p52-ZER6/DAZAP1 axis promotes ferroptosis resistance and colorectal cancer progression <i>via</i> regulating <i>SLC7A11</i> mRNA stabilization

p52-ZER6/DAZAP1 axis promotes ferroptosis resistance and colorectal cancer progression via regulating SLC7A11 mRNA stabilization

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Li Qiu^a^,^b, Wenfang Li^a^,^b, Lei Zhang^a^,^b, Xia Zhang^a^,^b, Hezhao Zhao^c, Makoto Miyagishi^d, Shourong Wu^a^,^b^,^*, Vivi Kasim^a^,^b^,^*

Acta Pharmaceutica Sinica B | 2025, 15(4) : 2039 - 2058

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Acta Pharmaceutica Sinica B | 2025, 15(4): 2039-2058

• ORIGINAL ARTICLE •

p52-ZER6/DAZAP1 axis promotes ferroptosis resistance and colorectal cancer progression via regulating SLC7A11 mRNA stabilization

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Li Qiu^a^,^b, Wenfang Li^a^,^b, Lei Zhang^a^,^b, Xia Zhang^a^,^b, Hezhao Zhao^c, Makoto Miyagishi^d, Shourong Wu^a^,^b^,^*, Vivi Kasim^a^,^b^,^*

Affiliations

^aKey Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China

^bThe 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044, China

^cDepartment of Gastrointestinal Surgery, Chongqing University Cancer Hospital, Chongqing University, Chongqing 400030, China

^dLife Science Innovation, School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan

About Author:

E-mail addresses: shourongwu@cqu.edu.cn (Shourong Wu)

vivikasim@cqu.edu.cn (Vivi Kasim)

Author contributions

Li Qiu: Writing – review & editing, Writing – original draft, Validation, Project administration, Methodology, Investigation, Formal analysis, Data curation. Wenfang Li: Validation, Software, Methodology, Investigation. Lei Zhang: Visualization, Methodology, Investigation, Formal analysis. Xia Zhang: Investigation, Formal analysis, Conceptualization. Hezhao Zhao: Visualization, Methodology, Conceptualization. Makoto Miyagishi: Methodology, Conceptualization. Shourong Wu: Writing – review & editing, Visualization, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization. Vivi Kasim: Writing – review & editing, Visualization, Supervision, Resources, Project administration, Investigation, Funding acquisition, Conceptualization.

doi: 10.1016/j.apsb.2025.02.013

Outline

Abstract

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Resistance to ferroptosis, a form of regulated cell death caused by disruptions in iron ion and intracellular redox homeostasis, is closely related to tumorigenesis and tumor drug resistance; therefore, targeting ferroptosis-related pathways has garnered attention as a potential antitumor therapeutic strategy. However, the molecular mechanisms underlying ferroptosis resistance in tumor cells remain unknown. Zinc-finger estrogen receptor interaction clone 6 (ZER6) consists of two isoforms with distinct N-termini, p52-ZER6 and p71-ZER6. ZER6 is upregulated in tumors and promotes tumorigenic potential; however, whether ZER6 is involved in tumor cell ferroptosis resistance remains unknown. Herein, we identified p52-ZER6 as a novel regulator of tumor cell ferroptosis resistance. p52-ZER6 promotes the transcriptional activity of DAZAP1, an RNA-binding protein. DAZAP1, in turn, enhances the stability of SLC7A11 mRNA by binding to its 3′-UTR region, thereby increasing SLC7A11 expression and cellular glutathione levels. This subsequently reduces lipid peroxide accumulation and enhances tumor cell ferroptosis resistance, eventually promoting tumorigenic potential. These findings reveal a new function of p52-ZER6 in regulating SLC7A11 mRNA stability via DAZAP1, ultimately leading to ferroptosis resistance and tumorigenic potential. Additionally, we also suggest targeting p52-ZER6 as a potential strategy to promote the efficacy of ferroptosis-based antitumor therapies.

Key words

Tumor cell ferroptosis / mRNA stabilization / RNA binding protein / Zinc-finger protein / ZER6 / p52-ZER6 / DAZAP1 / SLC7A11

Cite this Article

Li Qiu, Wenfang Li, Lei Zhang, Xia Zhang, Hezhao Zhao, Makoto Miyagishi, Shourong Wu, Vivi Kasim. p52-ZER6/DAZAP1 axis promotes ferroptosis resistance and colorectal cancer progression via regulating SLC7A11 mRNA stabilization[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (4) : 2039 -2058 . DOI: 10.1016/j.apsb.2025.02.013

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1.　Introduction

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Cell death resistance is one of the fourteen hallmarks of cancer¹. According to cell morphology and context, as well as the triggering stimuli, cell death can be classified as uncontrolled, accidental cell death and active, orderly processed, regulated cell death (RCD)^2-4. RCD, which mainly includes apoptosis, autophagy, necroptosis, ferroptosis, and pyroptosis, can function as a defense mechanism against malignancy; therefore, abnormalities in RCD can lead to tumor initiation, progression, and drug resistance⁵. In the early stages of cancer, mutations in tumor suppressor genes, such as BRCA1/2 and p53 can disrupt RCD pathways, thereby accelerating tumor initiation and promoting antitumor therapeutic resistance^6,7. With tumor development, cell death resistance supports sustained cell growth by overcoming the harsh tumor microenvironment, which becomes increasingly deficient in oxygen and nutrients^8,9. In later stages of cancer, cell death resistance helps tumor cells to survive and metastasize by circumventing the limitation of proliferative signaling at metastatic sites¹⁰. Hence, cell death resistance is crucial for every step of tumorigenesis.

Ferroptosis is an iron-dependent form of RCD triggered by the disruption of iron ion and redox homeostasis^11,12. Ferroptosis can occur via three main pathways: an imbalance in iron ion homeostasis, which results in the accumulation of lipid peroxides; impaired synthesis of glutathione (GSH), a cellular reductant that reduces lipid peroxides; and aberrant expression of glutathione peroxidase 4 (GPX4), an enzyme that reduces lipid peroxides using GSH^13-15. Recent studies have shown that ferroptosis plays crucial roles in tumor progression, drug resistance, and poor patient prognosis^16,17. Tumor cells often develop an iron dependency, leading to an increase in reactive oxygen species (ROS) levels during tumorigenesis, and therefore are prone to ferroptosis induction^18,19. Accumulating evidence demonstrates that inducing ferroptosis may be a potential antitumor therapeutic strategy, as even tumor cells that are resistant to conventional therapy may be sensitive to ferroptosis^20-22. However, the molecular mechanisms that enable tumor cells to resist ferroptosis remain largely unexplored.

Zinc-finger estrogen receptor interaction clone 6 (ZER6, also known as ZNF398) is a member of the Krüppel C2H2-type zinc-finger protein family. ZER6 comprises two isoforms with different N-termini: p52-ZER6 and p71-ZER6. While the N-terminus of p71-ZER6 has a full-length Krüppel-associated box (KRAB) and a HTLV-I U5RE-binding protein 1 (HUB-1) domains, that of p52-ZER6 has a truncated KRAB domain with only 30 C-terminal amino acids^23,24. Recent studies have revealed the tumorigenic functions of p52-ZER6, as it can promote cell cycle progression, tumor cell proliferation, and the pentose phosphate pathway^25-27. However, studies on the biological and pathological functions of ZER6 remain limited, and whether this protein is involved in ferroptosis resistance in tumor cells remains unknown.

Herein, using transcriptome analysis, we identified ZER6 as a novel ferroptosis regulator. Systematic investigations further revealed that this function is specific to the p52-ZER6 isoform, which improves solute carrier family 7 member 11 (SLC7A11) mRNA stability by promoting the transcriptional activity of the gene encoding deleted in azoospermia associated protein 1 (DAZAP1), an RNA-binding protein (RBP) crucial for regulating mRNA stability^28,29. This, in turn, enhances tumor cell ferroptosis resistance and subsequently, tumorigenic potential. These findings uncover a novel mechanism of tumor cell ferroptosis resistance and provide new insights into the role of p52-ZER6 in maintaining cellular redox homeostasis.

2.　Materials and methods

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2.1.　 Vectors construction

Short hairpin RNA (shRNA) expression vectors targeting ZER6, p52-ZER6, p71-ZER6, and SLC7A11 were constructed as described previously^25,30. The target sequences were as follows: shZER6-1: 5′-AGC CAG AAT CTG GAC CTA T-3′; shZER6-2: 5′-GTA TTA TGT TCC CCT TCT G-3′; shp52-1: 5′-GAG AGG ACG TAG TCT TGC T-3′; shp52-2: 5′-GAC TGA GAG GCA GCG AGA A-3′; shp71-1: 5′-GGG GAA GGC AGG GCC GGG T-3′; shp71-2: 5′-GCT TGG AGC CGG GCG CGG T-3′; shSLC7A11-1: 5′-GGA ACA ACT ATA AAG AAA T-3′; shSLC7A11-2: 5′-GGT CAA ACG CAG AAC TTT A-3′. Vectors overexpressing p52-ZER6, p71-ZER6, and SLC7A11 (pcp52-ZER6, pcp71-ZER6, and pcSLC7A11, respectively), as well as vectors overexpressing p52-ZER6 with full-length KRAB domain (pcFLAGp52^K), p52-ZER6 with full-length KRAB and HUB-1 domains (pcFLAGp52^KH), and p52-ZER6 without tKRAB domain (pcFLAGp52^tKdel) were constructed as described previously^25,30. For DAZAP1 overexpression vector (pcDAZAP1), the coding region of human DAZAP1 was obtained by reverse-transcribing total RNA extracted from HCT116^WT colorectal cancer (CRC) cells using the PrimeScript Reagent Kit with gDNA Eraser (Takara Bio, Dalian, China) and amplifying the corresponding regions using Takara Prime STAR Max DNA Polymerase (Takara Bio, Dalian, China). The amplicon was inserted into the BamHI and MluI sites of pcEF9-Puro vector bringing puromycin resistance gene³¹.

For reporter vector bringing −127 to +1520 region of the SLC7A11 promoter (SLC7A11-luc), as well as the −1621 to +1239 (DAZAP1-luc1), the −1274 to +1239 (DAZAP1-luc2), the +508 to +1239 (DAZAP1-luc3), the +741 to +1239 (DAZAP1-luc4), and the +867 to +1239 (DAZAP1-luc5) regions of the DAZAP1 promoter, human genome DNA extracted from HCT116^WT cells using Genomic DNA Kit (Tiangen Biotech, Beijing, China) was used as a template for amplifying the corresponding regions using Takara PrimeSTAR Max DNA Polymerase (Takara Bio, Dalian, China). For SLC7A11-luc, the amplicon was then cloned into the NheI and ApaI sites of the pGL4.13 vector (Promega, Madison, WI, USA); while for DAZAP1-lucs, the amplicons were cloned into the NheI and StuI sites of the pGL4.13 vector. For reporter vectors bringing the SLC7A11 5′-UTR (SLC7A11-5′-UTR-luc), SLC7A11 CDS (SLC7A11-CDS-luc), and SLC7A11 3′-UTR (SLC7A11-3′-UTR-luc-1: 1 to 1517; SLC7A11-3′-UTR-luc-2: 1491 to 3353; SLC7A11-3′-UTR-luc-3: 3296 to 4788; SLC7A11-3′-UTR-luc-4: 4735 to 6332; SLC7A11-3′-UTR-luc-5: 6325 to 7789; SLC7A11-3′-UTR-luc-4A: 4728 to 5023; SLC7A11-3′-UTR-luc-4B: 5110 to 6332), the corresponding regions were amplified from human genome DNA as described above, and the amplicons were cloned into the XhoI and NotI sites of the psiCHECK-2 vector (Promega, Madison, WI, USA). DAZAP1 luciferase reporter vector with mutated ZER6 binding site (DAZAP1-Luc^mut) and SLC7A11 luciferase reporter vector with mutated DAZAP1 binding site (SLC7A11-Luc^mut) were constructed from DAZAP1-luc4 and SLC7A11-3′UTR-luc-4A, respectively, by mutating the corresponding binding sites using Site-directed Mutagenesis Kit (Beyotime Biotechnology, Shanghai, China).

2.2.　 Cell lines and cell culture

Wild-type HCT116, LoVo, and HT29 CRC cell lines were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). p53-null HCT116 (HCT116^p53null) cell line was kindly provided by Dr. Bert Vogelstein at John Hopkins University School of Medicine. Wild-type HCT116 and HT29 cells were cultured in Dulbecco's modified Eagle's medium (Gibco, Life Technologies, Grand Island, NY, USA); while LoVo and HCT116^p53null cells were cultured in F12K Ham's Kaighn's modified medium (Macgene, Beijing, China) and McCoy's 5A medium (Gibco), respectively. All culture media were supplemented with 10% FBS (Biological Industries, Beith Haemek, Israel) and 1% penicillin–streptomycin. All cell lines were verified using short-tandem repeat profiling method and were tested periodically for mycoplasma contamination by using Mycoplasma Detection Kit–Quick Test (Biotool, Houston, TX, USA) routinely every 6 months. Transfection was performed using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. For gene-silencing and gene-overexpression experiments, cells were seeded in a 6-well plate and transfected with 2 μg of the indicated vector. After 24 h, cells were selected using puromycin (final concentration: 1.0 μg/mL) for 36 h to eliminate untransfected cells.

For establishing cell lines stably expressing indicated shRNA and/or overexpression vectors, HCT116^WT or HCT116^p53null cells were transfected with corresponding shRNA and/or overexpression vectors. Twenty-four hours later, puromycin selection (final concentration: 1 μg/mL) was for 7 days to eliminate untransfected cells.

For establishing ferroptosis-resistant HCT116 cells (HCT116^EraR), cells were seeded in 10 cm-well plates and cultured with medium containing a low-dose Erastin (MedChem Express, Monmouth Junction, NJ, USA; final concentration: 5 μmol/L) for 26 days.

For cell death inhibitor experiments, cells were treated with Ferrostatin-1 (Ferr-1; MedChem Express, Monmouth Junction, NJ; final concentration: 5 μmol/L), 3-methyladenine (3-MA; MedChem Express, Monmouth Junction, NJ, USA; final concentration: 10 μmol/L), Necrosulfonamide (NSA; MedChem Express, Monmouth Junction, NJ, USA; final concentration: 5 μmol/L), Z-VAD-FMK (Z-VAD; APExBIO, Houston, TX, USA; final concentration: 20 μmol/L) and Erastin (MedChem Express, Monmouth Junction, NJ, USA), treating cells with indicated concentrations for indicated times before detection.

2.3.　 Clinical human CRC specimens

Human CRC specimens were obtained from CRC patients undergoing surgery at Chongqing University Cancer Hospital (Chongqing, China) and stored in the Biological Specimen Bank of Chongqing University Cancer Hospital. Patients did not receive chemotherapy, radiotherapy, or other adjuvant therapies prior to surgery. The specimens were snap-frozen in liquid nitrogen. Prior patient's written informed consents were obtained. The experiments were approved by the Institutional Research Ethics Committee of Chongqing University Cancer Hospital (Permit No. CZLS2021292-A), and conducted in accordance with Declaration of Helsinki.

2.4.　 Animal experiment

For the in vivo tumor study, BALB/c-nu/nu mice (male, 6-weeks old, body weight: 18–22 g) were purchased from Chongqing Medical University (Chongqing, China). Animal studies were conducted in the Chongqing University Cancer Hospital, and approved by the Laboratory Animal Welfare and Ethics Committee of Chongqing University Cancer Hospital. All animal experiments conformed to the approved Guidelines for the Care and Use of Laboratory Animals of the Chongqing University Cancer Hospital (Permit No. SYXK-2021-0001). All efforts were made to minimize suffering.

For the xenograft experiment, mice were randomly divided into three groups (n = 6) and each group was injected subcutaneously with 5 × 10⁶ indicated stable cell lines. Tumor size (V) was evaluated by a caliper every 2 days with reference to Eq. (1):

V = a × b 2 / 2

(1)

where a and b are the major and minor axes of the tumor, respectively. The investigator was blinded to the group allocation and during the assessment.

2.5.　 RNA sequencing (RNA-seq) and transcriptomic analysis

HCT116^WT cells were transfected with shZER6 or control vector (shCon) as described above. RNA extraction and RNA-seq were performed by Novogene Technology Corporation (Beijing, China) using an Illumina HiSeq 2500 instrument (Illumina, San Diego, CA, USA; three repetitive for each group). Raw reads were preprocessed by filtering out rRNA reads, sequencing adapters, short-fragment reads, and other low-quality reads. Tophat v2.1.0 was used to map the clean reads to the human reference genome ensemble GRCh38 (hg38) with two mismatches. After genome mapping, Cufflinks v2.1.1 was run with reference annotations to generate fragments per kilobase per million mapped reads values for known gene models. Differentially expressed genes (DEGs) were identified using Cuffdiff software. The P-value significance threshold for DEGs in multiple tests was set based on a false discovery rate ≤0.05. The fold-changes were estimated according to the fragments per kilobase per million mapped reads in each sample.

2.6.　 Transmission electron microscopy

Cells were prepared as described above and fixed with 2.5% glutaraldehyde, washed in 0.1 mol/L phosphate buffer (pH 7.4), and post-fixed with 1% osmium 0.1 mol/L phosphate buffer. Samples were dehydrated in series acetone, infiltrated, and embedded in Epox812 before being cut into ultra-thin sections. Sections were stained with uranyl acetate and lead citrate prior to being examined using JEM-1400-FLASH transmission electron microscope (JEOL, Tokyo, Japan).

2.7.　 RNA immunoprecipitation (RIP) assay

Cells were lysed with RIP lysis buffer with protease inhibitor and RNase inhibitor (Beyotime Biotechnology, Shanghai, China). Cell lysates were then incubated with anti-DAZAP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or IgG antibody for 4 h before being immunoprecipitated with protein A/G beads (MCE, Shanghai, China). RNA was extracted with trizol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instruction, and reverse transcribed into cDNA using PrimeScript Reagent Kit with gDNA Eraser (Takara Bio, Dalian, China). qRT-PCR was performed using SYBR Premix ExTaq (Takara Bio, Dalian, China). The sequences of the primers used for qRT-PCR are shown in Supporting Information Table S1.

2.8.　 Biotinylated RNA pull-down assay

DNA fragments containing T7 RNA polymerase promoter sequences and sequences correspond to different regions of SLC7A11 mRNA were amplified using Takara PrimeSTAR Max DNA Polymerase (Takara Bio, Dalian, China) from human genome DNA as described above. The amplicons were then used as templates for in vitro transcription using RiboTM RNAmax-T7 (Ribo Bio, Guangzhou, China) to obtain biotinylated RNAs. Cells were lysed with RIP lysis buffer containing protease inhibitor and RNase inhibitor (Beyotime Biotechnology, Shanghai, China), and the cell lysates were incubated with biotinylated RNAs for 4 h at 4 ℃. The RNA-protein complexes were then added to streptavidin magnetic beads (MCE, Shanghai, China) overnight at 4 ℃, and the precipitants were then analyzed by Western blotting with an anti-DAZAP1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

2.9.　 Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed using the ChIP assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. Briefly, cells were lysed and chromatins were immunoprecipitated using protein A + G agarose/salmon sperm DNA and anti-ZER6 antibody, anti-histone H3 antibody, or normal rabbit IgG. Chromatin was then de-crosslinked for 4 h at 65 ℃ before being treated with 0.5 mol/L EDTA, 1 mol/L Tris (pH 6.5), and 20 mg/mL proteinase K. Immunoprecipitated chromatin was then subjected to PCR by using Primer STAR Max (Takara Bio, Dalian, China). Primer sequences for amplifying SLC7A11 promoter regions that contain the predicted p52-ZER6 binding site were as follows: for the +1310 to +1520 region (primer set 1): 5′-GCA ATT CTC CTG CCT AAG CCT -3′ (forward primer), and 5′- ATG GAA TTT GAG GAC CGG GC-3′ (reverse primer); for the +1230 to +1502 region (primer set 2): 5′- GAC AGA GAC TCG CTC TTT GCC -3′ (forward primer), and 5′- GCG CGG TGG CTC ATA TCT GTA -3′ (reverse primer). Primer sequences for amplifying the +742 to +948 region of DAZAP1 promoter, which contains the predicted p52-ZER6 binding site were: 5′-CAA AGT GGT AAG GTC GGG AGG -3′ (forward primer); and 5′-GCA GGT CAG GCT CCT ATA GGA -3′ (reverse primer).

2.10.　 Dual luciferase reporter assay

Cells were seeded into 24-well plates (5 × 10⁴ cells/well). Twenty-four hours later, cells were co-transfected with indicated vectors, reporter vector, and Renilla luciferase expression vector (pRL-SV40, Promega, Madison, WI, USA) as internal control. Forty-eight hours later, luciferase activities were measured using the Dual Luciferase Assay System (Promega, Madison, WI, USA). Relative light units of firefly luciferase were normalized to the corresponding Renilla luciferase activities. The results are shown as relative to the activities in the corresponding controls, which were assumed as 1.

2.11.　 In situ hybridization assay

Tissue sections were rehydrated and digested with protease K for 25 min at 37 ℃. After pre-hybridization at 37 ℃ for 1 h, hybridization was carried out by overnight incubation at 37 ℃ with 8 ng/μL digoxin-conjugated probe specific for the p52-ZER6 isoform (91–113 at the 5′-UTR of p52-ZER6 mRNA; TCT CGT CTT CGA CCG CAT CCC TC). After being washed with SSC washing buffer at 37 ℃, blocking was performed with bovine serum albumin for 30 min. Specifically bound probes were detected by anti-DIG-HRP antibody (Servicebio, Wuhan, China). Nuclei were stained with hematoxylin. Images were taken using Pannoramic Midi (3DHistech, Budapest, Hungary).

2.12.　 Immunohistochemistry and propidium iodide (PI) staining

Fresh human CRC tissues, normal adjacent tissues, and xenograft tumors were fixed using 4% paraformaldehyde overnight prior to being embedded in paraffin and sectioned at 4 μm thickness using a cryostat. Tissue sections were then incubated with primary antibodies for 1 h followed by incubation with corresponding secondary antibodies conjugated with horse-radish peroxidase or were stained with PI staining (Beyotime Biotechnology, Shanghai, China; final concentration: 4.5 μmol/L) for 10 min. Visualization was performed using a DAB Kit (DAKO, Beijing, China) under the microscope. The nuclei were then counterstained with hematoxylin, followed by dehydration and coverslip mounting. The antibodies used were listed in Supporting Information Table S2. Images were taken using Pannoramic Midi (3DHistech).

2.13.　 RNA extraction and quantitative reverse transcription-PCR (qRT-PCR)

Cells were prepared as described above. Total RNA was extracted with Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA samples were reverse transcribed into cDNA using PrimeScript Reagent Kit with gDNA Eraser (Takara Bio, Dalian, China). qRT-PCR was performed using SYBR Premix ExTaq (Takara Bio, Dalian, China). The sequences of the primers used for qRT-PCR were shown in Table S1. β-Actin was used to normalize sample amplification. The results are shown as relative to the expression level in the corresponding controls, which were assumed as 1.

For xenograft tissues and clinical CRC samples, tissues were frozen in liquid nitrogen and ground before being extracted with Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA extraction and qRT-PCR were performed as described above.

2.14.　 Western blotting

Cells were prepared as described above. Protein was extracted using RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science, Mannheim, Germany). Protein samples were separated by SDS–PAGE and transferred to PVDF membranes with 0.45 μm pores (Millipore, Billerica, MA, USA). Membranes were then incubated with primary antibodies followed by incubation with corresponding secondary antibodies. Antibodies used were listed in Table S2. Immunoblotting with anti-β-actin antibody was conducted to ensure equal protein loading. Signals were detected by using the SuperSignal West Femto Maximum Sensitivity Substrate detection system (Thermo Scientific, Waltham, MA, USA). Protein quantification was performed using Quantity One software.

For xenograft tissues and clinical CRC samples, tissues were frozen in liquid nitrogen and ground before being lysed with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science). Western blotting was performed as described above.

2.15.　 mRNA degradation analysis and protein degradation analysis

1×10⁶ cells were seeded in 6-well plates and treated with actinomycin D (final concentration: 5 μmol/L) or cycloheximide (CHX; final concentration: 30 μg/mL). At the indicated time points, samples were collected and then analyzed via qRT-PCR or Western blotting.

2.16.　 Lipid peroxidation analysis

Cells were prepared as described above before being incubated with 5 μmol/L BODIPY 581/591 C11 (Invitrogen Life Technologies, Carlsbad, CA, USA) in the dark for 20 min at 37 ℃. Cells were then harvested by trypsinization, washed twice with PBS, and re-suspended in 200 μL PBS. Fluorescence was analyzed by a flow cytometer equipped with a 488 nm laser for excitation (Beckman Coulter, Inc., CA, USA), and data were collected by the 530 nm band-pass filter.

2.17.　 GSH and cysteine levels analysis

Cells were prepared as described above, harvested by trypsinization, and collected by centrifugation. The pellets were then dissolved in protein stripping buffer. For xenograft tissues, tissues were homogenized with protein stripping buffer. Intracellular GSH and cysteine levels were then measured using the total GSH assay kit (Beyotime Biotechnology, Shanghai, China) and cysteine assay kit (Solarbio, Beijing, China), respectively. Values were normalized with total protein amount determined using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China).

2.18.　 Malondialdehyde (MDA) detection

Cells were prepared as described above, harvested by trypsinization, and collected by centrifugation. The pellets were then dissolved in protein stripping buffer. For xenograft tissues, tissues were homogenized with protein stripping buffer. MDA levels were measured by thiobarbituric acid method using the MDA assay kit (Beyotime Biotechnology, Shanghai, China). The values were normalized with total protein amount determined using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China).

2.19.　 Cell death assay

Cells were prepared as described above and re-seeded in 6-well plate at a density of 3 × 10⁵ cells per well. Twenty-four hours later, death cells were stained with PI (Beyotime Biotechnology, Shanghai, China; final concentration: 4.5 μmol/L) and analyzed using flow cytometry (Beckman Coulter, Inc.).

2.20.　 Cell viability

Cells were prepared as described above before being re-seeded into 96-well plates at a density of 5000 cells/well. Living cells were then counted at indicated time points using colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega, Madison, WI, USA) in accordance with the manufacturer's instructions.

2.21.　 Colony formation assay

Cells were prepared as described above before being re-seeded into 6-well plates at a density of 400 cells/well and cultured for 14 days. Cells were then fixed using 4% paraformaldehyde and stained with crystal violet staining solution (Beyotime Biotechnology, Shanghai, China). Quantification was performed by counting the number of colonies formed. Investigator was blinded during the assessment.

2.22.　 Statistical analysis

Quantification results were presented as the mean ± standard deviation (SD) (n = 3; unless otherwise indicated). Quantification data were analyzed by one-way ANOVA conducted using SPSS Statistics v17.0. (IBM, Chicago, IL, USA). A value of P < 0.05 were considered statistically significant.

3.　Results

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3.1.　 ZER6 inhibits ferroptosis

To investigate the role of ZER6 in CRC tumorigenesis, we first obtained the clinical data of 814 patients with CRC from 6 GEO datasets (GSE17538, GSE12945, GSE29621, GSE38832, GSE39582, and GSE41258). Subsequently, we conducted prognosis analyses of these patients using the Kaplan–Meier plotter database (http://kmplot.com/analysis/). The results showed that ZER6 expression negatively correlated with overall survival (Fig. 1A). Furthermore, the mRNA and protein expression levels of ZER6 were significantly higher in clinical CRC tissues than in corresponding normal adjacent tissues (Fig. 1B and C). We next investigated the role of ZER6 in CRC cell viability and colony formation potential using two shRNA expression vectors targeting different sites in ZER6 (Supporting Information Fig. S1A). Knockdown of ZER6 significantly suppressed its mRNA (Fig. S1B–S1D) and protein (Fig. S1E–S1G) expression levels in HCT116^WT, HT29, and LoVo cells. It also decreased their viability (Fig. S1H–S1J) and colony formation potential (Fig. S1K–S1M), highlighting the oncogenic role of ZER6.

To systematically examine the mechanism underlying the oncogenic function of ZER6, we performed RNA-seq-based transcriptome analysis to identify DEGs in ZER6-knocked down HCT116^WT cells. A total of 2069 DEGs were identified in ZER6-knocked down HCT116^WT cells, including 1151 upregulated and 918 downregulated genes (Fig. 1D). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed DEG enrichment in pathways related to ferroptosis in ZER6-knocked down HCT116^WT cells (Fig. 1E). We next validated the RNA-seq results by examining the effects of knocking down ZER6 on various types of cell death. ZER6 knockdown clearly increased the percentage of PI-positive cells (Fig. 1F), suggesting that ZER6 suppresses cell death. Treatment with the pan-caspase inhibitor Z-VAD slightly suppressed the cell death rate, whereas treatment with the ferroptosis inhibitor Ferr-1 robustly suppressed it (Fig. 1G). Further, Z-VAD slightly recovered the viability of ZER6-knocked down HCT116^WT cells, whereas Ferr-1 exerted a more significant effect (Fig. 1H). Meanwhile, treatments with the necroptosis inhibitor NSA and autophagy inhibitor 3-MA did not significantly affect the cell death and viability of ZER6-knocked down HCT116^WT cells. Moreover, ZER6 knockdown increased lipid ROS and MDA levels, which are characteristics of ferroptosis (Fig. 1I and J), whereas Ferr-1 treatment cancelled these effects (Fig. 1K and L). Taken together, these results suggested that ZER6 suppresses ferroptosis and positively contributes to tumorigenic potential.

To investigate which isoform regulates ferroptosis (Supporting Information Fig. S2A), we constructed shRNA expression vectors to specifically knock down p52-ZER6 or p71-ZER6 (Fig. S2B). shp52-ZER6 significantly suppressed the expression of p52-ZER6 in CRC cells at its mRNA (Fig. S2C–S2E) and protein (Fig. S2F–S2H) levels, but did not exert a significant effect on those of p71-ZER6, and vice versa (Fig. S2I–S2N). Similarly, overexpression of p52-ZER6 or p71-ZER6 did not affect the expression level of the other isoform (Fig. S2O). Together, these results indicate the efficacy and specificity of the vectors used. We next examined the effect of p52-ZER6 on ferroptosis characteristics. p52-ZER6 knockdown significantly enhanced lipid ROS and MDA levels in HCT116^WT cells (Fig. 1M and N), leading to an increase in the cell death rate (Fig. 1O). Similar results were obtained in HT29 and LoVo cells (Supporting Information Fig. S3A–S3D). Moreover, p52-ZER6 knockdown induced mitochondrial shrinkage, a key event in ferroptosis (Fig. 1P). Intriguingly, p71-ZER6 knockdown did not have a significant effect on lipid ROS and MDA levels in HCT116^WT cells (Fig. S3E and S3F), suggesting that the regulatory function of ZER6 in tumor cell ferroptosis is specific to its p52-ZER6 isoform.

To confirm this, we treated p52-ZER6-knocked down HCT116^WT cells with Ferr-1, which clearly suppressed lipid ROS levels and cell death induced by p52-ZER6 knockdown, thereby restoring the viability of p52-ZER6-knocked down HCT116^WT cells (Fig. S3G–S3I). In contrast, the addition of erastin, a ferroptosis inducer, significantly canceled the effect of p52-ZER6 overexpression in suppressing lipid ROS and cell death levels, thereby decreasing the viability of HCT116^WT cells (Fig. S3J–S3L). Given that HCT116^WT, HT29, and LoVo cells are naturally susceptible to ferroptosis (Supporting Information Fig. S4A), we established a HCT116^EraR by gradually acclimating the cells to a low-dose erastin for 26 days (Fig. S4B). Compared with HCT116^WT cells, HCT116^EraR cells demonstrated a less significant increase in lipid ROS and MDA levels as well as cell death rate upon erastin treatment (Fig. S4C–S4E); while the viability of these cells demonstrated the opposite trend (Fig. S4F). Meanwhile, knocking down p52-ZER6 significantly restored the effect of erastin in inducing lipid ROS and MDA levels, and subsequently, in enhancing the cell death rate and reducing the viability of the HCT116^EraR cells. These results further confirmed that p52-ZER6 is crucial for the regulation of CRC cell sensitivity to ferroptosis, and that p52-ZER6 enhances CRC cell viability by suppressing ferroptosis.

3.2.　 p52-ZER6 suppresses tumor cell ferroptosis by upregulating SLC7A11

To reveal the molecular mechanism through which p52-ZER6 suppresses ferroptosis, we analyzed the fold-changes of ferroptosis-related DEGs in ZER6-knocked down HCT116^WT cells using the RNA-seq data (Fig. 2A), and validated the results using qRT-PCR. ZER6-knockdown most significantly affected the mRNA expression level of SLC7A11, encoding a specific light-chain subunit of the cystine/glutamate antiporter (Fig. 2B). This regulatory effect was confirmed in CRC cells (Supporting Information Fig. S5) using another shRNA expression vector targeting a different site in ZER6. Consistent with the above finding that p52-ZER6, but not p71-ZER6, can inhibit ferroptosis, only knockdown of p52-ZER6 significantly suppressed SLC7A11 expression (Fig. 2C–E). Similarly, overexpression of p52-ZER6, but not p71-ZER6, increased SLC7A11 mRNA and protein expression levels in HCT116^WT cells (Fig. 2F–H). The positive correlation between p52-ZER6 and SLC7A11 expression was confirmed at the mRNA level in clinical CRC tissues using qRT-PCR (Fig. 2I), and at the protein level by Western blotting (Fig. 2J). This tendency was further confirmed by immunohistochemistry and in situ hybridization using serial sections (Fig. 2K). These results showed that both p52-ZER6 and SLC7A11 were highly expressed in CRC tissues compared to normal adjacent tissues (Fig. 2I–K).

SLC7A11 is the core subunit of system X_c⁻, which serves as a cystine-glutamate antiporter that mediates the exchange of intracellular glutamate and extracellular cystine. The imported cystine is rapidly reduced to cysteine, which is crucial for de novo GSH synthesis^32,33. Therefore, we next examined whether p52-ZER6 regulates the levels of intracellular cysteine and GSH. Cysteine and GSH levels remarkably decreased in p52-ZER6-knockdown HCT116^WT cells (Fig. 2L and M), whereas they significantly increased in p52-ZER6-overexpressing HCT116^WT cells (Fig. 2N and O). Together, these results clearly indicate that p52-ZER6 induces SLC7A11 expression, thereby increasing cystine uptake and de novo GSH synthesis.

To confirm the role of SLC7A11 in p52-ZER6-mediated tumor cell ferroptosis resistance, we constructed an SLC7A11 overexpression vector (Supporting Information Fig. S6A) and shRNA expression vectors targeting SLC7A11 (Fig. S6B and S6C). SLC7A11 overexpression significantly restored the levels of cysteine and GSH suppressed in p52-ZER6-knocked down HCT116^WT cells, suggesting that p52-ZER6 regulation of SLC7A11 expression is crucial for its role in promoting cystine uptake and GSH synthesis (Fig. 3A and B; Fig. S6D). Concomitantly, SLC7A11 overexpression cancelled the increases in lipid ROS and MDA levels (Fig. 3C and D), and restored mitochondrial morphology in p52-ZER6-knocked down HCT116^WT cells (Fig. 3E), thereby cancelling the increase in cell death and restoring the viability of p52-ZER6-knocked down HCT116^WT cells (Fig. 3F and G). In line herewith, knockdown of SLC7A11 canceled the effect of p52-ZER6 overexpression in promoting cystine uptake and GSH synthesis (Fig. S6E–S6G), thereby increasing lipid ROS and MDA levels suppressed by p52-ZER6 overexpression (Fig. S6H and S6I). This subsequently cancelled the effects of p52-ZER6 overexpression in decreasing the cell death rate and increasing cell viability (Fig. S6J and S6K). Together, these findings demonstrate that SLC7A11 is crucial for p52-ZER6-mediated ferroptosis resistance in tumor cells.

Next, to confirm the role of p52-ZER6/SLC7A11 axis in tumorigenesis, we performed xenograft experiments using a p52-ZER6-knocked down, SLC7A11 overexpressing HCT116^WT stable cell line (Fig. S6L). It is noteworthy that the mice body weight did not show significant differences among the groups used for xenograft experiments (Fig. S6M). As shown in Fig. 3H, knocking down p52-ZER6 robustly suppressed the tumorigenic potential of HCT116^WT cells, whereas SLC7A11 overexpression restored it. Western blotting, in situ hybridization, and immunohistochemistry staining results confirmed the downregulation of SLC7A11 expression in the xenograft tumors formed by p52-ZER6-knocked down HCT116^WT cells, as well as the restoration of its expression in those formed by p52-ZER6-knocked down, SLC7A11 overexpressing HCT116^WT cells (Fig. 3I and J). Concomitantly, SLC7A11 overexpression abrogated the effects of p52-ZER6 knockdown in decreasing intracellular cysteine and GSH levels (Fig. 3K and L), as well as in increasing MDA levels (Fig. 3M) and 4-hydroxynonenal (4-HNE) (Fig. 3N) in the xenograft tumor lesions. Subsequently, our results showed that SLC7A11 overexpression restored the tumorigenic potential of p52-ZER6-knocked down HCT116^WT cells by canceling the increase of their cell death rate, as indicated by the decrease in PI-positive cells (Fig. 3O) and restoring their proliferation potential, as indicated by the increase of Ki67-positive cells (Fig. 3N). Thus, these findings demonstrate that the p52-ZER6/SLC7A11 axis was critical for CRC cells tumorigenic potential by promoting ferroposis resistance.

3.3.　 p52-ZER6 enhances SLC7A11 mRNA stability and tumor cell ferroptosis resistance by promoting DAZAP1 expression

p52-ZER6 is a transcription factor with “GGTGGG” as the core binding sequence^23,26 (Supporting Information Fig. S7A). Prediction using the Eukaryotic Promoter Database (https://epd.expasy.org/epd/) revealed a potential ZER6-binding site in the +1453 to +1458 region of the SLC7A11 promoter (Fig. S7B). However, reporter assay results using a luciferase reporter vector containing the −127 to +1520 region of the SLC7A11 promoter (SLC7A11-luc) revealed that neither knockdown nor overexpression of p52-ZER6 exerted a significant effect on SLC7A11-luc activity (Fig. S7C and S7D). Similar results were obtained in HT29 and LoVo cells (Fig. S7E–S7H), indicating that p52-ZER6 may not regulate SLC7A11 transcriptional activity. To further confirm this, we performed ChIP assay with two primer sets flanking the predicted binding site to assess whether p52-ZER6 could bind to the SLC7A11 promoter. Specifically, these primer sets could amplify the +1310 to +1520 and the +1230 to +1502 regions of the SLC7A11, producing 210 bp and 272 bp amplicons, respectively. Both primer sets failed to amplify DNA fragment immunoprecipitated using anti-p52-ZER6 antibody, indicating that p52-ZER6 could not bind with SLC7A11 promoter (Fig. S7I). Intriguingly, in HCT116^WT cells treated with CHX which inhibits de novo protein synthesis, knocking down of p52-ZER6 also failed to affect the degradation rate and half-life of SLC7A11 protein, indicating that p52-ZER6 does not regulate its protein stability either (Fig. S7J). These results indicate that p52-ZER6 does not promote SLC7A11 expression via transcriptional or post-translational regulation.

Next, we analyzed Gene Ontology term enrichment of the DEGs in ZER6-knocked down HCT116^WT cells based on the RNA-seq data. The results showed DEG enrichment in “nuclear transcribed mRNA catabolic process”, “mRNA catabolic process”, “translation factor activity, RNA binding”, “mRNA 3′-UTR binding”, and “mRNA binding”, suggesting that p52-ZER6 may potentially regulate mRNA-binding proteins (Fig. 4A). As RBPs can regulate gene expression through post-transcriptional mechanisms, including mRNA stability^34,35, we next analyzed the effect of p52-ZER6 knockdown on SLC7A11 mRNA stability by treating p52-ZER6-knocked down HCT116^WT, HT29, and LoVo cells with the transcription inhibitor actinomycin D. The half-life of SLC7A11 mRNA was significantly reduced in p52-ZER6-knocked down HCT116^WT, HT29, and LoVo cells, suggesting that p52-ZER6 knockdown accelerated its degradation (Fig. 4B; Supporting Information Fig. S8A and S8B). Meanwhile, p52-ZER6 overexpression yielded opposite results (Fig. 4C; Fig. S8C and S8D). These results demonstrate that p52-ZER6 promotes SLC7A11 expression most plausibly by stabilizing its mRNA.

To explore which RBPs are involved in regulating SLC7A11 mRNA stability, we first predicted RBPs that can bind to SLC7A11 mRNA using RBPmap (http://rbpmap.technion.ac.il/). The results were then compared with the mRNA binding-related DEGs obtained using RNA-seq and ferroptosis-related genes obtained from FerrDbV2 (http://www.zhounan.org/ferrdb/). We found two potential ZER6-regulated RBP candidates: DAZAP1 and ZFP36 (Fig. 4D). Validation using qRT-PCR indicated that the mRNA level of DAZAP1, but not that of ZFP36, was significantly reduced in ZER6-knocked down HCT116^WT cells (Fig. 4E). Further analysis revealed that p52-ZER6 positively regulated DAZAP1 mRNA and protein expression levels (Fig. 4F–I). Furthermore, in situ hybridization, immunohistochemistry, and Western blotting results showed that both p52-ZER6 and DAZAP1 were upregulated in CRC tissues when compared with those in normal adjacent tissues (Fig. 4J and K). Moreover, correlation analysis using CRC clinical samples showed a positive correlation between p52-ZER6 and DAZAP1 mRNA expression in CRC (Fig. 4L). Together, these results suggested the possible regulation of DAZAP1 by p52-ZER6.

To explore whether DAZAP1 is involved in p52-ZER6 regulation of SLC7A11 mRNA stability, we performed rescue experiments by overexpressing DAZAP1 in p52-ZER6-knocked down HCT116^WT cells (Supporting Information Fig. S9A). DAZAP1 overexpression significantly reduced the SLC7A11 degradation rate in p52-ZER6-knocked down HCT116^WT cells (Fig. 4M), resulting in the restoration of SLC7A11 mRNA and protein expression levels (Fig. 4N and O). Concomitantly, DAZAP1 overexpression canceled the effects of p52-ZER6 knockdown in suppressing cellular cysteine and GSH levels (Fig. S9B and S9C), as well as in increasing lipid ROS and MDA levels (Fig. S9D and S9E), leading to the suppression of cell death and restoration of cell survival in p52-ZER6-knocked down HCT116^WT cells (Fig. S9F and S9G). Together, these results demonstrate that p52-ZER6 can stabilize SLC7A11 by promoting DAZAP1 expression, thereby promoting p52-ZER6/SLC7A11 axis-mediated tumor cell ferroptosis resistance.

3.4.　 DAZAP1 enhances SLC7A11 mRNA stability by direct binding to its 3′-UTR

RBPs regulate the stability of their target mRNAs by binding to specific sequences in the target mRNAs via their RNA-binding domain^36,37. A RIP assay using anti-DAZAP1 antibody showed that SLC7A11 mRNA could be enriched by co-precipitation with DAZAP1, suggesting that DAZAP1 can bind to SLC7A11 mRNA (Fig. 5A). Prediction using RBPmap (http://rbpmap.technion.ac.il/) revealed 16 potential DAZAP1-binding sites in the 3′-UTR of SLC7A11 mRNA (Supporting Information Table S3). To determine the specific site in SLC7A11 mRNA crucial for p52-ZER6/DAZAP1 regulation, we divided the full-length SLC7A11 mRNA into the following fragments: SLC7A11-5′-UTR, SLC7A11-CDS, and SLC7A11-3′-UTR-1 to SLC7A11-3′-UTR-5, comprising the 1 to 1517; 1491 to 3353; 3296 to 4788; 4735 to 6332; and 6325 to 7789 regions of SLC7A11 3′-UTR, respectively (Fig. 5B), and constructed corresponding luciferase reporter vectors. Luciferase assays revealed that while knockdown of p52-ZER6 suppressed the activity of SLC7A11-3′-UTR-4, it did not exert a significant effect on SLC7A11-5′-UTR, SLC7A11-CDS, SLC7A11-3′-UTR-1, SLC7A11-3′-UTR-2, SLC7A11-3′-UTR-3, and SLC7A11-3′-UTR-5 activity (Fig. 5C). Similarly, DAZAP1 overexpression restored the activity of SLC7A11-3′-UTR-4, while did not exert any significant effect on that of the other vectors, in p52-ZER6-knocked down HCT116^WT cells (Fig. 5D). Furthermore, RNA pull-down experiments corroborated that DAZAP1 could only bind to the 4735 to 6332 region of the 3′-UTR of SLC7A11 mRNA (Fig. 5E).

Next, we further divided the 4735–6332 region into two shorter regions, 4A (4728–5023) and 4B (5110–6332), and performed luciferase assays with corresponding reporter vectors. Although the p52-ZER6-knockdown did not affect the activity of SLC7A11-3′-UTR-4B, it significantly suppressed that of SLC7A11-3′-UTR-4A (Fig. 5F). RNA pull-down experiments revealed that DAZAP1 could only bind to SLC7A11-3′-UTR-4A, but not SLC7A11-3′-UTR-4B (Fig. 5G). Subsequently, we mutated the UAG sequences to CGA in the two predicted binding sites located within SLC7A11-3′-UTR-4A, specifically, at 4828 to 4830, and 4847 to 4849 regions (Fig. 5H). Luciferase reporter assay results showed that the knockdown of p52-ZER6 did not affect the activity of the Mut-2 vector, harboring the mutation in the 4847 to 4849 region, or that of the Mut-3 vector, encoding the mutation in both the 4828 to 4830, and 4847 to 4849 regions (Fig. 5I). Accordingly, RNA pull-down assays revealed that DAZAP1 could not bind to the SLC7A11-3′-UTR fragments with the sequence mutation in the 4847 to 4849 region (Fig. 5J). Together, these results indicate that p52-ZER6 promotes SLC7A11 mRNA stability most probably via DAZAP1, which binds to the 4847 to 4849 region of the 3′-UTR of SLC7A11 mRNA.

3.5.　 p52-ZER6 binds directly to the DAZAP1 promoter and promotes its transcription

We next explored the molecular mechanism underlying p52-ZER6 regulation of DAZAP1. As shown above, p52-ZER6 regulated the mRNA and protein expression levels of DAZAP1, suggesting a possible regulatory effect of p52-ZER6 on DAZAP1 transcription. Using the previously reported ZER6-binding motif²³ and the Eukaryotic Promoter Database (https://epd.expasy.org/epd/), we predicted four potential ZER6-binding sites in the DAZAP1 promoter, and constructed DAZ-luc-1 to DAZ-luc-5 luciferase reporter vectors carrying the −1621 to +1239, −1274 to +1239, +508 to +1239, +741 to +1239, and +867 to +1239 regions of the DAZAP1 promoter, respectively (Fig. 6A). Luciferase reporter assay results showed that p52-ZER6-knockdown significantly suppressed the activities of DAZ-luc-1 to DAZ-luc-4, but not that of DAZ-luc-5, suggesting that the +741 to +866 region of the DAZAP1 promoter is crucial for p52-ZER6-mediated transcriptional regulation (Fig. 6B).

To determine whether p52-ZER6 can bind directly to the DAZAP1 promoter at the predicted site, we performed a ChIP assay, confirming the binding of p52-ZER6 to the +742 to +948 region of the DAZAP1 promoter containing the predicted p52-ZER6-binding site (Fig. 6C). We then constructed a mutant DAZAP1 promoter luciferase reporter vector, wherein the GGTGGG sequence in the predicted p52-ZER6 core binding site was mutated into TTCTTT (DAZ-luc^mut). Knockdown of p52-ZER6 robustly suppressed the activity of DAZ-luc-4, but not that of DAZ-luc^mut (Fig. 6D). Taken together, these results revealed that p52-ZER6 could enhance the transcriptional activity of the DAZAP1 promoter by directly binding to its +844 to +849 region.

Intriguingly, p71-ZER6 failed to impact the transcriptional activity (Fig. 6E) and expression levels of DAZAP1 (Supporting Information Fig. S10). Given that p71-ZER6 differs from p52-ZER6 in its N terminus, we overexpressed FLAG-p52, FLAG-p71, and intermediate fragments between p52-ZER6 and p71-ZER6, including FLAG-conjugated p52-ZER6 with an additional 29 amino acids to form a complete KRAB domain (FLAG-p52^K) and FLAG-conjugated p52-ZER6 with an additional 138 amino acids to form complete KRAB and HUB-1 domains (FLAG-p52^KH) in their N termini (Fig. 6F), and performed ChIP assay using anti-FLAG antibodies. The results showed that only FLAG-p52 and FLAG-p52^K could bind directly to the DAZAP1 promoter (Fig. 6G). Similarly, while FLAG-p52 and FLAG-p52^K overexpression significantly induced DAZAP1-luc-1 reporter activity and DAZAP1 expression, FLAG-p52^KH and FLAG-p71 overexpression did not (Fig. 6H–J). Together with the above results showing that altering p71-ZER6 expression failed to affect SLC7A11 expression and ferroptotic characteristics, these results indicate that the presence of the HUB-1 domain in p71-ZER6 abrogated its function in promoting DAZAP1 transcriptional activity and SLCA711 expression, and subsequently, ferroptosis resistance.

3.6.　 p52-ZER6 promotes SLC7A11-mediated ferroptosis resistance in a p53-independent manner

Previous studies have shown that tumor suppressor p53 can induce ferroptosis via SLC7A11³⁸, and that p52-ZER6 can suppress p53 protein accumulation by promoting its ubiquitination/proteasomal degradation²⁵. Hence, we next explored whether p52-ZER6 enhances DAZAP1 and SLC7A11 expression levels by regulating p53. To this extent, we used HCT116^p53null cells, which showed a growth advantage compared with HCT116^WT cells (Supporting Information Fig. S11A and S11B). Similar to those in HCT116^WT cells, knocking down p52-ZER6 in HCT116^p53null cells significantly suppressed the mRNA and protein expression levels of DAZAP1 and SLC7A11 (Fig. 7A and B), whereas p52-ZER6 overexpression significantly increased them (Fig. S11C and S11D). Suppressing p52-ZER6 expression did not affect SLC7A11-luc activity, indicating that it did not affect the transcriptional activity of SLC7A11 (Fig. 7C); however, it significantly shortened the half-life of SLC7A11 mRNA (Fig. 7D), whereas p52-ZER6 overexpression clearly prolonged it (Fig. 7E). Together, these results clearly showed that p52-ZER6 could promote SLC7A11 mRNA stability in a p53-independent manner.

We next examined the effect of altering p52-ZER6 expression on ferroptosis in HCT116^p53null cells. Knockdown of p52-ZER6 in HCT116^p53null cells significantly reduced cellular cysteine and GSH levels (Fig. 7F and G), leading to increases in lipid ROS and MDA levels (Fig. 7H and I) and the cell death rate (Fig. 7J). In contrast, p52-ZER6 overexpression increased the levels of cellular cysteine and GSH in HCT116^p53null cells (Fig. S11E and S11F). Hence, p52-ZER6 increases cysteine uptake and GSH production, and eventually suppresses ferroptosis, in a p53-independent manner.

Next, we rescued SLC7A11 expression in p52-ZER6-knocked down HCT116^p53null cells (Fig. 7K), and analyzed its effect on ferroptosis. SLC7A11 overexpression clearly restored the cysteine and GSH levels suppressed by p52-ZER6 knockdown (Fig. 7L and M), thereby cancelling the increases in lipid ROS, MDA, and the cell death rate in p52-ZER6-knocked down HCT116^p53null cells (Fig. 7N–P). These tendencies were confirmed in SLC7A11-knocked down, p52-ZER6-overexpressing HCT116^p53null cells (Fig. S11G), as SLC7A11 knockdown abrogated the increases in cysteine uptake and GSH levels mediated by p52-ZER6 overexpression (Fig. S11H and S11I), leading to the restoration of the MDA level suppressed by p52-ZER6 in HCT116^p53null cells (Fig. S11J). These results suggested that p52-ZER6/SLC7A11 axis can promote tumor cell ferroptosis resistance in the absence of p53.

3.7.　 p52-ZER6/SC7A11 axis promotes tumorigenic potential in a p53-independent manner

Finally, we examined whether p52-ZER6/SLC7A11 axis could regulate tumorigenic potential in a p53-independent manner using xenograft experiments. To this end, we established a p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null stable cell line (Fig. 8A; Supporting Information Fig. S12A), and performed xenograft experiments. While did not significantly affect mice body weight (Fig. S12B), p52-ZER6 knockdown markedly suppressed the growth of tumors formed by HCT116^p53null cells, as shown by tumor morphology on day 22 and tumor volume (Fig. 8B). Meanwhile, SLC7A11 overexpression abrogated this downregulation effect and restored the tumorigenic potential. Western blotting results showed that SLC7A11 protein expression was suppressed in tumor tissues formed by p52-ZER6-knocked down HCT116^p53null cells, and, in accordance with their tumorigenic potential, was restored in tumor tissues formed by p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null cells (Fig. 8C). These tendencies were confirmed by in situ hybridization and immunohistochemical staining for p52-ZER6 and SLC7A11 using serial sections (Fig. 8D). Moreover, SLC7A11 overexpression abrogated the effects of knocking down p52-ZER6 in decreasing the levels of intracellular cysteine and GSH (Fig. 8E and F), as well as in increasing those of MDA and 4-HNE (Fig. 8G and H) in the xenograft tumor lesions. Subsequently, as indicated by the decrease of PI-positive cells (Fig. 8I) and the increase of Ki67-positive cells (Fig. 8H), our results clearly showed that SLC7A11 overexpression canceled the increase of cell death rate while restoring the proliferation potential of p52-ZER6-knocked down HCT116^p53null cells suppressed by p52-ZER6 knockdown. These results indicate that the p52-ZER6/SLC7A11 signaling axis also plays a crucial role in tumorigenesis by promoting ferroptosis resistance in CRC cells in a p53-independent manner.

In summary, we uncovered an unprecedented link between p52-ZER6 and ferroptosis based on the positive regulation exerted by the p52-ZER6/DAZAP1 axis on SLC7A11 mRNA stability, which in turn promotes ferroptosis resistance in tumor cells (Fig. 8J).

4.　Discussion

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Ferroptosis is a unique form of programmed cell death due to lipid peroxide accumulation³⁹. Ferroptosis is triggered by aberrant expression of iron accretion-related proteins, such as ferritin light chain 1, ferritin heavy chain 1, and solute carrier family 40 member 1, leading to iron accumulation and subsequently, lipid peroxidation. Defects in lipid peroxide reduction due to aberrant GSH synthesis and GPX4 lead to excessive lipid peroxide accumulation, which finally induces cell death⁴⁰. Ferroptosis resistance, which has been found in various cancers, including hepatocellular carcinoma (HCC), ovarian cancer, CRC, and lung cancer, plays a critical role in promoting tumorigenesis and tumor progression^41-44. Furthermore, tumor cells exhibit an increased dependence on iron, which promotes the production of ROS, markedly increasing their susceptibility and vulnerability to ferroptosis^45,46. Hence, targeting tumor cell ferroptosis resistance has attracted attention as an antitumor therapeutic strategy. Indeed, studies have shown that ferroptosis inducers, such as sorafenib and erastin, which target SLC7A11, can improve the efficacy of radiotherapy and chemotherapy, and are thus potential adjuvant treatments for cancer patients^47-49. In this study, we identified p52-ZER6 as a novel regulator of tumor cell ferroptosis resistance that maintains tumor cell redox homeostasis by promoting the transcription of the RBP DAZAP1 and stabilizing SLC7A11 mRNA, providing new insights into the regulatory mechanism of tumor cell redox homeostasis. Although the possible involvement of other cell death forms requires further investigation, our results indicate that p52-ZER6/DAZAP1/SLC7A11 axis-induced ferroptosis resistance plays crucial role in regulating CRC tumorigenesis.

SLC7A11, a specific light-chain subunit of the cystine/glutamate antiporter, contributes to redox homeostasis maintenance as it mediates the cellular uptake of cystine, which, after being reduced to cysteine, forms the cellular reductant GSH¹⁵. SLC7A11 overexpression has been observed in various cancers, including bladder⁵⁰, ovarian⁴³, and lung³² cancers. Previous studies have reported several regulatory mechanisms of SLC7A11 transcription in tumor cells. SLC7A11 transcription can be upregulated in tumor cells by imbalanced expression of its transcriptional suppressor, Yin Yang 2, which is downregulated in tumor cells, and its transcriptional activator Yin Yang 1, which is upregulated in tumor cells, or by the inactivation or downregulation of its transcriptional suppressor, p53^30,38. However, in our study, p52-ZER6 failed to bind to the SLC7A11 promoter and thus could not regulate its transcriptional activity. Our study revealed the post-transcriptional regulation of SLC7A11. We identified the p52-ZER6/DAZAP1 axis as a novel post-transcriptional regulatory pathway of SLC7A11 that promotes its mRNA stability and expression level, eventually enhancing tumor cell ferroptosis resistance. Hence, this study provided new insights into the regulatory mechanism of SLC7A11, and on GSH synthesis and ferroptosis resistance in tumor cells.

p52-ZER6 upregulation has been observed in various tumors including CRC, breast cancer, and HCC. Further, p52-ZER6 can promote tumorigenic potential by suppressing p53 protein accumulation. However, our knowledge of its biological and pathological functions was limited. The current study revealed that p52-ZER6 upregulation is crucial for tumor cell redox homeostasis and ferroptosis resistance. These findings confirm its role in promoting tumorigenesis and suggest its potential as a target for antitumor therapies. Furthermore, although knocking down p52-ZER6 exerts more significant effect in suppressing SLC7A11 expression and inducing ferroptosis in CRC cells with wild-type p53, we revealed that p52-ZER6 regulation of SLC7A11 and ferroptosis occurs irrespective of p53 status. In addition to the existing knowledge that p53 can suppress SLC7A11 expression by blocking its transcription³⁸ and p52-ZER6 can enhance p53 protein ubiquitination/proteasomal degradation²⁵, these findings show the presence of both p53-dependent and -independent pathways in p52-ZER6 regulation of SLC7A11, suggesting the possibility of targeting the p52-ZER6/SLC7A11 axis in cancer patients with p53 deletion and/or loss-of-function mutations, which account for more than 50% of cancer patients.

RBPs can interact with different classes of RNAs, such as mRNAs, tRNAs, as well as non-coding RNAs such as microRNAs (miRNAs), small nuclear RNAs, small nuclear RNAs, and small nucleolar RNAs. RBPs can regulate target gene expression in various post-transcriptional stages, including mRNA alternative splicing, mRNA degradation and stability, mRNA translation, and miRNA processing^51-53. Hence, RBPs potentially influence various physiological and pathological processes, including embryo development, brain development, muscle development, and particularly, cancer development^28,54-56. DAZAP1, a proline-rich RBP, was initially identified as a vital binding partner of deleted in azoospermia and deleted in azoospermia-like, and is associated with spermatogenesis and fertility⁵⁷. DAZAP1 regulates its target genes in the post-transcriptional stages, including mRNA stabilization, by binding to its target RNAs⁵⁸. Recent studies reported increased expression of DAZAP1 in tumor tissues, including HCC, multiple myeloma, and seminoma, and its function in regulating tumorigenic potential^59-61. However, the role of DAZAP1 in promoting tumorigenesis and the regulatory mechanism underlying its increase in tumor tissues was poorly understood. Our study revealed that p52-ZER6 can enhance DAZAP1 transcription, thereby promoting SLC7A11 mRNA stability, and subsequently, SLC7A11-mediated ferroptosis resistance and tumor cell redox homeostasis. These results not only elucidate a novel mechanism for the oncogenic function of DAZAP1, but also unravel the molecular mechanism underlying DAZAP1 upregulation in tumor cells.

5.　Conclusions

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In conclusion, we identified p52-ZER6 as a novel regulator of tumor cell ferroptosis resistance that activates DAZAP1 transcriptional activity. DAZAP1, in turn, enhances SLC7A11 mRNA stability by binding to its 3′-UTR, leading to increases in SLC7A11 expression and GSH synthesis, thus promoting ferroptosis resistance. These findings not only provide novel insights into the regulation of ferroptosis, but also uncover new physiological and pathological functions of p52-ZER6. Furthermore, our study suggests a potential strategy to promote the efficacy of ferroptosis-based anti-tumor therapeutics by targeting p52-ZER6.

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Year 2025 volume 15 Issue 4

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doi: 10.1016/j.apsb.2025.02.013

Receive Date：2024-07-16
Online Date：2026-09-17

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Received：2024-07-16
Revised：2024-09-27
Accepted：2024-11-05

Affiliations

^aKey Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China

^bThe 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044, China

^cDepartment of Gastrointestinal Surgery, Chongqing University Cancer Hospital, Chongqing University, Chongqing 400030, China

^dLife Science Innovation, School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan

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^* Corresponding authors.

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表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Figure 1 ZER6 positively regulates tumorigenesis by inhibiting ferroptosis. (A) Kaplan–Meier survival plots of clinical CRC patients with either low (black; n = 444) or high (red; n = 370) ZER6 expression. (B) ZER6 mRNA expression level in clinical human CRC and corresponding adjacent tissue samples (n = 22), as determined by quantitative reverse transcription-PCR (qRT-PCR). (C) Expression levels of ZER6 in clinical human CRC and adjacent tissue samples, as examined by immunohistochemistry staining using serial sections. Representative images (left; scale bar: 50 μm) and quantification results (right; n = 6) are shown. (D) Heatmap showing DEGs in ZER6-knocked down HCT116^WT cells (fold-change ≥1; P adj <0.05). (E) Kyoto Encyclopedia of Genes and Genomes analysis of DEGs in ZER6-knocked down HCT116^WT cells (P adj <0.05). (F) Cell death rate of ZER6-knocked down HCT116^WT cells. (G, H) Cell death rate (G) and relative cell viability (H) of ZER6-knocked down HCT116^WT cells treated with Ferr-1 (final concentration: 5 μmol/L), 3-MA (final concentration: 10 μmol/L), NSA (final concentration: 5 μmol/L), or Z-VAD (final concentration: 20 μmol/L) for 48 h. (I) Lipid ROS level in ZER6-knocked down HCT116^WT cells, as assessed by C11-BODIPY staining and flow cytometry. Representative images (left) and quantification results (right) are shown. (J) MDA level in ZER6-knocked down HCT116^WT cells. (K, L) Lipid ROS (K) and MDA (L) levels in ZER6-knocked down HCT116^WT cells treated with Ferr-1 (final concentration: 5 μmol/L). (M, N) Lipid ROS (M) and MDA (N) levels in p52-ZER6-knocked down HCT116^WT cells. (O) Cell death rate of p52-ZER6-knocked down HCT116^WT cells. (P) Transmission electron microscopy images of mitochondria in p52-ZER6-knocked down HCT116^WT cells. Arrows: mitochondria; scale bar: 1 μm. Cells transfected with shCon were used as controls. β-Actin was used for qRT-PCR normalization. Quantification data are expressed as mean ± SD (n = 3; unless otherwise indicated). P values were calculated using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; NS: not significant.

Figure 2 p52-ZER6 promotes SLC7A11 expression. (A, B) Heatmap (A; P adj <0.05; values are scaled as indicated from +1.5 to −1.5) and validation using qRT-PCR (B) of ferroptosis-related DEGs enriched in ZER6-knocked down HCT116^WT cells. (C) SLC7A11 mRNA expression level in p52-ZER6-knocked down or p71-ZER6-knocked down HCT116^WT cells, as determined using qRT-PCR. (D, E) SLC7A11 protein expression level in p52-ZER6-knocked down (D) or p71-ZER6-knocked down (E) HCT116^WT cells, as determined using Western blotting. (F) SLC7A11 mRNA expression level in p52-ZER6-overexpressing or p71-ZER6-overexpressing HCT116^WT cells, as determined using qRT-PCR. (G, H) SLC7A11 protein expression level in p52-ZER6-overexpressing (G) or p71-ZER6-overexpressing (H) HCT116^WT cells, as determined using Western blotting. (I) Correlation between p52-ZER6 and SLC7A11 mRNA expression levels in clinical human CRC and corresponding normal adjacent tissues, as determined using qRT-PCR. (J) p52-ZER6 and SLC7A11 protein expression levels in clinical human CRC and corresponding normal adjacent tissue, as determined using Western blotting. (K) p52-ZER6 and SLC7A11 expression levels in clinical human CRC and corresponding normal adjacent tissues, as examined by in situ hybridization assay and immunohistochemistry staining using serial sections, respectively (scale bar: 50 μm). (L, M) Cystein (L) and GSH (M) levels in p52-ZER6-knocked down HCT116^WT cells. (N, O) Cystein (N) and GSH (O) levels in p52-ZER6-overexpressing HCT116^WT cells. Cells transfected with shCon or pcCon were used as controls. β-Actin was used for qRT-PCR normalization and as Western blotting loading control. Quantification data are expressed as mean ± SD (n = 3). P values were calculated using one-way ANOVA. pcCon: pcEF9-Puro; **P < 0.01; ***P < 0.001; NS: not significant.

Figure 3 SLC7A11 is crucial for p52-ZER6 regulation on ferroptosis. (A, B) Cysteine (A) and GSH (B) levels in p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^WT cells. (C) Lipid ROS level in p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^WT cells, as assessed by C11-BODIPY staining and flow cytometry. Representative images (left) and quantification results (right; n = 3) are shown. (D) MDA level in p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^WT cells. (E) Transmission electron microscopy images of mitochondria in p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^WT cells. Arrows: mitochondria; scale bar: 1 μm. (F, G) Cell death rate (F) and viability (G) of p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^WT cells. (H) Tumorigenic potential of p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^WT stable cells, as examined in vivo by subcutaneous injection into BALB/c-nu/nu mice (n = 6). Morphological images of the tumors generated at 29 days after injection (left) and volume of the tumors formed at indicated time points (right) are shown. (I, J) p52-ZER6 and SLC7A11 expression levels in the xenograft tumors formed by HCT116^WT cells, as determined using Western blotting (I), as well as in situ hybridization and immunohistochemistry staining using serial sections, respectively (J); scale bar: 50 μm. (K–M) Cysteine (K), GSH (L), and MDA (M) levels in the xenograft tumors formed by p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^WT cells. (N) 4-HNE and Ki67 expression levels in the xenograft tumors formed by p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^WT cells, as examined by immunohistochemistry staining using serial sections (scale bar: 50 μm). (O) Cell death rate in the xenograft tumors formed by p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^WT cells, as determined by PI-staining (scale bar: 50 μm). Cells transfected with shCon and/or pcCon were used as controls. β-Actin was used as Western blotting loading control. Quantification data are expressed as mean ± SD (n = 3). P values were calculated using one-way ANOVA. pcCon: pcEF9-Puro; **P < 0.01; ***P < 0.001; NS: not significant.

Figure 4 p52-ZER6 enhances SLC7A11 mRNA stability and CRC cells ferroptotic resistance by promoting DAZAP1 expression. (A) Gene Ontology enrichment analysis of DEGs in ZER6-knocked down HCT116^WT cells (P adj< 0.05). (B, C) SLC7A11 mRNA expression levels in p52-ZER6-knocked down (B) and p52-ZER6-overexpressing (C) HCT116^WT cells at indicated time points after actinomycin D treatment, as determined using qRT-PCR. (D) Cross-omics analysis using DEGs in ZER6-knocked down HCT116^WT cells enriched in “mRNA binding”, RBP protein in RBPmap, and ferroptosis-related factor in FerrDb V2. (E) DAZAP1 and ZFP36 mRNA expression levels in ZER6-knocked down HCT116^WT cells, as determined using qRT-PCR. (F, G) DAZAP1 mRNA expression level in p52-ZER6-knocked down (F) and p52-ZER6-overexpressing (G) HCT116^WT cells, as determined using qRT-PCR. (H, I) DAZAP1 protein expression level in p52-ZER6-knocked down (H) and p52-ZER6-overexpressing (I) HCT116^WT cells, as determined by Western blotting. (J) p52-ZER6 and DAZAP1 expression levels in clinical human CRC and corresponding normal adjacent tissues, as examined by in situ hybridization assay and immunohistochemistry staining using serial sections, respectively (scale bar: 50 μm). (K) p52-ZER6 and DAZAP1 protein expression levels in clinical human CRC and corresponding normal adjacent tissues, as determined using Western blotting. (L) Correlation between p52-ZER6 and DAZAP1 mRNA expression levels in clinical human CRC and corresponding normal adjacent tissues, as determined using qRT-PCR. (M) SLC7A11 mRNA expression level in p52-ZER6-knocked down, DAZAP1-overexpressing HCT116^WT cells at indicated time points after actinomycin D treatment, as determined using qRT-PCR. (N, O) SLC7A11 mRNA (N) and protein (O) expression levels in p52-ZER6-knocked down, DAZAP1-overexpressing HCT116^WT cells, as determined using qRT-PCR and Western blotting, respectively. Cells transfected with shCon and/or pcCon were used as controls. β-Actin was used for qRT-PCR normalization and as Western blotting loading control. Quantification data are expressed as mean ± SD (n = 3). P values were calculated using one-way ANOVA. pcCon: pcEF9-Puro; ActD: actinomycin D (final concentration: 5 μmol/L); *P < 0.05; **P < 0.01; ***P < 0.001; NS: not significant.

Figure 5 DAZAP1 binds directly to SLC7A11 3′-UTR. (A) Binding capacity of DAZAP1 to SLC7A11 mRNA, as determined by RIP assay using anti-DAZAP1 antibody and qRT-PCR. (B) Schematic diagram of the regions of SLC7A11 mRNA. (C) Relative luciferase activities of SLC-5′-UTR, SLC-CDS, and SLC-3′-UTR-1 to SLC-3′-UTR-5 in p52-ZER6-knocked down HCT116^WT cells. (D) Relative luciferase activities of SLC-3′-UTR-1 to SLC-3′-UTR-5 in p52-ZER6-knocked down, DAZAP1 overexpressing HCT116^WT cells. (E) Binding capacity of DAZAP1 to SLC7A11 mRNA regions, as analyzed using RNA pull-down assay. (F) Relative luciferase activities of SLC-3′-UTR-4A and SLC-3′-UTR-4B in p52-ZER6-knocked down HCT116^WT cells. (G) Binding capacity of DAZAP1 to SLC7A11-3′-UTR-4 regions, as analyzed using RNA pull-down assay. (H) Schematic diagram of mutant SLC7A11-3′-UTR-luc reporter vectors. (I) Relative luciferase activities of mutant SLC7A11-3′-UTR-luc reporter vectors in p52-ZER6-knocked down HCT116^WT cells. (J) Binding capacity of DAZAP1 to the mutant SLC7A11-3′-UTR, as analyzed using RNA pull-down assay. Cells transfected with shCon or pcCon were used as controls. β-Actin was used as Western blotting loading control. Quantification data are expressed as mean ± SD (n = 3). P values were calculated using one-way ANOVA. pcCon: pcEF9-Puro; ***P < 0.001; NS: not significant.

Figure 6 p52-ZER6 promotes DAZAP1 transcriptional activity by binding directly to its promoter. (A) Schematic diagram of DAZAP1 reporter vectors (DAZ-lucs). (B) Relative luciferase activities of DAZ-luc-1 to DAZ-luc-5 in p52-ZER6-knocked down HCT116^WT cells. (C) Binding capacity of p52-ZER6 to the predicted region in DAZAP1 promoter, as examined using ChIP assay with anti-ZER6 antibody followed by PCR. The location of the primer set used for PCR is shown. Anti-histone H3 antibody was used as positive control. (D) Relative luciferase activity of DAZ-luc^mut in p52-ZER6-knocked down HCT116^WT cells. Schematic diagram (left) and luciferase activities (right) are shown. (E) Relative luciferase activities of DAZ-luc-1 to DAZ-luc-5 in p71-ZER6-knocked down HCT116^WT cells. (F) Schematic diagram of various fragments with intermediate length and sequences between p52-ZER6 and p71-ZER6 conjugated with FLAG. (G) Binding capacities of FLAG-p52, FLAG-p52^K, FLAG-p52^KH, and FLAG-p71 to the predicted region in the DAZAP1 promoter, as determined using ChIP assay. (H–J) Relative luciferase activity of DAZ-luc-1 (H), DAZAP1 mRNA (I), and DAZAP1 protein (J) levels in HCT116^WT cells overexpressing FLAG-p52, FLAG-p52^K, FLAG-p52^KH, or FLAG-p71, as determined using qRT-PCR (I) and Western blotting (J), respectively. Cells transfected with shCon or FLAG-Con were used as controls. β-Actin was used for qRT-PCR normalization and as Western blotting loading control. Quantification data are expressed as mean ± SD (n = 3). P values were calculated using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; NS: not significant.

Figure 7 p52-ZER6 suppresses ferroptosis by stabilizing SLC7A11 mRNA in a p53-independent manner. (A, B) DAZAP1 and SLC7A11 mRNA (A) and protein (B) expression levels in p52-ZER6-knocked down HCT116^p53null cells, as determined using qRT-PCR and Western blotting, respectively. (C) Relative luciferase activity of SLC7A11-luc in p52-ZER6-knocked down HCT116^p53null cells. (D, E) SLC7A11 mRNA expression levels in p52-ZER6-knocked down (D) and p52-ZER6-overexpressing (E) HCT116^p53null cells at indicated time points after actinomycin D treatment, as determined using qRT-PCR. (F, G) Cysteine (F) and GSH (G) levels in p52-ZER6-knocked down HCT116^p53null cells. (H, I) Lipid ROS (H) and MDA (I) levels in p52-ZER6-knocked down HCT116^p53null cells. (J) Cell death rate of p52-ZER6-knocked down HCT116^p53null cells. (K) p52-ZER6 and SLC7A11 protein expression levels in p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null cells, as determined using Western blotting. (L, M) Cysteine (L) and GSH (M) levels in p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null cells. (N, O) Lipid ROS (N) and MDA (O) levels in p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null cells. (P) Cell death rate of p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null Cells. Cells transfected with shCon and/or pcCon were used as controls. β-Actin was used for qRT-PCR normalization and as Western blotting loading control. Quantification data are expressed as mean ± SD (n = 3). P values were calculated using one-way ANOVA. pcCon: pcEF9-Puro; ActD: actinomycin D (final concentration: 5 μmol/L); *P < 0.05; **P < 0.01; ***P < 0.001; NS: not significant.

Figure 8 p52-ZER6 knockdown negatively regulates tumorigenesis by suppressing SLC7A11 in a p53-independent manner. (A) p52-ZER6 and SLC7A11 protein expression levels in p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null stable cells, as determined using Western blotting. (B) Tumorigenic potential of p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null stable cells, as examined in vivo by subcutaneous injection into BALB/c-nu/nu mice (n = 6). Morphological images of the tumors generated at 22 days after injection (left) and the volume of the tumors formed at indicated time points (right) are shown. (C) p52-ZER6 and SLC7A11 protein expression levels in the xenograft tumors formed by HCT116^p53null cells, as determined using Western blotting. (D) p52-ZER6 and SLC7A11 in the xenograft tumors formed by p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null cells, as examined by in situ hybridization and immunohistochemistry staining using serial sections, respectively (scale bar: 50 μm). (E–G) GSH (E), cysteine (F), and MDA (G) levels in the xenograft tumors formed by p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null cells. (H) 4-HNE and Ki67 expression levels in the xenograft tumors formed by p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null cells, as examined by immunohistochemistry staining using serial sections (scale bar: 50 μm). (I) Cell death rate in the xenograft tumors formed by p52-ZER6-knocked down, SLC7A11-overexpressing HCT116^p53null cells, as examined by PI-staining (scale bar: 50 μm). (J) Schematic diagram showing the molecular mechanism of p52-ZER6/DAZAP1 regulation on SLC7A11-mediated tumor cell ferroptosis resistance. Cells transfected with shCon and/or pcCon were used as controls. β-Actin was used as Western blotting loading control. Quantification data are expressed as mean ± SD (n = 3, unless otherwise indicated). P values were calculated using one-way ANOVA. pcCon: pcEF9-Puro; **P < 0.01; ***P < 0.001.

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