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NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4.

ABSTRACT: There is an urgent need to identify vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). PDAC cells acquire metabolic changes that augment NADPH production and cytosolic redox homeostasis. Here, we show that high NADPH levels drive activity of NADPH oxidase 4 (NOX4) expressed in the endoplasmic reticulum (ER) membrane. NOX4 produces H₂O₂ metabolized by peroxiredoxin 4 (PRDX4) in the ER lumen. Using functional genomics and subsequent in vitro and in vivo validations, we find that PDAC cell lines with high NADPH levels are dependent on PRDX4 for their growth and survival. PRDX4 addiction is associated with increased reactive oxygen species, a DNA-PKcs-governed DNA damage response and radiosensitivity, which can be rescued by depletion of NOX4 or NADPH. Hence, this study has identified NOX4 as a protein that paradoxically converts the reducing power of the cytosol to an ER-specific oxidative stress vulnerability in PDAC that may be therapeutically exploited by targeting PRDX4.

SUBMITTER: Jain P

PROVIDER: S-EPMC8104867 | biostudies-literature |

REPOSITORIES: biostudies-literature

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NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4

Project description:PDAC cells acquire metabolic changes that augment NADPH production and cytosolic redox homeostasis. Here we show that high NADPH levels drive activity of NADPH oxidase 4 (NOX4) expressed in the endoplasmic reticulum (ER) membrane. NOX4 produces H2O2 metabolized by Peroxiredoxin 4 (PRDX4) in the ER lumen. Using functional genomics and subsequent in vitro and in vivo validations, we find that PDAC cell lines with high NADPH levels are dependent on PRDX4 for their growth and survival. PRDX4 addiction is associated with increased reactive oxygen species, a DNA-PKcs-governed DNA damage response and radiosensitivity, which can be rescued by depletion of NOX4 or NADPH. As such, this study has identified NOX4 as a protein which paradoxically converts the reducing power of the cytosol to an ER-specific oxidative stress vulnerability in PDAC that may be therapeutically exploited by targeting PRDX4.

2021-05-25 | GSE166165 | GEO

NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4

Project description:NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4

| PRJNA699511 | ENA

RasGRF Couples Nox4-Dependent Endoplasmic Reticulum Signaling to Ras.

Project description:In response to endoplasmic reticulum (ER) stress, endothelial cells initiate corrective pathways such as the unfolded protein response. Recent studies suggest that reactive oxygen species produced on the ER may participate in homeostatic signaling through Ras in response to ER stress. We sought to identify mechanisms responsible for this focal signaling pathway.In endothelial cells, we found that ER stress induced by tunicamycin activates the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 focally on the ER surface but not on the plasma membrane. Ras activation is also restricted to the ER, occurs downstream of Nox4, and is required for activation of the unfolded protein response. In contrast, treatment with the growth factor VEGF (vascular endothelial growth factor) results in Ras activation and reactive oxygen species production confined instead to the plasma membrane and not to the ER, demonstrating local coupling of reactive oxygen species and Ras signals. We further identify the calcium-responsive, ER-resident guanyl exchange factors RasGRF1 and RasGRF2 as novel upstream mediators linking Nox4 with Ras activation in response to ER stress. Oxidation of the sarcoendoplasmic reticulum calcium ATPase and increases in cytosolic calcium caused by ER stress are blocked by Nox4 knockdown, and reduction in cytosolic free calcium prevents both Ras activation and the unfolded protein response.ER stress triggers a localized signaling module on the ER surface involving Nox4-dependent calcium mobilization, which directs local Ras activation through ER-associated, calcium-responsive RasGRF.

| S-EPMC5222703 | biostudies-literature

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity.

Project description:The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.

| S-EPMC7474598 | biostudies-literature

The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein.

Project description:Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.

| S-EPMC4807287 | biostudies-literature

Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL.

Project description:Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment.

| S-EPMC5026355 | biostudies-literature

FoxO1 links hepatic insulin action to endoplasmic reticulum stress.

Project description:Forkhead box O1 (FoxO1) is a transcription factor that mediates the inhibitory effect of insulin on target genes in hepatic metabolism. Hepatic FoxO1 activity is up-regulated to promote glucose production during fasting and is suppressed to limit postprandial glucose excursion after meals. Increased FoxO1 activity augments the expression of insulin receptor (IR) and IR substrate (IRS)2, which in turn inhibits FoxO1 activity in response to reduced insulin action. To address the underlying physiology of such a feedback loop for regulating FoxO1 activity, we delivered FoxO1-ADA by adenovirus-mediated gene transfer into livers of adult mice. FoxO1-ADA is a constitutively active allele that is refractory to insulin inhibition, allowing us to determine the metabolic effect of a dislodged FoxO1 feedback loop in mice. We show that hepatic FoxO1-ADA production resulted in significant induction of IR and IRS2 expression. Mice with increased FoxO1-ADA production exhibited near glycogen depletion. Unexpectedly, hepatic FoxO1-ADA production elicited a profound unfolded protein response, culminating in the induction of hepatic glucose-regulated protein 78 (GRP78) expression. These findings were recapitulated in primary human and mouse hepatocytes. FoxO1 targeted GRP78 gene for trans-activation via selective binding to an insulin responsive element in the GRP78 promoter. This effect was counteracted by insulin. Our studies underscore the importance of an IR and IRS2-dependent feedback loop to keep FoxO1 activity in check for maintaining hepatic glycogen homeostasis and promoting adaptive unfolded protein response in response to altered metabolism and insulin action. Excessive FoxO1 activity, resulting from a dislodged FoxO1 feedback loop in insulin resistant liver, is attributable to hepatic endoplasmic reticulum stress and metabolic abnormalities in diabetes.

| S-EPMC2940535 | biostudies-literature

Chaperone insufficiency links TLR4 protein signaling to endoplasmic reticulum stress.

Project description:Inflammation plays an important pathogenic role in a number of metabolic diseases such as obesity, type 2 diabetes, and atherosclerosis. The activation of inflammation in these diseases depends at least in part on the combined actions of TLR4 signaling and endoplasmic reticulum stress, which by acting in concert can boost the inflammatory response. Defining the mechanisms involved in this phenomenon may unveil potential targets for the treatment of metabolic/inflammatory diseases. Here we used LPS to induce endoplasmic reticulum stress in the human monocyte cell-line, THP-1. The unfolded protein response, produced after LPS, was dependent on CD14 activity but not on RNA-dependent protein kinase and could be inhibited by an exogenous chemical chaperone. The induction of the endoplasmic reticulum resident chaperones, GRP94 and GRP78, by LPS was of a much lower magnitude than the effect of LPS on TLR4 and MD-2 expression. In face of this apparent insufficiency of chaperone expression, we induced the expression of GRP94 and GRP78 by glucose deprivation. This approach completely reverted endoplasmic reticulum stress. The inhibition of either GRP94 or GRP78 with siRNA was sufficient to rescue the protective effect of glucose deprivation on LPS-induced endoplasmic reticulum stress. Thus, insufficient LPS-induced chaperone expression links TLR4 signaling to endoplasmic reticulum stress.

| S-EPMC3346100 | biostudies-literature

Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress and cell death in liver cells.

Project description:Chronic exposure to elevated free fatty acids, in particular long chain saturated fatty acids, provokes endoplasmic reticulum (ER) stress and cell death in a number of cell types. The perturbations to the ER that instigate ER stress and activation of the unfolded protein in response to fatty acids in hepatocytes have not been identified. The present study employed H4IIE liver cells and primary rat hepatocytes to examine the hypothesis that saturated fatty acids induce ER stress via effects on ER luminal calcium stores. Exposure of H4IIE liver cells and primary hepatocytes to palmitate and stearate reduced thapsigargin-sensitive calcium stores and increased biochemical markers of ER stress over similar time courses (6 h). These changes preceded cell death, which was only observed at later time points (16 h). Co-incubation with oleate prevented the reduction in calcium stores, induction of ER stress markers and cell death observed in response to palmitate. Inclusion of calcium chelators, BAPTA-AM or EGTA, reduced palmitate- and stearate-mediated enrichment of cytochrome c in post-mitochondrial supernatant fractions and cell death. These data suggest that redistribution of ER luminal calcium contributes to long chain saturated fatty acid-mediated ER stress and cell death.

| S-EPMC2899892 | biostudies-literature

ERO1-independent production of H2O2 within the endoplasmic reticulum fuels Prdx4-mediated oxidative protein folding.

Project description:The endoplasmic reticulum (ER)-localized peroxiredoxin 4 (PRDX4) supports disulfide bond formation in eukaryotic cells lacking endoplasmic reticulum oxidase 1 (ERO1). The source of peroxide that fuels PRDX4-mediated disulfide bond formation has remained a mystery, because ERO1 is believed to be a major producer of hydrogen peroxide (H2O2) in the ER lumen. We report on a simple kinetic technique to track H2O2 equilibration between cellular compartments, suggesting that the ER is relatively isolated from cytosolic or mitochondrial H2O2 pools. Furthermore, expression of an ER-adapted catalase to degrade lumenal H2O2 attenuated PRDX4-mediated disulfide bond formation in cells lacking ERO1, whereas depletion of H2O2 in the cytosol or mitochondria had no similar effect. ER catalase did not effect the slow residual disulfide bond formation in cells lacking both ERO1 and PRDX4. These observations point to exploitation of a hitherto unrecognized lumenal source of H2O2 by PRDX4 and a parallel slow H2O2-independent pathway for disulfide formation.

| S-EPMC4621842 | biostudies-other

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