PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo

Abstract

IntroductionMaintenance of cell life-death homeostasis by widely expressed cell surface death receptors carries potential risks of inadvertent apoptosis, thus death receptors must be tightly regulated. Fas (APO-1, CD95) is a potent death receptor that eliminates autoreactive lymphocytes during lymphocyte development, but is less known for its proliferative functions such as its ability to stimulate regeneration of liver tissue. 1 Levels of Fas expression in cancers vary, and Fas activation by ligand or agonistic antibodies is often blocked. A minority of cancer cells acquires disabling mutations of Fas or Fas signaling mediators, and various cancers rather express inhibitors of Fas signaling such as c-FLIP and other recently recognized Fas-associated inhibitors, for example, hepatocyte growth factor receptor and human herpesvirus 8 protein K1. [2][3][4] Defective Fas signaling is an important cause of cancer resistance to therapy. Many genotoxic therapies including radiation depend on intact Fas signaling to eradicate cancer cells. 5 For example, Fas defective cells are significantly hindered in undergoing apoptosis after treatment with conventional doses of chemotherapy and radiation. 6,7 Restoring Fas apoptosis or sensitizing cancer cells to Fas-mediated apoptosis would improve the efficacy of many cancer therapies. However, stimulation of Fas in cancer cells has also triggered apoptosis of noncancerous cells. 8 To elucidate a role for specific regulators of Fas signaling in cancer cells, we sought to identify potential modulators of Fas expressed in cancers and target them to selectively sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. This idea is appealing based on the assumption that cancer cells are abnormal in numerous aspects and, although poised to undergo apoptosis, survive through blockage of the apoptotic pathways.In this study, we screened cells for potential regulators of the Fas death receptor. Using mass spectrometric analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML) protein. PML is a tumor suppressor whose expression is ubiquitous, but it is significantly decreased in 60% of hematologic and epithelial cancers mostly because of enhanced degradation. 9 The dominant negative form of PML is the oncogenic promyelocytic leukemia-retinoic acid receptor ␣ (PMLRAR␣), formed by the translocation of chromosomes 15 and 17 t(15;17). 10-12 PMLRAR␣ has known proproliferative and antiapoptotic activities. 13 Thus, we investigated whether the dominant negative PMLRAR␣ regulates Fas-mediated apoptosis. We demonstrated that PMLRAR␣ binds to Fas in APL cell lines and primary cells, and blocks Fas-mediated apoptosis through recruitment of c-FLIP in several cell models and in mice, suggesting that PMLRAR␣ interrupts Fas-mediated apoptosis by binding directly to the Fas receptor complex.Submitted April 21, 2011; accepted July 9, 2011. Prepublished online as Blood First Edition paper, July 29, 2011; DOI 10.1182 DOI 10. /blood-2011 The ...

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex

“…16,29 Proteins were analyzed by IB with 1:1 000 dilution of primary antibodies in 5% blotting-grade blocker (BioRad, Hercules, CA) in phosphate-buffered saline with Tween 20: anti-Fas (B-10)-horseradish peroxidase (HRP), anti-Fas (N-18), anti-nucleolin MS3-HRP, anti-histone-3, anti-GST (Santa Cruz Biotechnologies), anti-Flag (M2)-HRP, anti-b-actin-HRP (Sigma Aldrich), anti-poly ADP ribose polymerase (PARP), anti-cleaved capase-3, anti-caspase-8, anti-Bcl-2 (Cell Signaling), and anti-DDK (Origene, Rockville, MD), followed by the corresponding HRP-conjugated secondary antibody when an unconjugated primary antibody was used. Visualization was achieved by Supersignal West Pico chemiluminescent substrate (Thermo Scientific).…”

“…16,17,29,31 C57BL/6 mice (6-to 8-weeks-old) (National Cancer Institute, Bethesda, MD) or (8-weeks-old) (Harlan Laboratories, Indianapolis, Indiana) were transfected with plasmids: pnuc-myc, pNK-FLAG pSG5, pSG5, NucleolinpCMVENTRY, NR123-pCMVENTRY, or pCMVENTRY. Mice were monitored for 8 hours post-challenge and scored for survival.…”

“…For coimmunoprecipitation (coIP), cells, homogenized livers, or primary lymphoma tissues were analyzed as previously described 16,29 and as detailed in the data supplements. Supernatants were incubated with 1 to 2 mg of the antiFas antibody (B-10) (Santa Cruz Biotechnologies, Santa Cruz, CA) or mouse IgG (Invitrogen, Carlsbad, CA) for 1 hour at 4°C.…”

“…BJAB cells and NHL primary cells were screened for potential binding partners of activation-resistant Fas as previously described 29 (and see the Blood website for data supplements).…”

“…6,8,9,37,38 Multiple chemotherapies, including the lymphoma therapies doxorubicin, methotrexate, mitoxantrone, bleomycin, and rituximab, have been shown to upregulate Fas and/or FasL to achieve high response rates. 3,29,42 In lymphomas, wildtype Fas is commonly expressed and Fas resistance cannot always be explained. 43 Recent reports have revealed that cell surface receptors HGFR/c-MET, human herpesvirus-8 K1, and CD44v6/v9, which have their own signaling functions, can also bind and block Fas.…”