SMURF1 attenuates endoplasmic reticulum stress by promoting the degradation of KEAP1 to activate NRF2 antioxidant pathway

Abstract

Cancer cells consistently utilize the unfolded protein response (UPR) to encounter the abnormal endoplasmic reticulum (ER) stress induced by the accumulation of misfolded proteins. Extreme activation of the UPR could also provoke maladaptive cell death. Previous reports have shown that NRF2 antioxidant signaling is activated by UPR and serves as noncanonical pathway to defense and reduce excessive ROS levels during ER stress. However, the mechanisms of regulating NRF2 signaling upon ER stress in glioblastoma have not been fully elucidated. Here we identify that SMURF1 protects against ER stress and facilitates glioblastoma cell survival by rewiring KEAP1-NRF2 pathway. We show that ER stress induces SMURF1 degradation. Knockdown of SMURF1 upregulates IRE1 and PERK signaling in the UPR pathway and prevents ER-associated protein degradation (ERAD) activity, leading to cell apoptosis. Importantly, SMURF1 overexpression activates NRF2 signaling to reduce ROS levels and alleviate UPR-mediated cell death. Mechanistically, SMURF1 interacts with and ubiquitinates KEAP1 for its degradation (NRF2 negative regulator), resulting in NRF2 nuclear import. Moreover, SMURF1 loss reduces glioblastoma cell proliferation and growth in subcutaneously implanted nude mice xenografts. Taken together, SMURF1 rewires KEAP1-NRF2 pathway to confer resistance to ER stress inducers and protect glioblastoma cell survival. ER stress and SMURF1 modulation may provide promising therapeutic targets for the treatment of glioblastoma.

Introduction

Glioblastoma is the most malignant primary brain tumor, characterized by high heterogeneity, resistance and relapse [1, 2]. The current treatment strategies including maximal-safe surgical resection and adjuvant radiation therapy with alkylating agent temozolomide (TMZ) treatment have shown limited survival benefits, with a median survival rate about 14.6 months [3, 4]. Glioblastoma cells have a high metabolic rate and produce high levels of reactive oxide species (ROS), which further disturbs the protein folding capacity [5]. Endoplasmic reticulum (ER) stress due to the accumulation of unfolded or misfolded proteins is present highly in glioblastoma [6, 7]. The mechanisms are involved in response to ER stress by the three UPR sensors, including activating transcription factor 6 alpha (ATF6α), inositol requiring enzyme 1 (IRE1) and protein kinase R (PKR)-like ER kinase (PERK). ER chaperone BiP/GRP78 constitutively binds to the three UPR sensors, resulting in their inactivation at the basal level. Under ER stress, BiP/GRP78 dissociates from the UPR sensors to activate IRE1, PERK and ATF6α by oligomerization and trans-autophosphorylation or secretion of ATF6α [8, 9]. UPR response is an adaptive mechanism to restore ER homeostasis through multiple pathways, including attenuating transcriptional signal, alleviating the accumulation of misfolded proteins via ER-associated protein degradation (ERAD) system and recycling of misfolded proteins through the induction of autophagy [8, 10,11,12]. Accumulating evidences have indicated that a high level of basal UPR is frequently found in primary human tumors including glioblastoma, and adaptive UPR promotes cancer survival upon adverse environments [13,14,15]. However, the aberrant activation of UPR also triggers cell death under the unresolved and extreme ER stress conditions [16, 17]. For instance, arginosuccinate synthase 1 treatment suppresses tumor progression and triggers pro-apoptotic ER stress responses in hepatocellular carcinoma through the hyperactivation of PERK-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP) arm of the UPR [18].

Misfolded protein accumulation and aggregation induce excessive production of ROS, which can activate nuclear factor erythroid 2-related factor 2 (NRF2). Many lines of evidences have shown that the activation of NRF2 pathway suppresses ER stress-induced apoptosis by regulating antioxidant synthesis and ROS eliminating enzymes expression [19, 20]. Under normal conditions, NRF2 is sequestered in the cytoplasm and degraded by its negative factor Kelch-like ECH-associated protein 1 (KEAP1) [21]. Whereas upon oxidative stress, the canonical KEAP1-NRF2 pathway activation is mediated by the release of NRF2 from KEAP1, then NRF2 translocates into the nucleus to activate antioxidant genes such as NAD(P)H quinone dehydrogenase 1 (NQO1) and HMOX1/HO-1 heme oxygenase 1 (HO1) [14, 22]. The noncanonical KEAP1-NRF2 pathway activation is mediated by p62/SQSTM1, an autophagy receptor protein that competitively binds with and degrades KEAP1 to activate NRF2 [22,23,24]. Indeed, NRF2-p62 system and selective autophagy are vital in tolerance of tumor microenvironmental stress [25, 26]. ER stress inducing agents, such as ER Ca²⁺ pump inhibitor TG (Thapsigargin) and the N-glycosylation inhibitor TM (Tunicamycin), increase ROS production and trigger tumor cell apoptosis [20]. NRF2 could be phosphorylated by PERK during ER stress, further triggering NRF2 dissociation from KEAP1 and induction of protective antioxidant response [27]. In addition, PERK-dependent activation of NRF2 attenuates accumulation of ROS triggering oxidative DNA damage and contributes to redox homeostasis and cell survival [28, 29]. Importantly, studies have reported that activation of NRF2 induces several components of the transcriptional UPR target genes, including XBP1 and ATF6α, to maintain ER integrity and protein homeostasis [30]. NRF2 interacts and activates ATF4 to induce the target genes expression to survive proteotoxic stress [31, 32]. Therefore, NRF2-UPR axis serves as a bidirectional signal for maintaining ER homeostasis. Accumulating evidences have suggested that sustained activation of NRF2 also induces pro-survival genes that promote cancer cell proliferation and chemoresistance [33]. Recent studies have shown that aberrantly high expression of NRF2 signaling is found in glioblastoma and promotes tumor cell mesenchymal transition, invasion and tumorigenesis [34]. However, the mechanisms of regulating NRF2 signaling in glioblastoma have not been well defined.

The HECT-type ubiquitin ligase (E3) Smad ubiquitination regulatory factor 1 (SMURF1) belongs to the Nedd4 family and mediates multiple biological processes, including cell growth and migration, and several physiological functions in bone formation, embryonic development, and tumorigenesis [35,36,37,38,39]. Our previous studies have demonstrated that SMURF1 is hyperactivated in glioblastoma and promotes tumor growth by ubiquitination and degradation of tumor suppresser phosphatase and tensin homolog (PTEN) [

Data availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

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