ABSTRACT
Initiation of B-cell receptor (BCR) 1 signaling, and subsequent antigen-encounter in germinal centers 2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) 4 and lymphoma 5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells 6-9 , the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ 10 - 12 , SHIP1 13,14 and SHP1 14, 15,16 , conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268 ). Genetic rescue experiments identified CD25-S 268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25 S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca 2+ -oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.
ABSTRACT
In most cell types, nuclear ß-catenin functions as prominent oncogenic driver and pairs with TCF7-family factors for transcriptional activation of MYC. Surprisingly, B-lymphoid malignancies not only lacked expression and activating lesions of ß-catenin but critically depended on GSK3ß for effective ß-catenin degradation. Our interactome studies in B-lymphoid tumors revealed that ß-catenin formed repressive complexes with lymphoid-specific Ikaros factors at the expense of TCF7. Instead of MYC-activation, ß-catenin was essential to enable Ikaros-mediated recruitment of nucleosome remodeling and deacetylation (NuRD) complexes for transcriptional repression of MYC. To leverage this previously unrecognized vulnerability of B-cell-specific repressive ß-catenin-Ikaros-complexes in refractory B-cell malignancies, we examined GSK3ß small molecule inhibitors to subvert ß-catenin degradation. Clinically approved GSK3ß-inhibitors that achieved favorable safety prof les at micromolar concentrations in clinical trials for neurological disorders and solid tumors were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of ß-catenin, repression of MYC and acute cell death. Preclinical in vivo treatment experiments in patient-derived xenografts validated small molecule GSK3ß-inhibitors for targeted engagement of lymphoid-specific ß-catenin-Ikaros complexes as a novel strategy to overcome conventional mechanisms of drug-resistance in refractory malignancies. HIGHLIGHTS: Unlike other cell lineages, B-cells express nuclear ß-catenin protein at low baseline levels and depend on GSK3ß for its degradation.In B-cells, ß-catenin forms unique complexes with lymphoid-specific Ikaros factors and is required for Ikaros-mediated tumor suppression and assembly of repressive NuRD complexes. CRISPR-based knockin mutation of a single Ikaros-binding motif in a lymphoid MYC superenhancer region reversed ß-catenin-dependent Myc repression and induction of cell death. The discovery of GSK3ß-dependent degradation of ß-catenin as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3ß-inhibitors for the treatment of refractory B-cell malignancies. GRAPHICAL ABSTRACT: Abundant nuclear ß-cateninß-catenin pairs with TCF7 factors for transcriptional activation of MYCB-cells rely on efficient degradation of ß-catenin by GSK3ßB-cell-specific expression of Ikaros factors Unique vulnerability in B-cell tumors: GSK3ß-inhibitors induce nuclear accumulation of ß-catenin.ß-catenin pairs with B-cell-specific Ikaros factors for transcriptional repression of MYC.
ABSTRACT
SYK and ZAP70 nonreceptor tyrosine kinases serve essential roles in initiating B-cell receptor (BCR) and T-cell receptor (TCR) signaling in B- and T-lymphocytes, respectively. Despite their structural and functional similarity, expression of SYK and ZAP70 is strictly separated during B- and T-lymphocyte development, the reason for which was not known. Aberrant co-expression of ZAP70 with SYK was first identified in B-cell chronic lymphocytic leukemia (CLL) and is considered a biomarker of aggressive disease and poor clinical outcomes. We recently found that aberrant ZAP70 co-expression not only functions as an oncogenic driver in CLL but also in various other B-cell malignancies, including acute lymphoblastic leukemia (B-ALL) and mantle cell lymphoma. Thereby, aberrantly expressed ZAP70 redirects SYK and BCR-downstream signaling from NFAT towards activation of the PI3K-pathway. In the sole presence of SYK, pathological BCR-signaling in autoreactive or premalignant cells induces NFAT-activation and NFAT-dependent anergy and negative selection. In contrast, negative selection of pathological B-cells is subverted when ZAP70 diverts SYK from activation of NFAT towards tonic PI3K-signaling, which promotes survival instead of cell death. We discuss here how both B-cell malignancies and autoimmune diseases frequently evolve to harness this mechanism, highlighting the importance of developmental separation of the two kinases as an essential safeguard.
Subject(s)
Autoimmune Diseases , Leukemia, Lymphocytic, Chronic, B-Cell , Adult , Autoimmunity , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Antigen, B-Cell , Syk Kinase , ZAP-70 Protein-Tyrosine KinaseABSTRACT
Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies.
Subject(s)
Autoimmunity , Neoplasms/enzymology , Neoplasms/prevention & control , Syk Kinase/metabolism , ZAP-70 Protein-Tyrosine Kinase/metabolism , Animals , Antigens, CD19/metabolism , B-Lymphocytes , Calcium/metabolism , Cell Differentiation , Cell Transformation, Neoplastic , Enzyme Activation , Humans , Immune Tolerance , Lymphoma, B-Cell/enzymology , Lymphoma, B-Cell/pathology , Mice , Models, Genetic , NFATC Transcription Factors/metabolism , Neoplasm Proteins , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Receptors, Antigen, B-Cell/metabolism , Signal TransductionABSTRACT
Unlike other cell types, developing B cells undergo multiple rounds of somatic recombination and hypermutation to evolve high-affinity antibodies. Reflecting the high frequency of DNA double-strand breaks, adaptive immune protection by B cells comes with an increased risk of malignant transformation. B lymphoid transcription factors (e.g., IKZF1 and PAX5) serve as metabolic gatekeepers by limiting glucose to levels insufficient to fuel transformation. We here identified aberrant expression of the lactonase PON2 in B cell acute lymphoblastic leukemia (B-ALL) as a mechanism to bypass metabolic gatekeeper functions. Compared to normal pre-B cells, PON2 expression was elevated in patient-derived B-ALL samples and correlated with poor clinical outcomes in pediatric and adult cohorts. Genetic deletion of Pon2 had no measurable impact on normal B cell development. However, in mouse models for BCR-ABL1 and NRASG12D-driven B-ALL, deletion of Pon2 compromised proliferation, colony formation, and leukemia initiation in transplant recipient mice. Compromised leukemogenesis resulted from defective glucose uptake and adenosine triphosphate (ATP) production in PON2-deficient murine and human B-ALL cells. Mechanistically, PON2 enabled glucose uptake by releasing the glucose-transporter GLUT1 from its inhibitor stomatin (STOM) and genetic deletion of STOM largely rescued PON2 deficiency. While not required for glucose transport, the PON2 lactonase moiety hydrolyzes the lactone-prodrug 3OC12 to form a cytotoxic intermediate. Mirroring PON2 expression levels in B-ALL, 3OC12 selectively killed patient-derived B-ALL cells but was well tolerated in transplant recipient mice. Hence, while B-ALL cells critically depend on aberrant PON2 expression to evade metabolic gatekeeper functions, PON2 lactonase activity can be leveraged as synthetic lethality to overcome drug resistance in refractory B-ALL.
Subject(s)
Aryldialkylphosphatase/metabolism , B-Lymphocytes/metabolism , Carcinogenesis/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Adenosine Triphosphate/metabolism , Animals , Aryldialkylphosphatase/genetics , Carcinogenesis/genetics , Cell Line, Tumor , Cells, Cultured , Glucose/metabolism , Glucose Transporter Type 1/metabolism , Humans , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein BindingABSTRACT
Interferon-induced transmembrane protein 3 (IFITM3) has previously been identified as an endosomal protein that blocks viral infection1-3. Here we studied clinical cohorts of patients with B cell leukaemia and lymphoma, and identified IFITM3 as a strong predictor of poor outcome. In normal resting B cells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the B cell receptor (BCR) induced both expression of IFITM3 and phosphorylation of this protein at Tyr20, which resulted in the accumulation of IFITM3 at the cell surface. In B cell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Ifitm3-/- naive B cells developed in normal numbers; however, the formation of germinal centres and the production of antigen-specific antibodies were compromised. Oncogenes that induce the development of leukaemia and lymphoma did not transform Ifitm3-/- B cells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant B cells. Mechanistic experiments revealed that IFITM3 functions as a PIP3 scaffold and central amplifier of PI3K signalling. The amplification of PI3K signals depends on IFITM3 using two lysine residues (Lys83 and Lys104) in its conserved intracellular loop as a scaffold for the accumulation of PIP3. In Ifitm3-/- B cells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60 lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after B cells encounter antigen induces a dynamic switch from antiviral effector functions in endosomes to a PI3K amplification loop at the cell surface. IFITM3-dependent amplification of PI3K signalling, which in part acts downstream of the BCR, is critical for the rapid expansion of B cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signalling complexes and amplify PI3K signalling for malignant transformation.
Subject(s)
B-Lymphocytes/metabolism , Membrane Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol Phosphates/metabolism , RNA-Binding Proteins/metabolism , Signal Transduction , Animals , Antigens, CD19/metabolism , B-Lymphocytes/enzymology , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Transformation, Neoplastic , Female , Germinal Center/cytology , Germinal Center/immunology , Germinal Center/pathology , Humans , Integrins/metabolism , Membrane Microdomains/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Models, Molecular , Phosphorylation , Receptors, Antigen, B-Cell/metabolismABSTRACT
Malignant transformation of cells typically involves several genetic lesions, whose combined activity gives rise to cancer1. Here we analyse 1,148 patient-derived B-cell leukaemia (B-ALL) samples, and find that individual mutations do not promote leukaemogenesis unless they converge on one single oncogenic pathway that is characteristic of the differentiation stage of transformed B cells. Mutations that are not aligned with this central oncogenic driver activate divergent pathways and subvert transformation. Oncogenic lesions in B-ALL frequently mimic signalling through cytokine receptors at the pro-B-cell stage (via activation of the signal-transduction protein STAT5)2-4 or pre-B-cell receptors in more mature cells (via activation of the protein kinase ERK)5-8. STAT5- and ERK-activating lesions are found frequently, but occur together in only around 3% of cases (P = 2.2 × 10-16). Single-cell mutation and phospho-protein analyses reveal the segregation of oncogenic STAT5 and ERK activation to competing clones. STAT5 and ERK engage opposing biochemical and transcriptional programs that are orchestrated by the transcription factors MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway comes at the expense of the principal oncogenic driver and reverses transformation. Conversely, deletion of divergent pathway components accelerates leukaemogenesis. Thus, persistence of divergent signalling pathways represents a powerful barrier to transformation, while convergence on one principal driver defines a central event in leukaemia initiation. Pharmacological reactivation of suppressed divergent circuits synergizes strongly with inhibition of the principal oncogenic driver. Hence, reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to enhance treatment responses.
Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Cell Transformation, Neoplastic , Leukemia, B-Cell/metabolism , Leukemia, B-Cell/pathology , Signal Transduction , Animals , B-Lymphocytes/pathology , Cell Line, Tumor , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Mice , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Proto-Oncogene Proteins c-bcl-6/metabolism , Proto-Oncogene Proteins c-myc/metabolism , STAT5 Transcription Factor/metabolismABSTRACT
Understanding cellular heterogeneity is a challenge for cell biology, particularly in the fields of stem cell and cancer biology. We recently established a reverse phase protein array (RPPA)-modified method, colony lysate array (CoLA), which enables proteomic profiling of individual subpopulations (i.e., colonies) derived from various cell proliferative conditions. Here we describe two independent CoLA assays for a functional subpopulation, drug-tolerant persisters (DTPs), which are considered to mimic the origin of chemotherapeutic cancer relapse. In the first study, we analyzed individual DTPs derived from different cell types grown in the presence of different drugs. A hierarchical clustering analysis of DTPs using CoLA revealed two types of clonal populations, including those that expressed stem cell-associated proteins as well as epithelium-associated proteins. Subsequent principal component analyses demonstrated that the DTPs clustered on the basis of their proteomic profiles, which could change in response to drugs and doses. Another study was designed to identify signaling pathways that may be associated with drug resistance using a pair of 5-fluorouracil-tolerant and parental gastric cancer cell lines. Although a hierarchical clustering analysis did not show any specific clusters for the tolerant line, a drug dose-response analysis revealed that phosphorylation of PI3K and AKT increased and decreased, respectively, specifically in the tolerant cell line. These frameworks that readily profile small subpopulations isolated from an external stresses may be applicable to a wide variety of heterogeneous cellular samples.
Subject(s)
Protein Array Analysis , Proteomics , Cell Line, Tumor , Humans , Neoplasm Recurrence, Local , Protein Array Analysis/methods , Proteomics/methods , Signal Transduction/geneticsABSTRACT
Functional heterogeneity of cancer cells is one of the key properties to understanding relapse after drug treatment. Hence, clarification is needed with regard to which types of subgroups of cancer cells dominantly contribute to the initiation of relapse. Recently, we established the colony lysate array (CoLA), which is a method that allows comparison of individual colonies at the protein level to assess the initiation of anticancer drug-tolerant persisters (DTPs) based on the reverse-phase protein array (RPPA) system. DTPs grow in various drug concentrations and types showing 2-dimensional growth (â¼1 mm) on a flat surface. The size of DTPs are larger than spheroids (â¼0.3 mm) in agarose gel, which makes them easy to handle for a number of assays. DTPs provide functional information during the process of their formation, initiating from the origin of a drug-tolerant single cell. Using >2000 DTPs generated from various drugs and doses profiled on the basis of 44 proteins, we demonstrate that the DTPs are clustered on the basis of their proteomic profiles changing in response to drugs and doses. Of interest, nine transcription factors in the DTPs, such as STAT3 and OCT4A, were identified as having decreased or increased levels of proteins in response to gefitinib. Importantly, these results can be obtained only by individual proteomic colony profiling, which may identify alternative therapeutic targets and biomarkers for DTPs that may harbor critical mechanisms for cancer relapse.
Subject(s)
Biological Assay/methods , Drug Resistance, Neoplasm/physiology , Neoplasms/metabolism , Proteomics/methods , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , HumansABSTRACT
Drug-tolerant cancer cell subpopulations are responsible for relapse after chemotherapy. By continuously exposing the gastric cancer cell line MKN45 to 5-FU for >100 passages, we established a 5-fluorouracil (5-FU)-tolerant line, MKN45/5FU. Orthotopic xenografts of MKN45/5FU cells in the stomach of nude mice revealed that these cells had a high potential to metastasize to sites such as the liver. Levels of phosphorylated phosphatidylinositide 3-kinase (PI3K) increased both in 5-FU-tolerant subpopulations according to the 5-FU dose, and in gastric submucosal orthotopic xenografts of MKN45/5FU cells. Sequential administration of 5-FU and a PI3K inhibitor, GDC-0941, targeted the downstream ribosomal S6 kinase phosphorylation to significantly suppress 5-FU-tolerant subpopulations and tumor propagation of orthotopic MKN45/5FU xenografts. These results suggest that administration of 5-FU followed by GDC-0941 may suppress disease relapse after 5-FU-based gastric cancer chemotherapy.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Drug Resistance, Neoplasm/drug effects , Fluorouracil/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/metabolism , Codon , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Genetic Variation , Heterografts , Humans , Mice , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Phenotype , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proteome , Proteomics/methods , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Signal TransductionABSTRACT
Tylosis is an inherited disorder characterized by abnormal palmoplantar skin thickening and a highly elevated risk of esophageal squamous cell carcinoma (ESCC). Analyses of tylosis in families have localized the responsible gene locus to a region of chromosome 17q25.1. Frequent loss of heterozygosity (LOH) in 17q25.1 was also observed in the sporadic form of ESCC. A putative tumor suppressor gene for ESCC may exist at this locus. We investigated the expression patterns of genes on 17q25.1 in tumor and corresponding normal tissues from patients with sporadic ESCC using RNA sequence analysis. For candidate genes, quantitative real-time reverse transcription-PCR (qRT-PCR), direct sequence, LOH and methylation analyses were performed using 93 clinical ESCC samples and 10 cell lines. A significant downregulation of ST6GALNAC1 was demonstrated in ESCC tissues compared to its expression in normal tissues by qRT-PCR (n=93, p<0.0001). Frequent LOH (17/27, 62.9%) and hypermethylation in ST6GALNAC1 were also observed in all cell lines. Our results indicated that ST6GALNAC1 was downregulated in sporadic ESCC via hyper-methylation and LOH, and it may be a candidate responsible gene for ESCC. Furthermore, recent studies suggest that multiple genes on chromosome 17q25 are involved in ESCC development.
Subject(s)
Carcinogenesis/genetics , Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Sialyltransferases/genetics , Aged , Chromosomes, Human, Pair 17/genetics , Down-Regulation , Esophageal Squamous Cell Carcinoma , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Humans , Loss of Heterozygosity , Male , Real-Time Polymerase Chain Reaction , TranscriptomeABSTRACT
Partial hepatectomy models in mice have been widely used for liver regeneration studies. A typical procedure removes ~2/3 of the liver by lobular ligation without tissue dissection. However, hepatectomy in humans involves physical damage (ie, physical partial hepatectomy, PPHx). Therefore, the liver regeneration process after PPHx should involve reactions to acute local injury followed by systematic remodeling. To clarify the liver regeneration process after PPHx, we used a murine liver injury model that mimics the actual human surgical procedure. A 20-30% PPHx was performed by transection of the left lobe of the liver using an ultrasonically activated scalpel in mice. Gene expression and morphological characteristics were analyzed during the liver regeneration process. Liver weight continuously increased by hypertrophic reaction of hepatocytes, whereas Ki67 staining showed hepatocyte proliferation. At the transected border, emergence of ductular reactions, a representative process of hepatic tissue remodeling that contain liver stem/progenitor cells, were observed. Gene expression of the transected border and non-damaged lobes revealed that inflammatory cytokine- and extracellular matrix-associated genes were significantly upregulated at the transected border. Our PPHx model triggered local extracellular matrix remodeling that resulted in ductular reactions. These processes occurred during the tissue repair process in local inflammatory responses as well as compensatory hepatocyte hypertrophy of the entire liver. These findings may provide insight for elucidating the mechanism of tissue repair and regeneration of the liver after PPHx.
Subject(s)
Hepatectomy , Liver Regeneration , Liver/pathology , Actins/metabolism , Animals , Cell Proliferation , Extracellular Matrix/physiology , Female , Gene Expression Profiling , Hepatic Stellate Cells/metabolism , Hepatocytes/physiology , Hypertrophy , I-kappa B Kinase/metabolism , Liver/enzymology , Liver/immunology , Matrix Metalloproteinases/metabolism , Mice, Inbred C57BL , Models, Animal , NF-kappa B/metabolism , Stem Cells/physiology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Cancer relapse occurs with substantial frequency even after treatment with curative intent. Here we studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Proteomic characterization of DTCs identified stemness- and epithelial-dominant subpopulations, but functional screening suggested that DTC formation was regulated at the transcriptional level independent from protein expression patterns. We consistently found that α-amanitin, an RNA polymerase II (RNAPII) inhibitor, effectively inhibited DTCs by suppressing TAF15 expression, which binds to RNA to modulate transcription and RNA processing. Sequential administration of α-amanitin and cisplatin extended overall survival in a cancer-relapse mouse model, namely peritonitis carcinomatosa. Therefore, post-treatment cancer relapse may occur through non-distinct subpopulations and may be effectively prevented by α-amanitin to disrupt transcriptional machinery, including TAF15.
Subject(s)
Alpha-Amanitin/administration & dosage , Drug Resistance/drug effects , Enzyme Inhibitors/administration & dosage , Peritoneal Neoplasms/drug therapy , TATA-Binding Protein Associated Factors/metabolism , Alpha-Amanitin/pharmacology , Animals , Cell Line, Tumor , Cisplatin/administration & dosage , Cisplatin/therapeutic use , Down-Regulation , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , HCT116 Cells , HT29 Cells , HeLa Cells , Humans , MCF-7 Cells , Mice , Peritoneal Neoplasms/genetics , Peritoneal Neoplasms/metabolism , Proteomics/methods , Secondary Prevention , Transcription, Genetic/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Information concerning to regulation of vegetative phase and floral initiation in herbaceous perennial plants has been limited to a few plant species. To know and compare flowering regulation in a wider range of plant species, we identified and characterized SHORT VEGETATIVE PHASE (SVP)-like genes (GtSVP-L1 and GtSVP-L2) from herbaceous perennial gentian (Gentiana triflora). Apple latent spherical virus (ALSV)-mediated silencing of the GtSVP-L1 in G. triflora seedlings resulted in early flowering and shortened vegetative phase by about one-third period of time, without vernalization. This indicated that GtSVP-L1 acts as a negative regulator of flowering and vegetative phase. Seasonal change in the expression of GtSVP was monitored in the overwinter buds (OWBs) of G. triflora. It was found that the levels of GtSVP-L1 mRNA in OWBs increased concomitantly with induction and/or maintenance of dormancy, then decreased toward release from dormancy, while that of GtSVP-L2 mRNA remained low and unchanged. These results implied that, in herbaceous perennial plants, SVP ortholog might concern to activity-dormancy control, as well as negative regulation in flowering. Practically, these results can be applicable to non-time-consuming technologies for breeding.
Subject(s)
Flowers/growth & development , Genes, Plant/physiology , Gentiana/physiology , Transcription Factors/physiology , Amino Acid Sequence , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Gene Silencing , Gentiana/genetics , Plant Dormancy/genetics , Plant Dormancy/physiology , Seasons , Seedlings/growth & development , Sequence Alignment , Transcription Factors/geneticsABSTRACT
BACKGROUND: Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile. METHODOLOGY: DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor. RESULTS: A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease. CONCLUSIONS: This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.
Subject(s)
Adenocarcinoma/blood , Colorectal Neoplasms/blood , DNA Mutational Analysis , DNA, Neoplasm/blood , Genes, Neoplasm , Point Mutation , Polymorphism, Single Nucleotide , Sequence Analysis, DNA/methods , Tumor Burden , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenocarcinoma/surgery , Adult , Aged , Alleles , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Colorectal Neoplasms/surgery , DNA Primers , Female , Humans , Leukocytes, Mononuclear/chemistry , Male , Middle Aged , Multiplex Polymerase Chain Reaction , Sequence Analysis, DNA/instrumentationABSTRACT
In this study we monitored protein dynamics in response to cisplatin, 5-fluorouracil, and irinotecan with different concentrations and administration modes using "reverse-phase" protein arrays (RPPAs) in order to gain comprehensive insight into the protein dynamics induced by genotoxic drugs. Among 666 protein time-courses, 38% exhibited an increasing trend, 32% exhibited a steady decrease, and 30% fluctuated within 24 h after drug exposure. We analyzed almost 12,000 time-course pairs of protein levels based on the geometrical similarity by correlation distance (dCor). Twenty-two percent of the pairs showed dCor > 0.8, which indicates that each protein of the pair had similar dynamics. These trends were disrupted by a proteasome inhibitor, MG132, suggesting that the protein degradation system was activated in response to the drugs. Among the pairs with high dCor, the average dCor of pairs with apoptosis-related protein was significantly higher than those without, indicating that regulation of protein levels was induced by the drugs. These results suggest that the levels of numerous functionally distinct proteins may be regulated by common degradation machinery induced by genotoxic drugs.
Subject(s)
Camptothecin/analogs & derivatives , Cisplatin/toxicity , Fluorouracil/toxicity , Mutagens/toxicity , Proteolysis/drug effects , Apoptosis , Camptothecin/toxicity , DNA Damage , HCT116 Cells , Humans , Irinotecan , Leupeptins/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Proteome/metabolismABSTRACT
Despite of remarkable improvement of postoperative 5-FU-based adjuvant chemotherapy, the relapse rate of gastric cancer patients who undergo curative resection followed by the adjuvant chemotherapy remains substantial. Therefore, it is important to identify prediction markers for the chemotherapeutic efficacy of 5-FU. We recently identified NF-κB as a candidate relapse prediction biomarker in gastric cancer. To evaluate the biological significance of NF-κB in the context of 5-FU-based chemotherapy, we analyzed the NF-κB-dependent biological response upon 5-FU treatment in gastric cancer cell lines. Seven genes induced by 5-FU treatment in an NF-κB-dependent manner were identified, five of which are known p53 targets. Knockdown of RELA, which encodes the p65 subunit of NF-κB, decreased both p53 and p53 target protein levels. In contrast, NF-κB was not affected by TP53 knockdown. We also demonstrated that cell lines bearing Pro/Pro homozygosity in codon72 of p53 exon4, which is important for NF-κB binding to p53, are more resistant to 5-FU than those with Arg/Arg homozygosity. We conclude that NF-κB plays an important role in the response to 5-FU treatment in gastric cancer cell lines, with a possible compensatory function of p53. These results suggest that NF-κB is a potential 5-FU-chemosensitivity prediction marker that may reflect 5-FU-induced stress-response pathways, including p53.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Fluorouracil/pharmacology , NF-kappa B/metabolism , Stomach Neoplasms/metabolism , Tumor Suppressor Protein p53/metabolism , Antimetabolites, Antineoplastic/therapeutic use , Cell Line, Tumor , Codon , Drug Resistance, Neoplasm/genetics , Fluorouracil/therapeutic use , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Protein Binding , Protein Transport , Stomach Neoplasms/drug therapy , Stomach Neoplasms/genetics , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
PURPOSE: Recent studies revealed that both disseminated tumor cells and noncancerous cells contributed to cancer progression cooperatively in the bone marrow. Here, RNA-seq analysis of bone marrow from gastric cancer patients was performed to identify prognostic markers for gastric cancer. EXPERIMENTAL DESIGN: Bone marrow samples from eight gastric cancer patients (stages I and IV: n = 4 each) were used for RNA-seq analysis. Results were validated through quantitative real-time PCR (qRT-PCR) analysis of HIST1H3D expression in 175 bone marrow, 92 peripheral blood, and 115 primary tumor samples from gastric cancer patients. miR-760 expression was assayed using qRT-PCR in 105 bone marrow and 96 primary tumor samples. Luciferase reporter assays were performed to confirm whether histone mRNAs were direct targets of miR-760. miR-760 expression was also evaluated in noncancerous cells from gastric cancer patients. RESULTS: RNA-seq analysis of bone marrow samples from gastric cancer patients revealed higher expression of multiple histone mRNAs in stage IV patients. HIST1H3D expression in the bone marrow, peripheral blood, and primary tumor of stage IV patients was higher than that in stage I patients (P = 0.0284, 0.0243, and 0.0006, respectively). In contrast, miR-760 was downregulated in the bone marrow and primary tumor of stage IV patients compared with stage I patients (P = 0.0094 and 0.0018, respectively). Histone mRNA and miR-760 interacted directly. Furthermore, miR-760 was downregulated in noncancerous mucosa in stage IV gastric cancer patients. CONCLUSION: Histone mRNA was upregulated, whereas miR-760 was downregulated in the bone marrow and primary tumor of advanced gastric cancer patients, suggesting that the histone mRNA/miR-760 axis had a crucial role in the development of gastric cancer.
Subject(s)
Adenocarcinoma/metabolism , Biomarkers, Tumor/metabolism , Histones/metabolism , MicroRNAs/metabolism , Stomach Neoplasms/metabolism , Adenocarcinoma/mortality , Adenocarcinoma/secondary , Aged , Biomarkers, Tumor/genetics , Bone Marrow/metabolism , Cell Line, Tumor , Female , Gastric Mucosa/metabolism , Gene Expression Regulation, Neoplastic , Histones/genetics , Humans , Kaplan-Meier Estimate , Lymphatic Metastasis , Male , MicroRNAs/genetics , Middle Aged , Multivariate Analysis , Neoplasm Staging , Oligonucleotide Array Sequence Analysis , Prognosis , Proportional Hazards Models , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNA , Stomach Neoplasms/mortality , Stomach Neoplasms/pathology , TranscriptomeABSTRACT
BACKGROUND: The use of standard chemotherapy regimens has changed the application of chemosensitivity tests from all chemotherapy-eligible patients to those who have failed standard chemotherapy, which includes patients with highly advanced, relapsed, or chemoresistant tumors. METHODS: We evaluated a total of 43 advanced primary and relapsed gastric cancers for chemosensitivity based on drug dose response curves to improve the objectivity and quality of quantitative measurements. The dose response curves were classified based on seven expected patterns. Instead of a binary chemosensitivity evaluation, we ranked drug sensitivity according to curve shapes and comparison with the peak plasma concentration (ppc) of each drug. RESULTS: A total of 193 dose response curves were obtained. The overall informative rate was 67.4%, and 85.3% for cases that had a sufficient number of cells. Paclitaxel (PXL)and docetaxel tended to show a higher rank, while cisplatin (CIS) and 5-fluorouracil (5-FU) tended to show resistance, particularly among the 20 cases (46.5%) that had recurrent disease after receiving chemotherapy with CIS and S-1 (5-FU). As such, we speculate that the resistant pattern of the chemosensitivity test suggests that cells with acquired drug resistance were selected by chemotherapy. Indeed, we observed a change in the chemosensitivity pattern of a sample before and after chemotherapy in terms of PXL sensitivity, which was used after primary chemotherapy. CONCLUSIONS: These results suggest that: (i) the dose-response pattern provides objective information for predicting chemosensitivity; and (ii) chemotherapy may select resistant cancer cell populations as a result of the therapy.