Regulator Of Endoplasmic Reticulum Homeostasis Research Articles

Targeting Integrin α3 Blocks β1 Maturation, Triggers Endoplasmic Reticulum Stress, and Sensitizes Glioblastoma Cells to TRAIL-Mediated Apoptosis.

Glioblastoma (GBM) is a devastating brain cancer for which new effective therapies are urgently needed. GBM, after an initial response to current treatment regimens, develops therapeutic resistance, leading to rapid patient demise. Cancer cells exhibit an inherent elevation of endoplasmic reticulum (ER) stress due to uncontrolled growth and an unfavorable microenvironment, including hypoxia and nutrient deprivation. Cancer cells utilize the unfolded protein response (UPR) to maintain ER homeostasis, and failure of this response promotes cell death. In this study, as integrins are upregulated in cancer, we have evaluated the therapeutic potential of individually targeting all αβ1 integrin subunits using RNA interference. We found that GBM cells are uniquely susceptible to silencing of integrin α3. Knockdown of α3-induced proapoptotic markers such as PARP cleavage and caspase 3 and 8 activation. Remarkably, we discovered a non-canonical function for α3 in mediating the maturation of integrin β1. In its absence, generation of full length β1 was reduced, immature β1 accumulated, and the cells underwent elevated ER stress with upregulation of death receptor 5 (DR5) expression. Targeting α3 sensitized TRAIL-resistant GBM cancer cells to TRAIL-mediated apoptosis and led to growth inhibition. Our findings offer key new insights into integrin α3's role in GBM survival via the regulation of ER homeostasis and its value as a therapeutic target.

Emergence of Lipid Droplets in the Mechanisms of Carcinogenesis and Therapeutic Responses.

Cancer shares common risk factors with cardiovascular diseases such as dyslipidemia, obesity and inflammation. In both cases, dysregulations of lipid metabolism occur, and lipid vesicles emerge as important factors that can influence carcinogenesis. In this review, the role of different lipids known to be involved in cancer and its response to treatments is detailed. In particular, lipid droplets (LDs), initially described for their role in lipid storage, exert multiple functions, from the physiological prevention of LD coalescence and regulation of endoplasmic reticulum homeostasis to pathological involvement in tumor progression and aggressiveness. Analysis of LDs highlights the importance of phosphatidylcholine metabolism and the diversity of lipid synthesis enzymes. In many cancers, the phosphatidylcholine pathways are disrupted, modifying the expression of genes coding for metabolic enzymes. Tumor microenvironment conditions, such as hypoxia, different types of stress or inflammatory conditions, are also important determinants of LD behavior in cancer cells. Therefore, LDs represent therapeutic targets in cancer, and many lipid mediators have emerged as potential biomarkers for cancer onset, progression, and/or resistance.

Nanocarrier-Assisted Delivery of Metformin Boosts Remodeling of Diabetic Periodontal Tissue via Cellular Exocytosis-Mediated Regulation of Endoplasmic Reticulum Homeostasis.

Endoplasmic reticulum (ER) dysfunction is a potential contributor to the impaired repair capacity of periodontal tissue in diabetes mellitus (DM) patients. Restoring ER homeostasis is thus critical for successful regenerative therapy of diabetic periodontal tissue. Recent studies have shown that metformin can modulate DM-induced ER dysfunction, yet its mechanism remains unclear. Herein, we show that high glucose elevates the intracellular miR-129-3p level due to exocytosis-mediated release failure and subsequently perturbs ER calcium homeostasis via downregulating transmembrane and coiled-coil domain 1 (TMCO1), an ER Ca2+ leak channel, in periodontal ligament stem cells (PDLSCs). This results in the degradation of RUNX2 via the ubiquitination-dependent pathway, in turn leading to impaired PDLSCs osteogenesis. Interestingly, metformin could upregulate P2X7R-mediated exosome release and decrease intracellular miR-129-3p accumulation, which restores ER homeostasis and thereby rescues the impaired PDLSCs. To further demonstrate the in vivo effect of metformin, a nanocarrier for sustained local delivery of metformin (Met@HALL) in periodontal tissue is developed. Our results demonstrate that compared to controls, Met@HALL with enhanced cytocompatibility and pro-osteogenic activity could boost the remodeling of diabetic periodontal tissue in rats. Collectively, our findings unravel a mechanism of metformin in restoring cellular ER homeostasis, enabling the development of a nanocarrier-mediated ER targeting strategy for remodeling diabetic periodontal tissue.

HBXIP is a novel regulator of the unfolded protein response that sustains tamoxifen resistance in ER+ breast cancer

Endocrine-therapy-resistant estrogen receptor–positive (ER+) breast cancer cells often exhibit an augmented capacity to maintain endoplasmic reticulum (EnR) homeostasis under adverse conditions. Oncoprotein hepatitis B X-interacting protein (HBXIP) is a known transcriptional coactivator that promotes cancer development. However, it is unclear whether HBXIP participates in maintaining EnR homeostasis and promoting drug resistance in ER+ breast cancer. Here, we report that tamoxifen-resistant (TmaR) breast cancer cells exhibit increased expression of HBXIP, which acts as an inactivator of the unfolded protein response (UPR) to diminish tamoxifen-induced EnR stress. We show that HBXIP deficiency promotes EnR-associated degradation, enhances UPR-element reporter activity and cellular oxidative stress, and ultimately attenuates the growth of TmaR cells in vitro and in vivo. Mechanistically, we demonstrate that HBXIP acts as a chaperone of UPR transducer inositol-requiring enzyme 1a and diminishes production of reactive oxygen species (ROS) in TamR breast cancer cells. Upon loss of HBXIP expression, tamoxifen treatment hyperactivates IRE1α and its downstream proapoptotic pathways and simultaneously induces accumulation of intracellular ROS. This elevated ROS programmatically activates the other two branches of the UPR, mediated by PKR-like ER kinase and activating transcription factor 6α. Clinical investigations and Kaplan–Meier plotter analysis revealed that HBXIP is highly expressed in TamR breast cancer tissues. Furthermore, reinforced HBXIP expression is associated with a high recurrence and poor relapse-free survival rates in tamoxifen monotherapy ER+ breast cancer patients. These findings indicate that HBXIP is a regulator of EnR homeostasis and a potential target for TamR breast cancer therapy.

P–424 Evaluating the relationship between endoplasmic reticulum stress and recurrent pregnancy loss in a young population cohort

Abstract Study question Does endoplasmic reticulum (ER) stress evaluated by X-box binding protein 1 (XBP–1) among patients aged 18–30 years play a role in recurrent pregnancy loss (RPL)? Summary answer High levels of XBP–1 observed in patients with known RPL indicate that ER stress plays an important role in RPL. What is known already Female reproductive tract undergoes dynamic changes during oogenesis which require protein synthesis, folding, maturation, which take place in the ER. ER is also responsible for destruction of unfolded/misfolded proteins. Excess accumulation of these faulty proteins leads to ER stress, which actives unfolded protein response (UPR). XBP–1 is a transcription factor involved in UPR and regulates ER stress-mediated apoptosis. Regulation of ER homeostasis is important in folliculogenesis, oocyte maturation and embryogenesis. It is also known that ER stress has a positive correlation with age and it is associated with age-related diseases. Study design, size, duration This prospective case-controlled study was conducted at University of Health Sciences Turkey, Istanbul Kanuni Sultan Suleyman Training and Research Hospital Department of Obstetrics and Gynecology between March 2020 – September 2020. A total of 70 subjects were included in the study. All patients gave their written informed consent before their enrollment in the study. Participants/materials, setting, methods 38 patients aged 18–30 years with a history of RPL were included in the study. Patients who had miscarriages due to fetal abnormalities, patients with infections, endocrine or genetic disorders, smokers, alcohol and/or drug abusers, with acute/chronic inflammatory diseases, patients using steroids, anti-inflammatory and antioxidant medications were excluded from the study. Age-matched 32 healthy subjects without RPL were included in the control group. XBP–1 levels were determined using Human XBP–1 ELISA Kit (Elabscience Co., USA). Main results and the role of chance The mean age in the control group and in the study group were 25.21±3.3 and 25.26±2.6, respectively and they were statistically similar (p = 0.324). When groups were compared according to thyroid stimulating hormone (TSH) levels and body mass index (BMI), which are additional risk factors of RPL both groups were statistically similar (p = 0.642 and 0.942, respectively). As expected gravidity and abortus numbers were significantly higher in the study group (p < 0.001). A mean XBP–1 level of 1233.41± 3902.97 was determined in the control group. The mean value of the study group was calculated to be 2251.49± 9621.12. Mean XBP–1 level in the study group was significantly high (p < 0.001). A receiver operating curve (ROC) analysis was conducted in the study group. The area under the curve was found to be 87% (95% CI: 79% - 95%). The specificity was 75%, sensitivity was 89%, positive LR was 3.5, negative LR was 0.15, positive predictive value was 80% and negative predictive value was 87% for the cut-off XBP–1 level at 1364.68 pg/mL. Limitations, reasons for caution Small sample size is an important limitation of this study. In addition, evaluating XBP–1 only in serum samples does not let us drive any conclusions on the local changes of ER stress. Studies with larger samples sizes and studying XBP–1 levels in tissue samples of endometrial material is needed. Wider implications of the findings: The significantly high levels of XBP–1 in RPL patients younger than 30 years, indicate higher ER stress in this group even when age dependent increase in ER stress is calculated out of the equation. XBP–1 can be a promising marker in evaluating patients with a fertility wish for RPL risk. Trial registration number NCT04455256

Hereditary Spastic Paraplegia and Future Therapeutic Directions: Beneficial Effects of Small Compounds Acting on Cellular Stress.

Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative conditions that share a characteristic feature of degeneration of the longest axons within the corticospinal tract, which leads to progressive spasticity and weakness of the lower limbs. Mutations of over 70 genes produce defects in various biological pathways: axonal transport, lipid metabolism, endoplasmic reticulum (ER) shaping, mitochondrial function, and endosomal trafficking. HSPs suffer from an adequate therapeutic plan. Currently the treatments foreseen for patients affected by this pathology are physiotherapy, to maintain the outgoing tone, and muscle relaxant therapies for spasticity. Very few clinical studies have been conducted, and it’s urgent to implement preclinical animal studies devoted to pharmacological test and screening, to expand the rose of compounds potentially attractive for clinical trials. Small animal models, such as Drosophila melanogaster and zebrafish, have been generated, analyzed, and used as preclinical model for screening of compounds and their effects. In this work, we briefly described the role of HSP-linked proteins in the organization of ER endomembrane system and in the regulation of ER homeostasis and stress as a common pathological mechanism for these HSP forms. We then focused our attention on the pharmacodynamic and pharmacokinetic features of some recently identified molecules with antioxidant property, such as salubrinal, guanabenz, N-acetyl cysteine, methylene blue, rapamycin, and naringenin, and on their potential use in future clinical studies. Expanding the models and the pharmacological screening for HSP disease is necessary to give an opportunity to patients and clinicians to test new molecules.

TRIM25 promotes the cell survival and growth of hepatocellular carcinoma through targeting Keap1-Nrf2 pathway

Tumor cells often exhibit augmented capacity to maintain endoplasmic reticulum (ER) homeostasis under adverse conditions, yet the underlying mechanisms are not well defined. Here, through the evaluation of all human TRIM proteins, we find that TRIM25 is significantly induced upon ER stress. Upregulation of TRIM25 ameliorates oxidative stress, promotes ER-associated degradation (ERAD), and reduces IRE1 signaling in the UPR pathway. In contrast, depletion of TRIM25 leads to ER stress and attenuates tumor cell growth in vitro and in vivo. Mechanistically, TRIM25 directly targets Keap1 by ubiquitination and degradation. This leads to Nrf2 activation, which bolsters anti-oxidant defense and cell survival. TRIM25 expression is positively associated with Nrf2 expression and negatively with Keap1 expression in hepatocellular carcinoma (HCC) xenografts and specimens. Moreover, high TRIM25 expression correlates with poor patient survival in HCC. These findings reveal TRIM25 as a regulator of ER homeostasis and a potential target for tumor therapy.

WDR45 contributes to neurodegeneration through regulation of ER homeostasis and neuronal death

ABSTRACT Mutations in the macroautophagy/autophagy gene WDR45 cause β-propeller protein-associated neurodegeneration (BPAN); however the molecular and cellular mechanism of the disease process is largely unknown. Here we generated constitutive wdr45 knockout (KO) mice that displayed cognitive impairments, abnormal synaptic transmission and lesions in several brain regions. Immunohistochemistry analysis showed loss of neurons in prefrontal cortex and basal ganglion in aged mice, and increased apoptosis in prefrontal cortex, recapitulating a hallmark of neurodegeneration. Quantitative proteomic analysis showed accumulation of endoplasmic reticulum (ER) proteins in KO mouse. At the cellular level, accumulation of ER proteins due to WDR45 deficiency resulted in increased ER stress and impaired ER quality control. The unfolded protein response (UPR) was elevated through ERN1/IRE1 or EIF2AK3/PERK pathway, and eventually led to neuronal apoptosis. Suppression of ER stress or activation of autophagy through MTOR inhibition alleviated cell death. Thus, the loss of WDR45 cripples macroautophagy machinery in neurons and leads to impairment in organelle autophagy, which provides a mechanistic understanding of cause of BPAN and a potential therapeutic strategy to treat this genetic disorder. Abbreviations 7-ADD: 7-aminoactinomycin D; ASD: autistic spectrum disorder; ATF6: activating transcription factor 6; ATG: autophagy-related; BafA1: bafilomycin A1; BCAP31: B cell receptor associated protein 31; BPAN: β-propeller protein-associated neurodegeneration; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CDIPT: CDP-diacylglycerol–inositol 3-phosphatidyltransferase (phosphatidylinositol synthase); DDIT3/CHOP: DNA-damage inducible transcript 3; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GFP: green fluorescent protein; HIP: hippocampus; HSPA5/GRP78: heat shock protein family A (HSP70) member 5; KO: knockout; LAMP1: lysosomal-associated membrane 1; mEPSCs: miniature excitatory postsynaptic currents; MG132: N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal; MIB: mid-brain; MTOR: mechanistic target of rapamycin kinase; PCR: polymerase chain reaction; PFA: paraformaldehyde; PFC: prefrontal cortex; PRM: parallel reaction monitoring; RBFOX3/NEUN: RNA binding protein, fox-1 homolog [C. elegans] 3; RTN3: reticulon 3; SEC22B: SEC22 homolog B, vesicle trafficking protein; SEC61B: SEC61 translocon beta subunit; SEM: standard error of the mean; SNR: substantia nigra; SQSTM1/p62: sequestosome 1; TH: tyrosine hydroxylase; Tm: tunicamycin; TMT: tandem mass tag; TUDCA: tauroursodeoxycholic acid; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling; UPR: unfolded protein response; WDR45: WD repeat domain 45; WT: wild type; XBP1: X-box binding protein 1.

Stearoyl CoA Desaturase Is Essential for Regulation of Endoplasmic Reticulum Homeostasis and Tumor Growth in Glioblastoma Cancer StemCells.

SummaryInherent plasticity and various survival cues allow glioblastoma stem-like cells (GSCs) to survive and proliferate under intrinsic and extrinsic stress conditions. Here, we report that GSCs depend on the adaptive activation of ER stress and subsequent activation of lipogenesis and particularly stearoyl CoA desaturase (SCD1), which promotes ER homeostasis, cytoprotection, and tumor initiation. Pharmacological targeting of SCD1 is particularly toxic due to the accumulation of saturated fatty acids, which exacerbates ER stress, triggers apoptosis, impairs RAD51-mediated DNA repair, and achieves a remarkable therapeutic outcome with 25%–100% cure rate in xenograft mouse models. Mechanistically, divergent cell fates under varying levels of ER stress are primarily controlled by the ER sensor IRE1, which either promotes SCD1 transcriptional activation or converts to apoptotic signaling when SCD1 activity is impaired. Taken together, the dependence of GSCs on fatty acid desaturation presents an exploitable vulnerability to target glioblastoma.

Lipin-1 determines lung cancer cell survival and chemotherapy sensitivity by regulation of endoplasmic reticulum homeostasis and autophagy.

Cancer cells undergo comprehensive metabolic reprogramming to meet the increased requirements of energy and building blocks for proliferation. Lipin‐1, a phosphatidic acid phosphatase converting phosphatidic acid (PA) to diacylglycerol (DAG), is upregulated in lung adenocarcinoma (LUAD) cell lines and tumor tissues. In this study, we reveal high lipin‐1 expression is correlated with poor prognosis of patients with LUAD. Knockdown of lipin‐1 decreases cell viability and proliferation in LUAD cells, whereas it has less effect on nontumorigenic lung cells. Autophagy and ER stress play important roles in tumor initiation and progression. Lipin‐1 knockdown induces the initiation of autophagy while disrupts formation of autolysosome. Lipin‐1 silencing induces the activation of ER stress through the IRE1α pathway. Furthermore, we demonstrate disrupted ER homeostasis contributes to the cell phenotype, and the elevated autophagy initiation is due to the ER stress in part. For the first time, we show lack of lipin‐1 enhances the sensitivity of LUAD cells to cisplatin treatment. Our results suggest that lipin‐1 is a potential target, alone or combined with other treatment, for lung cancer therapy.

GRP78 haploinsufficiency suppresses acinar-to-ductal metaplasia, signaling, and mutant Kras-driven pancreatic tumorigenesis in mice

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease in critical need of new therapeutic strategies. Here, we report that the stress-inducible 78-kDa glucose-regulated protein (GRP78/HSPA5), a key regulator of endoplasmic reticulum homeostasis and PI3K/AKT signaling, is overexpressed in the acini and PDAC of Pdx1-Cre;KrasG12D/+;p53f/+ (PKC) mice as early as 2 mo, suggesting that GRP78 could exert a protective effect on acinar cells under stress, as during PDAC development. The PKC pancreata bearing wild-type Grp78 showed detectable PDAC by 3 mo and rapid subsequent tumor growth. In contrast, the PKC pancreata bearing a Grp78f/+ allele (PKC78f/+ mice) expressing about 50% of GRP78 maintained normal sizes during the early months, with reduced proliferation and suppression of AKT, S6, ERK, and STAT3 activation. Acinar-to-ductal metaplasia (ADM) has been identified as a key tumor initiation mechanism of PDAC. Compared with PKC, the PKC78f/+ pancreata showed substantial reduction of ADM as well as pancreatic intraepithelial neoplasia-1 (PanIN-1), PanIN-2, and PanIN-3 and delayed onset of PDAC. ADM in response to transforming growth factor α was also suppressed in ex vivo cultures of acinar cell clusters isolated from mouse pancreas bearing targeted heterozygous knockout of Grp78 (c78f/+ ) and subjected to 3D culture in collagen. We further discovered that GRP78 haploinsufficiency in both the PKC78f/+ and c78f/+ pancreata leads to reduction of epidermal growth factor receptor, which is critical for ADM initiation. Collectively, our studies establish a role for GRP78 in ADM and PDAC development.

Maintenance of chondrocyte survival by PIT1/SLC20A1-mediated regulation of endoplasmic reticulum homeostasis

Maintenance of chondrocyte survival by PIT1/SLC20A1-mediated regulation of endoplasmic reticulum homeostasis

Methionine sulfoxide reductase B3 protects from endoplasmic reticulum stress in Drosophila and in mammalian cells

Methionine sulfoxide reductase B3A (MsrB3A), which catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine, is localized to the endoplasmic reticulum (ER). Here, we report a critical role of the ER-targeted MsrB3 in protection against ER stress in Drosophila and in mammalian cells. Flies overexpressing human MsrB3A exhibited significantly increased resistance to ER stress induced by dithiothreitol. These flies also showed slightly enhanced resistance to tunicamycin-induced ER stress. In addition, overexpression of MsrB3A in mammalian cells increased resistance to dithiothreitol- and thapsigargin-induced ER stresses. However, MsrB3A overexpression had no effect on the resistance to tunicamycin-induced ER stress. Knockdown of MsrB3A in mammalian cells led to a significant decrease in the resistance to thapsigargin-induced ER stress, but had no effects on the resistance to either dithiothreitol- or tunicamycin-induced ER stress. Collectively, our data provide evidence that the ER-type of MsrB3 plays an important role in protection against ER stress, suggesting that MsrB3 may be involved in the regulation of ER homeostasis.

Selenoprotein K Binds Multiprotein Complexes and Is Involved in the Regulation of Endoplasmic Reticulum Homeostasis

Selenoprotein K (SelK) is an 11-kDa endoplasmic reticulum (ER) protein of unknown function. Herein, we defined a new eukaryotic protein family that includes SelK, selenoprotein S (SelS), and distantly related proteins. Comparative genomics analyses indicate that this family is the most widespread eukaryotic selenoprotein family. A biochemical search for proteins that interact with SelK revealed ER-associated degradation (ERAD) components (p97 ATPase, Derlins, and SelS). In this complex, SelK showed higher affinity for Derlin-1, whereas SelS had higher affinity for Derlin-2, suggesting that these selenoproteins could determine the nature of the substrate translocated through the Derlin channel. SelK co-precipitated with soluble glycosylated ERAD substrates and was involved in their degradation. Its gene contained a functional ER stress response element, and its expression was up-regulated by conditions that induce the accumulation of misfolded proteins in the ER. Components of the oligosaccharyltransferase complex (ribophorins, OST48, and STT3A) and an ER chaperone, calnexin, were found to bind SelK. A glycosylated form of SelK was also detected, reflecting its association with the oligosaccharyltransferase complex. These data suggest that SelK is involved in the Derlin-dependent ERAD of glycosylated misfolded proteins and that the function defined by the prototypic SelK is the widespread function of selenium in eukaryotes.

Single nucleotide polymorphism of rs430397 in the fifth intron of GRP78 gene and clinical relevance of primary hepatocellular carcinoma in Han Chinese: Risk and prognosis

Large number of data showed that allele variants in certain genes are markers for hepatocellular carcinoma (HCC). GRP78 is a stress-associated protein which is a central regulator of endoplasmic reticulum homeostasis due to its multiple functional roles in the folding, maturation and transport of proteins. A case-control study was conducted on 576 HCC patients, and 539 age- and gender-matched healthy subjects to examine whether rs430397 polymorphism in the fifth intron of GRP78 gene is associated with the development and prognosis of HCC. Polymorphism in rs430397 was analyzed by resequencing and TaqMan real-time PCR. Allele A, genotype AA and combined genotypes (AG+AA) displayed significantly increased risk for HCC (OR = 1.48, 95%CI = 1.07-1.79, p = 0.010; OR = 2.25, 95%CI = 1.08-3.38, p = 0.019; and OR = 1.50, 95%CI = 1.09-1.85, p = 0.012, respectively). Genotypes AA and AG were mainly associated with HBV-related HCC (85.8%; p < 0.00001 versus HBV noncarriers with HCC) and cirrhosis-related HCC (90%; p = 0.011 versus noncirrhosis HCC). Patients carrying the AA genotype had a shorter survival time (median 23.0 months in all cases; median 21.0 months in the cases carrying HBsAg). Like HBV and cirrhosis, the rs430397 is an independent prognostic factor influencing the survival of HCC. In conclusion, allele A and genotypes AA and AG of rs430397 may represent high risk and poor prognosis for HCC.