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MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression.

ABSTRACT: BACKGROUND:MUC16 (CA125) is a large transmembrane mucin protein (> 200 kDa) aberrantly expressed in approximately 80% of human epithelial ovarian cancers (EOC). MUC16 expression in EOC cells is associated with increased tumorigenesis and inhibiton of genotoxic drug-induced apoptosis. However, the mechanism by which MUC16 mediates these effects is unknown. In the present study, we investigated the mechanisms by which MUC16 attenuates TRAIL-induced apoptosis. METHODS:MUC16 expression was down-regulated by stably expressing an anti-MUC16 single-chain antibody (scFv) targeted to the endoplasmic reticulum (ER), which prevents cell surface localization of MUC16 in OVCAR3 cells. We also generated a MUC16 C-terminal domain (MUC16CTD) construct that was stably expressed in MUC16 negative SKOV3 cells. RESULTS:We show that MUC16 attenuates apoptosis, activation of caspase-8 and mitochondria activation in EOC cells in response to TRAIL. MUC16 decreases TRAIL receptor R2 (DR5) expression and inhibits pro-caspase-8 activation at the death-inducing signaling complex (DISC). MUC16CTD expression is sufficient to attenuate the TRAIL signaling cascade. MUC16 knockdown decreases caspase-8 inhibitor cFLIP mRNA levels, increases cFLIP degradation, and consequently increases TRAIL-induced apoptosis. Down-regulation of cFLIP following treatment of MUC16-expressing OVCAR3 cells with cFLIP siRNA also increases TRAIL-induced apoptosis. CONCLUSIONS:These findings indicate that MUC16 protects EOC cells against TRAIL-induced apoptosis through multiple mechanisms including the blockade of TRAIL R2 expression and the regulation of cFLIP expression at both the transcriptional and the protein level.

SUBMITTER: Matte I

PROVIDER: S-EPMC4234371 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression.

Matte Isabelle I Lane Denis D Boivin Marianne M Rancourt Claudine C Piché Alain A

BMC cancer 20140401

<h4>Background</h4>MUC16 (CA125) is a large transmembrane mucin protein (> 200 kDa) aberrantly expressed in approximately 80% of human epithelial ovarian cancers (EOC). MUC16 expression in EOC cells is associated with increased tumorigenesis and inhibiton of genotoxic drug-induced apoptosis. However, the mechanism by which MUC16 mediates these effects is unknown. In the present study, we investigated the mechanisms by which MUC16 attenuates TRAIL-induced apoptosis.<h4>Methods</h4>MUC16 expressio ...[more]

PMID: 24690311

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Project description:TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2's pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1's DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1's recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)'s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.

Project description:The CA125 antigen is found in the serum of many patients with serous ovarian cancer and has been widely used as a disease marker. CA125 has been shown to be an independent factor for clinical outcome in this disease. In The Cancer Genome Atlas ovarian cancer project, MUC16 expression levels are frequently increased, and the highest levels of MUC16 expression are linked to a significantly worse survival. To examine the biologic effect of the proximal portion of MUC16/CA125, NIH/3T3 (3T3) fibroblast cell lines were stably transfected with the carboxy elements of MUC16. As few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the AKT and ERK pathways. MUC16 transformation was associated with up-regulation of a number of metastases and invasion gene transcripts, including IL-1β, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular "ectodomain" of MUC16. The biologic impact of MUC16 was also explored through the creation of a transgenic mouse model expressing 354 amino acids of the carboxy-terminal portion of MUC16 (MUC16c354). Under a CMV, early enhancer plus chicken β actin promoter (CAG) MUC16c354 was well expressed in many organs, including the brain, colon, heart, kidney, liver, lung, ovary, and spleen. MUC16c354 transgenic animals appear to be viable, fertile, and have a normal lifespan. However, when crossed with p53-deficient mice, the MUC16c354:p53+/- progeny displayed a higher frequency of spontaneous tumor development compared to p53+/- mice alone. We conclude that the carboxy-terminal portion of the MUC16/CA125 protein is oncogenic in NIH/3T3 cells, increases invasive tumor properties, activates the AKT and ERK pathways, and contributes to the biologic properties of ovarian cancer.

Dataset Information

MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression.

Publications

MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression.

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