FAS-Antisense 1 Lncrna and Production of Soluble Versus Membrane Fas in B-Cell Lymphoma
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Leukemia (2014) 28, 2376–2387 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE FAS-antisense 1 lncRNA and production of soluble versus membrane Fas in B-cell lymphoma L Sehgal, R Mathur, FK Braun, JF Wise, Z Berkova, S Neelapu, LW Kwak and F Samaniego 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. Leukemia (2014) 28, 2376–2387; doi:10.1038/leu.2014.126 INTRODUCTION The current challenge is to understand regulation of Fas Fas (APO-1, CD95 and TNFRSF6) is a member of the tumor necrosis signaling in lymphoma in order to overcome resistance to FasL factor receptor superfamily that has a major role in the extrinsic and chemotherapy. In this study, we investigated the regulation of pathway of apoptosis. Once Fas is activated by its ligand (FasL), a alternative splicing of Fas pre-mRNA that produces soluble decoy cascade of events leads to apoptosis.1 Fas receptor is expressed by Fas receptor known to be upregulated in lymphoma and a likely B- and T-cells, as well as numerous tumor cells and some normal source of chemoresistance. human tissues, yet sensitivity to Fas-mediated apoptosis does not always correlate with Fas expression levels.2 Defects of the Fas-mediated apoptotic pathway are often associated with MATERIALS AND METHODS lympho-proliferative disorders and autoimmune diseases.3–5 Overall survival of individuals with non-Hodgkin’s lymphoma Cells (NHL) has improved in recent years with advancements in Granta-519 cells were obtained from the German Collection of chemotherapy regimens.6 However, NHL still demonstrates Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). 6 Remaining cell lines were obtained from the American Type Culture frequent relapses and a high mortality rate of nearly 30%. One Collection (ATCC, Manassas, VA, USA). Cells were maintained in RPMI- of the likely mechanisms for NHL relapse is the survival and 1640 (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (Atlanta expansion of cells that are resistant to Fas-mediated apoptosis. It Biologicals, Flowery Branch, GA, USA) (RPMI-1640/10% FBS) in 5% CO2 has been shown that the cells lacking Fas or cells with defective atmosphere at 37 1C. Cell lines were authenticated by STR analysis (MD Fas signaling are resistant to conventional doses of chemotherapy Anderson Cancer Center Characterized Cell Line Core) and regularly and radiation.7–10 Fas levels and its signaling are thus important tested for mycoplasma (Lonza, Houston, TX, USA). determinants of the effectiveness of chemotherapy. Peripheral blood B-lymphocytes were isolated from healthy donors’ Tumor cells can evade apoptosis by several mechanisms; one of blood, obtained from Gulf Coast Blood Center (Houston, TX, USA), with them is the release of soluble decoy receptors. The Fas mRNA can CD19-positive magnetic beads and released with the competitive CD19 DETACHaBEAD (Invitrogen, Grand Island, NY, USA). be alternatively spliced during mRNA maturation to either include or exclude exon 6 that encodes the trans-membrane domain of Fas receptor.11–14 The serum levels of the soluble isoform of the RNA isolation and quantitative real-time PCR (qRT-PCR) Fas receptor (sFas) are elevated in patients with malignant lymphoma and chronic lymphocytic leukemia (CLL).15–18 Soluble Total cellular RNA was extracted with RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen Sciences, Valencia, CA, USA). The first- Fas blocks apoptosis induced by FasL in vitro, suggesting that strand cDNA was synthesized using a Superscript II reverse transcriptase kit production of sFas in lymphoma might be protective19 and 18 (Invitrogen) according to the manufacturer’s protocol. Samples were consistent with poor clinical outcomes. The overall survival and analyzed on 96-well microtiter plates using the StepOnePlus Real-Time PCR disease-free survival rates are significantly lower in lymphoma Systems (Applied Biosystems, Grand Island, NY, USA) with primers listed in patients if they express elevated serum sFas levels.18 the Supplementary Table S1 using 40 cycles of 95 1C for 15 s and 60 1C for Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Correspondence: Dr F Samaniego, Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, 7455 Fannin Street, Houston, TX 77054, USA. E-mail: [email protected] Received 4 October 2013; revised 10 February 2014; accepted 13 March 2014; accepted article preview online 3 April 2014; advance online publication, 9 May 2014 FAS-AS1 lncRNA sensitizes B-cell lymphomas to Fas-mediated apoptosis L Sehgal et al 2377 1 min and by SYBR green method for detection. qRT-PCR data were (Millipore, Billerica, MA, USA). Supernatants were incubated with 1–2 mgof analyzed by the Step-One software version 2.1. anti-RBM5 (Active motif) or mouse immunoglobulin G (IgG; Invitrogen) for 1 h at 4 1C followed by precipitation with protein A/G agarose (Pierce, Rockford, IL, USA). RNA immunoprecipitation (IP) and immunoblotting (IB) IB was performed according to the standard protocols as described For precipitation of RNA–protein complexes, 1 Â 107 cells were homo- previously.20,21 Proteins were detected by IB with anti-RBM5, anti-EZH2, genized using the EZ Magna RIP kit per the manufacturer’s protocol anti-histone-3 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-PARP, Figure 1. For caption see next page. & 2014 Macmillan Publishers Limited Leukemia (2014) 2376 – 2387 FAS-AS1 lncRNA sensitizes B-cell lymphomas to Fas-mediated apoptosis L Sehgal et al 2378 anti-cleaved capase-3 and anti-caspase-8 (Cell Signaling Technologies, thereby suggesting that the level of sFas might be important for Danvers, MA, USA) primary antibodies (1:1000 dilution in 5% nonfat milk), the evasion of apoptosis and chemoresistance.18 A qRT-PCR followed by the corresponding horseradish peroxidase-conjugated analysis using specific primers to quantify total Fas mRNA and secondary goat anti-mouse or anti-rabbit antibodies (Jackson alternatively spliced Fas mRNA encoding sFas revealed that ImmunoResearch, West Grove, PA, USA). Equal loading was verified by alternative splicing of Fas pre-mRNA to sFas isoform (skipping of blotting with anti-b-actin-horseradish peroxidase antibody (1:10 000; Sigma Aldrich, Buchs, Switzerland). Visualization was achieved by exon 6) is highly upregulated in lymphoma-derived cell lines and Supersignal West Pico chemiluminescent substrate (Pierce). tissues (Figure 1a and Supplementary Figures S1A and B). An antisense RNA transcribed from the opposite strand of the intron 1 of the human Fas gene, FAS-AS1, was identified Chromatin immunoprecipitation (ChIP) just recently.22 To test the hypothesis that FAS-AS1 might Cells were crosslinked by incubation with 1% formaldehyde (Sigma regulate exon 6 skipping, we determined the expression level of 1 Aldrich) at 37 C for 15 min. The reaction was stopped by addition of FAS-AS1 long-noncoding RNA (lncRNA) in lymphoma cell lines glycine (0.125 M final concentration; Sigma Aldrich) and processed for chromatin isolation as described in Supplementary Methods. and tissue samples by qRT-PCR. The levels of FAS-AS1 lncRNA were substantially reduced in primary lymphomas and lymphoma cell lines when compared with healthy donor CD19 þ Enzyme-linked immunosorbent assay (ELISA) 6 B lymphocytes (Figure 1b and Supplementary Figure S1C). We Cells (0.1 Â 10 /ml) were grown for 24 h as described above before plotted the levels of sFas mRNA and FAS-AS1 lncRNA in incubating with 1 mM DZNeP (3-Deazaneplanocin A; Cayman Chemical, lymphoma and other cell lines on x–y graph (Figure 1c) and Ann Arbor, MI, USA). Cell culture supernatants were collected 24 h later and 2 analyzed for sFas levels by ELISA (R&D Systems, Minneapolis, MN, USA). calculated Pearson correlation coefficient (r ¼0.58) that suggested their inverse correlation and thus a possible involvement of FAS-AS1 in regulation of alternative isoform Apoptosis induction and flow cytometry analysis expression as observed in other pairs of gene and antisense Cells were incubated with DZNeP or ibrutinib (PCI-32765; Selleckchem, mRNA regulatory models.23 We screened GEO Database Houston, TX, USA) in 1 ml of RPMI-1640/10% FBS for 24 h. Cell death was Profiles (http://www.ncbi.nlm.nih.gov/geo/) and confirmed the induced by incubation with the indicated dose of super FasL (Enzo, Farmingdale, NY, USA) in RPMI-1640/10% FBS for 18 h at 37 1C. Apoptosis abundance of FAS-AS1 lncRNA in normal tissues. was analyzed by flow cytometry using PI apoptosis kit (BD Bioscience, The gene expression is regulated by various genetic and San Jose, CA, USA) according to the manufacturer’s recommendations.