ABSTRACT
KRAS, NRAS, and BRAF mutations which activate p44/42 mitogen-activated protein kinase (MAPK) signaling are found in half of myeloma patients and contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms are not fully understood. We established myeloma cell lines expressing wild-type (WT), constitutively active (CA) (G12V/G13D/Q61H), or dominant-negative (DN) (S17N)-KRAS and -NRAS, or BRAF-V600E. Cells expressing CA mutants showed increased proteasome maturation protein (POMP) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) expression. This correlated with an increase in catalytically active proteasome subunit ß (PSMB)-8, PSMB9, and PSMB10, which occurred in an ETS transcription factor-dependent manner. Proteasome chymotrypsin-like, trypsin-like, and caspase-like activities were increased, and this enhanced capacity reduced PI sensitivity, while DN-KRAS and DN-NRAS did the opposite. Pharmacologic RAF or MAPK kinase (MEK) inhibitors decreased proteasome activity, and sensitized myeloma cells to PIs. CA-KRAS, CA-NRAS, and CA-BRAF down-regulated expression of endoplasmic reticulum (ER) stress proteins, and reduced unfolded protein response activation, while DN mutations increased both. Finally, a bortezomib (BTZ)/MEK inhibitor combination showed enhanced activity in vivo specifically in CA-NRAS models. Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue these mutations promote myeloma survival by reducing cellular stress, thereby distancing plasma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma.
Subject(s)
Endoplasmic Reticulum Stress , GTP Phosphohydrolases/metabolism , Membrane Proteins/metabolism , Multiple Myeloma/metabolism , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Apoptosis/drug effects , Bortezomib/pharmacology , Endoplasmic Reticulum Stress/drug effects , GTP Phosphohydrolases/genetics , Humans , Membrane Proteins/genetics , Multiple Myeloma/genetics , Multiple Myeloma/physiopathology , Mutation , Proteasome Endopeptidase Complex/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
Three proteasome inhibitors have garnered regulatory approvals in various multiple myeloma settings; but drug resistance is an emerging challenge, prompting interest in blocking upstream components of the ubiquitin-proteasome pathway. One such attractive target is the E1 ubiquitin-activating enzyme (UAE); we therefore evaluated the activity of TAK-243, a novel and specific UAE inhibitor. TAK-243 potently suppressed myeloma cell line growth, induced apoptosis, and activated caspases while decreasing the abundance of ubiquitin-protein conjugates. This was accompanied by stabilization of many short-lived proteins, including p53, myeloid cell leukemia 1 (MCL-1), and c-MYC, and activation of the activating transcription factor 6 (ATF-6), inositol-requiring enzyme 1 (IRE-1), and protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) arms of the ER stress response pathway, as well as oxidative stress. UAE inhibition showed comparable activity against otherwise isogenic cell lines with wild-type (WT) or deleted p53 despite induction of TP53 signaling in WT cells. Notably, TAK-243 overcame resistance to conventional drugs and novel agents in cell-line models, including bortezomib and carfilzomib resistance, and showed activity against primary cells from relapsed/refractory myeloma patients. In addition, TAK-243 showed strong synergy with a number of antimyeloma agents, including doxorubicin, melphalan, and panobinostat as measured by low combination indices. Finally, TAK-243 was active against a number of in vivo myeloma models in association with activation of ER stress. Taken together, the data support the conclusion that UAE inhibition could be an attractive strategy to move forward to the clinic for patients with relapsed and/or refractory multiple myeloma.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Multiple Myeloma/drug therapy , Proteasome Inhibitors/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Unfolded Protein Response/drug effects , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Drug Synergism , Endoplasmic Reticulum Stress/drug effects , Humans , Oxidative Stress/drug effects , Salvage Therapy/methods , Tumor Cells, Cultured , Tumor Suppressor Protein p53/drug effects , Tumor Suppressor Protein p53/metabolismABSTRACT
Various translocations and mutations have been identified in myeloma, and certain aberrations, such as t(4;14) and del17, are linked with disease prognosis. To investigate mutational prevalence in myeloma and associations between mutations and patient outcomes, we tested a panel of 41 known oncogenes and tumor suppressor genes in tumor samples from 133 relapsed myeloma patients participating in phase 2 or 3 clinical trials of bortezomib. DNA mutations were identified in 14 genes. BRAF as well as RAS genes were mutated in a large proportion of cases (45.9%) and these mutations were mutually exclusive. New recurrent mutations were also identified, including in the PDGFRA and JAK3 genes. NRAS mutations were associated with a significantly lower response rate to single-agent bortezomib (7% vs 53% in patients with mutant vs wild-type NRAS, P = .00116, Bonferroni-corrected P = .016), as well as shorter time to progression in bortezomib-treated patients (P = .0058, Bonferroni-corrected P = .012). However, NRAS mutation did not impact outcome in patients treated with high-dose dexamethasone. KRAS mutation did not reduce sensitivity to bortezomib or dexamethasone. These findings identify a significant clinical impact of NRAS mutation in myeloma and demonstrate a clear example of functional differences between the KRAS and NRAS oncogenes.
Subject(s)
Antineoplastic Agents/therapeutic use , Boronic Acids/therapeutic use , GTP Phosphohydrolases/genetics , Membrane Proteins/genetics , Multiple Myeloma/drug therapy , Mutation , Proto-Oncogene Proteins/genetics , Pyrazines/therapeutic use , ras Proteins/genetics , Bortezomib , Cohort Studies , Dose-Response Relationship, Drug , Humans , Multiple Myeloma/diagnosis , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Prognosis , Proto-Oncogene Proteins p21(ras) , Survival AnalysisABSTRACT
The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.
Subject(s)
Antigens, CD/metabolism , Epithelial Cells/metabolism , Integrin alpha3beta1/metabolism , Protein Tyrosine Phosphatases/metabolism , Animals , Cadherins/metabolism , Cell Adhesion/physiology , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Communication/physiology , Cell Membrane/metabolism , Cells, Cultured , Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , GTP-Binding Proteins , Integrin alpha3beta1/deficiency , Integrin alpha3beta1/genetics , Laminin/metabolism , Macromolecular Substances , Mice , Neoplasm Proteins/metabolism , Protein Kinase C/metabolism , Protein Kinase C beta , Receptor-Like Protein Tyrosine Phosphatases, Class 2 , Receptor-Like Protein Tyrosine Phosphatases, Class 8 , Receptors for Activated C Kinase , Receptors, Cell Surface , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tetraspanin 24 , Trans-Activators/metabolism , beta CateninABSTRACT
Proteasome inhibitors (PIs) are now standard of care for several cancers, and noninvasive biomarkers of treatment response are critically required for early patient stratification and treatment personalization. The present study evaluated whether chemical exchange (CEST) magnetic resonance imaging (MRI) can provide measurements that can be used as the noninvasive biomarkers of proteasome inhibition, alongside diffusion MRI and relaxometry. The sensitivity of human colorectal carcinoma cells to the PI Ixazomib was assessed via in vitro and in vivo dose-response experiments. Acute in vivo response to Ixazomib was assessed at three dosing concentrations, using CEST MRI (amide, amine, hydroxyl signals), diffusion MRI (ADC) and relaxometry (T1, T2). These responses were further evaluated with the known histological markers for Ixazomib and Bradford assay ex vivo. The CEST signal from amides and amines increased in proportion to Ixazomib dose in colorectal cancer xenografts. The cell lines differed in their sensitivity to Ixazomib, which was reflected in the MRI measurements. A mild stimulation in tumor growth was observed at low Ixazomib doses. Our results identify CEST MRI as a promising method for safely and noninvasively monitoring disrupted tumor protein homeostasis induced by proteasome inhibitor treatment, and for stratifying sensitivity between tumor types.
Subject(s)
Magnetic Resonance Imaging , Proteasome Inhibitors/pharmacology , Proteostasis/drug effects , Amides/analysis , Amines/analysis , Animals , Apoptosis/drug effects , Boron Compounds/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Colorectal Neoplasms/pathology , Diffusion Magnetic Resonance Imaging , Dose-Response Relationship, Drug , Female , Glycine/analogs & derivatives , Glycine/pharmacology , Humans , Image Interpretation, Computer-Assisted , Mice, Nude , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.
Subject(s)
Boron Compounds/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Colonic Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , Glycine/analogs & derivatives , Lung Neoplasms/drug therapy , Proteasome Inhibitors/therapeutic use , Proto-Oncogene Proteins p21(ras)/genetics , Amino Acids/metabolism , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Fatty Acids/metabolism , Glucose Transporter Type 4/biosynthesis , Glycine/therapeutic use , HCT116 Cells , Humans , Lung Neoplasms/metabolism , Metabolome/physiology , Mice , Oxidation-Reduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Matrixmetalloproteinases (MMPs) are a family of secreted or membrane-associated proteins capable of digesting extracellular matrix components. The importance of MMPs in tumor development and invasion is well known. Recent studies have strongly indicated the presence of a functional complex consisting of alpha(v)beta3 integrin, membrane type-1 metalloproteinase-2 (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2) on tumor cell surface, which helps the activation of MMP-2. In this article, we report on the association of active MMP-2 with the membrane fraction of human cervical cancer cells. The association of MMP-2 with alpha(v)beta3 integrin and the expression of membrane type MT1-MMP strongly indicate the role of alpha(v)beta3/MT1-MMP/TIMP-2 complex in the activation of MMP-2 in cervical cancer tissue membrane fraction. The membrane-associated activated MMP-2 may have a role in the migration of tumor cells during invasion.
Subject(s)
Cell Transformation, Neoplastic , Gene Expression Regulation, Neoplastic , Matrix Metalloproteinase 2/pharmacology , Tissue Inhibitor of Metalloproteinase-2/pharmacology , Uterine Cervical Neoplasms/enzymology , Uterine Cervical Neoplasms/physiopathology , Cell Membrane , Female , Humans , Neoplasm InvasivenessABSTRACT
Curcumin (diferuloyl methane), the major pigment from the rhizome of Curcuma longa L., has been widely studied for its tumor-inhibiting properties. Recent studies indicate that curcumin can modify cell receptor binding, it also affects intracellular signalling reactions. Curcumin-treated B16F10 melanoma cells formed eight-fold fewer lung metastases in C57BL6 mice. In the cell adhesion assays, curcumin-treated cells showed a dose-dependent reduction in their binding to four extracellular matrix (ECM) proteins. The binding to fibronectin, vitronectin, and collagen IV decreased by over 50% in 24 hours, and by 100% after 48 hours of curcumin treatment, it persisted at this level even after 15 days of cultivating cells in curcumin-free medium. Curcumin-treated cells showed a marked reduction in the expression of alpha5beta1 and alpha(v)beta3 integrin receptors. In addition, curcumin treatment inhibited pp125 focal adhesion kinase (FAK), tyrosine phosphorylation of a 120 kD protein, and collagenase activity. Curcumin enhances the expression of antimetastatic proteins, tissue inhibitor metalloproteinase (TIMP)-2, nonmetastatic gene 23 (Nm23), and E-cadherin. In this article we report on the effect of curcumin on the expression of integrin, TIMP-2, Nm23, E-cadherin, adhesion, and metalloproteinase activity.
Subject(s)
Antineoplastic Agents/pharmacology , Cadherins/biosynthesis , Collagenases/pharmacology , Curcumin/pharmacology , Integrins/metabolism , Lung Neoplasms/secondary , Melanoma/pathology , Skin Neoplasms/pathology , Animals , Cell Adhesion/drug effects , Collagenases/drug effects , Dose-Response Relationship, Drug , Mice , Mice, Inbred C57BL , Neoplasms, Experimental , Signal Transduction , Tissue Inhibitor of Metalloproteinase-3/biosynthesis , Tumor Cells, CulturedABSTRACT
Integrin receptors for the extracellular matrix and receptor tyrosine kinase growth factor receptors represent two of the major families of receptors that transduce into cells information about the surrounding environment. Wnt proteins are a major family of signaling molecules that regulate morphogenetic events. There is presently little understanding of how the expression of Wnt genes themselves is regulated. In this study, we demonstrate that alpha3beta1 integrin, a major laminin receptor involved in the development of the kidney, and c-Met, the receptor for hepatocyte growth factor, signal coordinately to regulate the expression of Wnt7b in the mouse. Wnt signals in turn appear to regulate epithelial cell survival in the papilla of the developing kidney, allowing for the elongation of epithelial tubules to form a mature papilla. Together, these results demonstrate how signals from integrins and growth factor receptors can be integrated to regulate the expression of an important family of signaling molecules so as to regulate morphogenetic events.
Subject(s)
Integrin alpha3beta1/metabolism , Kidney/embryology , Kidney/metabolism , Proto-Oncogene Proteins c-met/metabolism , Proto-Oncogene Proteins/metabolism , Wnt Proteins/metabolism , Animals , Base Sequence , Cell Survival , DNA Primers/genetics , Epithelium/embryology , Epithelium/metabolism , Female , Gene Expression Regulation, Developmental , Hepatocyte Growth Factor/antagonists & inhibitors , Hepatocyte Growth Factor/metabolism , Integrin alpha3beta1/deficiency , Integrin alpha3beta1/genetics , Kidney/cytology , Laminin/deficiency , Laminin/genetics , Laminin/metabolism , Mice , Mice, Knockout , Morphogenesis , Pregnancy , Proto-Oncogene Proteins/genetics , Signal Transduction , Wnt Proteins/geneticsABSTRACT
We report a case of systemic onset juvenile idiopathic arthritis (SOJIA), the manifestations of which started with fever and skin rash followed by arthritis within neonatal age. Such presentation is extremely rare in the newborn. After exclusion of closely mimicking conditions like congenital infections, neonatal onset multisystem inflammatory disease (NOMID), neonatal; lupus erythematosus (NLE) diagnosis of SOJIA may be entertained even in a neonate where arthritis, fever and rash are the presenting features.
Subject(s)
Arthralgia/diagnosis , Arthritis, Juvenile/diagnosis , Exanthema/diagnosis , Arthritis, Juvenile/drug therapy , Diagnosis, Differential , Drug Therapy, Combination , Fever/physiopathology , Follow-Up Studies , Humans , Ibuprofen/administration & dosage , Infant , Pain Measurement , Risk Assessment , Severity of Illness Index , Steroids/administration & dosage , Treatment OutcomeABSTRACT
Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disorder of the childhood and is manifested by synovitis with or without systemic features. Secondary vasculitis occurring in response to JIA is reflected clinically on different structures or systems of the body. Here is reported a rare case of systemic onset JIA (SOJIA) with vasculitis leading to peripheral gangrene.