CYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis of lung cancer cells via PERK-ATF4 and IRE1α-JNK pathway

CYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis of lung cancer cells via PERK-ATF4 and IRE1α-JNK pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis of lung cancer cells via PERK-ATF4 and IRE1α-JNK pathway Misun Won, Joo-Young Im, Soo Jin Kim, Jong-Lyul Park, Tae-Hee Han, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2810245/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jan, 2024 Read the published version in Experimental & Molecular Medicine → Version 1 posted 7 You are reading this latest preprint version Abstract Cytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth in vitro and in vivo . However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomics analysis revealed that CYB5R3 overexpression increased the production of NAD + and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress is induced by activating protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD + activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent-intrinsic cell death. Our findings highlight the significance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer. Health sciences/Diseases/Cancer/Tumour biomarkers Biological sciences/Molecular biology/Post-translational modifications/PolyADP-ribosylation Biological sciences/Biological techniques/Metabolomics Biological sciences/Computational biology and bioinformatics/Cellular signalling networks CYB5R3 DDIT3 PERK IRE1α PARP16 ER stress Lung cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Lung cancer is the leading cause of cancer-related mortality worldwide and has a 5-year survival rate of less than 20% 1, 2 . Lung cancer is histologically classified into two major types, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the most prevalent subtype, accounting for 80–85% of lung cancer patients 3 . Genomic studies have revealed multiple genetic alterations in oncogenes and tumor suppressor genes (TSGs) in lung cancer 4 , 5 . Although genetic alterations in TP53, EGFR, EML4-ALK, PIK3CA, and KRAS have been identified as biomarkers of lung cancer, difficulties remain in diagnosing, prognosis, and treating lung cancer patients. Therefore, novel therapeutic targets must be developed to improve lung cancer diagnosis and treatment. Cytochrome b5 reductase 3 (CYB5R3) catalyzes the one-electron transfer from NADH to electron acceptors, such as cytochrome b5 or coenzyme Q, producing NAD + 6 . CYB5R3 has two isoforms: soluble and membrane-bound. The soluble isoform expressed in erythrocytes has a truncated N-terminal binding domain and reduces methemoglobin 7 , and the membrane-bound isoform expressed in most cells is anchored to the mitochondrial outer membrane, endoplasmic reticulum (ER), and plasma membrane and participates in fatty acids elongation and desaturation 8 , 9 , cholesterol biosynthesis 10 , drug metabolism 11 , 12 , and aging 6 , 9 . CYB5R3 is a reductase of soluble guanylate cyclase (sGC) in vascular smooth muscle cells that regulates cGMP production, protein kinase G signaling, and hypertension 13 , 14 . CYB5R3 is transcriptionally regulated by FOXO3a and Nrf2, and controls both nutrient and oxidative stress responses 15 . CYB5R3 is a target of FoxO1 in β-cells and links mitochondrial dysfunction to β-cell failure 16 , and it also promotes the colonization and metastasis of estrogen receptor-negative breast cancer 17 . However, the functions and mechanisms of CYB5R3 in cancer biology have not yet been explored. The ER plays key roles in protein folding, transport, calcium homeostasis, and lipid synthesis. Alterations in cellular processes, such as protein folding and calcium regulation, lead to the activation of the unfolded protein response (UPR), a pro-survival response to recover normal ER function; however, prolonged ER stress ultimately triggers cell death 18 – 20 . UPR activation is mediated by three stress sensors: transmembrane receptors, protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 alpha (IRE1α) and activating transcription factor 6 (ATF6). These stress sensors bind to the ER chaperone GRP78 (also known as BiP) under non-stress conditions. ER stress, such as the accumulation of unfolded proteins, activates UPR by dissociating stress sensors from GRP78. Activated PERK phosphorylates eukaryotic translation initiation factor-2 (eIF2), thereby inhibiting protein translation and increasing the expression of activating transcription factor-4 (ATF4) and its downstream target C/EBP-homologous protein (CHOP; gene name DDIT3 ), which are involved in ER stress-induced apoptosis 21 . Similar to PERK, IRE1α splices the transcription factor X box-binding protein 1 (XBP1), and the spliced form of XBP1 induces the expression of genes involved in the UPR. When the recovery of ER function fails, active IRE1α recruits TNF receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase-1 (ASK1), which activates Jun-N-terminal kinase (JNK) and induces apoptosis 22 , 23 . The transcription factor ATF6 is cleaved by S1P and S2P endopeptidases in the Golgi apparatus, and the cleaved ATF6 induces UPR targets, including GRP78 and XBP1 21 . In this study, we provide evidence that CYB5R3 overexpression induces ER stress by promoting ADP-ribosylation of PERK and IRE1α, resulting in apoptosis of lung cancer cells. Our findings suggest that CYB5R3 functions as a tumor suppressor and can be utilized in the development of anti-cancer drugs for lung cancer. MATERIAL AND METHODS Reagents and antibodies Sulforhodamine B, tunicamycin, and GSH-EE were purchased from Sigma-Aldrich (St. Louis, MO, USA). Genomic DNA kit was obtained from Promega (Madison, WI, USA). The antibodies anti-β-Tubulin (#2128), anti-ATF3 (#18665), anti-GADD45A (#4632), anti-ATF6 (#65880), anti-CHOP (#2895), anti-GRP78 (#3177), anti-XBP1 (#40435), anti-IRE1α (#3294), anti-DR5 (#8074), anti-Bax (#2772), anti-ATF4 (#11815), anti-Puma (#4976), anti-PERK (#3192), anti-p-eIF2α (#9721), anti-eIF2α (#9722), anti-p-JNK (#9251), anti-JNK (#9252), anti-cytochrome c (#4272), anti-COXIV (#4850), anti-Calnexin (#2679), anti-PARP1 (#9542), anti-Poly/Mono-ADP Ribose (#83732), anti-Caspase 3 (#9662), anti-Caspase 8 (#9746), and anti-Caspase 9 (#9502) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-PARP16 (ab154510) was obtained from Abcam (Cambridge, UK); anti-Sestrin2 (10795-1-AP) was obtained from Proteintech (Rosemont, IL, USA); anti-CYB5R3 (BS-12162R) was obtained from Bioss Antibodies Inc. (Woburn, MA, USA); anti-GAPDH (LF-P-A0212) was obtained from AbFrontier (Seoul, Korea); anti-ARTC1 (SAB1300652) and anti-Flag (F1804) were obtained from Sigma-Aldrich; anti-mouse-FITC (sc-2010) and anti-rabbit-Rhodamine (sc-2492) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Cell culture and transfections Human lung fibroblasts (IMR-90 and WI-38) and NSCLC cell lines (NCI-H1299 (H1299), NCI-H1703 (H1703), NCI-H226 (H226), NCI-H23 (H23), NCI-H460 (H460), NCI-H2009 (H2009), HCC827, and A549) were purchased from the Korean Cell Line Bank (Seoul, Korea) and the KRIBB Cell Line Bank (Daejeon, Korea). IMR-90, WI-38, and A549 cells were cultured in Dulbecco′s modified Eagle′s medium (DMEM), while H226, H460, H2009, HCC827, H23, H1703, and H1299 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). All cell lines were tested for Mycoplasma contamination using an e-Myco™ VALiD Mycoplasma PCR Detection Kit (iNtRON Biotechnology, Gyeonggi-Do, Korea). siRNA pools (ON-TARGET-plus SMART pool, contains four different siRNAs that target a single gene) against ATF3 (L-008663), DDIT3/CHOP (L-004819), SESN2 (L-019134), ERN1/IRE1 (L-004951), EIF2AK3/PERK (L-004883), ATF6 (L-009917), ATF4 (L-005125), JNK1 (L-003514), JNK2 (L-003505), JNK3 (L-004324), ARTC1 (L-010387), PARP16 (L-020837), and non-targeting control (D-001810-10-05) were obtained from Dharmacon (Lafayette, CO, USA). CYB5R3 siRNA was purchased from Bioneer Corporation (Daejeon, Korea). The target sequences were as follows: siCYB5R3 #1: 5′-GUUUACUUCAAGGACACCCAU-3′, siCYB5R3 #2: 5′-AGAACCUCAGCAUUUCCUU-3′, siScrambled: 5′-CCUACGCCACCAAUUUCGU-3′. The cells were transfected with siRNAs (40 nM) using an electroporation (Neon, Invitrogen) according to the manufacturer′s instructions. Adenoviruses Adenoviral CYB5R3-Flag (Ad-CYB5R3) and empty-vector control (EV) were obtained from Vigene Biosciences (Rockville, MD, USA). Viral stocks were re-infected with AD293 cells to amplify the virus and purified by double cesium chloride gradient ultracentrifugation. The infectious viral particles in the cesium chloride gradient were collected, dialyzed against 10 mM Tris (pH 8.0), 2 mM MgCl 2 , and 5% sucrose solution, and stored in a deep freezer. Viral titers were determined using the Adeno-X™ Rapid Titer kit (Takara Bio USA, Inc., Mountain View, CA, USA) according to the manufacturer′s protocol. The cells were infected with adenovirus at a mode of infection (MOI) of 100. Animal experiments All mouse experiments were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee. Tumors were induced by 10 weekly intraperitoneal (IP) injection of 0.5 mg/g urethane (ethyl carbamate, Sigma-Aldrich) in C57BL/6 (Orient Bio, Gyeonggi-Do, Korea) mice 24 . The mice were sacrificed eight months after urethane injection. Lungs were fixed, embedded, stained with hematoxylin and eosin (H&E), and analyzed in an Olympus microscope (BX51, Tokyo, Japan). For the mouse xenograft assay, tumors were established by subcutaneously injecting NCI-H1299 cells (5×10 6 cells/mouse) into six-week-old BALB/c female nude mice (Orient Bio). Tumor volumes were estimated using the following formula: length (mm) × width (mm) × height (mm) / 2. When the average tumor volume reached 100 cm 3 , the mice were randomized into two groups (n = 8), and adenoviral EV or CYB5R3 (1 ×10 9 pfu per mouse) were administered intratumorally every three days three times. The mice were euthanized on day 18, and tumor weights were measured. Generation of CYB5R3 knockout mice CYB5R3 knockout mice were generated by GH Bio (Daejeon, Korea). Single-guide RNA (sgRNA) was designed using ZiFiT ( http://zifit.partners.org/ZiFiT/ ) program targeting N-terminal region of CYB5R3. The spacer sequences of sgRNAs are as follows; sgRNA1: 5′-CTTGATGTCGGGGTTCTCGA-3′, sgRNA2: 5′-AGACTCCGAGTAGCTGTTCC-3′, sgRNA3: 5′-TCTGAGGCTCATCGACAAGG-3′. The two complimentary oligonucleotides of each sgRNA were annealed and cloned in the pT7-gRNA vector, a vector designed for synthesizing sgRNA 25 . In vitro transcription of sgRNA for CYB5R3 and short RNA purification was performed using MEGAshortscript T7 kit (ThermoScientific, Waltham, MA, USA) according to the manufacturer′s instructions. Microinjection was performed in the fertilized eggs from C57BL/6N (Orient Bio) mice. The mixture of sgRNA (100 ng/µl) and Cas9 protein (80 ng/µl) (ToolGen Inc, Seoul, Korea) was injected into the cytoplasm of the pronuclear stage embryos. Injected embryos were cultured in the media overnight prior to embryo transfer into pseudo-pregnant mice (ICR strain). Genomic DNA from tails of the progenies were extracted and subjected to PCR using the primer sets; forward primer 5′-TGGAGTTCTCTGGTCAAGGC-3′, reverse primer 5′-TTGGCTGTCATTGTGCCTGA-3′. PCR products were analyzed by agarose gel electrophoresis and sequencing analysis to confirm the identity of every alleles. Immunohistochemistry Human tissue arrays were obtained from US Biomax (Rockville, MD, USA). Immunohistochemistry (IHC) was performed as previously described 26 . Briefly, the slides were incubated overnight with an anti-CYB5R3 antibody. After washing, the slides were incubated with a biotinylated HRP complex (Vector Laboratories, Burlingame, CA, USA), and 3,3-diaminobenzidine (DAB substrate kit; Vector Laboratories) was used for color development. Then, the slides were stained with hematoxylin and eosin (H&E). Cell viability and IncuCyte system Cell viability was determined using a sulforhodamine B assay, as previously described 27 . A cell growth inhibition assay (3000 cells/96-well plate) was performed in cells infected with adenoviral CYB5R3 and EV for 72 h. Cell death was analyzed with CellPlayer reagent-based annexin V (red) or caspase-3/7 (green), according to the manufacturer′s protocols (IncuCyte ZOOM System, Essen Bioscience, Ann Arbor, MI, USA). Images of green or red fluorescence and phase contrast were captured at 2 h intervals using a 10 × objective lens. RNA-sequencing (RNA-seq) analysis Total RNA was isolated using an mRNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Four micrograms of RNA was constructed using the TruSeq stranded mRNA LT Sample Prep Kit. The library was sequenced using the Illumina NovaSeq 6000 system (Illumina, San Diego, CA, USA) to generate 100 bp paired-end reads. The sequence reads were mapped to the human genome (GRCh38/hg38) using STAR (v.2.7.3a), and gene expression was quantified using the count module in STAR. A differentially expressed gene (DEG) was selected from RNA-seq count data using the edgeR package (v.3.32.0). Quantitative Reverse transcription polymerase chain reaction (QRT-PCR) Two micrograms of total isolated RNA were reverse transcribed into cDNA using a TOPscript RT kit (Enzynomics, Daejeon, Korea) according to the manufacturer′s protocol. Real-time qPCR was performed using the SYBR Green master mix kit (Qiagen) on a Rotor-Gene Q system (Qiagen). The primers were obtained from the Bioneer Corporation as follows: DUSP1 (P199349), DUSP2 (P259112), DUSP5 (P197350), DUSP10 (P210086), PPP1R15A (P192005), DDIT3 (P225750), ERN1 (P298108), XBP1 (190450), GADD45A (P230161), BBC3 (P150935), TNFRSF10B (P195586), TRAF1 (P141876), ATF3 (P292769), KLF4 (P154272), and SESN2 (P153243). All reactions were performed in triplicate and normalized to β-actin (Qiagen) as an internal control. Metabolomics analysis Metabolomics analysis was performed by Human Metabolome Technologies, Inc. (Tsuruoka, Japan) using the CARCINOSCOPE (C-SCOPE) platform. H1299 cells (2 × 10 6 cells) were seeded into a 100 mm dish the day before the assay and incubated with adenoviral EV or CYB5R3 for 24 or 36 h (n = 3/group). Metabolite extraction was performed according to the manufacture′s protocol (HMT), as described previously 28 . Absolute quantitative analysis of 116 metabolites (54 cations and 62 anions) was performed on 12 samples of harvested cells using capillary electrophoresis-mass spectrometry (CE-MS). Immunoprecipitation and Immunoblot analysis Cells were lysed with 1× RIPA buffer (Millipore, Temecula, CA, USA) containing 1 mM Na 3 VO 4 , 1 mM sodium fluoride, 1 mM PMSF, and a protease inhibitor cocktail (Roche, Basel, Switzerland), and the lysates were quantified using a the BCA protein assay kit (Thermo Scientific, 23227). For immunoprecipitation, 1 mg of lysates was incubated with 2 µg of the indicated antibodies or normal rabbit IgG at 4℃ overnight and then incubated with 20 µl of protein A/G plus agarose (sc-2003, Santa Cruz Biotechnology) at 4℃ for 1 h. The agarose was washed three times with wash buffer (0.1% NP40 in phosphate-buffered saline (PBS)). Mitochondrial isolation was performed using H1299 cells with a Mitochondrial Isolation Kit according to the manufacturer′s protocol (Thermo Scientific, 89874). The lysates were then subjected to immunoblotting using specific antibodies. Immunoblot signals were detected using an Enhanced Chemiluminescence (ECL) kit (Millipore). Immunofluorescence and DCF-DA staining Cells were plated onto a microslide 8-well plate (ibidi Inc., Fitchburg, WI, USA), fixed in 4% paraformaldehyde for 30 min, permeabilized in PBS with 0.3% Triton X-100 for 10 min, and blocked with 3% bovine serum albumin for 1 h at 25℃. The cells were incubated overnight with appropriate antibodies. For mitochondrial tracker staining, cells were incubated with the tracker for 30 min and washed with PBS. Cells were incubated with 10 µM H2DCFDA (Invitrogen) for 30 min and washed with PBS to measure ROS generation. Finally, the cells were counterstained with DAPI for 10 min to label the nuclei and then analyzed using a confocal microscope (LSM5 Live DuoScan, Carl Zeiss, Stuttgart, Germany). Generation of knockout cells with CRISPR-Cas9 strategy. sgRNA was cloned into a lentiCrispr V2 plasmid as previously reported 29 . Briefly, each sgRNA oligonucleotide annealed with the T4 PNK enzyme (#M0201, NEB, Ipswich, MA, USA) was cloned into lentiCrispr V2 vector-digested BsmBI (#R0739, NEB). To generate lentivirus, 293T cells in 100 mm tissue culture dishes were transfected with 6 µg of each sgRNA along with 3.34 µg of pLP1, 2.2 µg of pLP2, and 3.34 µg of VSVg packaging vectors using polyethyleneimine. Viral particles were produced and transduced into cells, followed by puromycin (3–10 µg/mL) selection. Gene targeting was confirmed using immunoblot analysis. Target sequences of the sgRNAs are listed below. Negative controls (sgNeg); 5′-GAAGATGGGCGGGAGTCTTC-3′, CYB5R3 #1; 5′-AGGCATCACCCCGATGCTGC-3′, CYB5R3 #2; 5′-GTGTATAGGGCCGGACGACC-3′, CYB5R3 #3; 5′-TCCCGGTCGATGAGCCGCAG-3′, CYB5R3 #4; 5′-GAAGACGAAGCAGCGCTCCG-3′. Statistical analysis All data represent at least three independent experiments. The results are presented as the means ± SD. Statistical analyses were performed using a two-tailed Student′s t -test using GraphPad Prism ver 9.0 software (GraphPad Software, Boston, MA, USA). Statistical significance was set at p < 0.05. RESULTS CYB5R3 expression is downregulated in lung cancer To investigate the relevance of CYB5R3 in tumorigenesis, we analyzed the CYB5R3 expression profile in The Cancer Genome Atlas (TCGA) cohort cBioPortal ( http://cbioportal.org ) using R software. TCGA data revealed that CYB5R3 mRNA expression was significantly decreased in 13 of 24 subtypes of the TCGA cancer patient tissues compared to normal tissues (Supplementary Fig. 1a). CYB5R3 expression was downregulated in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) (Fig. 1 a). Immunohistochemistry (IHC) was performed using a human tissue array of 32 normal lung tissues or adjacent normal lung tissues and 128 lung cancer samples. Consistent with public data, the IHC analysis revealed that CYB5R3 expression in lung cancer tissues was lower than that in normal tissues (Fig. 1 b and Supplementary Fig. 1b). We also found that 91% (29 of 32) of the normal lung tissues exhibited high CYB5R3 expression, while only 19% (24 of 128) of the lung cancer tissues showed high CYB5R3 expression (Fig. 1 c), indicating that CYB5R3 expression is associated with lung carcinogenesis. However, the expression levels of CYB5R1 and CYB5R2 were not downregulated in LUAD tissue (Supplementary Fig. 2a and b). Neither CYB5R1 nor CYB5R2 was detected in human lung cancer cells (Supplementary Fig. 2c). To examine the expression pattern of CYB5R3 in human lung cancer cells, we measured CYB5R3 mRNA levels using quantitative RT-PCR in two human lung fibroblast lines and eight NSCLC cell lines. The mRNA levels of CYB5R3 in the NSCLC cell lines A549, H1299, H1703, H226, H23, H460, H2009, and HCC827 were lower than those in normal lung fibroblast lines WI-38 and IMR-90 (Fig. 1 d). Similarly, CYB5R3 protein was relatively abundant in WI-38 and IMR-90 cells compared to that in the NSCLC cell lines (Fig. 1 e). To examine the effect of CYB5R3 depletion on cell growth, we performed CYB5R3 knockdown using two different siRNAs targeting CYB5R3 in WI-38 and IMR-90. CYB5R3 expression significantly decreased in CYB5R3-knockdown cells (Fig. 1 f, upper panel). CYB5R3 knockdown promoted cell growth compared with siScrambled (siScr) in WI-38 and IMR-90 cells (Fig. 1 f, lower panel). CYB5R3 is functionally associated with tumor suppressor in a mouse model We evaluated the role of CYB5R3 in tumorigenesis using an in vivo mouse model (Supplementary Fig. 3). First, we examined the expression profile of CYB5R3 in mouse tissues and found that it was abundantly expressed in the mouse lungs, liver, testes, and ovaries (Supplementary Fig. 3a). However, compared with mouse primary lung fibroblast, CYB5R3 protein was not detected in Lewis lung carcinoma (LLC), a murine lung cancer cell line (Supplementary Fig. 3b). CYB5R3 knockout (KO) mice were generated using the CRISPR-Cas9 system (Supplementary Fig. 3c). As expected, CYB5R3 protein expression was not detected in lungs, liver, spleen, or kidneys of CYB5R3 −/− mice (Supplementary Fig. 3d). We then evaluated the incidence of lung cancer in CYB5R3 −/− mice treated with urethane for eight months. H&E staining showed a higher incidence of tumors in the lung tissue of CYB5R3 −/− mice than CYB5R3 +/+ mice (Fig. 1 g). In the lung metastasis experiment using LLC cells, the tumor incidence and size were considerably increased in the lung tissues of CYB5R3 −/− mice than CYB5R3 +/+ mice (Supplementary Fig. 3e, f). These results suggest that CYB5R3 acts as a tumor suppressor in lung cancer. CYB5R3 overexpression leads to apoptosis of lung cancer To confirm the tumor-suppressive role of CYB5R3 in human lung cancer cells, we investigated the effects of CYB5R3 overexpression using Ad-CYB5R3, an adenovirus that expresses CYB5R3. CYB5R3 protein was detectable within 12 h and showed the highest values 24 h after infection with CYB5R3 (Fig. 2 a). CYB5R3 overexpression dramatically inhibited the growth of A549, H1299, H226, and H1703 cells compared to EV, but did not inhibit that of WI-38 and IMR-90 cells (Fig. 2 b). Cleavage of PARP1 and capase-3 was detected in H1299 or H1703 cells infected with Ad-CYB5R3 (Fig. 2 c). A caspase 3/7 activity assay and annexin V staining showed that CYB5R3 induced significant apoptosis in lung cancer cells (Fig. 2 d). To investigate the effect of CYB5R3 on tumor growth in vivo , we performed a xenograft assay using H1299 cells. Compared with EV-treated mice, CYB5R3-treated mice exhibited a 53.3% and 44.2% reduction in tumor volume and weight, respectively, without significant changes in body weight (Fig. 2 e-g). Immunoblot analysis of the resected tumors revealed that the protein level of CYB5R3 was dramatically increased in CYB5R3-treated tumors compared to EV-treated tumors (Fig. 2 h). These data implied that CYB5R3 functions as a tumor suppressor in lung cancer. CYB5R3 overexpression affects the transcriptome landscape of lung cancer cells To investigate the molecular mechanism by which CYB5R3 induces cancer cell death, we analyzed the gene expression pattern by CYB5R3 overexpression in H1299 cells. RNA sequencing analysis revealed 248 upregulated and 69 downregulated genes in cells infected with Ad-CYB5R3 compared with those infected with EV (Fig. 3 a). Genes in CYB5R3-overexpressing cells that are involved in mitogen‑activated protein kinase (MAPK) signaling, the TNF signaling pathway, pathways in cancer, protein processing in ER, and apoptosis pathway were upregulated (Fig. 3 b). The RNA sequencing data were verified using real-time PCR. The upregulated genes included DUSP1, DUSP2, DUSP5, and DUSP10 in MAPK signaling; DDIT3, ERN1, PPP1R15A, and XBP1 in protein processing in ER; BBC3, GADD45A, TNFRSF10B, and TRAF1 in apoptosis; and ATF3, KLF4, and SESN2 in other pathways (Fig. 3 c). Interestingly, the levels of CHOP (DDIT3), DR5 (TNFRSF10B), ATF3, and SESN2 proteins dramatically increased in CYB5R3-overexpressing H1299 and H1703 cells following Ad-CYB5R3 infection (Fig. 3 d). In contrast, the expression of XBP1, IRE1α (ERN1), PUMA (BBC3), and GADD45A proteins remained unchanged. To evaluate how genes upregulated by CYB5R3 overexpression are involved in cell death, we transfected siRNAs against ATF3, DDIT3, or SESN2 in H1299 cells. DDIT3 silencing overcame CYB5R3-induced cell death (Fig. 3 e, lower panel). The knockdown efficiency of each siRNA is shown in Fig. 3 e (upper panel). These data suggest that CHOP (DDIT3) induction is critical for CYB5R3-induced cell death in lung cancer cells. CYB5R3 triggers ER stress via PERK-ATF4 or IRE1α-JNK pathway Since CHOP (DDIT3) is a major hall marker of ER stress and induces ER stress-mediated apoptosis 30 , we examined the relationship between CYB5R3 overexpression and ER stress. Immunofluorescence staining showed that CYB5R3 co-localizes with calnexin, an ER marker, in CYB5R3-overexpressing H1299 cells (Fig. 4 a). CYB5R3 also localizes in the mitochondria, which is consistent with the results of previous studies 17 , 31 (Supplementary Fig. 4). To investigate whether CYB5R3 regulates ER function, we analyzed the expression levels of the ER stress sensors PERK, IRE1α, and ATF6 and their downstream targets. Similar to CHOP induction (Fig. 3 d), CYB5R3 overexpression increased the expression of ER stress signaling proteins, such as GRP78, p-eIF2α, and p-JNK, which triggered apoptosis in both H1299 and H1703 cells (Fig. 4 b). To assess the pathways involved in CYB5R3-induced cell death, we performed gene knockdown assays using siRNAs against PERK, IRE1α, or ATF6 in H1299 cells. Importantly, the silencing of PERK or IRE1α but not ATF6 overcame CYB5R3-induced cell death (Fig. 4 c). Moreover, the deletion of ATF4, which is a downstream target of PERK, or the deletion of JNK isoforms JNK1 or JNK2, which are downstream targets of IRE1α, rescued CYB5R3-induced cell death (Fig. 4 d, e). To examine the interplay between CYB5R3 and ER stress, we generated CYB5R3 knockout H1299 or H1703 cells using the CRISPR-Cas9 system. We used four different sgRNAs against CYB5R3 (sgCYB5R3) and found that sgCYB5R3 #3 and #4 completely suppressed CYB5R3 expression (Fig. 4 f, upper panel). As expected, CYB5R3 knockout cells displayed a significant increase in cell growth compared to control (sgNeg) cells (Fig. 4 f, lower panel), which is consistent with previous data from the normal lung fibroblasts WI38 and IMR-90 (Fig. 1 f). Following treatment with tunicamycin, an ER stress-inducing antitumor agent, the levels of GRP78, CHOP, and p-eIF2α were increased in H1299-sgNeg and H1703-sgNeg cells, whereas the induction of CHOP and p-eIF2α was attenuated in H1299-sgCYB5R3 or H1703-sgCYB5R3 cells (Fig. 4 g). These data suggest that CYB5R3-induced cell death is dependent on the PERK-ATF4 and IRE1α-JNK signaling pathway. CYB5R3 overexpression drives metabolic reprogramming Given that CYB5R3 functions in energy and lipid metabolism 9 , we speculated that CYB5R3 leads to metabolic alteration. To investigate the metabolic changes in H1299 cells infected with Ad-CYB5R3, we performed metabolomics analysis using capillary electrophoresis and time-of-flight mass spectrometry (CE-TOFMS). Principal component analysis (PCA) revealed that CYB5R3 overexpression resulted in marked differences in metabolic signatures (Fig. 5 a). Heatmap analysis revealed that EV and CYB5R3 were grouped into distinct metabolic clusters, and changes in 59 metabolites or 66 metabolites were observed in cells infected with CYB5R3 relative to that in cells infected with EV for 24 or 36 h, respectively (Fig. 5 b). Subsequently, we conducted a pathway enrichment analysis of CYB5R3-related metabolites using MetaboAnalyst 4.0, to comprehensively analyze metabolic changes. We found that CYB5R3-related metabolites showed significant increases in the Warburg effect and were significantly enriched in glutamate, purine metabolism, arginine and proline metabolism, aspartate metabolism, urea cycle, glycine and serine metabolism, and the citric acid cycle (Fig. 5 c, d). Redox homeostasis and purine metabolism were simultaneously altered in cells infected with CYB5R3 for 24 h and 36 h. Although reduced glutathione (GSH) was decreased, oxidized GSH (GSSG), NAD + , AMP, and ADP were remarkably increased 24 h after CYB5R3 infection (Fig. 5 e-g). These data suggested that CYB5R3 overexpression induces metabolic changes in lung cancer cells. CYB5R3 increases PARP16-mediated ADP-ribosylation of PERK and IRE1α Next, we explored how CYB5R3 activates PERK and IRE1α. NADH oxidation by CYB5R3 overexpression can affect NAD + -dependent signaling pathways, such as ADP-ribosylation and protein deacetylation 32 , 33 . Metabolomics revealed that ADP-ribose levels were higher in CYB5R3-overexpressing cells than in control cells (Fig. 6 a). A previous study demonstrated that ARTC1-mediated ADP-ribosylation of GRP78 is inactive and activates the ER stress response 34 . Moreover, ER-resident PARP16 activates PERK and IRE1α via ADP-ribosylation in the ER 35 . To investigate whether ADP-ribosylation is involved in the activation of PERK and IRE1α by CYB5R3, we examined total ADP-ribosylation, including mono-ADP-ribosylation (MAR) and poly-ADP-ribosylation (PAR). Indeed, CYB5R3 overexpression increased overall ADP-ribosylation (Fig. 6 b). We examined the effect of ARTC1 and PARP16 silencing on CYB5R3-induced cell death. Importantly, PARP16 depletion reduced CYB5R3-induced cell death (Fig. 6 c). We found that the increase in ADP-ribosylation in CYB5R3-overexpressing cells was attenuated by PARP16 knockdown (Fig. 6 d). Moreover, depletion of PARP16 decreased the expression of ER stress markers, such as CHOP, p-elF2a, and p-JNK, which were increased by CYB5R3 overexpression (Fig. 6 e). We further investigated the role of CYB5R3 in the ADP-ribosylation of PERK and IRE1α and found that it increased ADP-ribosylation of PERK and IRE1α (Fig. 6 f). Surprisingly, immunoprecipitates with anti-PERK or anti-IRE1α bound to CYB5R3-Flag (Fig. 6 f). Moreover, an immunoprecipitation assay using an anti-FLAG antibody showed that CYB5R3-Flag interacted with endogenous PERK or IRE1α in H1299 cells (Supplementary Fig. 5). These data suggest that CYB5R3 promotes the PARP16-mediated ADP-ribosylation of PERK and IRE1α to induce ER stress. CYB5R3 activates caspase-9 through oxidative stress Based on our finding of increased oxidized GSH (GSSG) in CYB5R3 overexpressed cells (Fig. 5 f), reactive oxygen species generation was detected in CYB5R3-overexpressing cells using DCF-DA staining. We observed the generation of ROS in CYB5R3-overexpressing H1299 cells (Fig. 7 a). GSH-ethyl ester (GSH-EE), a cell-permeable derivative of GSH, prevented CYB5R3-induced cell death (Fig. 7 b). To investigate whether CYB5R3 induced mitochondrial dysfunction, we fractionated the mitochondria and cytosol of CYB5R3-infected cells. The level of Bax increased in the mitochondrial fraction, whereas the level of cytochrome C increased in the cytosolic fraction (Fig. 7 c). Moreover, we found that the knockdown of caspase-9 or caspase-3 decreased CYB5R3-induced cell death (Fig. 7 d). These results suggested that CYB5R3 induces intrinsic apoptotic cell death through oxidative stress and caspase-9 activation. DISCUSSION Here, we showed that overexpression of CYB5R3 induces apoptosis in lung cancer in vitro and in vivo . In addition, the CYB5R3 KO mouse model study in vivo revealed the tumor-suppressive function of CYB5R3 against lung cancer. Transcriptome and metabolomics analyses indicated that CYB5R3 induces ER stress by activating PARP16-dependent ADP-ribosylation of PERK or IRE1α. Moreover, ROS generation by CYB5R3 activates caspase-9-mediated intrinsic apoptotic pathway. Accumulated data on CYB5R3 have provided evidence that CYB5R3 inhibits lung cancer growth. The CYB5R family has antioxidant properties and is expressed in several subcellular compartments, including ER, the mitochondrial outer membrane, and plasma membrane 36 – 39 . According to the Human Protein Atlas database, CYB5R1 localizes in the mitochondria and cytosol and is highly expressed in skeletal muscle. CYB5R2 localizes in the Golgi apparatus and nucleoplasm and is highly expressed in the testis. In CYB5R3-overexpressing H1299 cells, CYB5R3 was mainly located in the ER and undergoes post-translational modifications, leading to changes in subcellular localization 40 – 42 . Recent studies have suggested that prenylated CYB5R3 translocates from the mitochondria to the ER, while non-prenylated CYB5R3 localizes in the mitochondria 41 . In particular, geranylgeranyl diphosphate synthase (GGPPS), which is involved in CYB5R3 prenylation, is overexpressed in lung adenocarcinoma 40 . In addition, ufmylation of CYB5R3, which negative regulates its activity, occurs in the ER and ufmylated CYB5R3 is degraded in CDK5RAP3 (CDK5 Regulatory Subunit Associated Protein 3)-mediated macro-ER-phagy 42 . NAD-dependent signaling events regulate numerous biological processes, including transcription, DNA repair, apoptosis, and metabolism. NAD + is an important metabolite and enzyme substrate, such as poly(ADP-ribose) polymerases (PARPs) and sirtuins 32 . ADP-ribosylation is catalyzed by members of two different ADP-ribosyltransferase (ART) families, including clostridial-toxin-like ADP-ribosyltransferase (ARTCs) and diphtheria-toxin-like ARTs (ARTDs) 43 . The human ARTC family consists of four ecto-mono-ARTs: active mono-ARTs (ARTC1 and ARTC5) and inactive proteins (ARTC3 and ARTC4). The ARTD (also known as PARP) family contains 17 members: mono-ARTs (ARTD7-17), poly-ARTs (ARTD1-6), and inactive ARTD13, which have distant subcellular localizations and protein substrates 44 . Recently, ARTC1 (ART1) and ARTD15 (PARP16) have been identified as ER-resident ARTs that mediate the mono-ADP-ribosylation of their substrates 34 , 35 . Both ARTC1 and PARP16 are activated during ER stress and regulate the UPR in the ER. Mono-ADP-ribosylation of GRP78 by ARTC1 inactivates its chaperone activity and activates ER stress by dissociating it from its interactors, such as PERK, IRE1α, and ATF6 34 . Mono-ADP-ribosylation of PERK and IRE1α by PARP16 increases their enzyme activity and ER stress responses 35 . Our data demonstrated that PARP16 played a critical role in CYB5R3-induced lung cancer cell death by increasing ADP-ribosylation of PERK and IRE1α. Interestingly, we also found that CYB5R3 interacted with PERK and IRE1α. Therefore, further research is needed on whether the oxidation of PERK and IRE1α is required for their binding. Mitochondria are the main source of ROS. Recent studies have suggested that the ER regulates redox homeostasis and retains relatively high ROS levels 45 . Oxidative protein folding occurs in the ER and generates ROS by catalyzing disulfide bond formation between protein disulfide isomerase (PDI) and ERO1 during protein folding 46 – 48 . ER protein oxidation and mitochondrial oxidative phosphorylation are sources of ROS generated during ER stress 49 . ROS generation by NADPH oxidase 4 (NOX4) in the ER membrane can cause apoptosis 50 . Surprisingly, our data demonstrated that CYB5R3 dramatically decreased the ratio of GSH/GSSG and a cell-permeable GSH-EE attenuated apoptosis by CYB5R3 overexpression in lung cancer cells. In addition, we observed that CYB5R3 significantly altered metabolites, such as AMP and ADP, which induced the activation of the LKB1/AMPK pathway. Therefore, further studies are required to explore the role of CYB5R3 in tumor metabolism. Although the role of CYB5R3 in cancer remains controversial, a previous study showed that CYB5R3 promotes colonization and metastasis formation in estrogen receptor-negative breast cancer 17 . However, another study demonstrated that CYB5R3 overexpression protects against chemically induced liver cancer in CYB5R3 transgenic mice 9 . In clear cell renal cell carcinoma, HADHA overexpression inhibits tumor growth by increasing CYB5R3 or ACAT1 51 . The properties of CYB5R3 as a tumor suppressor in cancer cells can be exploited to develop anti-cancer drugs. CYB5R3-overexpressing vehicles, such as adenoviruses, lentiviruses, vaccinia viruses, and retroviruses for lung cancer, can be used to infect tumor cells. In addition, co-treatment with a CYB5R3-overexpressing vehicle and immune checkpoint inhibitors or cytokines can be developed to synergistically inhibit the growth of lung cancer. In conclusion, CYB5R3 deficiency promotes tumorigenesis and lung metastasis in mouse models. CYB5R3 overexpression induces apoptosis of lung cancer cells via ER stress and ROS generation, suggesting for the development of CYB5R3-based therapeutics for lung cancer. Declarations ACKNOWLEDGEMENTS This work was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5192322, IGM0481911) and Korea Institute for Advanced Technology (TGC1262011). CONFLICT OF INTEREST The authors declare that they have no conflict of interest. AUTHOR CONTRIBUTIONS J.Y.I. designed the study and wrote the manuscript. S.J.K., T.H.H., I.K., and H.S.B. performed the animal experiments and analyzed the data. J.Y.I., W.I.K., B.K., S.Y.C., M.J.K., and B.K.K. performed the biochemical and cell biological experiments. J.Y.I. and I.H. generated knockout cell lines. J.L.P., S.A.J., S.K.K., and S.Y.K. analyzed the RNA sequencing data and TCGA data. I.R. provided scientific input. M.W. supervised the study and revised the manuscript. References Bray, F. et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68, 394–424 (2018). Osmani, L., Askin, F., Gabrielson, E. & Li, Q.K. Current WHO guidelines and the critical role of immunohistochemical markers in the subclassification of non-small cell lung carcinoma (NSCLC): Moving from targeted therapy to immunotherapy. Semin Cancer Biol 52, 103–109 (2018). Pikor, L.A., Ramnarine, V.R., Lam, S. & Lam, W.L. Genetic alterations defining NSCLC subtypes and their therapeutic implications. Lung Cancer 82, 179–189 (2013). George, J. et al. Comprehensive genomic profiles of small cell lung cancer. Nature 524, 47–53 (2015). Lockwood, W.W. et al. Divergent genomic and epigenomic landscapes of lung cancer subtypes underscore the selection of different oncogenic pathways during tumor development. PLoS One 7, e37775 (2012). de Cabo, R., Siendones, E., Minor, R. & Navas, P. CYB5R3: a key player in aerobic metabolism and aging? Aging (Albany NY) 2, 63–68 (2009). Jaffe, E.R. Methemoglobin pathophysiology. Prog Clin Biol Res 51, 133–151 (1981). Oshino, N., Imai, Y. & Sato, R. A function of cytochrome b5 in fatty acid desaturation by rat liver microsomes. J Biochem 69, 155–167 (1971). Martin-Montalvo, A. et al. Cytochrome b5 reductase and the control of lipid metabolism and healthspan. NPJ Aging Mech Dis 2, 16006 (2016). Reddy, V.V., Kupfer, D. & Caspi, E. Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. J Biol Chem 252, 2797–2801 (1977). Hildebrandt, A. & Estabrook, R.W. Evidence for the participation of cytochrome b 5 in hepatic microsomal mixed-function oxidation reactions. Arch Biochem Biophys 143, 66–79 (1971). Sacco, J.C. & Trepanier, L.A. Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction. Pharmacogenet Genomics 20, 26–37 (2010). Rahaman, M.M. et al. Cytochrome b5 Reductase 3 Modulates Soluble Guanylate Cyclase Redox State and cGMP Signaling. Circ Res 121, 137–148 (2017). Durgin, B.G. et al. Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation. JCI Insight 4 (2019). Siendones, E. et al. Membrane-bound CYB5R3 is a common effector of nutritional and oxidative stress response through FOXO3a and Nrf2. Antioxid Redox Signal 21, 1708–1725 (2014). Fan, J. et al. Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with beta-cell failure. Mol Metab 34, 97–111 (2020). Lund, R.R. et al. NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer. Mol Cell Proteomics 14, 2988–2999 (2015). Szegezdi, E., Logue, S.E., Gorman, A.M. & Samali, A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep 7, 880–885 (2006). Tabas, I. & Ron, D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol 13, 184–190 (2011). Hetz, C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13, 89–102 (2012). Schroder, M. & Kaufman, R.J. The mammalian unfolded protein response. Annu Rev Biochem 74, 739–789 (2005). Urano, F. et al. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science 287, 664–666 (2000). Nishitoh, H. et al. ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16, 1345–1355 (2002). Miller, Y.E. et al. Induction of a high incidence of lung tumors in C57BL/6 mice with multiple ethyl carbamate injections. Cancer Lett 198, 139–144 (2003). Jao, L.E., Wente, S.R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A 110, 13904–13909 (2013). Im, J.Y. et al. DNA damage-induced apoptosis suppressor (DDIAS), a novel target of NFATc1, is associated with cisplatin resistance in lung cancer. Biochim Biophys Acta 1863, 40–49 (2016). Vichai, V. & Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1, 1112–1116 (2006). Kurashige, T. et al. Characterization of metabolic reprogramming by metabolomics in the oncocytic thyroid cancer cell line XTC.UC1. Sci Rep 13, 149 (2023). Sanjana, N.E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11, 783–784 (2014). Oyadomari, S. & Mori, M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11, 381–389 (2004). Yuan, S. et al. Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation. Redox Biol 47, 102166 (2021). Chiarugi, A., Dolle, C., Felici, R. & Ziegler, M. The NAD metabolome–a key determinant of cancer cell biology. Nat Rev Cancer 12, 741–752 (2012). Houtkooper, R.H., Pirinen, E. & Auwerx, J. Sirtuins as regulators of metabolism and healthspan. Nat Rev Mol Cell Biol 13, 225–238 (2012). Fabrizio, G. et al. ARTC1-mediated ADP-ribosylation of GRP78/BiP: a new player in endoplasmic-reticulum stress responses. Cell Mol Life Sci 72, 1209–1225 (2015). Jwa, M. & Chang, P. PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1alpha-mediated unfolded protein response. Nat Cell Biol 14, 1223–1230 (2012). De Cabo, R. et al. Calorie restriction attenuates age-related alterations in the plasma membrane antioxidant system in rat liver. Exp Gerontol 39, 297–304 (2004). Navarro, F., Villalba, J.M., Crane, F.L., Mackellar, W.C. & Navas, P. A phospholipid-dependent NADH-coenzyme Q reductase from liver plasma membrane. Biochem Biophys Res Commun 212, 138–143 (1995). Zhu, H. et al. NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum. J Biol Chem 279, 30316–30325 (2004). Zhao, X. et al. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes. Mol Cell Proteomics 10, M110 000299 (2011). Wang, X. et al. Overexpression of geranylgeranyl diphosphate synthase contributes to tumour metastasis and correlates with poor prognosis of lung adenocarcinoma. J Cell Mol Med 22, 2177–2189 (2018). Wei, L. et al. GGPP depletion initiates metaflammation through disequilibrating CYB5R3-dependent eicosanoid metabolism. J Biol Chem 295, 15988–16001 (2020). Ishimura, R. et al. The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3. Nat Commun 13, 7857 (2022). Di Girolamo, M. & Fabrizio, G. Overview of the mammalian ADP-ribosyl-transferases clostridia toxin-like (ARTCs) family. Biochem Pharmacol 167, 86–96 (2019). Vyas, S., Chesarone-Cataldo, M., Todorova, T., Huang, Y.H. & Chang, P. A systematic analysis of the PARP protein family identifies new functions critical for cell physiology. Nat Commun 4, 2240 (2013). Bhattarai, K.R., Riaz, T.A., Kim, H.R. & Chae, H.J. The aftermath of the interplay between the endoplasmic reticulum stress response and redox signaling. Exp Mol Med 53, 151–167 (2021). Enyedi, B., Varnai, P. & Geiszt, M. Redox state of the endoplasmic reticulum is controlled by Ero1L-alpha and intraluminal calcium. Antioxid Redox Signal 13, 721–729 (2010). Araki, K. et al. Ero1-alpha and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases. J Cell Biol 202, 861–874 (2013). Tu, B.P. & Weissman, J.S. Oxidative protein folding in eukaryotes: mechanisms and consequences. J Cell Biol 164, 341–346 (2004). Bhandary, B., Marahatta, A., Kim, H.R. & Chae, H.J. An involvement of oxidative stress in endoplasmic reticulum stress and its associated diseases. Int J Mol Sci 14, 434–456 (2012). Sciarretta, S. et al. Activation of NADPH oxidase 4 in the endoplasmic reticulum promotes cardiomyocyte autophagy and survival during energy stress through the protein kinase RNA-activated-like endoplasmic reticulum kinase/eukaryotic initiation factor 2alpha/activating transcription factor 4 pathway. Circ Res 113, 1253–1264 (2013). Liu, S. et al. HADHA overexpression disrupts lipid metabolism and inhibits tumor growth in clear cell renal cell carcinoma. Exp Cell Res 384, 111558 (2019). Additional Declarations (Not answered) Supplementary Files Suppl.1.tif Suppl.2.tif Suppl.3.tif Suppl.4.tif Suppl.5.tif Supplementaryinformation41223final.docx Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2024 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 12 Jun, 2023 Review # 1 received at journal 01 Jun, 2023 Reviewer # 1 agreed at journal 11 May, 2023 Reviewers invited by journal 11 May, 2023 Submission checks completed at journal 13 Apr, 2023 Editor assigned by journal 12 Apr, 2023 First submitted to journal 12 Apr, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2810245","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":191725569,"identity":"bce704fa-5ce8-4c6e-a8dd-0a0d82e461dc","order_by":0,"name":"Misun Won","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYDACZhBRkcDAJsED5hsQqeUMSVpAgLEtgYGBaC267ezPpAvnpeXzSfceYK6oYDA2byCgxewwj5n0zG05lm0y5xIYz5xhMJM5QFgLmzTvtgoDNokcA8bGNgYbCUIOMzsMdBjvHJiWf0RpYTCT5m3IgWppYDAjQguPsTXPsTQDNqBfDjYckzAmrOX88Ye3eWqSDeRn9x582FBjYziDkBYUcAAYOyRpGAWjYBSMglGAAwAAMfYysKBpvigAAAAASUVORK5CYII=","orcid":"","institution":"KRIBB","correspondingAuthor":true,"prefix":"","firstName":"Misun","middleName":"","lastName":"Won","suffix":""},{"id":191725570,"identity":"4073bcd3-cd07-48ce-b328-a58bfa27ee6a","order_by":1,"name":"Joo-Young Im","email":"","orcid":"https://orcid.org/0000-0001-6923-8189","institution":"KRIBB","correspondingAuthor":false,"prefix":"","firstName":"Joo-Young","middleName":"","lastName":"Im","suffix":""},{"id":191725571,"identity":"671ead3c-c71f-4df1-a7cb-db23cdc622bb","order_by":2,"name":"Soo Jin Kim","email":"","orcid":"","institution":"KRIBB","correspondingAuthor":false,"prefix":"","firstName":"Soo","middleName":"Jin","lastName":"Kim","suffix":""},{"id":191725572,"identity":"21e9517e-afc3-459c-9902-52d468d61fc5","order_by":3,"name":"Jong-Lyul Park","email":"","orcid":"","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Jong-Lyul","middleName":"","lastName":"Park","suffix":""},{"id":191725573,"identity":"b951070a-e773-4f92-a719-7480fe0a6102","order_by":4,"name":"Tae-Hee Han","email":"","orcid":"","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Tae-Hee","middleName":"","lastName":"Han","suffix":""},{"id":191725574,"identity":"b35182a4-a831-4759-9f7f-2f428184dda0","order_by":5,"name":"Woo-il Kim","email":"","orcid":"","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Woo-il","middleName":"","lastName":"Kim","suffix":""},{"id":191725575,"identity":"c68e6c85-0b5f-4cb2-86ab-dd6e849516ca","order_by":6,"name":"Inhyub Kim","email":"","orcid":"","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Inhyub","middleName":"","lastName":"Kim","suffix":""},{"id":191725576,"identity":"6dccc8c8-72ca-46d1-a63e-40f6322ed849","order_by":7,"name":"Bomin Ko","email":"","orcid":"","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Bomin","middleName":"","lastName":"Ko","suffix":""},{"id":191725577,"identity":"9db619f4-23c9-402a-ae0c-e8a7043e0bd9","order_by":8,"name":"So-Young Chun","email":"","orcid":"","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"So-Young","middleName":"","lastName":"Chun","suffix":""},{"id":191725578,"identity":"d8af5dbd-58cd-440f-8c62-38aaae7db28b","order_by":9,"name":"Mi-Jung Kang","email":"","orcid":"","institution":"KRIBB","correspondingAuthor":false,"prefix":"","firstName":"Mi-Jung","middleName":"","lastName":"Kang","suffix":""},{"id":191725579,"identity":"5a72278d-384d-4487-ac3d-d4c40d5467a9","order_by":10,"name":"Bo-Kyung Kim","email":"","orcid":"","institution":"KRIBB","correspondingAuthor":false,"prefix":"","firstName":"Bo-Kyung","middleName":"","lastName":"Kim","suffix":""},{"id":191725580,"identity":"b0859abe-eb6f-4cb9-a879-42d6157231df","order_by":11,"name":"Sol A Jeon","email":"","orcid":"","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Sol","middleName":"A","lastName":"Jeon","suffix":""},{"id":191725581,"identity":"4a9c13b0-a4da-4ee5-aceb-633091f758f2","order_by":12,"name":"Seon-Kyu Kim","email":"","orcid":"https://orcid.org/0000-0002-4176-5187","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Seon-Kyu","middleName":"","lastName":"Kim","suffix":""},{"id":191725582,"identity":"984c262f-bc7b-4065-8230-1625c82fca52","order_by":13,"name":"Incheol Ryu","email":"","orcid":"","institution":"6YD Global Life Science Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Incheol","middleName":"","lastName":"Ryu","suffix":""},{"id":191725583,"identity":"7954ed7b-b36b-416c-8aa4-f8bab11eeb3c","order_by":14,"name":"Seon-Young Kim","email":"","orcid":"https://orcid.org/0000-0002-1030-7730","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Seon-Young","middleName":"","lastName":"Kim","suffix":""},{"id":191725584,"identity":"e4ae8d26-168f-48b9-97cf-812cfb4a907e","order_by":15,"name":"Inah Hwang","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Inah","middleName":"","lastName":"Hwang","suffix":""},{"id":191725585,"identity":"f081cdfe-2940-433a-a3d6-f2f863b42813","order_by":16,"name":"Hyun Seung Ban","email":"","orcid":"","institution":"Korea Research Institute of Bioscience \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Seung","lastName":"Ban","suffix":""}],"badges":[],"createdAt":"2023-04-13 01:55:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2810245/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2810245/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-024-01155-9","type":"published","date":"2024-01-22T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":37058310,"identity":"1fc42e39-873f-4bcf-8be5-8acd27ea045f","added_by":"auto","created_at":"2023-05-16 00:52:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCYB5R3 is downregulated in lung cancer.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e CYB5R3 expression profile across lung tumor samples (T) and corresponding normal tissues (N) in the TCGA database. The expression data were collected from the TCGA cohort cBioPortal (http://cbioportal.org) and then visualized by ggplot2 (version 3.3.6). LUAD; lung adenocarcinoma, LUSC; lung squamous carcinoma. All statistical analyses were performed using R software. \u003cstrong\u003eb\u003c/strong\u003e IHC analysis of CYB5R3 in normal lung tissue and lung cancer patients. Representative images of CYB5R3 staining on tissue array were shown. Scale bar, 100 mm. \u003cstrong\u003ec\u003c/strong\u003e CYB5R3 expression levels of normal lung tissues and lung cancer tissues. CYB5R3\u003csup\u003eLow\u003c/sup\u003e indicates negative (0) or weak (1) IHC scores and CYB5R3\u003csup\u003eHigh\u003c/sup\u003e indicates moderate (2) or strong (3) IHC scores. \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e e\u003c/strong\u003e CYB5R3 expression in two human lung fibroblasts and eight lung cancer cell lines. mRNA (\u003cstrong\u003ed\u003c/strong\u003e) or protein levels (\u003cstrong\u003ee\u003c/strong\u003e) were evaluated by quantitative RT-PCR or immunoblot analysis. \u003cstrong\u003ef\u003c/strong\u003e Effects of CYB5R3 siRNA on the protein level of CYB5R3 (upper panel) and viability (lower panel) of WI-38 and IMR-90 cells. Values represent the mean ± SD of three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01. \u003cstrong\u003eg\u003c/strong\u003e H\u0026amp;E staining of lung tissues in CYB5R3\u003csup\u003e+/+\u003c/sup\u003e or CYB5R3\u003csup\u003e-/-\u003c/sup\u003e mice IP injected with 0.5 mg/g urethane for 8 months (n=3 per group). Scale bar, 100 mm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/93ad82ab6ee3ae712315d8c9.png"},{"id":37058824,"identity":"423cb3c6-5da2-41d2-8e65-6ca3d8f9bb30","added_by":"auto","created_at":"2023-05-16 01:00:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCYB5R3 overexpression induces cell death of lung cancer \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea \u003c/strong\u003eCells were infected with adenoviral EV or CYB5R3 at an MOI of 100 for the indicated times and cell lysates were analyzed by immunoblotting. \u003cstrong\u003eb \u003c/strong\u003eCells were infected with EV or CYB5R3 at an MOI (mode of infection) of 100 for 72 h. Cell viability was measured by SRB assay. Data represent the mean ± SD of three independent experiments. \u003cstrong\u003ec\u003c/strong\u003e Cleavage of PARP1 and caspase-3 in CYB5R3-infected H1299 or H1703 cells. \u003cstrong\u003ed\u003c/strong\u003e Caspase 3/7 activation and annexin staining in CYB5R3-infected H1299 cells. Representative images are shown. Scale bar, 100 mm. \u003cstrong\u003ee \u003c/strong\u003eTumor volume was measured at 2-3 days intervals in H1299 mouse xenograft (n=8 mice/group). \u003cstrong\u003ef\u003c/strong\u003e Tumor weight of xenograft (n=8 mice/group). \u003cstrong\u003eg\u003c/strong\u003e Body weight (n=8 mice/group). \u003cstrong\u003eh\u003c/strong\u003e CYB5R3 protein level in H1299 xenograft tissues. Data represent the mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/ba5f46b704b0a2022c59a4ba.png"},{"id":37058311,"identity":"52bb1151-c5a3-400d-b4c9-e29dc8ee4cbe","added_by":"auto","created_at":"2023-05-16 00:52:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCYB5R3 drive changes in gene expression profiles.\u003c/strong\u003e \u003cstrong\u003ea-c\u003c/strong\u003e RNA-seq analysis in H1299 cells infected with EV (Ad-EV) and CYB5R3 (Ad-CYB5R3). \u003cstrong\u003ea\u003c/strong\u003e Heat map depicts the differentially expressed genes (DEGs) from three independent samples treated with CYB5R3 versus EV or PBS. \u003cstrong\u003eb \u003c/strong\u003eKEGG database analysis of the enriched pathways of DEGs. The top twenty categories are exhibited. \u003cstrong\u003ec\u003c/strong\u003e qPCR analysis of the indicated genes in H1299 cells infected with CYB5R3 for 24 h. \u003cstrong\u003ed\u003c/strong\u003e Immunoblot analysis of proteins altered in H1299 or H1703 cells infected with CYB5R3 for 24 h.\u003cstrong\u003e e\u003c/strong\u003eH1299 cells were transfected with the indicated siRNA and infected with CYB5R3 for 48 h. Cell viability was measured by SRB assay (lower panel). Immunoblot analysis was performed to measure the indicated protein level (upper panel). The experiments were repeated three times. Data are shown as the mean ± SD. Student′s \u003cem\u003et-\u003c/em\u003etest. **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/31b58039c6dc45af82e9dd70.png"},{"id":37059341,"identity":"fde3b654-192f-470d-9e3b-1daa890960e4","added_by":"auto","created_at":"2023-05-16 01:16:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":64799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCYB5R3 induces ER stress.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Immunofluorescence staining of CYB5R3 (green) and calnexin (red) was carried out in H1299 cells infected with EV or CYB5R3. DAPI (blue) was used for nuclei staining. Scale bar, 20 mm. \u003cstrong\u003eb\u003c/strong\u003e Protein level of ER markers in H1299 cells infected with EV or CYB5R3 for 24 h. \u003cstrong\u003ec\u003c/strong\u003eH1299 cells were transfected with the indicated siRNA and infected with CYB5R3 for 48 h. Cell viability (left panel) or immunoblot analysis (right panel) was performed. \u003cstrong\u003ed\u003c/strong\u003e H1299 cells were transfected with siRNA against ATF4 and infected with CYB5R3 for 48 h. Cell viability (lower panel) or immunoblot analysis (upper panel) was performed. \u003cstrong\u003ee\u003c/strong\u003e H1299 cells were transfected with the indicated siRNAs and infected with CYB5R3 for 48 h. Cell viability (left panel) or immunoblot analysis (right panel) was performed. \u003cstrong\u003ef\u003c/strong\u003e Protein level of CYB5R3 (upper panel) and viability (lower panel) of CRISPR/Cas9-mediated CYB5R3-knockout H1299 or H1703 cells. \u003cstrong\u003eg\u003c/strong\u003e Protein levels of ER markers in tunicamycin-treated CYB5R3-knockout H1299 or H1703 cells. Values represent the mean ± SD of three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/da6c1b92dbecb618b9654bec.png"},{"id":37058314,"identity":"b63f68f6-446f-403a-b595-943add213bcb","added_by":"auto","created_at":"2023-05-16 00:52:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCYB5R3 leads to metabolic alterations.\u003c/strong\u003e Metabolomics analysis in H1299 cells infected with EV or CYB5R3 at an MOI 100 for 24 h or 36 h. \u003cstrong\u003ea\u003c/strong\u003e Principle component analysis (PCA) from three independent samples of four groups. A. EV for 24 h, B. CYB5R3 for 24 h, C. EV for 36 h, D. CYB5R3 for 36 h. \u003cstrong\u003eb\u003c/strong\u003e Heat map depicts the metabolites with statistically significant from three independent samples in CYB5R3 versus EV. \u003cstrong\u003ec\u003c/strong\u003e Venn diagram of the number of altered metabolites in EV vs CYB5R3 for 24 h or 36 h. \u003cstrong\u003ed\u003c/strong\u003e Enrichment analysis of the metabolites with statistically significant differences between EV and CYB5R3 for 24 h.\u003cstrong\u003e e-g \u003c/strong\u003eConcentration of altered metabolites in cells infected CYB5R3 compared to EV for 24 h.\u003cstrong\u003e \u003c/strong\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/31574f942040fe78eacc9a88.png"},{"id":37059380,"identity":"9e6b8370-045d-49cf-b806-6452b141561a","added_by":"auto","created_at":"2023-05-16 01:24:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCYB5R3 promotes ADP-ribosylation by PARP16.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e ADP-ribose level in H1299 cells infected CYB5R3. \u003cstrong\u003eb\u003c/strong\u003e MAR/PAR level in H1299 or H1703 cells infected with CYB5R3. \u003cstrong\u003ec\u003c/strong\u003eH1299 cells were transfected with the indicated siRNA and infected with CYB5R3 for 48 h. Cell viability (left panel) or immunoblot analysis (right panel) was performed. \u003cstrong\u003ed, e\u003c/strong\u003e H1299 cells were transfected with the PARP16 siRNA and infected with CYB5R3 for 48 h. Immunoblot analysis was performed using the indicated antibodies. \u003cstrong\u003ef\u003c/strong\u003e H1299 cells were infected with EV or CYB5R3 for 24 h, followed by immunoprecipitation with normal rabbit IgG, PERK, or IRE1a antibodies. The immunoprecipitates were analyzed by immunoblot analysis using the indicated antibodies.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/dea6aaf5170f34ce95668b40.png"},{"id":37058828,"identity":"4724a5e4-3ca4-43a4-b656-5069b5aaaef4","added_by":"auto","created_at":"2023-05-16 01:00:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":49714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCYB5R3 mediates intrinsic cell death through ROS generation and caspase-9.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e. DCF-DA staining in H1299 cells infected with CYB5R3 for 24 h. Scale bar, 50 mm. \u003cstrong\u003eb\u003c/strong\u003e. H1299 cells were pretreated with 1 mM GSH-EE for 1 h and infected with CYB5R3 for 72 h. Cell viability was performed using SRB assay. \u003cstrong\u003ec\u003c/strong\u003e Cell fractionation from H1299 cells infected with CYB5R3 for 24 h. Immunoblot analysis was performed using the indicated antibodies. \u003cstrong\u003ed\u003c/strong\u003e H1299 cells were transfected with the indicated siRNA and infected with CYB5R3 for 48 h. Cell viability (left panel) or immunoblot analysis (right panel) was performed. Values represent the mean ± SD of three independent experiments. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/6413ebd76051e8247db728fc.png"},{"id":37059067,"identity":"ed490367-935c-4486-9d4d-9b828c4fffe4","added_by":"auto","created_at":"2023-05-16 01:08:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":43027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic model for CYB5R3-induced lung cancer cell death.\u003c/strong\u003eIn lung cancer, CYB5R3 is downregulated and CYB5R3 overexpression promotes cell death by inducing ER stress and ROS generation. CYB5R3 activates PERK-ATF4-CHOP and IRE1a-JNK pathway through ADP-ribosylation by PARP16. ROS generation by CYB5R3 activates caspase-9-dependent cell death.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/811b52e72615d86881b58b0a.png"},{"id":50575830,"identity":"7e4d9d40-f922-43e3-9808-39513ef68e23","added_by":"auto","created_at":"2024-02-02 17:35:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2692481,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/eb0c2f6e-bb76-4bd9-8bc0-e545f5fbf093.pdf"},{"id":37058313,"identity":"76ab4aab-49a8-42ea-b1e6-49fa1684a831","added_by":"auto","created_at":"2023-05-16 00:52:27","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":249092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Suppl.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/f8e7c6efd9a98da08b91b2bc.tif"},{"id":37058321,"identity":"38a9266e-4530-4c8e-b197-72bfeaf7c402","added_by":"auto","created_at":"2023-05-16 00:52:27","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":197164,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/377b2db5e3c2e3541f3bc41e.tif"},{"id":37058323,"identity":"8d2aaec2-eeef-419b-adfe-fbbbe72db94e","added_by":"auto","created_at":"2023-05-16 00:52:27","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":442738,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/13241366515d4cde9738c23d.tif"},{"id":37058830,"identity":"26316685-5c05-427f-aa50-29b6bdff9727","added_by":"auto","created_at":"2023-05-16 01:00:27","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":126168,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/8f0315f310cd2551e3886b1b.tif"},{"id":37058319,"identity":"e52e945e-e0cb-4a12-a2d0-d745c59a2028","added_by":"auto","created_at":"2023-05-16 00:52:27","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":90124,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/0abadb9b88de045e999278d1.tif"},{"id":37058825,"identity":"84b05483-68b9-40e8-a952-7616f57cae79","added_by":"auto","created_at":"2023-05-16 01:00:27","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":20604,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation41223final.docx","url":"https://assets-eu.researchsquare.com/files/rs-2810245/v1/e6e9871dec6acf9646b61f7a.docx"}],"financialInterests":"(Not answered)","formattedTitle":"\u003cp\u003eCYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis of lung cancer cells via PERK-ATF4 and IRE1α-JNK pathway\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLung cancer is the leading cause of cancer-related mortality worldwide and has a 5-year survival rate of less than 20%\u003csup\u003e1, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Lung cancer is histologically classified into two major types, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the most prevalent subtype, accounting for 80\u0026ndash;85% of lung cancer patients\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Genomic studies have revealed multiple genetic alterations in oncogenes and tumor suppressor genes (TSGs) in lung cancer\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although genetic alterations in TP53, EGFR, EML4-ALK, PIK3CA, and KRAS have been identified as biomarkers of lung cancer, difficulties remain in diagnosing, prognosis, and treating lung cancer patients. Therefore, novel therapeutic targets must be developed to improve lung cancer diagnosis and treatment.\u003c/p\u003e \u003cp\u003eCytochrome b5 reductase 3 (CYB5R3) catalyzes the one-electron transfer from NADH to electron acceptors, such as cytochrome b5 or coenzyme Q, producing NAD\u003csup\u003e+\u0026thinsp;6\u003c/sup\u003e. CYB5R3 has two isoforms: soluble and membrane-bound. The soluble isoform expressed in erythrocytes has a truncated N-terminal binding domain and reduces methemoglobin\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, and the membrane-bound isoform expressed in most cells is anchored to the mitochondrial outer membrane, endoplasmic reticulum (ER), and plasma membrane and participates in fatty acids elongation and desaturation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, cholesterol biosynthesis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, drug metabolism\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and aging\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. CYB5R3 is a reductase of soluble guanylate cyclase (sGC) in vascular smooth muscle cells that regulates cGMP production, protein kinase G signaling, and hypertension\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. CYB5R3 is transcriptionally regulated by FOXO3a and Nrf2, and controls both nutrient and oxidative stress responses\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. CYB5R3 is a target of FoxO1 in β-cells and links mitochondrial dysfunction to β-cell failure\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and it also promotes the colonization and metastasis of estrogen receptor-negative breast cancer\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, the functions and mechanisms of CYB5R3 in cancer biology have not yet been explored.\u003c/p\u003e \u003cp\u003eThe ER plays key roles in protein folding, transport, calcium homeostasis, and lipid synthesis. Alterations in cellular processes, such as protein folding and calcium regulation, lead to the activation of the unfolded protein response (UPR), a pro-survival response to recover normal ER function; however, prolonged ER stress ultimately triggers cell death\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. UPR activation is mediated by three stress sensors: transmembrane receptors, protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 alpha (IRE1α) and activating transcription factor 6 (ATF6). These stress sensors bind to the ER chaperone GRP78 (also known as BiP) under non-stress conditions. ER stress, such as the accumulation of unfolded proteins, activates UPR by dissociating stress sensors from GRP78. Activated PERK phosphorylates eukaryotic translation initiation factor-2 (eIF2), thereby inhibiting protein translation and increasing the expression of activating transcription factor-4 (ATF4) and its downstream target C/EBP-homologous protein (CHOP; gene name \u003cem\u003eDDIT3\u003c/em\u003e), which are involved in ER stress-induced apoptosis\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Similar to PERK, IRE1α splices the transcription factor X box-binding protein 1 (XBP1), and the spliced form of XBP1 induces the expression of genes involved in the UPR. When the recovery of ER function fails, active IRE1α recruits TNF receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase-1 (ASK1), which activates Jun-N-terminal kinase (JNK) and induces apoptosis\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The transcription factor ATF6 is cleaved by S1P and S2P endopeptidases in the Golgi apparatus, and the cleaved ATF6 induces UPR targets, including GRP78 and XBP1\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we provide evidence that CYB5R3 overexpression induces ER stress by promoting ADP-ribosylation of PERK and IRE1α, resulting in apoptosis of lung cancer cells. Our findings suggest that CYB5R3 functions as a tumor suppressor and can be utilized in the development of anti-cancer drugs for lung cancer.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies\u003c/h2\u003e \u003cp\u003eSulforhodamine B, tunicamycin, and GSH-EE were purchased from Sigma-Aldrich (St. Louis, MO, USA). Genomic DNA kit was obtained from Promega (Madison, WI, USA). The antibodies anti-β-Tubulin (#2128), anti-ATF3 (#18665), anti-GADD45A (#4632), anti-ATF6 (#65880), anti-CHOP (#2895), anti-GRP78 (#3177), anti-XBP1 (#40435), anti-IRE1α (#3294), anti-DR5 (#8074), anti-Bax (#2772), anti-ATF4 (#11815), anti-Puma (#4976), anti-PERK (#3192), anti-p-eIF2α (#9721), anti-eIF2α (#9722), anti-p-JNK (#9251), anti-JNK (#9252), anti-cytochrome c (#4272), anti-COXIV (#4850), anti-Calnexin (#2679), anti-PARP1 (#9542), anti-Poly/Mono-ADP Ribose (#83732), anti-Caspase 3 (#9662), anti-Caspase 8 (#9746), and anti-Caspase 9 (#9502) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-PARP16 (ab154510) was obtained from Abcam (Cambridge, UK); anti-Sestrin2 (10795-1-AP) was obtained from Proteintech (Rosemont, IL, USA); anti-CYB5R3 (BS-12162R) was obtained from Bioss Antibodies Inc. (Woburn, MA, USA); anti-GAPDH (LF-P-A0212) was obtained from AbFrontier (Seoul, Korea); anti-ARTC1 (SAB1300652) and anti-Flag (F1804) were obtained from Sigma-Aldrich; anti-mouse-FITC (sc-2010) and anti-rabbit-Rhodamine (sc-2492) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfections\u003c/h2\u003e \u003cp\u003eHuman lung fibroblasts (IMR-90 and WI-38) and NSCLC cell lines (NCI-H1299 (H1299), NCI-H1703 (H1703), NCI-H226 (H226), NCI-H23 (H23), NCI-H460 (H460), NCI-H2009 (H2009), HCC827, and A549) were purchased from the Korean Cell Line Bank (Seoul, Korea) and the KRIBB Cell Line Bank (Daejeon, Korea). IMR-90, WI-38, and A549 cells were cultured in Dulbecco\u0026prime;s modified Eagle\u0026prime;s medium (DMEM), while H226, H460, H2009, HCC827, H23, H1703, and H1299 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). All cell lines were tested for \u003cem\u003eMycoplasma\u003c/em\u003e contamination using an e-Myco\u0026trade; VALiD \u003cem\u003eMycoplasma\u003c/em\u003e PCR Detection Kit (iNtRON Biotechnology, Gyeonggi-Do, Korea).\u003c/p\u003e \u003cp\u003esiRNA pools (ON-TARGET-plus SMART pool, contains four different siRNAs that target a single gene) against ATF3 (L-008663), DDIT3/CHOP (L-004819), SESN2 (L-019134), ERN1/IRE1 (L-004951), EIF2AK3/PERK (L-004883), ATF6 (L-009917), ATF4 (L-005125), JNK1 (L-003514), JNK2 (L-003505), JNK3 (L-004324), ARTC1 (L-010387), PARP16 (L-020837), and non-targeting control (D-001810-10-05) were obtained from Dharmacon (Lafayette, CO, USA). CYB5R3 siRNA was purchased from Bioneer Corporation (Daejeon, Korea). The target sequences were as follows: siCYB5R3 #1: 5\u0026prime;-GUUUACUUCAAGGACACCCAU-3\u0026prime;, siCYB5R3 #2: 5\u0026prime;-AGAACCUCAGCAUUUCCUU-3\u0026prime;, siScrambled: 5\u0026prime;-CCUACGCCACCAAUUUCGU-3\u0026prime;. The cells were transfected with siRNAs (40 nM) using an electroporation (Neon, Invitrogen) according to the manufacturer\u0026prime;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAdenoviruses\u003c/h2\u003e \u003cp\u003eAdenoviral CYB5R3-Flag (Ad-CYB5R3) and empty-vector control (EV) were obtained from Vigene Biosciences (Rockville, MD, USA). Viral stocks were re-infected with AD293 cells to amplify the virus and purified by double cesium chloride gradient ultracentrifugation. The infectious viral particles in the cesium chloride gradient were collected, dialyzed against 10 mM Tris (pH 8.0), 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 5% sucrose solution, and stored in a deep freezer. Viral titers were determined using the Adeno-X\u0026trade; Rapid Titer kit (Takara Bio USA, Inc., Mountain View, CA, USA) according to the manufacturer\u0026prime;s protocol. The cells were infected with adenovirus at a mode of infection (MOI) of 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003e All mouse experiments were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee. Tumors were induced by 10 weekly intraperitoneal (IP) injection of 0.5 mg/g urethane (ethyl carbamate, Sigma-Aldrich) in C57BL/6 (Orient Bio, Gyeonggi-Do, Korea) mice\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The mice were sacrificed eight months after urethane injection. Lungs were fixed, embedded, stained with hematoxylin and eosin (H\u0026amp;E), and analyzed in an Olympus microscope (BX51, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eFor the mouse xenograft assay, tumors were established by subcutaneously injecting NCI-H1299 cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mouse) into six-week-old BALB/c female nude mice (Orient Bio). Tumor volumes were estimated using the following formula: length (mm) \u0026times; width (mm) \u0026times; height (mm) / 2. When the average tumor volume reached 100 cm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, the mice were randomized into two groups (n\u0026thinsp;=\u0026thinsp;8), and adenoviral EV or CYB5R3 (1 \u0026times;10\u003csup\u003e9\u003c/sup\u003e pfu per mouse) were administered intratumorally every three days three times. The mice were euthanized on day 18, and tumor weights were measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of CYB5R3 knockout mice\u003c/h2\u003e \u003cp\u003eCYB5R3 knockout mice were generated by GH Bio (Daejeon, Korea). Single-guide RNA (sgRNA) was designed using ZiFiT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://zifit.partners.org/ZiFiT/\u003c/span\u003e\u003cspan address=\"http://zifit.partners.org/ZiFiT/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) program targeting N-terminal region of CYB5R3. The spacer sequences of sgRNAs are as follows; sgRNA1: 5\u0026prime;-CTTGATGTCGGGGTTCTCGA-3\u0026prime;, sgRNA2: 5\u0026prime;-AGACTCCGAGTAGCTGTTCC-3\u0026prime;, sgRNA3: 5\u0026prime;-TCTGAGGCTCATCGACAAGG-3\u0026prime;. The two complimentary oligonucleotides of each sgRNA were annealed and cloned in the pT7-gRNA vector, a vector designed for synthesizing sgRNA\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In vitro transcription of sgRNA for CYB5R3 and short RNA purification was performed using MEGAshortscript T7 kit (ThermoScientific, Waltham, MA, USA) according to the manufacturer\u0026prime;s instructions. Microinjection was performed in the fertilized eggs from C57BL/6N (Orient Bio) mice. The mixture of sgRNA (100 ng/\u0026micro;l) and Cas9 protein (80 ng/\u0026micro;l) (ToolGen Inc, Seoul, Korea) was injected into the cytoplasm of the pronuclear stage embryos. Injected embryos were cultured in the media overnight prior to embryo transfer into pseudo-pregnant mice (ICR strain). Genomic DNA from tails of the progenies were extracted and subjected to PCR using the primer sets; forward primer 5\u0026prime;-TGGAGTTCTCTGGTCAAGGC-3\u0026prime;, reverse primer 5\u0026prime;-TTGGCTGTCATTGTGCCTGA-3\u0026prime;. PCR products were analyzed by agarose gel electrophoresis and sequencing analysis to confirm the identity of every alleles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eHuman tissue arrays were obtained from US Biomax (Rockville, MD, USA). Immunohistochemistry (IHC) was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Briefly, the slides were incubated overnight with an anti-CYB5R3 antibody. After washing, the slides were incubated with a biotinylated HRP complex (Vector Laboratories, Burlingame, CA, USA), and 3,3-diaminobenzidine (DAB substrate kit; Vector Laboratories) was used for color development. Then, the slides were stained with hematoxylin and eosin (H\u0026amp;E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell viability and IncuCyte system\u003c/h2\u003e \u003cp\u003eCell viability was determined using a sulforhodamine B assay, as previously described\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. A cell growth inhibition assay (3000 cells/96-well plate) was performed in cells infected with adenoviral CYB5R3 and EV for 72 h. Cell death was analyzed with CellPlayer reagent-based annexin V (red) or caspase-3/7 (green), according to the manufacturer\u0026prime;s protocols (IncuCyte ZOOM System, Essen Bioscience, Ann Arbor, MI, USA). Images of green or red fluorescence and phase contrast were captured at 2 h intervals using a 10 \u0026times; objective lens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA-sequencing (RNA-seq) analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated using an mRNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Four micrograms of RNA was constructed using the TruSeq stranded mRNA LT Sample Prep Kit. The library was sequenced using the Illumina NovaSeq 6000 system (Illumina, San Diego, CA, USA) to generate 100 bp paired-end reads. The sequence reads were mapped to the human genome (GRCh38/hg38) using STAR (v.2.7.3a), and gene expression was quantified using the count module in STAR. A differentially expressed gene (DEG) was selected from RNA-seq count data using the edgeR package (v.3.32.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Reverse transcription polymerase chain reaction (QRT-PCR)\u003c/h2\u003e \u003cp\u003eTwo micrograms of total isolated RNA were reverse transcribed into cDNA using a TOPscript RT kit (Enzynomics, Daejeon, Korea) according to the manufacturer\u0026prime;s protocol. Real-time qPCR was performed using the SYBR Green master mix kit (Qiagen) on a Rotor-Gene Q system (Qiagen). The primers were obtained from the Bioneer Corporation as follows: DUSP1 (P199349), DUSP2 (P259112), DUSP5 (P197350), DUSP10 (P210086), PPP1R15A (P192005), DDIT3 (P225750), ERN1 (P298108), XBP1 (190450), GADD45A (P230161), BBC3 (P150935), TNFRSF10B (P195586), TRAF1 (P141876), ATF3 (P292769), KLF4 (P154272), and SESN2 (P153243). All reactions were performed in triplicate and normalized to β-actin (Qiagen) as an internal control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMetabolomics analysis\u003c/h2\u003e \u003cp\u003eMetabolomics analysis was performed by Human Metabolome Technologies, Inc. (Tsuruoka, Japan) using the CARCINOSCOPE (C-SCOPE) platform. H1299 cells (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) were seeded into a 100 mm dish the day before the assay and incubated with adenoviral EV or CYB5R3 for 24 or 36 h (n\u0026thinsp;=\u0026thinsp;3/group). Metabolite extraction was performed according to the manufacture\u0026prime;s protocol (HMT), as described previously\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Absolute quantitative analysis of 116 metabolites (54 cations and 62 anions) was performed on 12 samples of harvested cells using capillary electrophoresis-mass spectrometry (CE-MS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation and Immunoblot analysis\u003c/h2\u003e \u003cp\u003eCells were lysed with 1\u0026times; RIPA buffer (Millipore, Temecula, CA, USA) containing 1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 1 mM sodium fluoride, 1 mM PMSF, and a protease inhibitor cocktail (Roche, Basel, Switzerland), and the lysates were quantified using a the BCA protein assay kit (Thermo Scientific, 23227). For immunoprecipitation, 1 mg of lysates was incubated with 2 \u0026micro;g of the indicated antibodies or normal rabbit IgG at 4℃ overnight and then incubated with 20 \u0026micro;l of protein A/G plus agarose (sc-2003, Santa Cruz Biotechnology) at 4℃ for 1 h. The agarose was washed three times with wash buffer (0.1% NP40 in phosphate-buffered saline (PBS)).\u003c/p\u003e \u003cp\u003eMitochondrial isolation was performed using H1299 cells with a Mitochondrial Isolation Kit according to the manufacturer\u0026prime;s protocol (Thermo Scientific, 89874). The lysates were then subjected to immunoblotting using specific antibodies. Immunoblot signals were detected using an Enhanced Chemiluminescence (ECL) kit (Millipore).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence and DCF-DA staining\u003c/h2\u003e \u003cp\u003eCells were plated onto a microslide 8-well plate (ibidi Inc., Fitchburg, WI, USA), fixed in 4% paraformaldehyde for 30 min, permeabilized in PBS with 0.3% Triton X-100 for 10 min, and blocked with 3% bovine serum albumin for 1 h at 25℃. The cells were incubated overnight with appropriate antibodies. For mitochondrial tracker staining, cells were incubated with the tracker for 30 min and washed with PBS. Cells were incubated with 10 \u0026micro;M H2DCFDA (Invitrogen) for 30 min and washed with PBS to measure ROS generation. Finally, the cells were counterstained with DAPI for 10 min to label the nuclei and then analyzed using a confocal microscope (LSM5 Live DuoScan, Carl Zeiss, Stuttgart, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of knockout cells with CRISPR-Cas9 strategy.\u003c/b\u003e \u003c/p\u003e \u003cp\u003esgRNA was cloned into a lentiCrispr V2 plasmid as previously reported\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Briefly, each sgRNA oligonucleotide annealed with the T4 PNK enzyme (#M0201, NEB, Ipswich, MA, USA) was cloned into lentiCrispr V2 vector-digested BsmBI (#R0739, NEB). To generate lentivirus, 293T cells in 100 mm tissue culture dishes were transfected with 6 \u0026micro;g of each sgRNA along with 3.34 \u0026micro;g of pLP1, 2.2 \u0026micro;g of pLP2, and 3.34 \u0026micro;g of VSVg packaging vectors using polyethyleneimine. Viral particles were produced and transduced into cells, followed by puromycin (3\u0026ndash;10 \u0026micro;g/mL) selection. Gene targeting was confirmed using immunoblot analysis. Target sequences of the sgRNAs are listed below. Negative controls (sgNeg); 5\u0026prime;-GAAGATGGGCGGGAGTCTTC-3\u0026prime;, CYB5R3 #1; 5\u0026prime;-AGGCATCACCCCGATGCTGC-3\u0026prime;, CYB5R3 #2; 5\u0026prime;-GTGTATAGGGCCGGACGACC-3\u0026prime;, CYB5R3 #3; 5\u0026prime;-TCCCGGTCGATGAGCCGCAG-3\u0026prime;, CYB5R3 #4; 5\u0026prime;-GAAGACGAAGCAGCGCTCCG-3\u0026prime;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data represent at least three independent experiments. The results are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analyses were performed using a two-tailed Student\u0026prime;s \u003cem\u003et\u003c/em\u003e-test using GraphPad Prism ver 9.0 software (GraphPad Software, Boston, MA, USA). Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCYB5R3 expression is downregulated in lung cancer\u003c/h2\u003e \u003cp\u003eTo investigate the relevance of CYB5R3 in tumorigenesis, we analyzed the CYB5R3 expression profile in The Cancer Genome Atlas (TCGA) cohort cBioPortal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cbioportal.org\u003c/span\u003e\u003cspan address=\"http://cbioportal.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using R software. TCGA data revealed that CYB5R3 mRNA expression was significantly decreased in 13 of 24 subtypes of the TCGA cancer patient tissues compared to normal tissues (Supplementary Fig.\u0026nbsp;1a). CYB5R3 expression was downregulated in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Immunohistochemistry (IHC) was performed using a human tissue array of 32 normal lung tissues or adjacent normal lung tissues and 128 lung cancer samples. Consistent with public data, the IHC analysis revealed that CYB5R3 expression in lung cancer tissues was lower than that in normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;1b). We also found that 91% (29 of 32) of the normal lung tissues exhibited high CYB5R3 expression, while only 19% (24 of 128) of the lung cancer tissues showed high CYB5R3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), indicating that CYB5R3 expression is associated with lung carcinogenesis. However, the expression levels of CYB5R1 and CYB5R2 were not downregulated in LUAD tissue (Supplementary Fig.\u0026nbsp;2a and b). Neither CYB5R1 nor CYB5R2 was detected in human lung cancer cells (Supplementary Fig.\u0026nbsp;2c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the expression pattern of CYB5R3 in human lung cancer cells, we measured CYB5R3 mRNA levels using quantitative RT-PCR in two human lung fibroblast lines and eight NSCLC cell lines. The mRNA levels of CYB5R3 in the NSCLC cell lines A549, H1299, H1703, H226, H23, H460, H2009, and HCC827 were lower than those in normal lung fibroblast lines WI-38 and IMR-90 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Similarly, CYB5R3 protein was relatively abundant in WI-38 and IMR-90 cells compared to that in the NSCLC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). To examine the effect of CYB5R3 depletion on cell growth, we performed CYB5R3 knockdown using two different siRNAs targeting CYB5R3 in WI-38 and IMR-90. CYB5R3 expression significantly decreased in CYB5R3-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, upper panel). CYB5R3 knockdown promoted cell growth compared with siScrambled (siScr) in WI-38 and IMR-90 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, lower panel).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCYB5R3 is functionally associated with tumor suppressor in a mouse model\u003c/h2\u003e \u003cp\u003eWe evaluated the role of CYB5R3 in tumorigenesis using an \u003cem\u003ein vivo\u003c/em\u003e mouse model (Supplementary Fig.\u0026nbsp;3). First, we examined the expression profile of CYB5R3 in mouse tissues and found that it was abundantly expressed in the mouse lungs, liver, testes, and ovaries (Supplementary Fig.\u0026nbsp;3a). However, compared with mouse primary lung fibroblast, CYB5R3 protein was not detected in Lewis lung carcinoma (LLC), a murine lung cancer cell line (Supplementary Fig.\u0026nbsp;3b). CYB5R3 knockout (KO) mice were generated using the CRISPR-Cas9 system (Supplementary Fig.\u0026nbsp;3c). As expected, CYB5R3 protein expression was not detected in lungs, liver, spleen, or kidneys of CYB5R3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Supplementary Fig.\u0026nbsp;3d). We then evaluated the incidence of lung cancer in CYB5R3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice treated with urethane for eight months. H\u0026amp;E staining showed a higher incidence of tumors in the lung tissue of CYB5R3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than CYB5R3\u003csup\u003e+/+\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). In the lung metastasis experiment using LLC cells, the tumor incidence and size were considerably increased in the lung tissues of CYB5R3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than CYB5R3\u003csup\u003e+/+\u003c/sup\u003e mice (Supplementary Fig.\u0026nbsp;3e, f). These results suggest that CYB5R3 acts as a tumor suppressor in lung cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCYB5R3 overexpression leads to apoptosis of lung cancer\u003c/h2\u003e \u003cp\u003eTo confirm the tumor-suppressive role of CYB5R3 in human lung cancer cells, we investigated the effects of CYB5R3 overexpression using Ad-CYB5R3, an adenovirus that expresses CYB5R3. CYB5R3 protein was detectable within 12 h and showed the highest values 24 h after infection with CYB5R3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). CYB5R3 overexpression dramatically inhibited the growth of A549, H1299, H226, and H1703 cells compared to EV, but did not inhibit that of WI-38 and IMR-90 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Cleavage of PARP1 and capase-3 was detected in H1299 or H1703 cells infected with Ad-CYB5R3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). A caspase 3/7 activity assay and annexin V staining showed that CYB5R3 induced significant apoptosis in lung cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). To investigate the effect of CYB5R3 on tumor growth \u003cem\u003ein vivo\u003c/em\u003e, we performed a xenograft assay using H1299 cells. Compared with EV-treated mice, CYB5R3-treated mice exhibited a 53.3% and 44.2% reduction in tumor volume and weight, respectively, without significant changes in body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-g). Immunoblot analysis of the resected tumors revealed that the protein level of CYB5R3 was dramatically increased in CYB5R3-treated tumors compared to EV-treated tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). These data implied that CYB5R3 functions as a tumor suppressor in lung cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCYB5R3 overexpression affects the transcriptome landscape of lung cancer cells\u003c/h2\u003e \u003cp\u003eTo investigate the molecular mechanism by which CYB5R3 induces cancer cell death, we analyzed the gene expression pattern by CYB5R3 overexpression in H1299 cells. RNA sequencing analysis revealed 248 upregulated and 69 downregulated genes in cells infected with Ad-CYB5R3 compared with those infected with EV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Genes in CYB5R3-overexpressing cells that are involved in mitogen‑activated protein kinase (MAPK) signaling, the TNF signaling pathway, pathways in cancer, protein processing in ER, and apoptosis pathway were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The RNA sequencing data were verified using real-time PCR. The upregulated genes included DUSP1, DUSP2, DUSP5, and DUSP10 in MAPK signaling; DDIT3, ERN1, PPP1R15A, and XBP1 in protein processing in ER; BBC3, GADD45A, TNFRSF10B, and TRAF1 in apoptosis; and ATF3, KLF4, and SESN2 in other pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Interestingly, the levels of CHOP (DDIT3), DR5 (TNFRSF10B), ATF3, and SESN2 proteins dramatically increased in CYB5R3-overexpressing H1299 and H1703 cells following Ad-CYB5R3 infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In contrast, the expression of XBP1, IRE1α (ERN1), PUMA (BBC3), and GADD45A proteins remained unchanged. To evaluate how genes upregulated by CYB5R3 overexpression are involved in cell death, we transfected siRNAs against ATF3, DDIT3, or SESN2 in H1299 cells. DDIT3 silencing overcame CYB5R3-induced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, lower panel). The knockdown efficiency of each siRNA is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee (upper panel). These data suggest that CHOP (DDIT3) induction is critical for CYB5R3-induced cell death in lung cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCYB5R3 triggers ER stress via PERK-ATF4 or IRE1α-JNK pathway\u003c/h2\u003e \u003cp\u003eSince CHOP (DDIT3) is a major hall marker of ER stress and induces ER stress-mediated apoptosis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, we examined the relationship between CYB5R3 overexpression and ER stress. Immunofluorescence staining showed that CYB5R3 co-localizes with calnexin, an ER marker, in CYB5R3-overexpressing H1299 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). CYB5R3 also localizes in the mitochondria, which is consistent with the results of previous studies\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether CYB5R3 regulates ER function, we analyzed the expression levels of the ER stress sensors PERK, IRE1α, and ATF6 and their downstream targets. Similar to CHOP induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), CYB5R3 overexpression increased the expression of ER stress signaling proteins, such as GRP78, p-eIF2α, and p-JNK, which triggered apoptosis in both H1299 and H1703 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). To assess the pathways involved in CYB5R3-induced cell death, we performed gene knockdown assays using siRNAs against PERK, IRE1α, or ATF6 in H1299 cells. Importantly, the silencing of PERK or IRE1α but not ATF6 overcame CYB5R3-induced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Moreover, the deletion of ATF4, which is a downstream target of PERK, or the deletion of JNK isoforms JNK1 or JNK2, which are downstream targets of IRE1α, rescued CYB5R3-induced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e).\u003c/p\u003e \u003cp\u003eTo examine the interplay between CYB5R3 and ER stress, we generated CYB5R3 knockout H1299 or H1703 cells using the CRISPR-Cas9 system. We used four different sgRNAs against CYB5R3 (sgCYB5R3) and found that sgCYB5R3 #3 and #4 completely suppressed CYB5R3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, upper panel). As expected, CYB5R3 knockout cells displayed a significant increase in cell growth compared to control (sgNeg) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, lower panel), which is consistent with previous data from the normal lung fibroblasts WI38 and IMR-90 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Following treatment with tunicamycin, an ER stress-inducing antitumor agent, the levels of GRP78, CHOP, and p-eIF2α were increased in H1299-sgNeg and H1703-sgNeg cells, whereas the induction of CHOP and p-eIF2α was attenuated in H1299-sgCYB5R3 or H1703-sgCYB5R3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). These data suggest that CYB5R3-induced cell death is dependent on the PERK-ATF4 and IRE1α-JNK signaling pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCYB5R3 overexpression drives metabolic reprogramming\u003c/h2\u003e \u003cp\u003eGiven that CYB5R3 functions in energy and lipid metabolism\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, we speculated that CYB5R3 leads to metabolic alteration. To investigate the metabolic changes in H1299 cells infected with Ad-CYB5R3, we performed metabolomics analysis using capillary electrophoresis and time-of-flight mass spectrometry (CE-TOFMS). Principal component analysis (PCA) revealed that CYB5R3 overexpression resulted in marked differences in metabolic signatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Heatmap analysis revealed that EV and CYB5R3 were grouped into distinct metabolic clusters, and changes in 59 metabolites or 66 metabolites were observed in cells infected with CYB5R3 relative to that in cells infected with EV for 24 or 36 h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Subsequently, we conducted a pathway enrichment analysis of CYB5R3-related metabolites using MetaboAnalyst 4.0, to comprehensively analyze metabolic changes. We found that CYB5R3-related metabolites showed significant increases in the Warburg effect and were significantly enriched in glutamate, purine metabolism, arginine and proline metabolism, aspartate metabolism, urea cycle, glycine and serine metabolism, and the citric acid cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). Redox homeostasis and purine metabolism were simultaneously altered in cells infected with CYB5R3 for 24 h and 36 h. Although reduced glutathione (GSH) was decreased, oxidized GSH (GSSG), NAD\u003csup\u003e+\u003c/sup\u003e, AMP, and ADP were remarkably increased 24 h after CYB5R3 infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-g). These data suggested that CYB5R3 overexpression induces metabolic changes in lung cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eCYB5R3 increases PARP16-mediated ADP-ribosylation of PERK and IRE1α\u003c/h2\u003e \u003cp\u003eNext, we explored how CYB5R3 activates PERK and IRE1α. NADH oxidation by CYB5R3 overexpression can affect NAD\u003csup\u003e+\u003c/sup\u003e-dependent signaling pathways, such as ADP-ribosylation and protein deacetylation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Metabolomics revealed that ADP-ribose levels were higher in CYB5R3-overexpressing cells than in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). A previous study demonstrated that ARTC1-mediated ADP-ribosylation of GRP78 is inactive and activates the ER stress response\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Moreover, ER-resident PARP16 activates PERK and IRE1α via ADP-ribosylation in the ER\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. To investigate whether ADP-ribosylation is involved in the activation of PERK and IRE1α by CYB5R3, we examined total ADP-ribosylation, including mono-ADP-ribosylation (MAR) and poly-ADP-ribosylation (PAR). Indeed, CYB5R3 overexpression increased overall ADP-ribosylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). We examined the effect of ARTC1 and PARP16 silencing on CYB5R3-induced cell death. Importantly, PARP16 depletion reduced CYB5R3-induced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). We found that the increase in ADP-ribosylation in CYB5R3-overexpressing cells was attenuated by PARP16 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Moreover, depletion of PARP16 decreased the expression of ER stress markers, such as CHOP, p-elF2a, and p-JNK, which were increased by CYB5R3 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). We further investigated the role of CYB5R3 in the ADP-ribosylation of PERK and IRE1α and found that it increased ADP-ribosylation of PERK and IRE1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Surprisingly, immunoprecipitates with anti-PERK or anti-IRE1α bound to CYB5R3-Flag (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Moreover, an immunoprecipitation assay using an anti-FLAG antibody showed that CYB5R3-Flag interacted with endogenous PERK or IRE1α in H1299 cells (Supplementary Fig.\u0026nbsp;5). These data suggest that CYB5R3 promotes the PARP16-mediated ADP-ribosylation of PERK and IRE1α to induce ER stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eCYB5R3 activates caspase-9 through oxidative stress\u003c/h2\u003e \u003cp\u003eBased on our finding of increased oxidized GSH (GSSG) in CYB5R3 overexpressed cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), reactive oxygen species generation was detected in CYB5R3-overexpressing cells using DCF-DA staining. We observed the generation of ROS in CYB5R3-overexpressing H1299 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). GSH-ethyl ester (GSH-EE), a cell-permeable derivative of GSH, prevented CYB5R3-induced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). To investigate whether CYB5R3 induced mitochondrial dysfunction, we fractionated the mitochondria and cytosol of CYB5R3-infected cells. The level of Bax increased in the mitochondrial fraction, whereas the level of cytochrome C increased in the cytosolic fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Moreover, we found that the knockdown of caspase-9 or caspase-3 decreased CYB5R3-induced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). These results suggested that CYB5R3 induces intrinsic apoptotic cell death through oxidative stress and caspase-9 activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere, we showed that overexpression of CYB5R3 induces apoptosis in lung cancer \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. In addition, the CYB5R3 KO mouse model study \u003cem\u003ein vivo\u003c/em\u003e revealed the tumor-suppressive function of CYB5R3 against lung cancer. Transcriptome and metabolomics analyses indicated that CYB5R3 induces ER stress by activating PARP16-dependent ADP-ribosylation of PERK or IRE1α. Moreover, ROS generation by CYB5R3 activates caspase-9-mediated intrinsic apoptotic pathway. Accumulated data on CYB5R3 have provided evidence that CYB5R3 inhibits lung cancer growth.\u003c/p\u003e \u003cp\u003eThe CYB5R family has antioxidant properties and is expressed in several subcellular compartments, including ER, the mitochondrial outer membrane, and plasma membrane\u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. According to the Human Protein Atlas database, CYB5R1 localizes in the mitochondria and cytosol and is highly expressed in skeletal muscle. CYB5R2 localizes in the Golgi apparatus and nucleoplasm and is highly expressed in the testis. In CYB5R3-overexpressing H1299 cells, CYB5R3 was mainly located in the ER and undergoes post-translational modifications, leading to changes in subcellular localization\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Recent studies have suggested that prenylated CYB5R3 translocates from the mitochondria to the ER, while non-prenylated CYB5R3 localizes in the mitochondria\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In particular, geranylgeranyl diphosphate synthase (GGPPS), which is involved in CYB5R3 prenylation, is overexpressed in lung adenocarcinoma\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In addition, ufmylation of CYB5R3, which negative regulates its activity, occurs in the ER and ufmylated CYB5R3 is degraded in CDK5RAP3 (CDK5 Regulatory Subunit Associated Protein 3)-mediated macro-ER-phagy\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNAD-dependent signaling events regulate numerous biological processes, including transcription, DNA repair, apoptosis, and metabolism. NAD\u003csup\u003e+\u003c/sup\u003e is an important metabolite and enzyme substrate, such as poly(ADP-ribose) polymerases (PARPs) and sirtuins\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. ADP-ribosylation is catalyzed by members of two different ADP-ribosyltransferase (ART) families, including clostridial-toxin-like ADP-ribosyltransferase (ARTCs) and diphtheria-toxin-like ARTs (ARTDs)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The human ARTC family consists of four ecto-mono-ARTs: active mono-ARTs (ARTC1 and ARTC5) and inactive proteins (ARTC3 and ARTC4). The ARTD (also known as PARP) family contains 17 members: mono-ARTs (ARTD7-17), poly-ARTs (ARTD1-6), and inactive ARTD13, which have distant subcellular localizations and protein substrates\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Recently, ARTC1 (ART1) and ARTD15 (PARP16) have been identified as ER-resident ARTs that mediate the mono-ADP-ribosylation of their substrates\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Both ARTC1 and PARP16 are activated during ER stress and regulate the UPR in the ER. Mono-ADP-ribosylation of GRP78 by ARTC1 inactivates its chaperone activity and activates ER stress by dissociating it from its interactors, such as PERK, IRE1α, and ATF6\u003csup\u003e34\u003c/sup\u003e. Mono-ADP-ribosylation of PERK and IRE1α by PARP16 increases their enzyme activity and ER stress responses\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Our data demonstrated that PARP16 played a critical role in CYB5R3-induced lung cancer cell death by increasing ADP-ribosylation of PERK and IRE1α. Interestingly, we also found that CYB5R3 interacted with PERK and IRE1α. Therefore, further research is needed on whether the oxidation of PERK and IRE1α is required for their binding.\u003c/p\u003e \u003cp\u003eMitochondria are the main source of ROS. Recent studies have suggested that the ER regulates redox homeostasis and retains relatively high ROS levels\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Oxidative protein folding occurs in the ER and generates ROS by catalyzing disulfide bond formation between protein disulfide isomerase (PDI) and ERO1 during protein folding\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. ER protein oxidation and mitochondrial oxidative phosphorylation are sources of ROS generated during ER stress\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. ROS generation by NADPH oxidase 4 (NOX4) in the ER membrane can cause apoptosis\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Surprisingly, our data demonstrated that CYB5R3 dramatically decreased the ratio of GSH/GSSG and a cell-permeable GSH-EE attenuated apoptosis by CYB5R3 overexpression in lung cancer cells. In addition, we observed that CYB5R3 significantly altered metabolites, such as AMP and ADP, which induced the activation of the LKB1/AMPK pathway. Therefore, further studies are required to explore the role of CYB5R3 in tumor metabolism.\u003c/p\u003e \u003cp\u003eAlthough the role of CYB5R3 in cancer remains controversial, a previous study showed that CYB5R3 promotes colonization and metastasis formation in estrogen receptor-negative breast cancer\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, another study demonstrated that CYB5R3 overexpression protects against chemically induced liver cancer in CYB5R3 transgenic mice\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In clear cell renal cell carcinoma, HADHA overexpression inhibits tumor growth by increasing CYB5R3 or ACAT1\u003csup\u003e51\u003c/sup\u003e. The properties of CYB5R3 as a tumor suppressor in cancer cells can be exploited to develop anti-cancer drugs. CYB5R3-overexpressing vehicles, such as adenoviruses, lentiviruses, vaccinia viruses, and retroviruses for lung cancer, can be used to infect tumor cells. In addition, co-treatment with a CYB5R3-overexpressing vehicle and immune checkpoint inhibitors or cytokines can be developed to synergistically inhibit the growth of lung cancer.\u003c/p\u003e \u003cp\u003eIn conclusion, CYB5R3 deficiency promotes tumorigenesis and lung metastasis in mouse models. CYB5R3 overexpression induces apoptosis of lung cancer cells via ER stress and ROS generation, suggesting for the development of CYB5R3-based therapeutics for lung cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5192322, IGM0481911) and Korea Institute for Advanced Technology (TGC1262011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.Y.I. designed the study and wrote the manuscript. S.J.K., T.H.H., I.K., and H.S.B. performed the animal experiments and analyzed the data. J.Y.I., W.I.K., B.K., S.Y.C., M.J.K., and B.K.K. performed the biochemical and cell biological experiments. J.Y.I. and I.H. generated knockout cell lines. J.L.P., S.A.J., S.K.K., and S.Y.K. analyzed the RNA sequencing data and TCGA data. I.R. provided scientific input. M.W. supervised the study and revised the manuscript. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray, F. \u003cem\u003eet al.\u003c/em\u003e Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68, 394\u0026ndash;424 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsmani, L., Askin, F., Gabrielson, E. \u0026amp; Li, Q.K. Current WHO guidelines and the critical role of immunohistochemical markers in the subclassification of non-small cell lung carcinoma (NSCLC): Moving from targeted therapy to immunotherapy. Semin Cancer Biol 52, 103\u0026ndash;109 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePikor, L.A., Ramnarine, V.R., Lam, S. \u0026amp; Lam, W.L. Genetic alterations defining NSCLC subtypes and their therapeutic implications. Lung Cancer 82, 179\u0026ndash;189 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeorge, J. \u003cem\u003eet al.\u003c/em\u003e Comprehensive genomic profiles of small cell lung cancer. Nature 524, 47\u0026ndash;53 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLockwood, W.W. \u003cem\u003eet al.\u003c/em\u003e Divergent genomic and epigenomic landscapes of lung cancer subtypes underscore the selection of different oncogenic pathways during tumor development. PLoS One 7, e37775 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Cabo, R., Siendones, E., Minor, R. \u0026amp; Navas, P. CYB5R3: a key player in aerobic metabolism and aging? Aging (Albany NY) 2, 63\u0026ndash;68 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaffe, E.R. Methemoglobin pathophysiology. Prog Clin Biol Res 51, 133\u0026ndash;151 (1981).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOshino, N., Imai, Y. \u0026amp; Sato, R. A function of cytochrome b5 in fatty acid desaturation by rat liver microsomes. J Biochem 69, 155\u0026ndash;167 (1971).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin-Montalvo, A. \u003cem\u003eet al.\u003c/em\u003e Cytochrome b5 reductase and the control of lipid metabolism and healthspan. NPJ Aging Mech Dis 2, 16006 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy, V.V., Kupfer, D. \u0026amp; Caspi, E. Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. J Biol Chem 252, 2797\u0026ndash;2801 (1977).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHildebrandt, A. \u0026amp; Estabrook, R.W. Evidence for the participation of cytochrome b 5 in hepatic microsomal mixed-function oxidation reactions. Arch Biochem Biophys 143, 66\u0026ndash;79 (1971).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSacco, J.C. \u0026amp; Trepanier, L.A. Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction. Pharmacogenet Genomics 20, 26\u0026ndash;37 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahaman, M.M. \u003cem\u003eet al.\u003c/em\u003e Cytochrome b5 Reductase 3 Modulates Soluble Guanylate Cyclase Redox State and cGMP Signaling. Circ Res 121, 137\u0026ndash;148 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDurgin, B.G. \u003cem\u003eet al.\u003c/em\u003e Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation. JCI Insight 4 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiendones, E. \u003cem\u003eet al.\u003c/em\u003e Membrane-bound CYB5R3 is a common effector of nutritional and oxidative stress response through FOXO3a and Nrf2. Antioxid Redox Signal 21, 1708\u0026ndash;1725 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan, J. \u003cem\u003eet al.\u003c/em\u003e Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with beta-cell failure. Mol Metab 34, 97\u0026ndash;111 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLund, R.R. \u003cem\u003eet al.\u003c/em\u003e NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer. Mol Cell Proteomics 14, 2988\u0026ndash;2999 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzegezdi, E., Logue, S.E., Gorman, A.M. \u0026amp; Samali, A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep 7, 880\u0026ndash;885 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabas, I. \u0026amp; Ron, D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol 13, 184\u0026ndash;190 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHetz, C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13, 89\u0026ndash;102 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchroder, M. \u0026amp; Kaufman, R.J. The mammalian unfolded protein response. Annu Rev Biochem 74, 739\u0026ndash;789 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrano, F. \u003cem\u003eet al.\u003c/em\u003e Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science 287, 664\u0026ndash;666 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishitoh, H. \u003cem\u003eet al.\u003c/em\u003e ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16, 1345\u0026ndash;1355 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller, Y.E. \u003cem\u003eet al.\u003c/em\u003e Induction of a high incidence of lung tumors in C57BL/6 mice with multiple ethyl carbamate injections. Cancer Lett 198, 139\u0026ndash;144 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJao, L.E., Wente, S.R. \u0026amp; Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A 110, 13904\u0026ndash;13909 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIm, J.Y. \u003cem\u003eet al.\u003c/em\u003e DNA damage-induced apoptosis suppressor (DDIAS), a novel target of NFATc1, is associated with cisplatin resistance in lung cancer. Biochim Biophys Acta 1863, 40\u0026ndash;49 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVichai, V. \u0026amp; Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1, 1112\u0026ndash;1116 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurashige, T. \u003cem\u003eet al.\u003c/em\u003e Characterization of metabolic reprogramming by metabolomics in the oncocytic thyroid cancer cell line XTC.UC1. Sci Rep 13, 149 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanjana, N.E., Shalem, O. \u0026amp; Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11, 783\u0026ndash;784 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyadomari, S. \u0026amp; Mori, M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11, 381\u0026ndash;389 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan, S. \u003cem\u003eet al.\u003c/em\u003e Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation. Redox Biol 47, 102166 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiarugi, A., Dolle, C., Felici, R. \u0026amp; Ziegler, M. The NAD metabolome\u0026ndash;a key determinant of cancer cell biology. Nat Rev Cancer 12, 741\u0026ndash;752 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoutkooper, R.H., Pirinen, E. \u0026amp; Auwerx, J. Sirtuins as regulators of metabolism and healthspan. Nat Rev Mol Cell Biol 13, 225\u0026ndash;238 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFabrizio, G. \u003cem\u003eet al.\u003c/em\u003e ARTC1-mediated ADP-ribosylation of GRP78/BiP: a new player in endoplasmic-reticulum stress responses. Cell Mol Life Sci 72, 1209\u0026ndash;1225 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJwa, M. \u0026amp; Chang, P. PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1alpha-mediated unfolded protein response. Nat Cell Biol 14, 1223\u0026ndash;1230 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Cabo, R. \u003cem\u003eet al.\u003c/em\u003e Calorie restriction attenuates age-related alterations in the plasma membrane antioxidant system in rat liver. Exp Gerontol 39, 297\u0026ndash;304 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavarro, F., Villalba, J.M., Crane, F.L., Mackellar, W.C. \u0026amp; Navas, P. A phospholipid-dependent NADH-coenzyme Q reductase from liver plasma membrane. Biochem Biophys Res Commun 212, 138\u0026ndash;143 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, H. \u003cem\u003eet al.\u003c/em\u003e NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum. J Biol Chem 279, 30316\u0026ndash;30325 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, X. \u003cem\u003eet al.\u003c/em\u003e Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes. Mol Cell Proteomics 10, M110 000299 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X. \u003cem\u003eet al.\u003c/em\u003e Overexpression of geranylgeranyl diphosphate synthase contributes to tumour metastasis and correlates with poor prognosis of lung adenocarcinoma. J Cell Mol Med 22, 2177\u0026ndash;2189 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei, L. \u003cem\u003eet al.\u003c/em\u003e GGPP depletion initiates metaflammation through disequilibrating CYB5R3-dependent eicosanoid metabolism. J Biol Chem 295, 15988\u0026ndash;16001 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshimura, R. \u003cem\u003eet al.\u003c/em\u003e The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3. Nat Commun 13, 7857 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Girolamo, M. \u0026amp; Fabrizio, G. Overview of the mammalian ADP-ribosyl-transferases clostridia toxin-like (ARTCs) family. Biochem Pharmacol 167, 86\u0026ndash;96 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVyas, S., Chesarone-Cataldo, M., Todorova, T., Huang, Y.H. \u0026amp; Chang, P. A systematic analysis of the PARP protein family identifies new functions critical for cell physiology. Nat Commun 4, 2240 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhattarai, K.R., Riaz, T.A., Kim, H.R. \u0026amp; Chae, H.J. The aftermath of the interplay between the endoplasmic reticulum stress response and redox signaling. Exp Mol Med 53, 151\u0026ndash;167 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEnyedi, B., Varnai, P. \u0026amp; Geiszt, M. Redox state of the endoplasmic reticulum is controlled by Ero1L-alpha and intraluminal calcium. Antioxid Redox Signal 13, 721\u0026ndash;729 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAraki, K. \u003cem\u003eet al.\u003c/em\u003e Ero1-alpha and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases. J Cell Biol 202, 861\u0026ndash;874 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTu, B.P. \u0026amp; Weissman, J.S. Oxidative protein folding in eukaryotes: mechanisms and consequences. J Cell Biol 164, 341\u0026ndash;346 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhandary, B., Marahatta, A., Kim, H.R. \u0026amp; Chae, H.J. An involvement of oxidative stress in endoplasmic reticulum stress and its associated diseases. Int J Mol Sci 14, 434\u0026ndash;456 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSciarretta, S. \u003cem\u003eet al.\u003c/em\u003e Activation of NADPH oxidase 4 in the endoplasmic reticulum promotes cardiomyocyte autophagy and survival during energy stress through the protein kinase RNA-activated-like endoplasmic reticulum kinase/eukaryotic initiation factor 2alpha/activating transcription factor 4 pathway. Circ Res 113, 1253\u0026ndash;1264 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, S. \u003cem\u003eet al.\u003c/em\u003e HADHA overexpression disrupts lipid metabolism and inhibits tumor growth in clear cell renal cell carcinoma. Exp Cell Res 384, 111558 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CYB5R3, DDIT3, PERK, IRE1α, PARP16, ER stress, Lung cancer","lastPublishedDoi":"10.21203/rs.3.rs-2810245/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2810245/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomics analysis revealed that CYB5R3 overexpression increased the production of NAD\u003csup\u003e+\u003c/sup\u003e and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress is induced by activating protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD\u003csup\u003e+\u003c/sup\u003e activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent-intrinsic cell death. Our findings highlight the significance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer.\u003c/p\u003e","manuscriptTitle":"CYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis of lung cancer cells via PERK-ATF4 and IRE1α-JNK pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-16 00:52:22","doi":"10.21203/rs.3.rs-2810245/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-06-12T22:50:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-06-01T14:18:33+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-05-11T12:46:17+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-05-11T10:31:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-04-13T23:27:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-04-13T01:51:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2023-04-13T01:51:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9e4a64e3-fdb3-428a-8a48-9e308713797c","owner":[],"postedDate":"May 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":21534596,"name":"Health sciences/Diseases/Cancer/Tumour biomarkers"},{"id":21534597,"name":"Biological sciences/Molecular biology/Post-translational modifications/PolyADP-ribosylation"},{"id":21534598,"name":"Biological sciences/Biological techniques/Metabolomics"},{"id":21534599,"name":"Biological sciences/Computational biology and bioinformatics/Cellular signalling networks"}],"tags":[],"updatedAt":"2024-02-02T17:35:43+00:00","versionOfRecord":{"articleIdentity":"rs-2810245","link":"https://doi.org/10.1038/s12276-024-01155-9","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2024-01-22 05:00:00","publishedOnDateReadable":"January 22nd, 2024"},"versionCreatedAt":"2023-05-16 00:52:22","video":"","vorDoi":"10.1038/s12276-024-01155-9","vorDoiUrl":"https://doi.org/10.1038/s12276-024-01155-9","workflowStages":[]},"version":"v1","identity":"rs-2810245","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2810245","identity":"rs-2810245","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}