ABSTRACT
Anthracyclines have been a cornerstone in the cure of diffuse large B-cell lymphoma (DLBCL) and other hematological cancers. The ability of anthracyclines to eliminate DLBCL depends on the presence of topoisomerase-II-alpha (TopIIA), a DNA repair enzyme complex. We identified nucleolin as a novel binding partner of TopIIA. Abrogation of nucleolin sensitized DLBCL cells to TopIIA targeting agents (doxorubicin/etoposide). Silencing nucleolin and challenging DLBCL cells with doxorubicin enhanced the phosphorylation of H2AX (γH2AX-marker of DNA damage) and allowed DNA fragmentation. Reconstitution of nucleolin expression in nucleolin-knockdown DLBCL cells prevented TopIIA targeting agent-induced apoptosis. Nucleolin binding to TopIIA was mapped to RNA-binding domain 3 of nucleolin, and this interaction was essential for blocking DNA damage and apoptosis. Nucleolin silencing decreased TopIIA decatenation activity, but enhanced formation of TopIIA-DNA cleavable complexes in the presence of etoposide. Moreover, combining nucleolin inhibitors: aptamer AS1411 or nucant N6L with doxorubicin reduced DLBCL cell survival. These findings are of clinical importance because low nucleolin levels versus high nucleolin levels in DLBCL predicted 90-month estimated survival of 70% versus 12% (P<0.0001) of patients treated with R-CHOP-based therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Lymphoma, Large B-Cell, Diffuse/metabolism , Phosphoproteins/antagonists & inhibitors , RNA-Binding Proteins/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , DNA Damage , DNA Topoisomerases, Type II/metabolism , Female , Gene Expression , Humans , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/mortality , Male , Molecular Targeted Therapy , Phosphoproteins/genetics , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , Poly-ADP-Ribose Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , NucleolinABSTRACT
Impaired Fas-mediated apoptosis is associated with poor clinical outcomes and cancer chemoresistance. Soluble Fas receptor (sFas), produced by skipping of exon 6, inhibits apoptosis by sequestering Fas ligand. Serum sFas is associated with poor prognosis of non-Hodgkin's lymphomas. We found that the alternative splicing of Fas in lymphomas is tightly regulated by a long-noncoding RNA corresponding to an antisense transcript of Fas (FAS-AS1). Levels of FAS-AS1 correlate inversely with production of sFas, and FAS-AS1 binding to the RBM5 inhibits RBM5-mediated exon 6 skipping. EZH2, often mutated or overexpressed in lymphomas, hyper-methylates the FAS-AS1 promoter and represses the FAS-AS1 expression. EZH2-mediated repression of FAS-AS1 promoter can be released by DZNeP (3-Deazaneplanocin A) or overcome by ectopic expression of FAS-AS1, both of which increase levels of FAS-AS1 and correspondingly decrease expression of sFas. Treatment with Bruton's tyrosine kinase inhibitor or EZH2 knockdown decreases the levels of EZH2, RBM5 and sFas, thereby enhancing Fas-mediated apoptosis. This is the first report showing functional regulation of Fas repression by its antisense RNA. Our results reveal new therapeutic targets in lymphomas and provide a rationale for the use of EZH2 inhibitors or ibrutinib in combination with chemotherapeutic agents that recruit Fas for effective cell killing.
Subject(s)
Lymphoma, B-Cell/blood , Lymphoma, B-Cell/genetics , RNA, Antisense/genetics , RNA, Long Noncoding/genetics , fas Receptor/blood , fas Receptor/genetics , Adenine/analogs & derivatives , Alternative Splicing , Apoptosis/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enhancer of Zeste Homolog 2 Protein , Fas Ligand Protein/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Histones/metabolism , Humans , Introns , Lymphoma, B-Cell/metabolism , Models, Biological , Piperidines , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Protein Binding , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Despite recent therapeutic improvements, the prognosis for patients suffering from Sézary syndrome (SS), a disseminated form of cutaneous T-cell lymphomas, is still poor. We identified bi- and monoallelic deletions of the tumor necrosis factor-α-induced protein 3 gene (TNFAIP3; A20) in a high proportion of SS patients as well as biallelic A20 deletion in the SS-derived cell line SeAx. Furthermore, we demonstrate that inhibition of A20 activates the NF-κB pathway thereby increasing the proliferation of normal T lymphocytes. On the other hand, the reconstitution of A20 expression slowed down the cell cycle in SeAx cells. Recently A20 inactivation has been reported in various B-cell lymphomas. In this study, we show that A20 is also a putative tumor suppressor in the T-cell malignancy-SS.
Subject(s)
Gene Deletion , Genes, Tumor Suppressor , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Sezary Syndrome/genetics , Skin Neoplasms/genetics , Aged , Aged, 80 and over , Blotting, Western , Cell Cycle , Comparative Genomic Hybridization , DNA Methylation , DNA-Binding Proteins , Female , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Activation , Male , Middle Aged , NF-kappa B/genetics , NF-kappa B/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sezary Syndrome/metabolism , Sezary Syndrome/pathology , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , T-Lymphocytes/metabolism , Tumor Cells, Cultured , Tumor Necrosis Factor alpha-Induced Protein 3ABSTRACT
Stimulation of the TNF receptors CD30 and CD95 exerts opposite effects. Crosstalk of both receptors is unknown. We aimed to reveal regulatory mechanisms of CD30-induced effects on CD95 signaling of cALCL cell lines. "CD30/CD95 crosstalk analysis" was performed in cALCL cell lines by comparison of CD30 or CD95 stimulation and CD30/CD95 costimulation. Receptor expression and induction of apoptosis was investigated by flow cytometry. mRNA expression of CD30-inducible genes (cFLIP, TRAF1, cIAP2, and A20) was compared by semiquantitative reverse transcription (RT-RQ-) PCR in stimulated and unstimulated cells. Protein expression of IκBα, p100/p52, caspase-8, caspase-3, and cFLIP was analyzed by immunoblotting. A lentiviral-based shRNA-mediated approach was used to inhibit cFLIP expression. CD30/CD95 crosstalk experiments revealed that CD30 ligation leads to NFκB-mediated cFLIP upregulation in cALCL cells, which in turn enhanced resistance to CD95-mediated apoptosis. This effect is based on the CD30-induced upregulation of cFLIP. Knockdown of cFLIP restores sensitivity to CD95-mediated apoptosis. We conclude that the anti-apoptotic function of CD30 antibodies should be kept in mind if CD30 antibody-based therapeutic concepts for ALCL lymphoma are considered.
