PERK induces resistance to cell death elicited by endoplasmic reticulum stress and chemotherapy.
ABSTRACT: Nutrient deprivation, hypoxia, radiotherapy and chemotherapy induce endoplasmic reticulum (ER) stress, which activates the so-called unfolded protein response (UPR). Extensive and acute ER stress directs the UPR towards activation of death-triggering pathways. Cancer cells are selected to resist mild and prolonged ER stress by activating pro-survival UPR. We recently found that drug-resistant tumor cells are simultaneously resistant to ER stress-triggered cell death. It is not known if cancer cells adapted to ER stressing conditions acquire a chemoresistant phenotype.To investigate this issue, we generated human cancer cells clones with acquired resistance to ER stress from ER stress-sensitive and chemosensitive cells.ER stress-resistant cells were cross-resistant to multiple chemotherapeutic drugs: such multidrug resistance (MDR) was due to the overexpression of the plasma-membrane transporter MDR related protein 1 (MRP1). Gene profiling analysis unveiled that cells with acquired resistance to ER stress and chemotherapy share higher expression of the UPR sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK), which mediated the erythroid-derived 2-like 2 (Nrf2)-driven transcription of MRP1. Disrupting PERK/Nrf2 axis reversed at the same time resistance to ER stress and chemotherapy. The inducible silencing of PERK reduced tumor growth and restored chemosensitivity in resistant tumor xenografts.Our work demonstrates for the first time that the adaptation to ER stress in cancer cells produces a MDR phenotype. The PERK/Nrf2/MRP1 axis is responsible for the resistance to ER stress and chemotherapy, and may represent a good therapeutic target in aggressive and resistant tumors.
Project description:Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)-scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy.
Project description:Malignant carcinomas that recur following therapy are typically de-differentiated and multi-drug resistant (MDR). De-differentiated cancer cells acquire MDR by upregulating reactive oxygen species (ROS)-scavenging enzymes and drug efflux pumps, but how these genes are upregulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already upregulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular response to oxidative stress, is pre-activated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a non-canonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells upregulate MDR genes via PERK-Nrf2 signaling, and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy. Differentiated human breast epithelial cells (HMLE-shGFP) or de-differentiated cells (HMLE-Twist) were treated in culture with 40uM menadione for 2 hours or 1uM PERK inhibitor for 48 hours and compared to control DMSO-treated cells.
Project description:BACKGROUND:Standard treatment for advanced malignant pleural mesothelioma (MPM) is a cisplatin/pemetrexed (MTA) regimen; however, this is confronted by drug resistance. Proteotoxic stress in the endoplasmic reticulum (ER) is a hallmark of cancer and some rely on this stress signalling in response to cytotoxic chemotherapeutics. We hypothesise that ER stress and the adaptive unfolded protein response (UPR) play a role in chemotherapy resistance of MPM. METHODS:In vitro three-dimensional (3D) and ex vivo organotypic culture were used to enrich a chemotherapy-resistant population and recapitulate an in vivo MPM microenvironment, respectively. Markers of ER stress, the UPR and apoptosis were assessed at mRNA and protein levels. Cell viability was determined based on acid phosphatase activity. RESULTS:MPM cells with de novo and/or acquired chemotherapy resistance displayed low ER stress, which rendered the cells hypersensitive to agents that induce ER stress and alter the UPR. Bortezomib, an FDA-approved proteasome inhibitor, selectively impairs chemotherapy-resistant MPM cells by activating the PERK/eIF2?/ATF4-mediated UPR and augmenting apoptosis. CONCLUSIONS:We provide the first evidence for ER stress and the adaptive UPR signalling in chemotherapy resistance of MPM, which suggests that perturbation of the UPR by altering ER stress is a novel strategy to treat chemotherapy-refractory MPM.
Project description:Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress-induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop((-)/(-)) cells are partially resistant to ER stress-induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2? and promotes XIAP degradation through ATF4. Of interest, PERK's down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress-induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a "two-hit" model of ER stress-induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.
Project description:An important event in the unfolded protein response (UPR) is activation of the endoplasmic reticulum (ER) kinase PERK. The PERK signalling branch initially mediates a prosurvival response, which progresses to a proapoptotic response upon prolonged ER stress. However, the molecular mechanisms of PERK-mediated cell death are not well understood. Here we show that expression of the primary miR-17-92 transcript and mature miRNAs belonging to the miR-17-92 cluster are decreased during UPR. We found that miR-17-92 promoter reporter activity was reduced during UPR in a PERK-dependent manner. Furthermore, we show that activity of the miR-17-92 promoter is repressed by ectopic expression of ATF4 and NRF2. Promoter deletion analysis mapped the region responding to UPR-mediated repression to a site in the proximal region of the miR-17-92 promoter. Hypericin-mediated photo-oxidative ER damage reduced the expression of miRNAs belonging to the miR-17-92 cluster in wild-type but not in PERK-deficient cells. Importantly, ER stress-induced apoptosis was inhibited upon miR-17-92 overexpression in SH-SY5Y and H9c2 cells. Our results reveal a novel function for ATF4 and NRF2, where repression of the miR-17-92 cluster plays an important role in ER stress-mediated apoptosis. Mechanistic details are provided for the potentiation of cell death via sustained PERK signalling mediated repression of the miR-17-92 cluster.
Project description:Multidrug resistance (MDR) is a phenotype of cancer cells with reduced sensitivity to a wide range of unrelated drugs. P-glycoprotein (P-gp)-a drug efflux pump (ABCB1 member of the ABC transporter gene family)-is frequently observed to be a molecular cause of MDR. The drug-efflux activity of P-gp is considered as the underlying mechanism of drug resistance against P-gp substrates and results in failure of cancer chemotherapy. Several pathological impulses such as shortages of oxygen and glucose supply, alterations of calcium storage mechanisms and/or processes of protein N-glycosylation in the endoplasmic reticulum (ER) leads to ER stress (ERS), characterized by elevation of unfolded protein cell content and activation of the unfolded protein response (UPR). UPR is responsible for modification of protein folding pathways, removal of misfolded proteins by ER associated protein degradation (ERAD) and inhibition of proteosynthesis. However, sustained ERS may result in UPR-mediated cell death. Neoplastic cells could escape from the death pathway induced by ERS by switching UPR into pro survival mechanisms instead of apoptosis. Here, we aimed to present state of the art information about consequences of P-gp expression on mechanisms associated with ERS development and regulation of the ERAD system, particularly focused on advances in ERS-associated therapy of drug resistant malignancies.
Project description:Although cancer stem cells (CSC) have been implicated in the development of resistance to anti-cancer therapy including chemotherapy, the mechanisms underlying chemo-resistance by CSC have not yet been elucidated. We herein isolated sphere-forming (cancer stem-like) cells from the cervical cancer cell line, SiHa, and examined the unfolded protein reaction (UPR) to chemotherapeutic-induced endoplasmic reticulum (ER) stress. We revealed that tunicamycin-induced ER stress-mediated apoptosis occurred in monolayer, but not sphere-forming cells. Biochemical assays demonstrated that sphere-forming cells were shifted to pro-survival signaling through the inactivation of IRE1 (XBP-1 splicing) and activation of PERK (elF2? phosphorylation) branches under tunicamycin-induced ER stress conditions. The proportion of apoptotic cells among sphere-forming cells was markedly increased by the tunicamycin+PERK inhibitor (PERKi) treatment, indicating that PERKi sensitized sphere-forming cells to tunicamycin-induced apoptosis. Cisplatin is also known to induce ER stress-mediated apoptosis. A low concentration of cisplatin failed to shift sphere-forming cells to apoptosis, although IRE1 branch, but not PERK, was activated. ER stress-mediated apoptosis occurred in sphere-forming cells by the cisplatin+IRE1? inhibitor (IRE1i) treatment. IRE1i, synergistic with cisplatin, up-regulated elF2? phosphorylation, and this was followed by the induction of CHOP in sphere-forming cells. The results of the present study demonstrated that the inhibition of ER stress sensors, combined with ER stress-inducible chemotherapy, shifted cancer stem-like cells to ER stress-mediated apoptosis.
Project description:The water-soluble Nrf2 (nuclear factor, erythroid 2-like 2, also called Nfe2l2) is accepted as a master regulator of antioxidant responses to cellular stress, and it was also identified as a direct target of the endoplasmic reticulum (ER)-anchored PERK (protein kinase RNA-like endoplasmic reticulum kinase). However, the membrane-bound Nrf1 (nuclear factor, erythroid 2-like 1, also called Nfe2l1) response to ER stress remains elusive. Herein, we report a unity of opposites between these two antioxidant transcription factors, Nrf1 and Nrf2, in coordinating distinct cellular responses to the ER stressor tunicamycin (TU). The TU-inducible transcription of Nrf1 and Nrf2, as well as GCLM (glutamate cysteine ligase modifier subunit) and HO-1 (heme oxygenase 1), was accompanied by activation of ER stress signaling networks. Notably, the unfolded protein response (UPR) mediated by ATF6 (activating transcription factor 6), IRE1 (inositol requiring enzyme 1) and PERK was significantly suppressed by Nrf1-specific knockout, but hyper-expression of Nrf2 and its target genes GCLM and HO-1 has retained in Nrf1a-/- cells. By contrast, Nrf2-/-?TA cells with genomic deletion of its transactivation (TA) domain resulted in significant decreases of GCLM, HO-1 and Nrf1; this was accompanied by partial decreases of IRE1 and ATF6, rather than PERK, but with an increase of ATF4 (activating transcription factor 4). Interestingly, Nrf1 glycosylation and its trans-activity to mediate the transcriptional expression of the 26S proteasomal subunits, were repressed by TU. This inhibitory effect was enhanced by Nrf1a-/- and Nrf2-/-?TA, but not by a constitutive activator caNrf2?N (that increased abundances of the non-glycosylated and processed Nrf1). Furthermore, caNrf2?N also enhanced induction of PERK and IRE1 by TU, but reduced expression of ATF4 and HO-1. Thus, it is inferred that such distinct roles of Nrf1 and Nrf2 are unified to maintain cell homeostasis by a series of coordinated ER-to-nuclear signaling responses to TU. Nrf1 (i.e., a full-length form) acts in a cell-autonomous manner to determine the transcription of most of UPR-target genes, albeit Nrf2 is also partially involved in this process. Consistently, transactivation of ARE (antioxidant response element)-driven BIP (binding immunoglobulin protein)-, PERK- and XBP1 (X-box binding protein 1)-Luc reporter genes was mediated directly by Nrf1 and/or Nrf2. Interestingly, Nrf1 is more potent than Nrf2 at mediating the cytoprotective responses against the cytotoxicity of TU alone plus tBHQ (tert-butylhydroquinone). This is also further supported by the evidence that the intracellular reactive oxygen species (ROS) levels are increased in Nrf1a-/- cells, but rather are, to our surprise, decreased in Nrf2-/-?TA cells.
Project description:Endoplasmic reticulum (ER) stress leads to activation of the unfolded protein response (UPR) signaling cascade and induction of an apoptotic cell death, autophagy, oncogenesis, metastasis, and/or resistance to cancer therapies. Mechanisms underlying regulation of ER transmembrane proteins PERK, IRE1?, and ATF6?/?, and how the balance of these activities determines outcome of the activated UPR, remain largely unclear. Here, we report a novel molecule transmembrane protein 33 (TMEM33) and its actions in UPR signaling. Immunoblotting and northern blot hybridization assays were used to determine the effects of ER stress on TMEM33 expression levels in various cell lines. Transient transfections, immunofluorescence, subcellular fractionation, immunoprecipitation, and immunoblotting were used to study the subcellular localization of TMEM33, the binding partners of TMEM33, and the expression of downstream effectors of PERK and IRE1?. Our data demonstrate that TMEM33 is a unique ER stress-inducible and ER transmembrane molecule, and a new binding partner of PERK. Exogenous expression of TMEM33 led to increased expression of p-eIF2? and p-IRE1? and their known downstream effectors, ATF4-CHOP and XBP1-S, respectively, in breast cancer cells. TMEM33 overexpression also correlated with increased expression of apoptotic signals including cleaved caspase-7 and cleaved PARP, and an autophagosome protein LC3II, and reduced expression of the autophagy marker p62. TMEM33 is a novel regulator of the PERK-eIE2?-ATF4 and IRE1-XBP1 axes of the UPR signaling. Therefore, TMEM33 may function as a determinant of the ER stress-responsive events in cancer cells.
Project description:The endoplasmic reticulum (ER) localized unfolded protein response (UPR) sensors, IRE1?, PERK, and ATF6?, are activated by the accumulation of misfolded proteins in the ER. It is unclear how the endogenous UPR sensors are regulated by both ER stress and the ER luminal chaperone BiP, which is a negative regulator of UPR sensors. Here we simultaneously examined the changes in the endogenous complexes of UPR sensors by blue native PAGE immunoblotting in unstressed and stressed cells. We found that all three UPR sensors exist as preformed complexes even in unstressed cells. While PERK complexes shift to large complexes, ATF6? complexes are reduced to smaller complexes on ER stress. In contrast, IRE1? complexes were not significantly increased in size on ER stress, unless IRE1? is overexpressed. Surprisingly, depletion of BiP had little impact on the endogenous complexes of UPR sensors. In addition, overexpression of BiP did not significantly affect UPR complexes, but suppressed ER stress mediated activation of IRE1?, ATF6? and, to a lesser extent, PERK. Furthermore, we captured the interaction between IRE1? and misfolded secretory proteins in cells, which suggests that the binding of unfolded proteins to preformed complexes of UPR sensors may be crucial for activation.