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COMMD10 inhibits HIF1α/CP loop to enhance ferroptosis and radiosensitivity by disrupting Cu-Fe balance in hepatocellular carcinoma

  • Author Footnotes
    # Mi Yang, Xixi Wu and Jinlong Hu contributed equally to this work.
    Mi Yang
    Footnotes
    # Mi Yang, Xixi Wu and Jinlong Hu contributed equally to this work.
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Author Footnotes
    # Mi Yang, Xixi Wu and Jinlong Hu contributed equally to this work.
    Xixi Wu
    Footnotes
    # Mi Yang, Xixi Wu and Jinlong Hu contributed equally to this work.
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Author Footnotes
    # Mi Yang, Xixi Wu and Jinlong Hu contributed equally to this work.
    Jinlong Hu
    Footnotes
    # Mi Yang, Xixi Wu and Jinlong Hu contributed equally to this work.
    Affiliations
    Department of Pathology, Nanfang Hospital and Basic Medical College, Southern Medical University, Guangzhou, Guangdong, China

    Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China

    Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong, China
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  • Yingqiao Wang
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Yin Wang
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Longshan Zhang
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Weiqiang Huang
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Xiaoqing Wang
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Nan Li
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Liwei Liao
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Min Chen
    Affiliations
    Department of Radiation Oncology, Peking University Shenzhen Hospital, Shenzhen, China
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  • Nanjie Xiao
    Affiliations
    Department of Radiation Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, China
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  • Yongmei Dai
    Affiliations
    Department of Oncology, Provincial Clinical College of Fujian Medical University, Fujian Provincial Hospital, Fujian, China
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  • Huazhen Liang
    Affiliations
    The First Tumor Department, Maoming People’s Hospital, Maoming, China
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  • Wenqi Huang
    Affiliations
    Chronic Airways Diseases Laboratory, Department of Respiratory and Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Lu Yuan
    Affiliations
    Chronic Airways Diseases Laboratory, Department of Respiratory and Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Hua Pan
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Lu Li
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Longhua Chen
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Laiyu Liu
    Correspondence
    Chronic Airways Diseases Laboratory, Department of Respiratory and Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, China. Tel: 86-20-61641575.
    Affiliations
    Chronic Airways Diseases Laboratory, Department of Respiratory and Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Li Liang
    Correspondence
    Department of Pathology, Nanfang Hospital and Basic Medical College, Southern Medical University, Guangzhou, Guangdong, 510515, China; Guangdong Province Key Laboratory of Molecular Tumor Pathology, Guangzhou, Guangdong, 510515, China. Tel: 86-20-62787274.
    Affiliations
    Department of Pathology, Nanfang Hospital and Basic Medical College, Southern Medical University, Guangzhou, Guangdong, China

    Guangdong Province Key Laboratory of Molecular Tumor Pathology, Guangzhou, Guangdong, China
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  • Jian Guan
    Correspondence
    Corresponding authors: Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, China. Tel: 86-20-61642136.
    Affiliations
    Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
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  • Author Footnotes
    # Mi Yang, Xixi Wu and Jinlong Hu contributed equally to this work.
Open AccessPublished:January 28, 2022DOI:https://doi.org/10.1016/j.jhep.2022.01.009

      Highlights

      • Decreased COMMD10 induces radioresistance through intracellular Cu accumulation in HCC.
      • COMMD10 inhibits HIF1α/CP positive feedback loop to enhance radiosensitivity by disrupting Cu-Fe balance.
      • Cu accumulation upregulates CP to reduce Fe concentration and inhibit lipid peroxidation and ferroptosis.
      • COMMD10, HIF1α, CP and SLC7A11 might be potential new targets and predictive biomarkers of radioresistant HCC.

      Background & Aims

      Copper (Cu) is an essential trace element whose serum levels have been reported to act as an effective indicator of the efficacy of radiotherapy. However, little is known about the role of Cu in radiotherapy. In this study we aimed to determine this role and investigate the precise mechanism by which Cu or Cu-related proteins regulate the radiosensitivity of hepatocellular carcinoma (HCC).

      Methods

      The expression and function of Cu and copper metabolism MURR1 domain 10 (COMMD10) were assessed via a Cu detection assay, immunostaining, real-time PCR, western blot, a radiation clonogenic assay and a 5-ethynyl-2'-deoxyuridine assay. Ferroptosis was determined by detecting glutathione, lipid peroxidation, malondialdehyde and ferrous ion (Fe) levels. The in vivo effects of Cu and COMMD10 were examined with Cu/Cu chelator treatment or lentivirus modification of COMMD10 expression in radiated mouse models.

      Results

      We identified a novel role of Cu in promoting the radioresistance of HCC cells. Ionizing radiation (IR) induced a reduction of COMMD10, which increased intracellular Cu and led to radioresistance of HCC. COMMD10 enhanced ferroptosis and radiosensitivity in vitro and in vivo. Mechanistically, low expression of COMMD10 induced by IR inhibited the ubiquitin degradation of HIF1α (by inducing Cu accumulation) and simultaneously impaired its combination with HIF1α, promoting HIF1α nuclear translocation and the transcription of ceruloplasmin (CP) and SLC7A11, which jointly inhibited ferroptosis in HCC cells. In addition, elevated CP promoted HIF1α expression by reducing Fe, forming a positive feedback loop.

      Conclusions

      COMMD10 inhibits the HIF1α/CP loop to enhance ferroptosis and radiosensitivity by disrupting Cu-Fe homeostasis in HCC. This work provides new targets and treatment strategies for overcoming radioresistance in HCC.

      Lay summary

      Radiotherapy benefits patients with unresectable or advanced hepatocellular carcinoma (HCC), but its effectiveness is hampered by radioresistance. Herein, we uncovered a novel role for copper in promoting the radioresistance of HCCs. This work has revealed new targets and potential treatment strategies that could be used to sensitize HCC to radiotherapy.

      Graphical abstract

      Keywords

      Introduction

      Cu, an essential micronutrient metabolized and excreted by the liver, is involved in many biological processes such as cell respiration, Fe homeostasis, anti-oxidation defense and neuropeptide processing.
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      Current biomedical use of copper chelation therapy.
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      The physiological function of copper, the etiological role of copper excess and deficiency.
      However, excessively elevated Cu levels lead to tumorigenesis, chemotherapy resistance and poor prognosis in patients with hepatocellular carcinoma (HCC).
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      Overcoming platinum drug resistance with copper-lowering agents.
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      Radiotherapy benefits patients with unresectable or advanced HCC by improving the local control ratio, but the efficacy of radiotherapy is hampered by radioresistance.
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      It has been reported that the level of serum Cu was negatively associated with radiotherapy response in patients with cancer, which indicated that serum Cu was an effective monitoring index for the efficacy of radiotherapy.
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      These clues indicated that modifying Cu or targeting Cu-related proteins may become new strategies for radiosensitization, however, the potential mechanisms by which Cu or Cu-related proteins regulate responses to radiotherapy remain unknown.
      The copper metabolism MURR1 domain (COMMD) protein family, including 10 members (COMMD1-COMMD10), has been reported to play a critical role in Cu metabolism.
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      COMMD1: A multifunctional regulatory protein.
      The expression of COMMD family members (COMMD9 as an exception) was significantly decreased in Cu-stimulated HEK293T cells, indicating that except from COMMD9, the COMMD family engages in the regulation of Cu metabolism.
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      Gene expression profiling of liver cells after copper overload in vivo and in vitro reveals new copper-regulated genes.
      Besides, COMMD proteins have been increasingly reported as suppressors and prognostic factors in multiple tumors, but little is known about the role of COMMD proteins in radiotherapy for HCC.
      Resisting cell death is one of the most important characteristics of cancer cells and is a major reason for therapeutic resistance.
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      • Weinberg R.A.
      Hallmarks of cancer: the next generation.
      Ferroptosis is a newly identified form of cell death characterized by accumulation of Fe and lipid peroxidation,
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      Ferroptosis, a new form of cell death, and its relationships with tumourous diseases.
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      Lei and his colleagues
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      • et al.
      The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression.
      reported that radiation induced ferroptosis in lung cancer, breast cancer, fibrosarcoma and esophageal cancer by inducing lipid peroxidation. In addition, ferroptosis inducers synergized with radiation to enhance the antitumor activity of radiation in a murine xenograft model and in patient-derived models of lung adenocarcinoma and glioma.
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      • et al.
      Radiation-induced lipid peroxidation triggers ferroptosis and synergizes with ferroptosis inducers.
      Depletion of SLC7A11 (or GPX4) could boost radiosensitivity by inducing ferroptosis.
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      • Zhou H.
      • et al.
      Erastin decreases radioresistance of NSCLC cells partially by inducing GPX4-mediated ferroptosis.
      Otherwise, it has been reported that HIF1α stabilization by hypoxia promotes fatty acid uptake and lipid storage by transcriptional upregulation of fatty acid-binding proteins
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      • et al.
      Fatty acid uptake and lipid storage induced by HIF-1α contribute to cell growth and survival after hypoxia-reoxygenation.
      and inhibits ferroptosis in human fibrosarcoma or lung cancer cells by increasing lipid droplet formation.
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      Clockophagy is a novel selective autophagy process favoring ferroptosis.
      ,
      • Chen X.
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      • Kroemer G.
      • Tang D.
      Broadening horizons: the role of ferroptosis in cancer.
      These results indicate the possibility of inducing ferroptosis to overcome radioresistance. However, more investigations are required to uncover the relationship (and underlying molecular mechanisms) between ferroptosis and radiotherapy.
      In this study, we demonstrated a novel role of Cu in promoting radioresistance of HCCs. IR induced a reduction in COMMD10 (but not other COMMD family numbers), which increased intracellular Cu and inhibited ferroptosis, leading to radioresistance in HCC. This work provided new targets and treatment strategies for radioresistance.

      Materials and methods

      The details regarding the materials and methods, please refer to the CTAT table and supplementary information.

      Results

      Increased intracellular Cu contributes to radioresistance of HCC

      IR-resistant HepG2 and MHCC-97H cells were applied to evaluate the relationship between Cu and IR response in HCCs and their radioresistant characteristics were confirmed by radiation clonogenic assays (Fig. 1A-C and Fig. S1A-B). The radiobiological parameters such as D0 (the dose that lowers the survival rate by 37%), SF2 (the survival fraction at 2 Gy) and N value (a factor that reflects the repair ability of cells after IR injury) are common indicators positively associated with radioresistance. The D0, SF2 and N value in IR-resistant HepG2 and MHCC-97H cells were significantly higher than those in parental cells (Fig. 1D and Fig. S1C). In addition, a significantly higher level of Cu was detected in IR-resistant cells than parental cells, which suggested a potential role of increased Cu in inducing radioresistance (Fig. 1E). In a microarray dataset (GSE9539)
      • Song M.O.
      • Li J.
      • Freedman J.H.
      Physiological and toxicological transcriptome changes in HepG2 cells exposed to copper.
      that profiled transcriptome changes in HepG2 cells exposed to increasing doses of Cu for different times, low Cu concentration was related to IR response (Fig. 1F). Cell Counting Kit-8 (CCK-8) showed that toxicological responses occurred in both HepG2 and HCCLM3 cells when exposed to Cu levels of 300 μM to 400 μM, while radioresistance was observed in cells exposed to a Cu level lower than 200 μM (Fig. 1G). In addition, an EDU (5-ethynyl-2'-deoxyuridine) assay confirmed that the increased intracellular Cu (≤200 μM) of HepG2 cells promoted proliferation, regardless of radiation exposure (Fig. 1H). Furthermore, the serum Cu concentration and subcutaneous tumor volume and weight were significantly increased after IR in mice fed with Cu compared to in those not fed with Cu (Fig. 1I-K). Tetraethylenepentamine (TEPA), a Cu chelator, significantly inhibited serum Cu and enhanced radiosensitivity of subcutaneous tumors in mice fed with Cu (Fig. 1I-K). However, compared with the IR group, TEPA had no effect on radiosensitivity of tumors derived from HepG2 and HCCLM3 cells in mice not fed with Cu (Fig. S1D-H). To further explore whether the radiosensitive effect of TEPA was Cu-dependent, in vitro experiments revealed that TEPA could not affect the proliferation of MHCC-97H, HepG2 and HCCLM3 cells exposed to IR without Cu treatment (Fig. S1I). The above findings confirmed that increased Cu contributes to radioresistance and TEPA was a Cu-dependent selective radiosensitizer in HCC.
      Figure thumbnail gr1
      Fig. 1Increased intracellular Cu contributes to IR resistance of HCC.
      (A) Schematic of IR-resistant HepG2 cell construction. (B) Radiosensitivity of IR-resistant and parental HepG2 cells were assessed via radiation clonogenic survival assay, (C) survival fraction with multi-target single-hit model, and (D) radiobiological parameters. (E) Cu concentration in IR-resistant and parental HepG2 cells. (F) Gene set enrichment analysis of GSE9539 dataset. (G) IR response of HepG2 and HCCLM3 cells treated with Cu for 24 and 48 hours were evaluated via CCK-8 assay. P value vs. without Cu treatment group. (H) IR response of Cu-treated HepG2 cells were assessed via EdU assay. (I) Cu concentration of serum in indicated mice. (J-K) Volumes and weight of subcutaneous tumors in indicated mice. Data represents the mean ± SD; ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. Unpaired t test was used unless otherwise stated. EdU, 5-ethynyl-2’-deoxyuridine; HCC, hepatocellular carcinoma; IR, ionizing radiation; TEPA, tetraethylenepentamine.

      COMMD10 enhances radiosensitivity of HCCs by decreasing intracellular Cu

      Having established Cu as a contributor to radioresistance in HCC, we explored candidate genes responsible for the regulation of Cu metabolism and radiosensitivity. Reportedly, COMMD family genes decreased in HEK293T cells treated with Cu, which indicated their important roles in the regulation of Cu metabolism.
      • Riera-Romo M.
      COMMD1: A multifunctional regulatory protein.
      To verify which COMMD protein member is involved in regulating radiosensitivity, HepG2 cells were exposed to 0, 2, 4, 6 and 8 Gy radiation respectively for 24 hours and a real-time PCR assay was applied to indicate the expression level of all COMMD family members. Results showed that COMMD10 was the unique gene that was significantly decreased in an IR dosage-dependent manner in HepG2 cells (Fig. 2A). To explore the potential role of COMMD10 in regulating IR response, we constructed COMMD10 overexpressed and depleted HCCs based on endogenous COMMD10 level (Fig. S2A-E). The CCK-8 assay showed that COMMD10 overexpression significantly inhibited the proliferation of HCCLM3 and MHCC-97H cells exposed to IR (Fig. 2B), while COMMD10 depletion enhanced the proliferation of HepG2 and Huh7 cells (Fig. S2F). Consistently, radiation clonogenic assays showed that COMMD10 overexpression increased radiosensitivity in MHCC-97H and HCCLM3 cells (Fig. 2C-E), while COMMD10 depletion induced radioresistance in HepG2 cells (Fig. S2G-I). Moreover, COMMD10 significantly decreased in the IR-resistant HepG2 and MHCC-97H cells (Fig. 2F and Fig. S2J-K), and radiosensitivity could be reversed in the COMMD10 overexpressed condition (Fig. 2G and Fig. S2L-M).
      Figure thumbnail gr2
      Fig. 2COMMD10 enhances radiosensitivity of HCCs by decreasing intracellular Cu.
      (A) Expression of COMMD family mRNAs in HepG2 cells exposed to increasing dosage of IR (One-way ANOVA). (B) The proliferation of COMMD10-overexpressing and control MHCC-97H and HCCLM3 cells after IR were detected by CCK-8 assay. (C) The radiosensitivity of COMMD10-overexpressing and control MHCC-97H and HCCLM3 cells were detected using a radiation clonogenic survival assay, (D) survival fraction with multi-target single-hit model, and (E) radiobiological parameters. (F) COMMD10 mRNA and protein expression in IR-resistant and parental HepG2 cells. (G) Radiosensitivity in COMMD10-treated IR-resistant HepG2 cells were evaluated by CCK-8 assay. (H) The RSI of COMMD10 low and high groups was calculated using the data from TCGA and GEO database. (I) Representative IHC images of COMMD10 expression are shown in IR-sensitive and IR-resistant clinical HCC samples (Mann-Whitney U test). (J) Expression of COMMD10 mRNA and protein in indicated HepG2 cells. (K) Cu concentration in COMMD10-depleted and control HepG2 cells. (L) The proliferation of IR-exposed COMMD10-depleted HepG2 and Huh7 treated with TEPA or not was detected via CCK-8 assay. Data represents the mean ± SD; ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. Unpaired t test was used unless otherwise stated. HCC, hepatocellular carcinoma; IHC, immunohistochemical; IR, ionizing radiation; RSI, radiosensitivity index; TEPA, tetraethylenepentamine.
      We further explored the correlation between COMMD10 and radiosensitivity in clinical data accessed from TCGA and GEO databases. The radiosensitivity index (RSI) is a genome-based model used to estimate the intrinsic radiosensitivity of tumors, which negatively correlated with radiosensitivity.
      • Qi W.
      • Li Z.
      • Xia L.
      • Dai J.
      • Zhang Q.
      • Wu C.
      • et al.
      LncRNA GABPB1-AS1 and GABPB1 regulate oxidative stress during erastin-induced ferroptosis in HepG2 hepatocellular carcinoma cells.
      Results suggested that COMMD10 low-expressing HCC were generally predicted to be more radioresistant than COMMD10 high-expressing HCC (Fig. 2H). In addition, immunohistochemistry staining revealed that the expression of COMMD10 in radiosensitive HCC clinical samples was higher than that in radioresistant samples (Fig. 2I), which suggested that COMMD10 may be an effective predictor of radiosensitivity. Notably, COMMD10 low expression also predicted radioresistance in a wide spectrum of cancers in TCGA database (Fig. S3A-F) and contributed to radioresistance in nasopharyngeal carcinoma cells (CNE2: Fig. S3G and S3I) and colorectal cancer cells (SW480: Fig. S3H and S3J), implying that COMMD10 might be a broad-spectrum predictor of tumor radiosensitivity.
      As a member of the COMMD family, which is associated with Cu metabolism, we observed an interesting relationship between COMMD10 and Cu. The expression of COMMD10 in HepG2 cells was decreased by Cu treatment and further decreased by Cu treatment + IR (Fig. 2J). Moreover, the concentration of intracellular Cu was significantly increased in COMMD10-depleted cells compared to control cells (Fig. 2K). Importantly, the Cu chelator TEPA could reverse COMMD10 depletion-induced proliferation of HepG2 and Huh7 cells exposed to IR (Fig. 2L). The above findings indicated that COMMD10 enhanced radiosensitivity by decreasing intracellular Cu in HCCs.

      COMMD10 induces ferroptosis to enhance radiosensitivity of HCC

      To explore how COMMD10 increases the radiosensitivity of HCC, we conducted proteomic analysis using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry to analyze differential proteins of COMMD10-depleted and control HepG2 cells (Fig. S4A). Gene ontology analysis showed that differentially expressed proteins were significantly enriched in ‘regulation of cell death’ (Fig. 3A).
      Figure thumbnail gr3
      Fig. 3COMMD10 induces ferroptosis to enhance radiosensitivity of HCC.
      (A) Gene ontology analysis of differentially expressed proteins between COMMD10-depleted and control HepG2 cells. (B) Proliferation of IR-exposed COMMD10-overexpressing and control HCCLM3 cells, treated with or without cell death inhibitors, were detected by CCK-8 assay (p value vs. IR alone). (C-D) The effect of COMMD10 overexpression or combined Fer-1 on the level of lipid peroxidation and MDA in IR-exposed HCCLM3 and MHCC-97H cells. (E) Schematic of IR model construction of subcutaneous tumor treated with COMMD10 overexpression or combined with Fer-1. (F) The volumes of subcutaneous tumors in indicated mice. (G) The lipid peroxidation level of subcutaneous tumor in indicated mice. Unpaired t test was used unless otherwise stated. Data represents the mean ± SD; ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. Fer-1, ferrostatin-1; HCC, hepatocellular carcinoma; IHC, immunohistochemical; IR, ionizing radiation; MDA, malondialdehyde.
      To further clarify their relationship, we treated COMMD10-overexpressing and -depleted HCCs with several cell death inhibitors. Compared with the cells treated with ZVAD-FMK (an apoptosis inhibitor), chloroquine (an autophagy inhibitor), belnacasan (a pyroptosis inhibitor) and necrosulfonamide (a necroptosis inhibitor), the ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) significantly increased growth in all cell groups (Fig. 3B and Fig. S4B). Therefore, COMMD10 might sensitize HCCs to radiation by inducing ferroptosis.
      It has been reported that chemical drugs, such as erastin, RSL-3 and sorafenib can induce ferroptosis in HCC,
      • Qi W.
      • Li Z.
      • Xia L.
      • Dai J.
      • Zhang Q.
      • Wu C.
      • et al.
      LncRNA GABPB1-AS1 and GABPB1 regulate oxidative stress during erastin-induced ferroptosis in HepG2 hepatocellular carcinoma cells.
      • Sun X.
      • Niu X.
      • Chen R.
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      Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis.
      • Nie J.
      • Lin B.
      • Zhou M.
      • Wu L.
      • Zheng T.
      Role of ferroptosis in hepatocellular carcinoma.
      but whether IR could induce ferroptosis in HCCs remains unclear. Here, IR significantly increased the level of lipid peroxidation and malondialdehyde (MDA) in HCCLM3 and HepG2 cells (Fig. S4C-E) and this phenomenon could be significantly reversed by ferrostatin-1. These results demonstrated that IR induced ferroptosis in HCC cells. In addition, a ferroptosis inducer (erastin) sensitized HCCLM3 and MHCC-97H cells exposed to IR (Fig. S4F), suggesting that combining a ferroptosis inducer and radiotherapy may be an effective therapeutic strategy for HCC.
      We further studied whether COMMD10 modulated radiosensitization in HCCs by regulating ferroptosis. The level of lipid peroxidation and MDA were significantly elevated in COMMD10-overexpressing cells exposed to IR and this elevation could be significantly attenuated by ferrostatin-1 (Fig. 3C-D). Next, a subcutaneous tumor in nude mice model (Fig. 3E) was constructed to explore whether COMMD10 enhances the radiosensitivity of HCC by promoting ferroptosis. Subcutaneous tumors derived from the COMMD10-overexpressing group were much smaller, with a higher level of lipid peroxidation, than those in the Mock group of HCCLM3 cells. However, this effect could be reversed by treating mice with ferrostatin-1 (Fig. 3F-G). These results suggested that COMMD10 enhanced radiosensitization by promoting ferroptosis.

      COMMD10 inhibits SLC7A11-mediated ferroptosis by binding to and suppressing HIF1α in HCC

      To determine the underlying mechanism of ferroptosis induced by COMMD10, we examined the genes associated with ferroptosis. Among these genes, only the SLC7A11 mRNA level decreased obviously in COMMD10-overexpressing cells and increased in COMMD10-depleted cells under IR exposure (Fig. 4A). Furthermore, COMMD10 depletion increased SLC7A11 expression while overexpression of COMMD10 inhibited SLC7A11 expression by western blot (Fig. 4B).
      Figure thumbnail gr4
      Fig. 4COMMD10 inhibits SLC7A11-mediated ferroptosis by binding to and suppressing HIF1α in HCC.
      (A) The expression of ferroptosis related genes were detected in IR-exposed COMMD10-depleted HepG2 and COMMD10-overexpressing HCCLM3 cells. (B) SLC7A11 expression in IR-exposed COMMD10-depleted HepG2 and -overexpressing HCCLM3 cells. (C) GSH levels were detected in IR-exposed COMMD10 overexpressed and COMMD10-SLC7A11 co-expressed HCCLM3 cells. (D) Potential interacting proteins of COMMD10 analyzed in STRING database. (E) Enrichment of COMMD10-related proteins in STRING database analyzed by KEGG pathway. (F) The binding between COMMD10 and HIF1α detected by immunoprecipitation under normoxic and hypoxic conditions. (G) The binding between truncation mutants of COMMD10 and HIF1α was detected by GST-pulldown assay. (H) Expression of HIF1α in the nucleus and cytoplasm of COMMD10-overexpressing HCCLM3 cells under IR, normoxic, and hypoxic conditions. (I-J) The effect of YC-1 on the expression of SLC7A11 in COMMD10-depleted HepG2 cells. Unpaired t test was used unless otherwise stated. Data represents the mean ± SD; ∗∗∗p <0.001. GSH, glutathione; HCC, hepatocellular carcinoma; IR, ionizing radiation; YC-1, lificiguat.
      SLC7A11 is a light chain protein of glutamate-cystine reverse transporter and plays an important role in glutathione synthesis.
      • Friedmann Angeli J.P.
      • Krysko D.V.
      • Conrad M.
      Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion.
      Reduced glutathione synthesis can increase lipid peroxidation and promote ferroptosis. To explore whether SLC7A11 is a downstream target of COMMD10 involved in the induction of ferroptosis, we constructed COMMD10/SLC7A11 co-expression cells (Fig. S5A-B) to detect the levels of glutathione. The results showed that COMMD10 overexpression decreased the level of glutathione compared to mock cells, and SLC7A11 expression attenuated COMMD10-induced inhibition of glutathione (Fig. 4C), suggesting that COMMD10 enhanced ferroptosis in response to IR by suppressing SLC7A11 expression in HCCs.
      We further explored how COMMD10 inhibited SLC7A11 expression. The STRING database predicted 20 proteins that could potentially interact with COMMD10 (Fig. 4D), and KEGG pathway analysis showed that these proteins were mainly associated with the HIF1 signaling pathway and ubiquitin-mediated proteolysis (Fig. 4E). Thus, we verified whether COMMD10 interacted with HIF1α using immunoprecipitation, and results showed that COMMD10 combined with HIF1α in normoxic and hypoxic conditions (Fig. 4F). We further constructed COMMD10-N terminal (1-132) and COMMD10-C terminal (133-202) truncation mutants to determine the binding site of HIF1α in COMMD10. GST-pulldown revealed that COMMD10-N terminus could combine with HIF1α (Fig. 4G). Moreover, in both IR and hypoxic conditions, the nuclear translocation of HIF1α was promoted in COMMD10-depleted cells and was inhibited in COMMD10-overexpressing cells (Fig. 4H and Fig. S5C-E). Interestingly, we found the binding sites of HIF1α in the promoter of SLC7A11 (Fig. S5F). Overexpression of HIF1α significantly increased the expression of SLC7A11 at both mRNA and protein levels (Fig. S5G-H). Furthermore, COMMD10 depletion could increase HIF1α and SLC7A11 expression (Fig. 4I-J). Meanwhile, the HIF1α inhibitor lificiguat (YC-1) could reverse the upregulation of HIF1α and SLC7A11 via COMMD10 depletion (Fig. 4I-J), suggesting that COMMD10 inhibited SLC7A11 by binding to and suppressing the nuclear translocation of HIF1α in HCCs.

      COMMD10 inhibits HIF1α stability by decreasing intracellular Cu to enhance ferroptosis in HCC

      It has been reported that intracellular Cu could stabilize the HIF1α protein,
      • Martin F.
      • Linden T.
      • Katschinski D.M.
      • Oehme F.
      • Flamme I.
      • Mukhopadhyay C.K.
      • et al.
      Copper-dependent activation of hypoxia-inducible factor (HIF)-1: implications for ceruloplasmin regulation.
      and a microarray dataset (GSE9539) indicated that intracellular Cu was positively correlated with HIF1α in HepG2 cells (Fig. 5A). Thus, we hypothesized that COMMD10 might modulate HIF1α protein stability by regulating Cu. HIF1α expression significantly increased and the half-life of HIF1α was significantly prolonged in COMMD10-depleted cells under normoxic and hypoxic conditions, while TEPA could reverse this effect (Fig. 5B-C). Furthermore, COMMD10 depletion could inhibit the ubiquitination and degradation of HIF1α proteins, an effect that could also be reversed by TEPA (Fig. 5D). The above results indicated that COMMD10 depletion stabilized the HIF1α protein by augmenting intracellular Cu.
      Figure thumbnail gr5
      Fig. 5COMMD10 inhibits HIF1α stability by decreasing intracellular Cu to enhance ferroptosis in HCC.
      (A) The relationship between the Cu and HIF1α analyzed by gene-set enrichment analysis in the GSE9539 dataset. (B) The effect of TEPA on HIF1α expression in COMMD10-depleted HepG2 cells under normoxic and hypoxic conditions. (C) The half-life of HIF1α protein in COMMD10-depleted cells treated with TEPA or without treatment under normoxic and hypoxic conditions. (D) The ubiquitination of HIF1α protein in COMMD10-depleted cells treated with TEPA or without treatment under normoxic and hypoxic conditions. (E-F) The effect of HIF1α on the level of lipid peroxidation and MDA in COMMD10-overexpressing and control HCCLM3 cells. (G) Schematic of IR model construction of COMMD10-depleted subcutaneous tumors treated with YC-1 or without treatment. (H-I) The volumes of subcutaneous tumors in indicated mice. (J) The lipid peroxidation level of subcutaneous tumors in indicated mice. (K) The death area and Ki67-positive cell ratio of subcutaneous tumors in indicated mice. Unpaired t test was used unless otherwise stated. Data represents the mean ± SD; ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. CHX, cycloheximide; HCC, hepatocellular carcinoma; IR, ionizing radiation; MG132, proteasome inhibitor; TEPA, tetraethylenepentamine; YC-1, lificiguat.
      The effect of COMMD10/HIF1α on IR-induced ferroptosis was further confirmed in vitro and in vivo. COMMD10 overexpression obviously elevated the level of lipid peroxidation and MDA while HIF1α could reverse this effect in IR-exposed HCCLM3 cells (Fig. 5E-F). Furthermore, tumors derived from COMMD10-depleted cells were larger and had lower lipid peroxidation than the control group under IR exposure in vivo. Conversely, intraperitoneal injection of YC-1 could reverse the tumor-promoting effect (Fig. 5G-J). Consistently, the COMMD10-depleted group showed fewer cell death areas and a higher ratio of Ki67-positive malignant cells than the control group under IR exposure, while YC-1 injection could reverse this effect (Fig. 5K). Above all, COMMD10 inhibited HIF1α stability by decreasing intracellular Cu to enhance ferroptosis in HCC.

      COMMD10 suppresses ceruloplasmin (CP) expression by decreasing intracellular Cu to enhance ferroptosis

      Fe can trigger ferroptosis by inducing Fenton reactions and promoting lipid peroxidation, but whether Fe is involved in IR-induced ferroptosis in HCC remains unclear.
      • Stockwell B.R.
      • Jiang X.
      The chemistry and biology of ferroptosis.
      Our results showed that the level of lipid peroxidation and MDA was significantly increased in IR-exposed HCCs and could be further increased by Fe stimulation. In contrast, the Fe chelator desferrioxamine significantly decreased the level of lipid peroxidation and MDA in HCCs exposed to IR compared to HCCs exposed to Fe and/or IR treatment (Fig. S6A-B). These results suggested that Fe played a critical role in IR-induced ferroptosis in HCC. Fe detected by FerroOrange fluorescence probe was decreased in COMMD10-depleted cells and increased in COMMD10-overexpressing cells (Fig. 6A), indicating that COMMD10 could increase the level of Fe in HCCs in response to IR.
      Figure thumbnail gr6
      Fig. 6COMMD10 suppresses CP expression by decreasing intracellular Cu to enhance ferroptosis.
      (A) The effect of COMMD10 on the Fe content of IR-exposed HepG2 and HCCLM3 cells detected by FerroOrange fluorescence probe. (B-C) The effect of COMMD10 on CP expression in IR-exposed HepG2 and HCCLM3 cells. (D-F) The effect of CP expression on the content of Fe, lipid peroxidation and MDA in IR-exposed COMMD10-overexpressing HCCLM3 cells. Unpaired t test was used unless otherwise stated. Data represents the mean ± SD; ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. IR, ionizing radiation; MDA, malondialdehyde; TEPA, tetraethylenepentamine.
      CP, whose synthesis depends on Cu, is an important regulator of iron metabolism.
      • Collins J.F.
      • Prohaska J.R.
      • Knutson M.D.
      Metabolic crossroads of iron and copper.
      Thus, we speculated that COMMD10 might regulate CP expression to induce ferroptosis in HCCs. Our results showed that COMMD10 negatively regulated the level of CP expression in HCCs (Fig. 6B-C), and TEPA could restore CP expression caused by COMMD10 depletion (Fig. 6B). Using COMMD10/CP-expressing HCCLM3 cells (Fig. S6C), we demonstrated that their co-expression could restore the increase of Fe, lipid peroxidation and MDA caused by COMMD10 overexpression (Fig. 6D-F). These results indicated that COMMD10 suppressed CP expression by decreasing intracellular Cu to enhance ferroptosis.

      COMMD10 inhibits HIF1α/CP loop to enhance ferroptosis and radiosensitivity by disrupting Cu-Fe homeostasis in HCC

      HIF1α has been reported to promote CP transcription.
      • Martin F.
      • Linden T.
      • Katschinski D.M.
      • Oehme F.
      • Flamme I.
      • Mukhopadhyay C.K.
      • et al.
      Copper-dependent activation of hypoxia-inducible factor (HIF)-1: implications for ceruloplasmin regulation.
      To clarify their relationship, we analyzed the binding profile of HIF1α and the CP promoter region using the JASPAR website. The results showed that HIF1α bound to the CP promoter (Fig. S7) and HIF1α overexpression increased the mRNA and protein levels of CP in HCCLM3 cells (Fig. 7A-B), which suggested that HIF1α promoted CP transcription and translation. Moreover, YC-1 could recover the increased CP expression induced by COMMD10 depletion (Fig. 7C), suggesting that COMMD10 depletion promoted CP expression by enhancing HIF1α in HCCs in response to IR.
      Figure thumbnail gr7
      Fig. 7COMMD10 inhibits HIF1α/CP loop to enhance ferroptosis and radiosensitivity by disrupting Cu-Fe homeostasis in HCC.
      (A-B) The effect of HIF1α on the expression of CP in HCCLM3 cells detected by real-time PCR and western blot. (C) The effect of YC-1 on CP expression in IR-exposed COMMD10-depleted HepG2 cells detected by western blot. (D) The effect of CP on the expression of HIF1α in HCCLM3 cells detected by western blot. (E) The effect of CP on HIF1α expression in IR-exposed COMMD10-overexpressing HCCLM3 cells detected by western blot. (F) The expression of COMMD10, HIF1α, CP, SLC7A11, and 4-HNE in IR-sensitive and IR-resistant HCC specimens using IHC assay (Mann-Whitney U test). (G) The effect of IR-resistant HepG2 cells treated with increasing doses of TEPA and YC-1 for 24 hours. (H) The volumes and weight of radioresistant subcutaneous tumors in indicated mice. (K) The expression of 4-HNE and Ki67-positive cell ratios of radioresistant subcutaneous tumors in indicated mice (Mann-Whitney U test was used to analyze 4-HNE expression). Unpaired t test was used unless otherwise stated. Data represents the mean ± SD; ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. 4-HNE, 4-hydroxynonenal; HCC, hepatocellular carcinoma; IHC, immunohistochemical; IR, ionizing radiation; TEPA, tetraethylenepentamine; YC-1, lificiguat.
      Proline hydroxylase (PHD) can hydroxylate proline residues of HIF1α and then ubiquitinate HIF1α. Considering that Fe is an important cofactor for PHD activity, we hypothesized that CP could promote HIF1α expression by inhibiting Fe.
      • Nandal A.
      • Ruiz J.C.
      • Subramanian P.
      • Ghimire-Rijal S.
      • Sinnamon R.A.
      • Stemmler T.L.
      • et al.
      Activation of the HIF prolyl hydroxylase by the iron chaperones PCBP1 and PCBP2.
      Indeed, CP overexpression increased HIF1α expression (Fig. 7D), and COMMD10/CP co-expression could reverse the decreased HIF1α expression induced by COMMD10 overexpression in response to IR (Fig. 7E), indicating that COMMD10 inhibited HIF1α expression by inhibiting CP expression. Overall, COMMD10 increased ferroptosis and radiosensitivity via inhibition of the HIF1α/CP loop in HCC. The relationship between COMMD10, HIF1α and ferroptosis was further explored in clinical HCC specimens. The immunohistochemical (IHC) staining scores of HIF1α and SLC7A11 in COMMD10 low-expressing HCC specimens were higher than those in COMMD10 high-expressing specimens (Fig. S8A-B). Besides, COMMD10 expression was negatively correlated with the expression of HIF1α and SLC7A11 (Table S1-2). 4-Hydroxynonenal (4-HNE) is a lipid peroxidation product and widely used as a marker of lipid peroxidation. The IHC staining score of 4-HNE in COMMD10 high-expressing HCC specimens was significantly higher than that in COMMD10 low-expressing specimens (Fig. S8C). Meanwhile, COMMD10 expression showed a positive correlation with the expression of 4-HNE (Table S3). Moreover, the IHC staining scores of COMMD10 and 4-HNE in radiosensitive HCC specimens were significantly higher than those in radioresistant specimens (Fig. 7F). In contrast, the IHC staining scores of HIF1α, CP, and SLC7A11 in radiosensitive HCC specimens were significantly lower than those in radioresistant specimens (Fig. 7F). These results revealed that COMMD10, HIF1α, CP, SLC7A11, and 4-HNE might be used as predictors of HCC radiosensitivity.
      To further explore the clinical application of TEPA and YC-1, we treated IR-resistant HepG2 cells with increasing doses of TEPA (25 μM, 50 μM,100 μM) and YC-1(1 μM, 5 μM,10 μM), and found that YC-1 and TEPA significantly reduced the proliferation of IR-resistant cells (Fig. 7G). The effect of TEPA and YC-1 on radiosensitivity was further investigated in a radioresistant mouse model. The results revealed that both TEPA and YC-1 treatment could significantly decrease the volume and weight of radioresistant tumors (Fig. 7H-J). Moreover, the IHC score of 4-HNE in tumors treated with TEPA and YC-1 was higher than that in the control group. In contrast, the ratio of Ki67-positive malignant cells of tumors treated with TEPA and YC-1 was reduced (Fig. 7K). The above results suggest that the Cu chelator TEPA and the HIF1α inhibitor YC-1 could be potential radiotherapy sensitizers in radioresistant patients.

      Discussion

      Radiotherapy benefits patients with unresectable or advanced HCC by improving local tumor control,
      • Ishida S.
      • McCormick F.
      • Smith-McCune K.
      • Hanahan D.
      Enhancing tumor-specific uptake of the anticancer drug cisplatin with a copper chelator.
      but its effectiveness is hampered by radioresistance.
      • Krisnawan V.E.
      • Stanley J.A.
      • Schwarz J.K.
      • DeNardo D.G.
      Tumor microenvironment as a regulator of radiation therapy: new insights into stromal-mediated radioresistance.
      It was reported that the imbalance of Cu homeostasis is closely related to the occurrence, proliferation and progression of cancer.
      • Chen H.H.
      • Kuo M.T.
      Overcoming platinum drug resistance with copper-lowering agents.
      • Fang A.P.
      • Chen P.Y.
      • Wang X.Y.
      • Liu Z.Y.
      • Zhang D.M.
      • Luo Y.
      • et al.
      Serum copper and zinc levels at diagnosis and hepatocellular carcinoma survival in the Guangdong Liver Cancer Cohort.
      • Lu H.
      • Samanta D.
      • Xiang L.
      • Zhang H.
      • Hu H.
      • Chen I.
      • et al.
      Chemotherapy triggers HIF-1-dependent glutathione synthesis and copper chelation that induces the breast cancer stem cell phenotype.
      In this study, we identified COMMD10, a member of the newly discovered copper metabolism-related proteins family which was involved in the regulation of sodium transport, NF-κB activity, cell cycle, immunomodulation and tumor progression,
      • Maine G.N.
      • Burstein E.
      COMMD proteins and the control of the NF kappa B pathway.
      • Ware A.W.
      • Cheung T.T.
      • Rasulov S.
      • Burstein E.
      • McDonald F.J.
      Epithelial Na(+) channel: reciprocal control by COMMD10 and Nedd4-2.
      • Ben Shlomo S.
      • Mouhadeb O.
      • Cohen K.
      • Varol C.
      • Gluck N.
      COMMD10-Guided phagolysosomal maturation promotes clearance of Staphylococcus aureus in macrophages.
      • Mouhadeb O.
      • Ben Shlomo S.
      • Cohen K.
      • Farkash I.
      • Gruber S.
      • Maharshak N.
      • et al.
      Impaired COMMD10-mediated regulation of Ly6C(hi) monocyte-driven inflammation disrupts gut barrier function.
      as a novel contributor to radiosensitivity in HCC. Mechanistically, low expression of COMMD10 induced intracellular Cu accumulation, HIF1α upregulation and nuclear translocation by inhibiting ubiquitination and degradation of HIF1α, thereby upregulating downstream CP and SLC7A11 target genes, which promoted intracellular glutathione synthesis and reduced the level of lipid peroxidation, inhibiting ferroptosis in HCC. Conversely, a high concentration of intracellular Cu upregulated CP expression to reduce the intracellular Fe concentration, which inhibited ferroptosis by directly decreasing the level of lipid peroxidation. Upregulation of CP could also enhance HIF1α expression, forming a HIF1α/CP positive feedback regulatory loop and promoting HCC radioresistance.
      Copper, a micronutrient essential for basic biological processes, has been recognized as a vital factor involved in tumor growth, angiogenesis, metastasis and treatment response.
      • Baldari S.
      • Di Rocco G.
      • Toietta G.
      Current biomedical use of copper chelation therapy.
      ,
      • Vetlényi E.
      • Rácz G.
      The physiological function of copper, the etiological role of copper excess and deficiency.
      Physiologic Cu concentrations in healthy adults mostly fluctuated in the range of 70∼110 ug/dl.
      • Feng Y.
      • Zeng J.W.
      • Ma Q.
      • Zhang S.
      • Tang J.
      • Feng J.F.
      Serum copper and zinc levels in breast cancer: a meta-analysis.
      The serum Cu concentration in patients with HCC was significantly higher than that in the chronic hepatitis control group (137±24 vs. 107±15 μg/dl, p = 0.0030).
      • Song M.O.
      • Li J.
      • Freedman J.H.
      Physiological and toxicological transcriptome changes in HepG2 cells exposed to copper.
      ,
      • Voli F.
      • Valli E.
      • Lerra L.
      • Kimpton K.
      • Saletta F.
      • Giorgi F.M.
      • et al.
      Intratumoral copper modulates PD-L1 expression and influences tumor immune evasion.
      A higher serum Cu level strongly associated with worse liver cancer-specific survival and overall survival.
      • Fang A.P.
      • Chen P.Y.
      • Wang X.Y.
      • Liu Z.Y.
      • Zhang D.M.
      • Luo Y.
      • et al.
      Serum copper and zinc levels at diagnosis and hepatocellular carcinoma survival in the Guangdong Liver Cancer Cohort.
      Using Cu feeding according to the published study,
      • Brady D.C.
      • Crowe M.S.
      • Turski M.L.
      • Hobbs G.A.
      • Yao X.
      • Chaikuad A.
      • et al.
      Copper is required for oncogenic BRAF signalling and tumorigenesis.
      we successfully establish a liver cancer mouse model with high serum Cu levels of 137.4±0.05 μg/dl, which was comparable to serum copper concentrations in patients with HCC.
      Actually, the antitumoral effect of Cu chelators, such as TEPA,
      • Voli F.
      • Valli E.
      • Lerra L.
      • Kimpton K.
      • Saletta F.
      • Giorgi F.M.
      • et al.
      Intratumoral copper modulates PD-L1 expression and influences tumor immune evasion.
      ,
      • Ding X.
      • Xie H.
      • Kang Y.J.
      The significance of copper chelators in clinical and experimental application.
      penicillamine, trientine, and tetrathiomolybdate, has been known for many years, leading to several clinical trials.
      • Baldari S.
      • Di Rocco G.
      • Toietta G.
      Current biomedical use of copper chelation therapy.
      Without any serious side effects, most of these therapies are already used to treat Wilson’s disease, a rare inherited disease that causes systemic Cu accumulation with neurological and hepatic presentations.
      • Lucena-Valera A.
      • Perez-Palacios D.
      • Muñoz-Hernandez R.
      • Romero-Gómez M.
      • Ampuero J.
      Wilson's disease: revisiting an old friend.
      Therefore, Cu chelators might be an effective new strategy for future HCC treatment.
      In this study, we surprisingly found that the effects of TEPA and YC-1, as potential radiosensitive agents for HCC, were selective. They might only increase radiosensitivity to originally IR-resistant HCCs, which are characterized by significant accumulation of intracellular Cu. This interesting finding emphasized that it would be very important to precisely screen the potential beneficiaries before radiotherapy in future clinical practice.
      To further evaluate the biomarkers of radioresistance, we conducted IHC staining in patients with HCC and found that COMMD10 along with key downstream markers, such as HIF1α, CP, SLC7A11 and 4-HNE, were all significantly correlated with radiosensitivity in HCC. This gives us confidence that these factors could be potential biomarkers for predicting radiosensitivity, for precisely screening beneficiaries and for developing new strategies for precision radiotherapy in HCC. Moreover, we recently reported that COMMD10 adds value to BCLC staging for the prediction of overall survival in HCC, which also provides evidence for the identification of potential therapeutic targets.
      • Yang M.
      • Wu X.
      • Li L.
      • Li S.
      • Li N.
      • Mao M.
      • et al.
      COMMD10 inhibits tumor progression and induces apoptosis by blocking NF-κB signal and values up BCLC staging in predicting overall survival in hepatocellular carcinoma.
      Meanwhile, we tried to check the Cu content in paraffin slices using a rhodanine stain, which is the most commonly used method. However, few cases are positive despite a positive internal standard. Previously, it was reported that negative staining might be shown even in documented cases of Wilson’s disease, whether in early or advanced stages. A negative Cu stain does not exclude the possibility of this disease.
      • Iezzoni J.C.
      Diagnostic histochemistry in hepatic pathology.
      Therefore, we believe that the Cu staining in paraffin slices is not yet suitable as a screening indicator for radioresistance in HCC.
      Furthermore, we have successfully compared the individual antitumor effects of IR alone with IR+TEPA, IR+YC-1 and IR+COMMD10 both in vitro and in vivo using radioresistant HCC models, which validated the therapeutic potential of TEPA and YC-1 to improve radiosensitivity in HCC. Besides, considering these agents act selectively on IR-resistant HCCs with significant intracellular accumulation of Cu, they might be safe to normal cells and be generally well tolerated. Meanwhile, we have also developed a COMMD10 nano-particle that is undergoing preclinical validation and which could have potential clinical applications.
      In our study, it was found that ferroptosis was not the only, but was the primary mode of cell death caused by radiotherapy regardless of control, or COMMD10 overexpression or depletion. It was shown that the ferroptosis inhibitors significantly restored cell viability following IR treatment when compared with the cells treated with apoptosis inhibitors, autophagy inhibitors, pyroptosis inhibitors and necroptosis inhibitors. Therefore, it should be emphasized that ferroptosis was the leading pattern of cell death induced by radiotherapy in HCCs.
      In this study, we used RSI to preliminarily predict the correlation between COMMD10 and radiosensitivity. RSI is a model based on the expression of 10 specific genes used to estimate the intrinsic radiosensitivity of tumors (high RSI = radioresistance).
      • Eschrich S.
      • Zhang H.
      • Zhao H.
      • Boulware D.
      • Lee J.H.
      • Bloom G.
      • et al.
      Systems biology modeling of the radiation sensitivity network: a biomarker discovery platform.
      It was initially developed in a panel of 48 human cancer cell lines and has been validated in multiple independent clinical cohorts including rectal, esophageal, head and neck, breast, colon, glioblastoma, pancreas, prostate and metastatic liver malignancies,
      • Eschrich S.
      • Zhang H.
      • Zhao H.
      • Boulware D.
      • Lee J.H.
      • Bloom G.
      • et al.
      Systems biology modeling of the radiation sensitivity network: a biomarker discovery platform.
      ,
      • Scott J.G.
      • Berglund A.
      • Schell M.J.
      • Mihaylov I.
      • Fulp W.J.
      • Yue B.
      • et al.
      A genome-based model for adjusting radiotherapy dose (GARD): a retrospective, cohort-based study.
      which provided a basis for predicting tumor radiosensitivity with RSI. In our study, we firstly tried to verify the accuracy of RSI with HCC radiotherapy data in multiple datasets. Unfortunately, only 8 HCC cases were obtained with radiotherapeutic response in TCGA dataset, and we were unable to show the evidence that RSI really correlated with radiosensitivity in HCC. Therefore, we could only use RSI for a preliminarily prediction of the correlation between COMMD10 and radiosensitivity. Though 3 independent datasets showed similar results, the differences in RSI between the COMMD10-overexpressing and -depleted groups were indeed faint. We recently reported that COMMD10 was significantly downregulated in HCCs compared to normal liver cells.
      • Yang M.
      • Wu X.
      • Li L.
      • Li S.
      • Li N.
      • Mao M.
      • et al.
      COMMD10 inhibits tumor progression and induces apoptosis by blocking NF-κB signal and values up BCLC staging in predicting overall survival in hepatocellular carcinoma.
      This implies that it would be difficult to find obvious differences in HCC tissues with mostly originally low expression of COMMD10. Thus, it was urgent to further validate the correlation between COMMD10 and radiosensitivity in HCC using clinical samples. We further performed IHC staining in 22 radiosensitive patients and 20 radioresistant ones. It was confirmed that COMMD10 expression was significantly lower in radioresistant HCC samples than that in radiosensitive ones, indicating a potential role of COMMD10 in predicting radiosensitivity.
      Furthermore, it was validated that in the IR-resistant HCCs, COMMD10 was significantly decreased compared to parental HCCs in a dose-dependent manner, namely, the higher the IR dosage, the lower the expression level of COMMD10. In addition, COMMD10 depletion induced radioresistance in HCCs, while COMMD10 overexpression fully restored radiosensitivity in IR-resistant HCCs, highlighting the crucial role of COMMD10 in radiosensitivity.
      Taken together, our study provides a detailed and novel mechanistic work-up of how COMMD10 is involved in radiosensitivity. COMMD10 inhibits the HIF1α/CP loop to enhance ferroptosis and radiosensitivity mediated by Cu-Fe balance in HCCs. Given the significant proportion of patients with radioresistant HCC exhibiting COMMD10 depletion, targeting COMMD10 and downstream signaling may provide avenues to create novel biomarkers and therapeutics to overcome radioresistance.

      Abbreviations

      4-HNE, 4-hydroxynonenal; CCK-8, cell counting kit-8; COMMD, copper metabolism MURR1 domain; COMMD10, copper metabolism MURR1 domain 10; CP, ceruloplasmin; Cu, Copper; Edu, 5-ethynyl-2'-deoxyuridine; Fe, iron, ferrous ion; HCC, hepatocellular carcinoma; HCCs, HCC cells; IHC, immunohistochemical; IR, ionizing radiation; MDA, malondialdehyde; PHD, proline hydroxylase; RSI, radiosensitivity index; TEPA, tetraethylenepentamine; YC-1, Lificiguat.

      Financial support

      This work was supported by the National Natural Science Foundation of China (NO. 81602685, 81870026, 82102839); the Natural Science Foundation of Guangdong Province (NO. 2017A030313486); the Science and Technology Projects in Guangdong Province (NO. 2018-1201-SF-0019); Health & Medical Collaborative Innovation Project of Guangzhou City, China (NO. 201803040003); China Postdoctoral Science Foundation (NO. 2020M682815); Guangdong Basic and Applied Basic Research Foundation (NO. 2020A1515110787).

      Authors’ contributions

      Jian Guan conceived the idea, wrote the manuscript; Laiyu Liu and Li Liang analyzed data; Mi Yang wrote the manuscript; Mi Yang, Xixi Wu, Jinlong Hu, Yingqiao Wang and Yin Wang performed most experiments; Longshan Zhang, Weiqiang Huang, Xiaoqing Wang, Min Chen, Nanjie Xiao, Liwei Liao and Yongmei Dai aided in clinical sample collection; Nan Li, Huazhen Liang, Wenqi Huang, Lu Yuan, Hua Pan, Lu Li and Longhua Chen analyzed the data from public database; All authors were involved in final approval of the submitted and published versions.

      Data availability statement

      All data in the article as well as in the Supplementary Information file are available upon reasonable request.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      We thank State Key Laboratory of Oncology in Southern Medical University for providing experimental platform. We thank Dr. Yi Ding for providing the IR-resistant MHCC-97H and parental cell lines.

      Supplementary data

      The following are the supplementary data to this article:

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