The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity

Article4 January 2021free access Source DataTransparent process The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity Jia Song Jia Song orcid.org/0000-0003-1418-3656 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Tongtong Liu Tongtong Liu orcid.org/0000-0003-4979-8220 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Yue Yin Yue Yin orcid.org/0000-0002-7998-8597 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Wei Zhao Wei Zhao orcid.org/0000-0003-2498-1272 Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China Search for more papers by this author Zhiqiang Lin Zhiqiang Lin orcid.org/0000-0003-1834-2060 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Yuxin Yin Corresponding Author Yuxin Yin [email protected] orcid.org/0000-0003-4102-0043 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Dan Lu Corresponding Author Dan Lu [email protected] orcid.org/0000-0002-3000-5094 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Fuping You Corresponding Author Fuping You [email protected] orcid.org/0000-0002-7444-729X Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Jia Song Jia Song orcid.org/0000-0003-1418-3656 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Tongtong Liu Tongtong Liu orcid.org/0000-0003-4979-8220 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Yue Yin Yue Yin orcid.org/0000-0002-7998-8597 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Wei Zhao Wei Zhao orcid.org/0000-0003-2498-1272 Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China Search for more papers by this author Zhiqiang Lin Zhiqiang Lin orcid.org/0000-0003-1834-2060 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Yuxin Yin Corresponding Author Yuxin Yin [email protected] orcid.org/0000-0003-4102-0043 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Dan Lu Corresponding Author Dan Lu [email protected] orcid.org/0000-0002-3000-5094 Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Fuping You Corresponding Author Fuping You [email protected] orcid.org/0000-0002-7444-729X Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Search for more papers by this author Author Information Jia Song1,2, Tongtong Liu1, Yue Yin1, Wei Zhao3, Zhiqiang Lin1, Yuxin Yin *,1, Dan Lu *,1 and Fuping You *,1 1Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China 2Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China 3Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China *Corresponding author. Tel: +86 010 82805570; E-mail: [email protected] *Corresponding author. Tel: +86 010 82805807; E-mail: [email protected] *Corresponding author. Tel: +86 010 82805340; E-mail: [email protected] EMBO Reports (2021)22:e51162https://doi.org/10.15252/embr.202051162 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Although iron is required for cell proliferation, iron-dependent programmed cell death serves as a critical barrier to tumor growth and metastasis. Emerging evidence suggests that iron-mediated lipid oxidation also facilitates immune eradication of cancer. However, the regulatory mechanisms of iron metabolism in cancer remain unclear. Here we identify OTUD1 as the deubiquitinase of iron-responsive element-binding protein 2 (IREB2), selectively reduced in colorectal cancer. Clinically, downregulation of OTUD1 is highly correlated with poor outcome of cancer. Mechanistically, OTUD1 promotes transferrin receptor protein 1 (TFRC)-mediated iron transportation through deubiquitinating and stabilizing IREB2, leading to increased ROS generation and ferroptosis. Moreover, the presence of OTUD1 promotes the release of damage-associated molecular patterns (DAMPs), which in turn recruits the leukocytes and strengthens host immune response. Reciprocally, depletion of OTUD1 limits tumor-reactive T-cell accumulation and exacerbates colon cancer progression. Our data demonstrate that OTUD1 plays a stimulatory role in iron transportation and highlight the importance of OTUD1-IREB2-TFRC signaling axis in host antitumor immunity. SYNOPSIS OTUD1 stabilizes IREB2 and enhances TFRC-mediated iron uptake in intestinal epithelial cells. OTUD1 increases cell susceptibility to ferroptosis and is essential for T cell surveillance against cancer. OTUD1 acts as a deubiquitinase for IREB2 and stabilizes IREB2 by removing K48-linked ubiquitin modifications. OTUD1 augments TFRC-mediated iron transportation and increases intracellular ROS production. OTUD1-deficient colon cancer cells are resistant to immune attack by limiting ferroptosis and subsequent DAMPs release. Introduction Intact ion transport is essential for host immune response. Uncontrolled accumulation of extracellular potassium in tumor-surrounding area leads to T-cell dysfunction (Eil et al, 2016; Vodnala et al, 2019). Overabundance of sodium promotes pathogenic Th17 cell polarization and exacerbates colon inflammation (Kleinewietfeld et al, 2013; Wilck et al, 2017). Although iron deficiency and consequent anemia are frequent complications in patients with cancer (Ludwig et al, 2015), the role of iron metabolism within tumor microenvironment is largely unknown. Iron is the essential element for DNA synthesis, metabolism, and cell proliferation (Raza et al, 2014). Cellular iron uptake is mainly dependent on iron transporters, such as transferrin receptor protein 1 (TFRC) and divalent metal transporter 1 (DMT1/SLC11A2) (Arosio et al, 2017; Gao et al, 2019). Because excessive cellular iron is toxic, iron concentration must be tightly orchestrated. As the iron sensor, iron-responsive element-binding protein 2 (IREB2) is involved in modulation of iron transporters (Zumbrennen et al, 2009). IREB2 directly binds to the RNA stem-loop structures in the 3′-untranslated region (UTR) of mRNA and stabilizes transcripts of TFRC or DMT1, thereby increasing intracellular iron concentration (Samaniego et al, 1994). When an iron excess occurs, ubiquitin E3 ligase F-box/LRR-repeat protein 5 (FBXL5) promotes ubiquitination and consequent degradation of IREB2, which in turn limits iron absorption and utilization (Salahudeen et al, 2009). On the other hand, a high-level expression of IREB2 is required for regulating absorption of iron when cytosolic iron is not enough. However, the key factors and regulatory mechanism responsible for maintaining stability of IREB2 under iron-deficient conditions remain elusive. Iron is also a pro-oxidant agent, which can react with the hydrogen peroxide to produce reactive oxygen species (ROS) (Sousa et al, 2020). When the antioxidant system is saturated, excess of lipid ROS causes cellular changes, eventually leading to ferroptosis, a non-apoptotic cell death (Torti et al, 2018; Friedmann Angeli et al, 2019; Hassannia et al, 2019). Although the mechanism by which iron is related to ferroptosis is not yet well elucidated, the stimulatory effects of TFRC or DMT1 on ferroptosis have already been established (Stockwell et al, 2017; Lei et al, 2020). Unlike apoptosis that was considered as a physiological non-immunogenic cell death, other types of cell death, such as necrosis and pyroptosis, have been related with immunogenic cell death (ICD) (Minagawa et al, 2020; Zhang et al, 2020). Depending on the emission of a specific panel of danger-associated molecular patterns (DAMPs), ICD triggers host adaptive immunity in the context of cancers or infectious diseases (Galluzzi et al, 2017). Reciprocally, neoplastic cells have developed an arsenal of strategies to curb danger signaling and thereby escape from immune detection (Bidwell et al, 2012). Despite ferroptosis has been implicated in T cell-mediated cytotoxicity (Wang et al, 2019), it is still elusive whether ferroptosis can cause ICD and strengthen host antitumor immunity. In the present study, we identify that OTUD1 acts as a deubiquitinase of IREB2 and blocks its degradation, thereby promoting TFRC expression and augmenting cellular iron uptake. OTUD1 is mainly expressed in intestinal epithelial cells and selectively downregulated during tumorigenesis. Clinical analysis reveals that reduced OTUD1 is highly correlated with poor outcome of colorectal cancers. Activation of OTUD1-IREB2-TFRC pathway increases intracellular iron concentration and enhances cell sensitivity to ferroptosis, which in turn leads to immunogenic cell death and reinforces host antitumor immunity. Our data thus demonstrate that the tumor suppressor OTUD1 promotes iron absorption and potentiates host antitumor immunity. Results Cellular iron uptake is impaired during colon cancer development Although iron is an essential element for multiple biological processes, it is still elusive whether iron metabolism is involved in colorectal cancer development. To this end, we analyzed the dataset from patients with colorectal cancer (CRC) (GDS4382) and found that, along with dysregulation of ion homeostasis, genes related with iron transportation were reduced in tumor as relative to normal tissues, such as LTF, CP, FTH1, and TFRC (Fig 1A). We next interrogated gene expression data in colon cancer tissues from TCGA database and found that dysregulation of iron metabolism was highly correlated with poor prognosis of CRC patients (Figs 1B, and EV1A and B). To further confirm these results, we measured the status of transferrin receptor protein 1 (TFRC) in patients with colorectal cancer, which is essential for iron delivery from transferrin to cells. As shown in Fig EV1C, TFRC is remarkably downregulated in CRCs compared with their matched normal tissues. Moreover, the intracellular iron sensor IREB2, which can facilitate iron absorption via promoting of TFRC mRNA stability, was also downregulated during colon cancer development (Fig 1C). Notably, the protein level of IREB2 rather than its mRNA was reduced in colon cancers (Figs 1C, and EV1D and E). These results thus indicate that iron transportation is impaired during colon tumorigenesis. Figure 1. OTUD1 stabilizes IREB2 and promotes TFRC expression Gene set enrichment analysis (GSEA) of differentially expressed genes in colorectal cancer from the GEO database (GDS4382). ES, enrichment score; NES, normalized enrichment score. n = 3 biological samples. Kaplan–Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset (n = 590 samples, P = 0.016, log-rank (Mantel–Cox) test). “Normal” represents the patients with unaltered expression of iron homoeostasis-related genes, including LTF, CP, FTH1, SLC30A1, and TFRC; “Dysfunctional iron homoeostasis” represents the patients with altered expression of iron homoeostasis-related genes. Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues (left), the relative protein level of IREB2 was assessed by ImageJ software (right) (n = 8 human samples, *P = 0.0114, two-tailed paired Student’s t-test). Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue, and was subsequently analyzed by anti-ubiquitin antibody for the assessment of ubiquitination. Mass spectrum analysis of IREB2-associated proteins. Mock or IREB2-FLAG was transfected into HEK293T cells, and FLAG-tagged proteins were enriched by anti-FLAG M2 beads and incubated with lysates from normal mice colon tissues. Five matched peptides corresponding to OTUD1 were shown on the right panel. In vivo ubiquitination assay of IREB2. HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti-FLAG antibody followed by Western blot analysis. Half-life analysis of IREB2 in wild-type (WT) and OTUD1−/− NCM460 cells. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated times for Western blot analysis (up), and the relative protein level of IREB2 was assessed by ImageJ software (down) (n = 3 biological replicates, mean ± s.e.m., **P = 0.0049, ***P < 0.0001, two-tailed unpaired Student’s t-test). Flow cytometric analysis of TFRC expression in mock- and OTUD1-expressing CT26 cells with AFC (50 μM) and hemin (100 μM) treatment. MFI, mean fluorescence intensity. Control uses isotype-matched control antibody (n = 4 biological replicates, mean ± s.e.m., **P = 0.0010, ***P = 0.0008, two-tailed unpaired Student’s t-test). Flow cytometric analysis of TFRC expression in wild-type (WT) and OTUD1−/− NCM460 cells with DFO (100 μM) treatment. MFI, mean fluorescence intensity (n = 4 biological replicates, mean ± s.e.m., ***P = 0.0001, two-tailed unpaired Student’s t-test). Source data are available online for this figure. Source Data for Figure 1 [embr202051162-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Iron transportation signaling is impaired in colorectal cancers A, B. Kaplan–Meier survival curves for patients with colon cancer containing low or high expression of CP (low expression of CP, n = 49 patients; high expression of CP, n = 116 patients) and TFRC (low expression of TFRC, n = 74 patients; high expression of TFRC, n = 91 patients) mRNA in the GSE dataset. C. RT–qPCR analysis of TFRC mRNA levels in colon tumors and matched adjacent normal tissues (n = 101 human samples, ***P < 0.001, two-tailed paired Student’s t-test). D. The mRNA level of IREB2 in normal (n = 17) or colorectal cancer (n = 16) tissues from the GEO database (GDS4382) (mean ± s.e.m., ns, not significant (P > 0.05), two-tailed unpaired Student’s t-test). E. The relationship of protein level and mRNA level of IREB2 in colon cancer (n = 8 samples, P = 0.4185, two-tailed Pearson correlation analysis). Download figure Download PowerPoint OTUD1 acts as a deubiquitinase of IREB2 The above results indicated that posttranslational modification might be involved in modulation of IREB2 expression in colon. To test whether ubiquitin–proteasome system is involved in this process, we used anti-IREB2 antibody to pull down the endogenous IREB2 from primary colon cancer and normal tissues. As shown in Fig 1D, the ubiquitination of IREB2 was increased in colon cancer tissues as compared with their matched normal tissues. To investigate the mechanism by which IREB2 ubiquitination is modulated in colon, we thus performed immunoprecipitation followed by mass spectrometry (MS) to identify the interactome of IREB2 in colon. A list of IREB2 interacting proteins was identified. Among them, the deubiquitinase OTUD1 is of interest and might be involved in regulation of IREB2 ubiquitination and stability (Fig 1E). Through analysis of single-cell transcriptomic data in human colon, we found that OTUD1 was exclusively expressed in intestinal epithelial cells and goblet cells rather than gut-resident lymphocytes (Appendix Fig S1A). Ensued co-immunoprecipitation and confocal assays further confirmed this result and revealed that N-terminal of OTUD1 was required for its association with IREB2 (Fig EV2A and B). Notably, supplementation of the iron chelation deferoxamine (DFO) strengthened the association of IREB2 with OTUD1 compared with the treatment with hemin, an iron-containing porphyrin (Fig EV2C). Click here to expand this figure. Figure EV2. OTUD1 deubiquitinases and stabilizes IREB2 Confocal examination of OTUD1 and IREB2 colocalization in HEK293T cells ectopically expressing GFP-tagged OTUD1. The scale bars represent 10 μm. HEK293T cells were co-transfected with IREB2-FLAG and S-tagged OTUD1 and its truncations. At 24 h later, cell lysates were immunoprecipitated with S-protein agarose and analyzed by immunoblot with anti-FLAG antibody. Co-immunoprecipitation analysis of IREB2-FLAG together with OTUD1-GFP with treatment of hemin (100 μM) or DFO (100 μM). In vivo ubiquitination assay of IREB2. HEK293T cells were co-transfected with indicated plasmids and subjected to immunoprecipitation with anti-FLAG antibody followed by Western blot analysis. In vitro ubiquitination assay of IREB2. IREB2 was enriched by anti-FLAG beads and incubated with purified OTUD1 and OTUD1C320S protein in deubiquitination buffer followed by Western blot analysis. Half-life analysis of IREB2 in the presence or absence of OTUD1 in CT26 cells. Cells were treated with CHX for indicated times and analyzed by Western blot (up), and the relative protein level of IREB2 was assessed by ImageJ software (down) (n = 3 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student’s t-test). Analysis of endogenous IREB2 and TFRC level in wild-type (WT) or Ireb2−/− CT26 cells with or without OTUD1 overexpressing. Flow cytometric analysis of TFRC expression in wild-type (WT) or Ireb2−/− CT26 cells with or without OTUD1 overexpression treated with AFC (50 μM). MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P = 0.0001, two-tailed unpaired Student’s t-test). Source data are available online for this figure. Download figure Download PowerPoint To determine whether OTUD1 can deubiquitinate IREB2, we co-transfected wild-type (WT) or enzyme-dead mutant (C320S) OTUD1 with IREB2 into HEK293T cells. As shown in Fig EV2D, ectopic expression of wild-type OTUD1 rather than C320S reduced IREB2 ubiquitination. Similar result was detected by in vitro ubiquitination assay (Fig EV2E), which further verified that OTUD1 directly deubiquitinated IREB2. As the treatment of ammonium iron citrate (AFC) stimulates IREB2 ubiquitination and degradation, we detected the ubiquitination level of IREB2 with or without AFC stimulation. As shown in Fig 1F and Appendix Fig S1B, OTUD1 dramatically restrained iron-induced IREB2 ubiquitination. Additionally, we found that OTUD1 effectively removed both Lys 48- and Lys 63-linked poly-ubiquitination of IREB2 (Appendix Fig S1C). Since Lys 48-linked poly-ubiquitination is critical for protein stability, we thus treated cells with the protein synthesis inhibitor cycloheximide (CHX) to determine the effects of OTUD1 on IREB2 stability. As shown in Figs 1G and EV2F, the half-life of IREB2 was shortened in cells depleted of OTUD1, but prolonged in cells overexpressing OTUD1. Furthermore, flow cytometry assay showed that treatment of hemin or AFC damped the expression of TFRC, however, significantly rescued by overexpression of OTUD1 (Fig 1H and Appendix Fig S2A). In line with this, loss of OTUD1 limited the DFO-induced TFRC upregulation (Fig 1I and Appendix Fig S2B). To ascertain that the stimulatory effects of OTUD1 on TFRC expression are in an IREB2-dependent manner, we used CRISPR-Cas9 to delete the Ireb2 in CT26 cells. As shown in Fig EV2G and H as well as Appendix Fig S2C, overexpression of OTUD1 selectively promoted TFRC expression in wild-type rather than Ireb2−/− CT26 cells, which further confirms that IREB2 is required for the modulation of TFRC by OTUD1. Our data thus demonstrate that OTUD1 functions as a bona fide deubiquitinase of IREB2 in colon. OTUD1 is selectively reduced in colon cancer We next assessed the status of OTUD1 during colon cancer development. As shown in Fig 2A, the mRNA level of OTUD1 was remarkably reduced in colon cancer tissues as relative to their matched adjacent normal tissues. In accordance to the above data that OTUD1 promotes IREB2-TFRC signaling activation, the transcription of OTUD1 was highly correlated with TFRC expression in CRCs (Fig 2B). Subsequent Western blot assay further confirmed this result (Fig 2C). To determine whether loss of OTUD1 is associated with clinic pathological features, we examined OTUD1 protein expression in a total of 50 paired tissue sections by immunohistochemistry. Consistent to the bioinformatics data that late-stage tumors expressed lower level of OTUD1 compared with early-stage tumors did (Appendix Fig S3), decreased OTUD1 was highly correlated with cancer progression (Fig 2D and E). In addition, we noticed that age rather than gender or histological grade was related with OTUD1 reduction in CRCs (Table 1). To assess the status of OTUD1 in other colorectal diseases, we detected OTUD1 expression in colitis, adenomatoid polypus as well as benign adenoma. As shown in Fig 2F, OTUD1 was selectively downregulated in colorectal cancers. Figure 2. OTUD1 is selectively downregulated in colon cancers RT–qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues (n = 101 human samples, ***P < 0.001, two-tailed paired Student’s t-test). Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer (n = 101 human samples, two-tailed Pearson correlation analysis, ****P < 0.0001). Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues (n = 4 human samples), the protein levels relative to GAPDH are marked. Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue (the scale bars represent 1,000 μm). Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05-118e. Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a (the scale bars represent 1,000 μm). Source data are available online for this figure. Source Data for Figure 2 [embr202051162-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Table 1. Information on microarray of samples from patients with colon cancer. CoI05-118e Low Medium High P value Sex Male 16 5 3 0.778801 Female 16 5 5 Age >50 20 8 6 0.022371 <50 12 2 2 TNM T2NxM0 3 0 1 0.000341 T3NxM0 16 8 4 T4NxM0 13 2 3 Stage I–IIA 9 7 4 0.007255 IIB–IIIA 6 1 3 IIIB–C 17 2 1 Grade G1 9 4 2 0.353049 G2 13 5 4 G3 10 1 2 Deletion of OTUD1 restricts iron transportation To further determine the role of OTUD1 in iron transportation, we treated cells with hemin or AFC and consequently assessed the intracellular iron concentration. As shown in Fig 3A, overexpression of OTUD1 rather than its catalytically inactive (C320S) mutant promoted cellular uptake of iron. In contrast to the stimulatory effects of OTUD1 on iron transportation in wild-type cells, OTUD1 presence exerts little effects on iron absorption in Ireb2−/− cells (Fig EV3A), which further confirms the essential role of IREB2 in regulation of TFRC by OTUD1. Reciprocally, loss of OTUD1 restricted intracellular iron concentration upon hemin or AFC stimulation (Fig 3B). To further confirm this result in vivo, we employed CRISPR-Cas9 technique to generate Otud1−/− mice (Fig EV3B and C). Consistent with the data that OTUD1 stabilized IREB2 protein, loss of OTUD1 weakened the expression of IREB2 and TFRC but not FBXL5 in colon (Fig 3C). We next compared the iron absorption between wild-type and Otud1−/− mice after oral administration of ferrous gluconate for 1 week. As shown in Fig 3D, loss of OTUD1 remarkably limited the intestinal iron concentration compared with their littermate controls did. Furthermore, we also challenged wild-type and Otud1−/− mice with iron-deficient diets. As shown in Fig 3E–G, iron-deficient diets for 2 weeks decreased mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in Otud1−/− mice compared with wild-type mice. Moreover, compared with wild-type mice, Otud1−/− mice had lower amounts of the red blood cell (RBC) counts, the hemoglobin (HB) as well as hematocrits (HCTs) 4 weeks after treatment with iron-deficient diets (Figs 3H and I, and EV3D). Our data thus indicate that loss of OTUD1 curbs iron transportation and enhances susceptibility to iron-deficient anemia. Figure 3. OTUD1 promotes iron transportation A, B. Intracellular iron concentration was measured in CT26 cells stably expressing mock, OTUD1, or OTUD1C320S (n = 2 biological replicates) (A), wild-type (WT) or OTUD1−/− NCM460 cells (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student’s t-test) (B). C. The protein levels of IREB2, TFRC, and OTUD1 were assessed by Western blot in colon tissues from wild-type (WT) or Otud1−/− mice. D. Intracellular iron concentration was measured in colon tissues from wild-type (WT) or Otud1−/− mice with supplementation of ferrous gluconate (n = 3 biological replicates, mean ± s.e.m., ***P = 0.0007, two-tailed unpaired Student’s t-test). E–I. Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0472, two-tailed unpaired Student’s t-test) (E), mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0048, two-tailed unpaired Student’s t-test) (F), mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0068, two-tailed unpaired Student’s t-test) (G), red blood cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0049, two-tailed unpaired Student’s t-test) (H) and hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0036, ***P = 0.0002, two-tailed unpaired Student’s t-test) (I) in wild-type (WT) and Otud1−/− mice with or without low-iron diets for indicated times. Source data are available online for this figure. Source Data for Figure 3 [embr202051162-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Strategy of generation of Otud1−/− mice Intracellular iron concentration was measured in wild-type (WT) and Ireb2−/− CT26 cells with or without OTUD1 overexpression treated with AFC (50 µM) (n = 4 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P = 0.0006, two-tailed unpaired Student’s t-test). Targeting strategy for generation of Otud1−/− mice (up) and sequence for Otud1−/− mice genomic DNA (down). Validation of Otud1−/− mice by PCR. Hemoglobin and hematocrits (HCTs) in wild-type (WT) and Otud1−/− mice with or without low-iron diets (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P < 0.01, two-tailed unpaired Student’s t-test). Download figure Download PowerPoint OTUD1 activates IREB2-TFRC signaling in cancer and suppresses tumor growth To study the biological function of OTUD1 in tumorigenesis, we reconstituted the expression of OTUD1 in colon cancer cells. Unexpectedly, enforced expression of OTUD1 exhibited little effects on tumor cell proliferation in vitro (Fig EV4A). Moreover, enforced expression of OTUD1 hardly affected tumor growth in NOD-SCID immune-deficient mice (Fig EV4B and C). To test whether OTUD1 influences host immune surveillance against cancer, we subcutaneously transplanted equal numbers of mock-expressing, OTUD1-expressing, or the catalytically inactive mutant OTUD1C320S-expressing CT26 cells into the back flanks of BALB/c mice and monitored tumor growth. As shown in Fig 4A and B, enforced expression of OTUD1 rather than OTUD1C320S elicited suppressive effects on tumor growth in vivo. Si