore, associated with poor prognosis primary and secondary translocations and the molecular mechanisms involved in generating them.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Multiple Myeloma/genetics , Translocation, Genetic/genetics , Adult , Aged , Aged, 80 and over , Cytidine Deaminase/genetics , Female , Gene Expression Profiling , Humans , MafB Transcription Factor/genetics , Male , Middle Aged , Minor Histocompatibility Antigens , Mutation , Prognosis , Proto-Oncogene Proteins c-maf/genetics , Proto-Oncogene Proteins c-myc/geneticsABSTRACT
Carfilzomib, the next generation of proteasome inhibitor, may increase osteoblast-related markers in patients with multiple myeloma, but the molecular mechanism of its effect on mesenchymal stem cell differentiation to osteoblasts remains unknown. Herein, we demonstrated that carfilzomib significantly promoted mesenchymal stem cell differentiation into osteoblasts. In osteoprogenitor cells and primary mesenchymal stem cells from patients with myeloma, carfilzomib induced increases in alkaline phosphatase activity, matrix mineralization, and calcium deposition via Wnt-independent activation of ß-catenin/TCF signaling. Using affinity pull-down assays with immunoblotting analysis and immunofluorescence, we found that carfilzomib induced stabilization of both free and active forms of ß-catenin in a time- and dose-dependent manner that was not associated with ß-catenin transcriptional regulation. Nuclear translocation of ß-catenin protein was associated with TCF transcriptional activity that was independent of the effects of GSK3ß-activation and of signaling induced by 19 Wnt ligands, 10 Frizzled receptors, and LRP5/6 co-receptors. Blocking activation of ß-catenin/TCF signaling by dominant negative TCF1 or TCF4 attenuated carfilzomib-induced matrix mineralization. Thus, carfilzomib induced osteoblast differentiation via Wnt-independent activation of the ß-catenin/TCF pathway. These results provide a novel molecular mechanism critical to understanding the anabolic role of carfilzomib on myeloma-induced bone disease.
Subject(s)
Multiple Myeloma/drug therapy , Oligopeptides/therapeutic use , beta Catenin/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Multiple Myeloma/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , beta Catenin/geneticsABSTRACT
Bone disease in patients with multiple myeloma (MM) is characterized by increase in the numbers and activity of bone-resorpting osteoclasts and decrease in the number and function of bone-formation osteoblasts. MM-triggered inhibition of bone formation may stem from suppression of Wnt/ß-catenin signaling, a pivotal pathway in the differentiation of mesenchymal stem cells (MSC) into osteoblasts, and regulating production of receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) axis by osteoblasts. Proteasome inhibitors (PIs), such as bortezomib (Bz), induce activation of Wnt/ß-catenin pathway and MSC differentiation toward osteoblasts. PIs also suppress osteoclastogenesis, possibly through regulating multiple pathways including NF-κB, Bim, and the ratio of RANKL/OPG. The critical role of PI in increasing osteoblast function and suppression of osteoclast activity is highlighted by clinical evidence of increases in bone formation and decreases in bone resorption makers. This review will discuss the function of PIs in stimulating bone formation and suppression of bone resorption, and the mechanism underlying this process that leads to inhibition bone disease in MM patients.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bone Diseases/drug therapy , Multiple Myeloma/drug therapy , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/therapeutic use , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bone Diseases/complications , Bone Diseases/enzymology , Bone Diseases/pathology , Humans , Multiple Myeloma/complications , Multiple Myeloma/enzymology , Multiple Myeloma/pathology , Osteoclasts/drug effects , Osteoclasts/pathology , Proteasome Inhibitors/pharmacologyABSTRACT
We recently showed that increasing Wnt/beta-catenin signalling in the bone marrow microenvironment or in multiple myeloma (MM) cells clearly suppresses osteoclastogenesis in SCID-hu mice; however, this regulation of osteoclastogenesis could result directly from activation of Wnt/beta-catenin signalling in osteoclasts or indirectly from effects on osteoblasts. The present studies characterized Wnt/beta-catenin signalling and its potential role in osteoclasts. Systematic analysis of expression of WNT, FZD, LRP and TCF gene families demonstrated that numerous Wnt-signalling components were expressed in human osteoclasts from patients with MM. Functional Wnt/beta-catenin signalling was identified by accumulation of total and active beta-catenin and increases in Dvl-3 protein in response to Wnt3a or LiCl. Furthermore, Wnt-induced increases in beta-catenin and Dvl-3 were attenuated by Wnt antagonists Dkk1 and sFRP1. Finally, Wnt3a-induced TCF/LEF transcriptional activity suggests that canonical Wnt signalling is active in osteoclasts. Supernatants from dominant-negative-beta-catenin-expressing osteoblast clones significantly stimulated tartrate-resistant acid phosphatase-positive osteoclast formation from primary MM-derived osteoclasts, compared with supernatants from control cells. These results suggested that Wnt/beta-catenin signalling is active in osteoclasts in MM and is involved in osteoclastogenesis in bone marrow, where it acts as a negative regulator of osteoclast formation in an osteoblast-dependent manner in MM.
Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Multiple Myeloma/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line, Tumor , Dishevelled Proteins , Frizzled Receptors/genetics , Frizzled Receptors/metabolism , Genes, Reporter/physiology , Humans , Intercellular Signaling Peptides and Proteins/genetics , Luciferases/metabolism , Mice , Multiple Myeloma/genetics , Osteogenesis/physiology , Phosphoproteins/metabolism , Signal Transduction/physiology , Wnt Proteins/antagonists & inhibitors , Wnt Proteins/genetics , beta Catenin/geneticsABSTRACT
Inhibition of Wnt/beta-catenin/T-cell factor (TCF) signaling induces proliferation of mesenchymal stem cells and/or suppresses their differentiation into osteoblasts (OBs). Osteolysis in multiple myeloma (MM) is related to the suppression of canonical Wnt signaling caused by DKK1, a soluble inhibitor of this pathway secreted by MM cells. Bortezomib (Bzb) can induce OB differentiation in vitro and in vivo and its anti-MM efficacy linked to bone anabolic effects. However, the molecular basis of the action of Bzb on bone is not completely understood. In the present study, we show that Bzb promotes matrix mineralization and calcium deposition by osteoprogenitor cells and primary mesenchymal stem cells via Wnt-independent activation of beta-catenin/TCF signaling. Using affinity pull-down assays with immunoblotting and immunofluorescence, we found that Bzb induced stabilization of beta-catenin. Nuclear translocation of stabilized beta-catenin was associated with beta-catenin/TCF transcriptional activity that was independent of the effects of Wnt ligand-receptor-induced signaling or GSK3beta activation. Blocking the activation of beta-catenin/TCF signaling by dominant negative TCF attenuated Bzb-induced matrix mineralization. These results provide evidence that Bzb induces OB differentiation via Wnt-independent activation of beta-catenin/TCF pathway and suggest that proteasome inhibition therapy in MM may function in part by subverting tumor-induced suppression of canonical Wnt signaling in the bone microenvironment.
Subject(s)
Boronic Acids/pharmacology , Cell Differentiation/drug effects , Osteoblasts/cytology , Pyrazines/pharmacology , Signal Transduction , TCF Transcription Factors/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , Animals , Bortezomib , Cadherins/metabolism , Calcification, Physiologic , Calcium/metabolism , Cell Nucleus/metabolism , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/metabolism , Fluorescent Antibody Technique , Humans , Immunoblotting , Luciferases/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Osteoblasts/metabolism , Protease Inhibitors/pharmacology , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/metabolism , TCF Transcription Factors/genetics , Wnt Proteins/genetics , beta Catenin/geneticsABSTRACT
Wnt/beta-catenin signaling is central to bone development and homeostasis in adulthood and its deregulation is associated with bone pathologies. Dickkopf-1 (DKK1), a soluble inhibitor of Wnt/beta-catenin signaling required for embryonic head development, regulates Wnt signaling by binding to the Wnt coreceptor lipoprotein-related protein-5 (LRP5)/Arrow. LRP5 mutations causing high bone mass syndromes disrupt DKK1-mediated regulation of LRP5. Forced overexpression of Dkk1 in osteoblasts causes osteopenia, disruption of the hematopoietic stem cell (HSC) niche, and defects in HSC function. Dkk1 also inhibits fracture repair. Studies suggest that DKK1 activation in osteoblasts is the underlying cause of glucocorticoid- and estrogen deficiency-mediated osteoporosis, and at least partially underlies the teratogenic effects of thalidomide on limb development. DKK1 induces proliferation of mesenchymal stem cells (MSC) in vitro and may play a role in the development of high-grade undifferentiated pleomorphic sarcomas derived from MSC and osteosarcomas. DKK1 has been implicated in causing erosive arthritis, the osteolytic phenotypes of multiple myeloma and metastatic breast cancer, and osteoblastic metastases of prostate cancer. Preclinical studies have shown that neutralizing DKK1/Dkk1 and/or enhancing Wnt/beta-catenin signaling may prove effective in treating bone pathologies. Here, we review the rapidly growing body of literature defining a pivotal role for DKK1 in bone health and disease.
Subject(s)
Bone Development/physiology , Bone Diseases/physiopathology , Bone and Bones/physiology , Homeostasis/physiology , Intercellular Signaling Peptides and Proteins/physiology , Animals , HumansABSTRACT
Canonical Wnt signaling is central to normal bone homeostasis, and secretion of Wnt signaling inhibitors by multiple myeloma (MM) cells contributes to MM-related bone resorption and disease progression. The aim of this study was to test the effect of Wnt3a on bone disease and growth of MM cells in vitro and in vivo. Although Wnt3a activated canonical signaling in the majority of MM cell lines and primary cells tested, Wnt3a had no effect on MM cell growth in vitro. Moreover, forced expression of Wnt3a in H929 MM cells conferred no growth advantage over empty vector-transfected cells in vitro or importantly when grown subcutaneously in severe combined immunodeficient (SCID) mice. Importantly, although H929 cells stably expressing an empty vector injected into human bone grew rapidly and induced a marked reduction in bone mineral density, bones engrafted with Wnt3a-expressing H929 cells were preserved, exhibited increased osteoblast-to-osteoclast ratios, and reduced tumor burden. Likewise, treatment of myelomatous SCID-hu mice, carrying primary disease, with recombinant Wnt3a stimulated bone formation and attenuated MM growth. These results provide further support of the potential anabolic and anti-MM effects of enhancing Wnt signaling in the bone.
Subject(s)
Bone Diseases/pathology , Multiple Myeloma/pathology , Signal Transduction , Wnt Proteins/metabolism , Animals , Bone and Bones/metabolism , Humans , Mice , Mice, SCID , Multiple Myeloma/complications , Transplantation, Heterologous , Wnt3 Protein , Wnt3A ProteinABSTRACT
Multiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell-derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios.
Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Multiple Myeloma/complications , Multiple Myeloma/metabolism , Osteoblasts/metabolism , Osteolysis/etiology , Osteolysis/metabolism , Osteoprotegerin/biosynthesis , RANK Ligand/biosynthesis , Wnt Proteins/metabolism , Animals , Base Sequence , Cell Differentiation , Cell Line , Cell Line, Tumor , Coculture Techniques , DNA Primers/genetics , Gene Expression , Gene Silencing , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/pharmacology , Mice , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Osteoblasts/pathology , Osteolysis/genetics , Osteolysis/pathology , Osteoprotegerin/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tumor Cells, Cultured , Wnt3 Protein , Wnt3A ProteinABSTRACT
Expression of the Wnt signaling inhibitor, DKK1 by multiple myeloma cells is correlated with lytic bone disease in multiple myeloma. However, the mechanism(s) by which DKK1 contributes to this process is not clear. Herein, we analyzed the functional role of canonical Wnt signaling and Dkk1 inhibition of this pathway in bone morphogenic protein (BMP)-2-induced osteoblast differentiation. Osteoblast differentiation was measured by alkaline phosphatase (ALP) activity in murine (C2C12) and human pre-osteoblast (hFOB1.19) and osteoblast-like (Saos-2 and MG63) cell lines. Cytoplasmic beta-catenin protein was separated by E-cadherin-GST pull-down assay and analyzed by Western blotting. A dominant negative form of beta-catenin, Dkk1 and TCF reporter constructs were transfected into C2C12 cells. C2C12 cells were also transfected with siRNA specific to LRP5/6 to knockdown receptor expression. Canonical Wnt signaling was activated in these cell lines in response to Wnt3a as assessed by increased cytoplasmic, non-phosphorylated beta-catenin and TCF/LEF transcription activity. Recombinant Dkk1 and plasma from MM patients containing high levels of Dkk1 blocked Wnt3a-induced beta-catenin accumulation. Importantly, Dkk1 abrogated BMP-2 mediated osteoblast differentiation. The requirement for Wnt signaling in osteoblast differentiation was confirmed by the following observations: 1) overexpression of Dkk1 decreased endogenous beta-catenin and ALP activity; 2) silencing of Wnt receptor mRNAs blocked ALP activity; and 3) a dominant negative form of beta-catenin eliminated BMP-2-induced ALP activity. Furthermore, Wnt3a did not increase ALP activity nor did BMP-2 treatment result in beta-catenin stabilization indicating that cooperation between these two pathways is required, but they are not co-regulated by either ligand. These studies have revealed that autocrine Wnt signaling in osteoblasts is necessary to promote BMP-2-mediated differentiation of pre-osteoblast cells, while Wnt signaling alone is not capable of inducing such differentiation. Dkk1 inhibits this process and may be a key factor regulating pre-osteoblast differentiation and myeloma bone disease.
Subject(s)
Bone Diseases, Metabolic/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Multiple Myeloma/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Wnt Proteins/metabolism , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/metabolism , Cell Differentiation , Cell Line , Core Binding Factor Alpha 1 Subunit/metabolism , Humans , Intercellular Signaling Peptides and Proteins/genetics , LDL-Receptor Related Proteins/genetics , LDL-Receptor Related Proteins/metabolism , Low Density Lipoprotein Receptor-Related Protein-5 , Low Density Lipoprotein Receptor-Related Protein-6 , Mice , RNA, Small Interfering/genetics , Signal Transduction , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolism , beta Catenin/metabolismABSTRACT
Multiple myeloma is an incurable form of lymphoid cancer characterized by accumulation of neoplastic plasma cells in the bone marrow cavity. Little is known about the mechanisms regulating myeloma cell movement within the bone marrow and metastasis to secondary sites. Herein, we identify multiple members of the wingless/int (Wnt) family as promoters of myeloma cell migration/invasion. Wnt-mediated migration was associated with the Wnt/RhoA pathway and did not necessitate signaling through beta-catenin. Activation of both RhoA and members of the protein kinase C (PKC) family, including PKCalpha, PKCbeta, and PKCmu, were required for induction of migration. Activated RhoA and PKCalpha, PKCbeta, and PKCmu appear to assemble in macromolecular signaling complexes that are associated with the cell membrane. These results suggest that Wnt responsiveness of myeloma plasma cells may be a significant factor in disease progression.
Subject(s)
Cell Movement/drug effects , Intercellular Signaling Peptides and Proteins/pharmacology , Multiple Myeloma/pathology , Neoplasm Invasiveness/physiopathology , Cell Line, Tumor , Cell Movement/physiology , Humans , Intercellular Signaling Peptides and Proteins/physiology , Macromolecular Substances/metabolism , Multiple Myeloma/metabolism , Protein Kinase C/physiology , Signal Transduction/drug effects , Signal Transduction/physiology , Wnt Proteins , rhoA GTP-Binding Protein/metabolismABSTRACT
Wnt signaling has been shown to be critical for proper embryonic development as well as growth regulation of certain adult tissues. Defects in Wnt pathways have additionally been associated with a number of human cancers. However, it is only recently that a role for Wnts in the immune system has come to be appreciated. Wnts have now been shown to play significant roles in early stage development of both B and T lineage cells. Current studies suggest that proliferation and/or survival of these cells is associated with activation of the 'canonical' Wnt/beta-catenin pathway. Functional Wnt signaling appears to also occur in end stage B (plasma) cells where both the 'canonical' and the Wnt/RhoA pathways are activated. Herein, we review the current understanding of Wnt signaling in B and T cell development and the potential involvement of Wnt cascades in lymphoid neoplasia.
Subject(s)
B-Lymphocytes/physiology , Proto-Oncogene Proteins/physiology , Signal Transduction/physiology , T-Lymphocytes/physiology , Zebrafish Proteins , Animals , Humans , Wnt ProteinsABSTRACT
Multiple myeloma (MM) is an incurable form of cancer characterized by accumulation of malignant plasma cells in the bone marrow. During the course of this disease, tumor cells cross endothelial barriers and home to the bone marrow. In latter stages, myeloma cells extravasate through blood vessels and may seed a variety of organs. Insulin-like growth factor I (IGF-I) is one of several growth factors shown to promote the growth of MM cells. In the current study, we have assessed the ability of IGF-I to serve additionally as a chemotactic factor affecting the mobility and invasive properties of these cells. Results indicate that IGF-I promotes transmigration through vascular endothelial cells and bone marrow stromal cell lines. Analysis of endogenous signaling pathways revealed that protein kinase D/protein kinase Cmicro (PKD/PKCmicro) and RhoA were both activated in a phosphatidylinositol 3-kinase (PI-3K)-dependent manner. Inhibition of PI-3K, PKCs, or Rho-associated kinase by pharmacologic inhibitors abrogated migration, whereas mitogen-activated protein kinase (MAPK), Akt, and p70S6 kinase inhibitors had no effect. These results suggest that IGF-I promotes myeloma cell migration by activation of PI-3K/PKCmicro and PI-3K/RhoA pathways independent of Akt. The identification of IGF-I as both a proliferative and migratory factor provides a rational basis for the development of targeted therapeutic strategies directed at IGF-I in the treatment of MM.
Subject(s)
Insulin-Like Growth Factor I/pharmacology , Multiple Myeloma/pathology , 3-Phosphoinositide-Dependent Protein Kinases , Cell Division/drug effects , Cell Division/physiology , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/physiology , Enzyme Inhibitors/pharmacology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Insulin-Like Growth Factor I/physiology , Multiple Myeloma/metabolism , Neoplasm Invasiveness/physiopathology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , rhoA GTP-Binding Protein/biosynthesisABSTRACT
Wnts comprise a family of secreted proteins that interact with receptors consisting of a Frizzled (Fz) family member alone or complexed with LDL receptor-related proteins (LRP5/6). Wnt signaling plays a crucial role in both development and differentiation, and activation of a 'canonical' Wnt pathway resulting in beta-catenin stabilization is associated with several types of human cancers. To date, little is known about potential Wnt signaling in mature lymphocytes or lymphoid neoplasia. Herein, we have analysed Wnt signaling in mature B cells (lymphomas) and plasma cells (multiple myeloma). Both Fz and LRP5/6 mRNAs were expressed in myeloma lines, but LRP5/6 were not observed in lymphomas. In myelomas, a canonical Wnt signaling pathway was activated following treatment with Wnt-3a as assessed by accumulation of beta-catenin, but beta-catenin levels actually decreased in lymphoma cells. Wnt-3a treatment further led to striking morphological changes in myeloma cells accompanied by rearrangement of the actin cytoskeleton. Morphological changes were associated with a second Wnt pathway dependent on Rho activation. These results suggest that Wnt responsiveness is a stage-specific phenomenon in B-cell development and that the morphological changes associated with Wnt signaling may play a role in the motility and metastatic potential of myeloma cells.