Skip to main content
Download PDF

β-catenin-IRP2-primed iron availability to mitochondrial metabolism is druggable for active β-catenin-mediated cancer

Journal of Translational Medicine volume 21, Article number: 50 (2023) Cite this article

Abstract

Background

Although β-catenin signaling cascade is frequently altered in human cancers, targeting this pathway has not been approved for cancer treatment.

Methods

High-throughput screening of an FDA-approved drug library was conducted to identify therapeutics that selectively inhibited the cells with activated β-catenin. Efficacy of iron chelator and mitochondrial inhibitor was evaluated for suppression of cell proliferation and tumorigenesis. Cellular chelatable iron levels were measured to gain insight into the potential vulnerability of β-catenin-activated cells to iron deprivation. Extracellular flux analysis of mitochondrial function was conducted to evaluate the downstream events of iron deprivation. Chromatin immunoprecipitation, real-time quantitative PCR and immunoblotting were performed to identify β-catenin targets. Depletion of iron-regulatory protein 2 (IRP2), a key regulator of cellular iron homeostasis, was carried out to elucidate its significance in β-catenin-activated cells. Online databases were analyzed for correlation between β-catenin activity and IRP2-TfR1 axis in human cancers.

Results

Iron chelators were identified as selective inhibitors against β-catenin-activated cells. Deferoxamine mesylate, an iron chelator, preferentially repressed β-catenin-activated cell proliferation and tumor formation in mice. Mechanically, β-catenin stimulated the transcription of IRP2 to increase labile iron level. Depletion of IRP2-sequered iron impaired β-catenin-invigorated mitochondrial function. Moreover, mitochondrial inhibitor S-Gboxin selectively reduced β-catenin-associated cell viability and tumor formation.

Conclusions

β-catenin/IRP2/iron stimulation of mitochondrial energetics is targetable vulnerability of β-catenin-potentiated cancer.

Background

WNT/β-catenin pathway plays critical roles in cellular physiology and pathology. Upon WNT activation, β-catenin escapes from its degradation complex and translocates to nucleus. Nuclear β-catenin interacts with transcription factors and chromatin modifiers such as T cell factor (TCF) and cyclic adenosine monophosphate response element binding protein-binding protein (CBP) to stimulate target gene expression [1]. By promoting the expression of cyclin D1, fibroblast growth factor, glutamate-ammonia ligase (GLUL), peroxisome proliferator activated receptor alpha (PPARα), and telomerase reverse transcriptase, β-catenin regulates cell proliferation, survival, metabolism and genetic stability [2,3,4,5,6]. Activation of WNT/β-catenin pathway is prevalent in human cancers including hepatic, colorectal, endometrial, and pancreatic cancers [7,8,9,10]. Active mutations of β-catenin, encoded by catenin beta 1 (CTNNB1), or loss-of-function mutations of its destruction complex, such as adenomatous polyposis coli (APC), AXIN1 and AXIN2, lead to β-catenin stabilization and eventually unrestrained transcriptional activity [11,12,13]. According to cBioPortal database, APC is the top 1 mutated tumor suppressor gene in colon adenocarcinoma (COAD, 68.6% mutation rate) and CTNNB1 (30.6% mutation rate) is the most frequently mutated proto-oncogene in hepatocellular carcinoma (HCC) [14, 15]. In uterine corpus endometrial carcinoma (UCEC), CTNNB1 is also highly altered (34.6% mutation rate) [14, 15]. The importance of β-catenin in carcinogenesis has provided rationale for the study of its oncogenic mechanisms and the development of therapeutics that selectively inhibits β-catenin-activated tumors.

Various compounds are investigated to intervene WNT/β-catenin pathway for cancer treatment. A β-catenin degrader, proteolysis targeting chimeras, suppress WNT-dependent colorectal cancer (CRC) in mice [16].Targeting β-catenin-TCF transcription complex with CCT036477, iCRT3, iCRT5, iCRT14 or PKF118-310 preferentially represses the survival of malignant cells in preclinical studies [17,18,19,20]. Pri-724, a first-in-class antagonist that blocks β-catenin/CBP interaction, is being studied in clinical trials to treat CRC, pancreatic cancer, acute myeloid leukemia and chronic myelogenous leukemia [21, 22]. Besides, inhibitions of β-catenin downstream AKT Serine/Threonine Kinase 2 (AKT2)-stimulated pyrimidine synthesis axis or PPARα-driven fatty acid oxidation (FAO) blocked β-catenin-activated tumor formation [5, 23]. Despite these substantial efforts, none of them have been approved for clinical application.

Given the escalating cost, slow pace, and high attrition rates of translating new drugs into clinical practice, repurposing of old drugs is an attractive proposition [24]. Taking an unbiased screening of compound library of approved drugs, we identified iron chelators as selective inhibitors of β-catenin-activated cell proliferation and tumorigenesis. β-catenin-stimulated IRP2 (iron regulating protein 2)-iron homeostasis was essential for mitochondrial function. Targeting β-catenin-IRP2-iron-mitochondria cascade is an appealing therapeutic strategy for β-catenin-activated tumors.

Methods

Reagents

Deferoxamine mesylate (DFO, #D9533), ferric ammonium citrate (FAC, #F3388), and insulin (#I9278) were purchased from Sigma-Aldrich (St. Louise, MO, USA). Deferasirox (DFX, #S1712), oligomycin A (#S1478), hydrocortisone hemisuccinate (#S5972), and pri-724 (#S8968) were purchased from Selleck Chemicals (Houston, TX, USA). VLX600 (#T8500) was purchased from Target Molecule (Boston, MA, USA). S-Gboxin was purchased from BiochemPartner Biotech (Shanghai, China). Lipofectamine 2000 (#11668019), TRIzol (#15596018), trypsin–EDTA (#25200056), DMEM (#11995–065), RPMI-1640 (#72400047), fetal bovine serum (FBS, #10100147C), nonessential amino acid solution (NEAA, #11140050), penicillin G and streptomycin (#15140163, P/S), glutamax (#35050061), and sodium pyruvate (#11360070) were purchased from Thermo Fisher (Carlsbad, CA, USA). Horse serum was purchased from Procell (#164215, Wuhan, Hubei, China). Puromycin (#P8230), 0.1% crystal violet (#G1063), and polybrene (#H8761) were purchased from Solarbio (Beijing, China). 4% paraformaldehyde (PFA) was purchased from Servicebio (#G1101, Wuhan, Hubei, China). Cell counting kit-8 (CCK8) was purchased from Yeasen Biotech (#40203ES60, Shanghai, China). β-cateninmut plasmid was purchased from Addgene (#24204, Watertown, MA, USA).

High-throughput drug screening

A panel of 1088 compounds from an FDA-approved drug library (Selleck chemicals) was screened for drugs which preferentially target β-catenin-activated cells. In brief, wildtype (WT) or β-cateninΔ(ex3)/+ mouse embryonic fibroblasts (MEFs) were plated in 384-well plates with 500 cells per well and treated with DMSO or 10 μM compounds using Echo® 550 Liquid Handler (Beckman counter, Brea, CA, USA). After 72 h incubation, cell viability was determined with CCK8 assay using EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). The high-throughput screening was repeated for 3 times. Inhibition rate was normalized against DMSO treatment.

Cell lines

WT, β-cateninΔ(ex3)/+ MEFs and MHCC97H were previously reported [23]. Cells were maintained at 37 °C incubator with 5% CO2. The supplier and culture medium of hepatic cell lines were shown as follow:

Cell lines

Supplier

Culture medium

HepaRG

Thermo Fisher

DMEM + 10% FBS + 1% NEAA + 1% P/S + 1% glutamax + 5 μg/ml insulin + 50 μM hydrocortisone hemisuccinate

293FT

Abclonal (Wuhan, Hubei, China)

DMEM + 10% FBS + 1% P/S

CCC-HEL-1

China Infrastructure of Cell Line Resource (Beijing, China)

DMEM + 20% FBS + 1% P/S

NCTC1469

China Infrastructure of Cell Line Resource

DMEM + 10% horse Serum + 1% P/S

HepG2

China Infrastructure of Cell Line Resource

DMEM + 10% FBS + 1% P/S

SNU886

Cobioer biosciences (Nanjing, Jiangsu, China)

RPMI-1640 + 10% FBS + 1% P/S

HuH7

Procell

RPMI-1640 + 10% FBS + 1% P/S

Hepa 1–6

Procell

DMEM + 10% FBS + 1 mM Sodium Pyruvate + 1% P/S

SNU182

Procell

RPMI-1640 + 20% FBS + 1% P/S

SNU398

Sagen tech (Guangzhou, Guangdong, China)

DMEM + 10% FBS + 1% P/S

HCC-LM3

China Infrastructure of Cell Line Resource

DMEM + 10% FBS + 1% P/S

siRNA and shRNA transfections

Cells were transfected with β-catenin siRNA or Irp2 shRNA (Tsingke Biotech, Beijing, China) using lipofectamine 2000 according to the manufacturer’s instructions. Sequences were followed:

si-Human β-catenin-1: 5′-TTGTTATCAGAGGACTAAAT-3′.

si-Human β-catenin-2: 5′-TCTAACCTCACTTGCAATAAT-3′.

si-Mouse Tcf7l2-1: 5′-CCTTAGCGTAAGCATCTTATA-3′.

si-Mouse Tcf7l2-2: 5′-GCTCCGAAAGTTTCCGAGATA-3′.

si-Human TCF7L2-1: 5′-GCGGGATAACTATGGAAAGAA-3′.

si-Human TCF7L2-2: 5′-CGAACCTATCTCCAGATGAAA-3′.

sh-Irp2-1:5′-CCGGCCTGCCAGTTACTCTTACTTTCTCGAGAAAGTAAGAGTAACTGGCAGGTTTTTT-3′.

sh-Irp2-2:5′-CCGGCGATAGAACTACCATAGCAAACTCGAGTTTGCTATGGTAGTTCTATCGTTTTTT-3′.

Cell viability and colony formation

For cell viability, MEFs and hepatic cell lines were seeded in 96-well plates at a density of 3000 to 6000 cells per well. Cells were treated with different concentrations of compounds for indicated times and then incubated with CCK8 for 4 h at 37 °C. The absorbance at 450 nm wavelength was measured by microplate reader (Agilent, Santa Clara, CA, USA). For colony formation, 200 cells were plated in six-well plates. Cells were fixed in 4% PFA for 15 min and then stained with 0.1% crystal violet for half-hour.

Extracellular flux analysis of oxygen consumption rate (OCR) and ATP production rate

OCR and ATP analysis was performed using Seahorse Xfe 24 flux analyzer (Agilent). 10,000 cells were seeded in 24-well microplates. Before measurement, cell medium was replaced by XF Assay Medium supplemented with pyruvate, glucose, and glutamine (pH 7.4). Measurement was done at baseline and sequential injections of 1 μM oligomycin, 2 μM FCCP and 0.5 μM rot/antimycin A for OCR, or 1.5 μM oligomycin and 0.5 μM rot/antimycin A for ATP respectively. Data were represented as normalization to cell numbers.

Labile iron pool (LIP) measurement

Intracellular labile iron pool analysis was performed with Phen Green™ SK (PGSK) fluorescent probe (#P14313, Thermo Fisher). For flow cytometry analysis, cells were harvested and washed with PBS after incubating with 2 μM PGSK for 15 min. Samples were then analyzed on BD Accuri C6 or C6 plus (Franklin Lakes, NJ, USA). For fluorescent images, cells incubated with 2 μM PGSK were fixed in 4% PFA for 15 min and washed with PBS for 3 times before observation by LSM780 (Zeiss, Oberkochen, Germany).

Real-time quantitative PCR

Total RNA was extracted from cells with TRIzol reagent. cDNA was generated using Hifair® II 1st Strand cDNA Synthesis Kit (gDNA digester plus) (#11121ES60, Yeasen) according to the manufacturer’s protocol. Realtime-PCR was performed using SYBR High-Sensitivity qPCR SuperMix (#abs60086, Absin Biosciences, Shanghai, China). β-actin transcripts were used to normalize the expression of each gene. The primer sequences were as follows:

Mouse Irp2-F: 5′-TTCTGCCTTACTCAATACGGGT-3′.

Mouse Irp2-R: 5′-AGGGCACTTCAACATTGCTCT-3′.

Mouse Glul-F: 5′-TGAACAAAGGCATCAAGCAAATG-3′.

Mouse Glul-R: 5′-CAGTCCAGGGTACGGGTCTT-3′.

Mouse β-actin-F: 5′-AGAGGGAAATCGTGCGTGAC-3′.

Mouse β-actin-R: 5′-CAATAGTGATGACCTGGCCGT-3′.

Human IRP2-F: 5′-GGAATTCCATATGATACAGAATGCACCAAAT-3′.

Human IRP2-R: 5′-CGGGATCCTCATGTTTCAGGTTCAGCCAC-3′.

Human β-actin-F: 5′-CCTGGCACCCAGCACAAT-3′.

Human β-actin-R: 5′-GCCGATCCACACGGAGTACT-3′.

Immunoblotting

Cell proteins were extracted from SDS loading buffer with Protease and Phosphatase Inhibitor Cocktail (EDTA-Free) (#P002, New cell & molecular, Suzhou, Jiangsu, China). Immunoblotting was conducted as previously described [23]. Membranes were first incubated with primary antibodies against β-catenin (#9587, Cell Signaling Technology, Danvers, MA, USA), Cyclin D1 (#A19038, Abclonal), IRP2 (#A6382, Abclonal), IRP1 (#A7867, Abclonal), TfR1 (#A5865, Abclonal), TCF7L2 (#A20770, Abclonal), FBXL5 (#A5602, Abclonal) or GAPDH (#AC002, Abclonal) overnight at 4 °C, and then incubated with secondary IRDye 680RD goat anti-rabbit (#68071, LI-COR Biosciences, Lincoln, USA) or IRDye 800 CW goat anti-mouse (#32210, LI-COR Biosciences) antibodies for 2 h at room temperature. Protein bands were detected by LI-COR Odyssey Infrared Scanner.

Chromatin immunoprecipitation (ChIP)

Β-catenin ChIP-seq data analysis was conducted using our published datasets (GSE165853). For the bioinformatic analysis of ChIP-seq data, Bowtie2 was used for alignment, MACS2 with FDR < 0.01 was for enriched peaks identification, and ChIPseeker was for peaks annotation. Peaks enrichment distribution analysis was performed by Deeptools as previously described [23]. ChIP assays were performed for β-cateninΔ(ex3)/+ MEFs using SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (#9005, Cell Signaling Technology). Briefly, cells were seeded on 15 cm plates, cross-linked with 1% formaldehyde, and stopped with glycine. Cells were harvested with lysis buffer containing protease inhibitor complex. The lysates were digested by micrococcal nuclease and then sonicated to shear the DNA. Chromatins were immunoprecipitated with 20 μl anti-β-catenin (106 μg/ml), 10 μl anti-Histone H3 (269 μg/ml, positive control) or 2 μl anti-IgG (1 mg/ml) antibody overnight at 4 °C on a rotating wheel according to manufacturer’s instructions. The ChIP-enriched DNA samples were purified and then amplified by real-time PCR using primers as followed, and data were expressed as a percentage of the total input chromatin by the formula: percent Input = 2% × 2(C[T] 2%Input Sample − C[T] IP Sample), C[T] = Threshold cycle of PCR reaction.

Site1-F: 5′-GTGAGCGCTGTGATGCAATA-3′.

Site1-R: 5′-CCTTGGTGCTGTTGGTATCTG-3′.

Site2-F: 5′-TAGCCAACGGTGTACAGGAG-3′.

Site2-R: 5′-TGAGTCTGGATTTGCGATGC-3′.

Site3-F: 5′-GCATCGCAAATCCAGACTCA-3′.

Site3-R: 5′-TTGATCCACTGGCTTCCTGAC-3′.

Site4-F: 5′-GATGCCTGTGGTAACAAGCG-3′.

Site4-R: 5′-ATTGGCTCCAGCTATCCGTT-3′.

Site5-F: 5′-GCAAGAGCTGGGGTATTAGCA-3′.

Site5-R: 5′-GGAGTTGTCTGGTTTGCCCT-3′.

Subcutaneous tumor formation

All animal studies were performed following the protocols approved by the Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College (ethical code: ACUC-A02-2022-057). Female BALB/c nude mice (4–6 weeks) were purchased from HFK Bio-Technology (Beijing, China). 1 × 106 β-cateninΔ(ex3)/+ MEFs suspended in 100 μL PBS or 2 × 106 HuH7 in 100 μL PBS containing 50% Matrigel (#354234, BD Biosciences, San Jose, CA, USA) were subcutaneously inoculated in the right dorsal flank of nude mice. When tumor volumes reached 100 mm3, mice were randomly assigned to different groups. The treatment group was intraperitoneally injected with 10 mg/kg S-Gboxin, or 400 mg/kg DFO every other day and control group was injected with equal volume of vehicle. Tumor volumes and body weights were measured every other day and tumor volumes were calculated by the formula: volume = length × width2/2.

Database analysis

For gene expression correlation between CTNNB1 and IRP2 or TFRC, cell line analysis was conducted with Dependency Map (DepMap; https://depmap.org/portal/interactive), and human sample analysis was performed using Gene Expression Profiling Interactive Analysis 2.0 (GEPIA2; http://gepia.cancer-pku.cn/detail.php) [25]. UALCAN was analyzed for protein expression (http://ualcan.path.uab.edu/analysis-prot.html) [26]. JASPAR database was analyzed for TCF7L2 binding motif at IRP2 promoter (https://jaspar.genereg.net) [27].

Statistical analysis

Data were expressed as mean ± SD. All data were independently repeated for at least 3 times. Statistical analysis was carried out using a two-tailed unpaired t-test with GraphPad Prism software. P < 0.05 was considered as statistically significant.

Results

β-catenin-activated cells are more susceptible to iron chelators

To identify candidate molecules that preferentially inhibit β-catenin-activated tumors and could be readily translated into clinical practice, we first deleted exon 3, the most altered site in CTNNB1 gene, to establish constitutive β-catenin-activated MEFs (β-cateninΔ(ex3)/+) [23]. We then conducted a high-throughput screening with a chemical library containing 1088 FDA-approved drugs for molecules that selectively inhibited the β-catenin-activated cells but not WT MEFs (Fig. 1A). Among the top hits that preferentially suppressed β-catenin-activated cells, iron chelator DFX was chosen for validation. DFX specifically decreased β-cateninΔ(ex3)/+ cell viability (Fig. 1B). We confirmed the efficacy of iron chelating with other two iron chelators, DFO and VLX600. Consistently, both DFO and VLX600 preferentially reduced β-cateninΔ(ex3)/+ MEF viability (Fig. 1C). In addition, iron supplementation with FAC rescued DFO, DFX and VLX600-mediated cell proliferation arrest (Fig. 1D). Therefore, activated β-catenin cells are sensitive to iron depletion.

Fig. 1
figure 1

Identification of iron chelators as selective inhibitors for β-catenin-activated cells. A, Schematic illustration of drug screening and validation. Wildtype (WT) and β-cateninΔ(ex3)/+ MEFs were treated with a chemical library containing 1088 FDA-approved drugs at a concentration of 10 μM for 72 h. Cell viability was measured. Candidate hits were verified in MEFs, hepatic cells and subcutaneous tumors of mice. B–D, Cell viability. WT and β-cateninΔ(ex3)/+ MEFs were treated with DFX (B), DFO (C, left) and VLX600 (C, right) at different concentrations for 48 h. β-cateninΔ(ex3)/+ MEFs were treated with DFX, DFO or VLX600 in the presence or absence of FAC for 48 h (D); n = 3. Data were shown as mean ± SD and analysis was performed using t test. **p < 0.01, ***p < 0.001

Full size image

Depleting iron suppresses β-catenin-activated tumor growth

To investigate the efficacy of iron deprivation in liver cancer treatment, DFO was chosen for further study as it less likely causes rashes, gastrointestinal symptoms, and severe adverse events than DFX does [28]. We treated 10 hepatic cell lines which consisted of 2 mouse-derived (1 immortalized, 1 malignant) and 8 human-derived (1 immortalized, 7 malignant) liver cell lines (Additional file 1: Table S1). β-catenin mutant cell lines were more sensitive to DFO than WT ones (Fig. 2A). Knocking down β-catenin compromised the sensitivity of CTNNB1-mutated MHCC97H and SNU398 cells to DFO treatment (Fig. 2B, C). In contrast, overexpression of oncogenic β-cateninmut which harbored S33A, S37A, T41A and S45A mutations sensitized SNU886 and HuH7 cells to DFO treatment (Fig. 2D, E). Cellular sensitivity to DFO is thus positively correlated with β-catenin activity. DFO inhibited tumorigenicity of β-cateninΔ(ex3)/+ MEFs, manifesting as reduced tumor volumes and tumor weights, with negligible impact on body weights in nude mice (Fig. 2F–H). HuH7 cells transfected with oncogenic β-cateninmut were more tumorigenic than the cells transfected with vector alone in nude mice. The tumorigenesis of oncogenic β-catenin-expressed cells was blunted by DFO treatment (Fig. 2I, J), while DFO had marginal impact on body weights of treated mice (Fig. 2K). Collectively, iron chelator repressed β-catenin-activated tumor formation.

Fig. 2
figure 2

DFO preferentially suppresses β-catenin-activated cell proliferation and tumor formation. A–E, Viability of cells treated with DFO at different concentrations for 48 h. 10 hepatic cell lines (A); MHCC97H (B) or SNU398 (C) transfected with control or β-catenin siRNA; and SNU886 (D) or HuH7 (E) transfected with vector or β-cateninmut plasmid. Protein abundances were analyzed (B, up; C, up; D, left; E, left). F–H, Nude mice subcutaneously inoculated with β-cateninΔ(ex3)/+ MEFs were administered intraperitoneally with PBS or DFO (400 mg/kg) every other day. Tumor growth was calculated as the mean value in tumor volume (F). Tumor images, tumor weights (G), and body weights (H) were plotted at the end of treatment; n = 6. I–K, Nude mice were subcutaneously inoculated with HuH7 transfected with vector or β-cateninmut plasmid. These mice were administered intraperitoneally with PBS or DFO (400 mg/kg) every other day. Tumor growth was calculated as the mean value in tumor volumes (I). Tumor images, tumor weights (J), and body weights (K) were plotted at the end of treatment; n = 6. Data were shown as mean ± SD and analysis was performed using t test. *p < 0.05, ***p < 0.001

Full size image

Since iron plays multifaceted roles in cancer biology, addition of excessive iron and induction of iron-dependent ferroptosis have been reported to treat cancer [29, 30]. To check whether β-catenin-activated cells were liable to these interventions, we treated MEFs with iron supplements or ferroptosis inducer. Supplementation of FeCl2 or FAC didn’t alter cell viability of both WT and β-cateninΔ(ex3)/+ MEFs (Additional file 1: Fig. S1A, B). β-cateninΔ(ex3)/+ MEFs were more resistant to erastin-induced ferroptosis (Additional file 1: Fig. S2), which was consistent with glutathione peroxidase 4-mediated ferroptosis resistance in β-catenin-activated cancers [31]. These data suggest that iron chelation but not iron addition and ferroptosis induction is promising for β-catenin-activated cancer treatment.

β-catenin increases cellular labile iron pool

Either addiction to excessive iron or clinging to limited iron might render CTNNB1 mutant cells susceptible to iron chelators. We checked chelatable or LIP abundance in CTNNB1 mutant cells with a fluorescent indicator PGSK. PGSK fluorescence is quenched upon interaction with LIP. Compared to WT MEFs, β-cateninΔ(ex3)/+ MEFs had higher LIP (Fig. 3A, B). β-catenin inhibitor pri-724 reduced LIP in β-cateninΔ(ex3)/+ MEFs (Fig. 3C). More LIP was detected in β-catenin-activated liver cancer cells than in the WT ones (Fig. 3D). Oncogenic β-catenin overexpression enhanced cellular LIP in CTNNB1 WT cells (Fig. 3E). Moreover, pri-724 decreased LIP in CTNNB1-mutated HepG2, HCCM97H and Hepa 1–6 cells (Fig. 3F). All these data indicate that β-catenin raises intracellular LIP.

Fig. 3
figure 3

Oncogenic β-catenin elevates cellular LIP. A, Representative confocal images of WT and β-cateninΔ(ex3)/+ MEFs using iron-sensitive fluorescent PGSK dye. B–F, Flow cytometry analysis of PGSK intensity in WT and β-cateninΔ(ex3)/+ MEFs (B); β-cateninΔ(ex3)/+ MEFs treated with DMSO or 20 μM pri-724 for 48 h (C); 7 hepatic cancer cell lines (D); HuH7 and SNU886 transfected with vector or β-cateninmut plasmid (E); HepG2, MHCC97H and Hepa 1–6 treated with DMSO or 20 μM pri-724 for 48 h (F); n = 3. Data were shown as mean ± SD and analysis was performed using t test. *p < 0.05, ***p < 0.001

Full size image

β-catenin stimulates IRP2 transcription to increase cellular LIP

Β-catenin is a transcriptional cofactor that binds transcription factor 7 like 2 (TCF7L2, also known as TCF4) to drive target gene expression [1]. We therefore analyzed β-catenin ChIP-seq data to dissect the molecular mechanism responsible for β-catenin-boosted LIP [23]. β-catenin was enriched in the promoter region of Irp2 gene (Fig. 4A), which is critical for cellular iron homeostasis via post-transcriptional stabilization of transferrin receptor 1 (TfR1, TFRC) mRNA [29]. JASPAR database analysis revealed the TCF7L2 binding motif, which is conserved between human and mouse, in IRP2 gene (Fig. 4B) [27, 32]. ChIP-PCR assay indeed detected the interaction of β-catenin with Irp2 promoter regions (Fig. 4C). Moreover, β-catenin-activated cells had more Irp2 mRNA (Fig. 4D), which was attenuated by pri-724 treatment or Tcf7l2 knockdown (Fig. 4E, F). IRP2 and TfR1 proteins were enriched in β-catenin-activated cells (Fig. 4G, H) and decreased in pri-724-treated cells (Fig. 4I). TCF7L2 silence decreased IRP2 protein levels in β-cateninΔ(ex3)/+ MEFs and HepG2 (Fig. 4J). Therefore, β-catenin may bind to TCF7L2 to stimulates IRP2 transcription. Unlike IRP2, IRP1 proteins was not changed in WT and β-cateninΔ(ex3)/+ MEFs (Fig. 4G). As an iron sensor, F-box/LRR-repeat protein 5 (FBXL5) is degraded in low-iron conditions caused by DFO [33]. Suppression of FBXL5 decreases ubiquitylation and degradation of IRP2 proteins [34]. Since the protein abundances of FBXL5 did not differ between WT and β-cateninΔ(ex3)/+ MEFs (Fig. 4G), β-catenin increased IRP2 through TCF7L2-mediated transcription activation but not inhibition of FBXL5-mediated degradation.

Fig. 4
figure 4

β-catenin transcriptionally stimulates IRP2 to boost LIP and cell proliferation. A, Genome browser track of β-catenin ChIP-seq profiles at Irp2 loci. B, TCF7L2 binding motif analysis using JASPAR. C, β-catenin antibody-precipitated DNA was PCR amplified for Irp2 promoter regions; n = 3. D–F, Irp2 mRNA levels in WT and β-cateninΔ(ex3)/+ MEFs (D), β-cateninΔ(ex3)/+ MEFs treated with DMSO or 20 μM pri-724 for 48 h (E), and β-cateninΔ(ex3)/+ MEFs or HepG2 cells transfected with control or Tcf7l2 siRNA (F); n = 3. G–J, Immunoblotting of WT and β-cateninΔ(ex3)/+ MEFs (G); HuH7 transfected with vector or β-cateninmut plasmid (H); β-cateninΔ(ex3)/+ MEFs, HepG2 and SNU398 treated with DMSO or 20 μM pri-724 for 48 h (I); β-cateninΔ(ex3)/+ MEFs and HepG2 cells transfected with control or Tcf7l2 siRNA (J). K–N, β-cateninΔ(ex3)/+ MEFs were transfected with control or Irp2 shRNA. Analysis of IRP2 abundance (K), cellular PGSK intensity (L), cell proliferation (M), and colony formation (N) were performed; n = 3. O, P, Correlation between CTNNB1 and IRP2 mRNA abundance. Data of cancer cell lines were generated from DepMap database (O). Data of HCC, COAD and UCEC were from GEPIA database (P). Data were shown as mean ± SD and analysis was performed using t test. *p < 0.05, **p < 0.01, ***p < 0.001

Full size image

To elucidate the significance of IRP2 in β-catenin-activated cells, we silenced Irp2 in β-cateninΔ(ex3)/+ MEFs (Fig. 4K). IRP2 knockdown dropped cellular LIP levels (Fig. 4L) and weakened cell viabilities (Fig. 4M, N). In addition, we validated the relationship between IRP2-TfR1 axis and β-catenin activity in human cancers. β-catenin mRNA was positively correlated with mRNAs of IRP2 and TfR1 in 1406 cancer cell lines (Fig. 4O, Additional file 1: Fig. S3A) and β-catenin-frequently activated human HCC, COAD and UCEC samples (Fig. 4P, Additional file 1: Fig. S3B). WNT/β-catenin-altered tumors contained more IRP2 and TfR1 than WNT/β-catenin-unaltered tumors (Additional file 1: Fig. S3C, D). β-catenin is thus positively correlated with IRP2-TfR1 signaling cascade in human cancers. Taken together, β-catenin-activated tumors potentiate Irp2 transcription to enhance cellular LIP levels and cell proliferation.

Iron depletion impairs mitochondrial function to suppress β-catenin-mediated tumor formation

Because iron-sulfur mediates electron transfer in mitochondrial respiratory chain, iron plays critical roles in maintenance of mitochondrial function [35]. Mitochondrial activity is enhanced in β-catenin-activated cancer to fuel tumorigenesis [5]. IRP2 knockdown dropped cellular OCR levels (Fig. 5A) and mitochondrial ATP production (Fig. 5B) in these cells. DFO also reduced OCR (Fig. 5C), mitochondrial ATP production and total ATP production (Fig. 5D) of β-cateninΔ(ex3)/+ MEFs. Similar to iron chelators, direct suppression of mitochondrial metabolism with oligomycin (mitochondrial complex I inhibitor) or S-Gboxin (ATP synthetase inhibitor) preferentially hindered β-cateninΔ(ex3)/+ MEF proliferation (Fig. 5E). In addition, S-Gboxin suppressed tumorigenicity of β-cateninΔ(ex3)/+ MEFs with minimal impact on mouse body weights (Fig. 5F–H), suggesting iron-mediated mitochondrial metabolism is a targetable vulnerability for β-catenin activated tumors.

Fig. 5
figure 5

Iron deprivation blunts β-catenin-activated tumor formation via impairing mitochondrial function A, B, oxygen consumption rate (OCR) (A, left) and ATP production rate (B, left) in β-cateninΔ(ex3)/+ MEFs transfected with control or IRP2 shRNA. OCR for basal and maximal respiration (A, right); Glycolytic and mitochondrial ATP production rates (B, right); n = 3. C, D, OCR (C, left) and ATP production rate (D, left) in WT and β-cateninΔ(ex3)/+ MEFs treated with control or 5 μM DFO for 48 h. OCR for basal and maximal respiration (C, right); Glycolytic and mitochondrial ATP production rates (D, right); n = 3. E, Viability of WT and β-cateninΔ(ex3)/+ MEFs treated with S-Gboxin or oligomycin A at different concentrations for 48 h; n = 5. F–H, Nude mice subcutaneously inoculated with β-cateninΔ(ex3)/+ MEFs were treated with PBS (n = 6) or S-Gboxin (10 mg/kg) every other day, n = 7. Tumor growth was calculated as the mean value in tumor volumes (F). Tumor images, tumor weights (G), and body weights (H) were plotted at the end of treatment. Data were shown as mean ± SD and analysis was performed using t test. *p < 0.05, **p < 0.01, ***p < 0.001

Full size image

Discussion

Β-catenin signaling pathway plays important roles in regulation of cell growth and proliferation. Gain-of-function mutations of β-catenin are common in human cancers. In this study, we found that oncogenic β-catenin augmented intracellular iron to promote cell proliferation and tumorigenesis. Mechanistically, β-catenin transcriptionally stimulated IRP2 expression to enhance cellular LIP which contributed to the enhanced mitochondrial function. The boosted IRP2-iron-mitochondrial function is druggable for β-catenin-activated tumors (Fig. 6).

Fig. 6
figure 6

Illustration of β-catenin-IRP2-iron stimulation of mitochondrial metabolism promotes cell proliferation

Full size image

No approved target therapy is available for β-catenin-activated tumor. Repurposing existing drugs, with the advantages of lower risk, shorter timelines, and less investment, expedites drug discovery and development. For example, Paxlovid is quickly approved for COVID-19 treatment [36]. Through screening of 1088 approved drugs, we discovered that iron chelators selectively suppressed β-catenin-activated cell viability. The first approved iron chelator for iron-overload-related disease, DFO, with higher safety and lower price than DFX [37], deterred β-catenin-activated tumorigenesis.

DFO exhibits anticancer effect in breast cancer cell lines and lung cancer cell-derived xenograft tumor [38, 39]. In combination with cytosine arabinoside, DFO causes striking cytoreduction in a patient with acute leukemia [40]. However, limited activity (20% overall response rate) was observed in 10 DFO-treated patients with treatment-refractory metastatic HCC [41]. 70% hepatic cancer without CTNNB1 mutation may contribute to the low response rate of genetically unclassified HCC to DFO [14, 15].

It is known that iron activates Wnt/β-catenin signaling [42,43,44,45]. We found elevated chelatable iron in β-catenin-activated cells. A potential positive feedback loop between β-catenin activation and iron overload may contribute to tumor development. Iron overload is associated with increased risk for cancers, particularly gastrointestinal cancers and HCC [46,47,48]. Excess iron accelerates HCC and CRC development in mice, whereas iron deprivation blocks myeloma cell growth and mouse CRC development [49,50,51,52]. In patients with chronic hepatitis C, long-term low-iron diet decreases the risk of HCC development [53]. Because iron chelation selectively reduced β-catenin-activated cell viability and tumor formation, we suggest that increased LIP is necessary in oncogenic β-catenin-induced cancer development.

β-catenin promotes stabilization and nuclear translocation of hypoxia inducible factor 1α (HIF-1α) [55]. Nuclear β-catenin cooperates with HIF-1α at the promoter regions of HIF-1α target genes to enhance HIF-1α-mediated transcription [56]. In addition, HIF-1α stimulates cellular iron uptake by inducing TfR1 expression [57,58,59,60]. Therefore, β-catenin enhancement of HIF-1α may contribute to β-catenin-stimulated iron accumulation we identified in this study. Intracellular iron homeostasis is strictly secured by IRP1/2 [54]. We demonstrated that transcriptional activation of IRP2 by β-catenin also contributed to the elevated LIP. Despite the well-established function of IRP2 in securing iron metabolism, little is known about its role in cancer development. We found that IRP2 knockdown not only decreased LIP but also blocked proliferation of β-catenin-activated cells.

Iron plays various roles such as mitochondrial metabolism, DNA synthesis, cell cycle progression and redox homeostasis, deprivation of iron may cause various pathological changes [29]. Iron chelating causes redox damage, cell cycle arrest, and DNA replication impairment to repress cell proliferation and tumorigenesis [50, 61,62,63]. We demonstrated that iron deprivation impaired β-catenin-invigorated mitochondrial activity. Therefore, iron chelators may play anti-tumor roles through multiple mechanisms in addition to mitochondrial dysfunction we presented here. Mitochondria bestow flexibility on tumor cells to adapt to cellular and environmental alterations [54]. Unlike most tumors that use the Warburg effect, β-catenin-activated tumors are addicted to mitochondria-supplied energy [5]. In this study, pharmaceutical inhibition of mitochondrial function by S-Gboxin hindered oncogenic β-catenin-mediated cell viability and tumor development. Thus, suppressing mitochondrial function is a promising therapeutic strategy for β-catenin-activated tumors.

Conclusions

Β-catenin-activated cells exhibit an augmented dependency on iron availability. Oncogenic β-catenin transactivates IRP2 to enhance mitochondrial function via raising intracellular LIP. Β-catenin/IRP2/iron-boosted mitochondrial energetics is essential for cell proliferation and tumor growth. Intervention of iron, IRP2 or mitochondrial function abolishes β-catenin-activated cancers. Since DFO is an old drug, its efficacy can be readily tested for the treatment of oncogenic β-catenin-related tumors.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Abbreviations

AKT2:

AKT Serine/Threonine Kinase 2

APC:

Adenomatous polyposis coli

CBP:

Cyclic adenosine monophosphate response element binding protein-binding protein

COAD:

Colon adenocarcinoma

CRC:

Colorectal cancer

CCK8:

Cell Counting Kit-8

DFO:

Deferoxamine mesylate

DFX:

Deferasirox

FAC:

Ferric ammonium citrate

FAO:

Fatty acid oxidation

FBXL5:

F-box/LRR-repeat protein 5

FGF:

Fibroblast growth factor

GLUL:

Glutamate-ammonia ligase

HCC:

Hepatocellular carcinoma

HIF-1α:

Hypoxia-inducible factor-1α

IRP2:

Iron-regulatory protein 2

LIP:

Labile iron pool

MEFs:

Mouse embryonic fibroblasts

OCR:

Oxygen consumption rate

PFA:

Paraformaldehyde

PGSK:

Phen Green™ SK

PK:

Pharmacokinetics

PPAP:

Peroxisome proliferator activated receptor alpha

TCF:

T cell factor

TCF7L2:

Transcription factor 7 like 2

TERT:

Telomerase reverse transcriptase

UCEC:

Uterine corpus endometrial carcinoma

WT:

Wildtype

References

  1. R Nusse H Clevers 2017 Wnt/beta-catenin signaling, disease, and emerging therapeutic modalities Cell 169 985 999

    Article  CAS  Google Scholar 

  2. O Tetsu F McCormick 1999 Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells Nature 398 422 426

    Article  CAS  Google Scholar 

  3. H Paek JY Hwang RS Zukin JM Hebert 2011 beta-Catenin-dependent FGF signaling sustains cell survival in the anterior embryonic head by countering Smad4 Dev Cell 20 689 699

    Article  CAS  Google Scholar 

  4. S Sekine BY Lan M Bedolli S Feng M Hebrok 2006 Liver-specific loss of beta-catenin blocks glutamine synthesis pathway activity and cytochrome p450 expression in mice Hepatology 43 817 825

    Article  CAS  Google Scholar 

  5. N Senni M Savall D Cabrerizo Granados MC Alves-Guerra C Sartor I Lagoutte A Gougelet B Terris H Gilgenkrantz C Perret 2019 beta-catenin-activated hepatocellular carcinomas are addicted to fatty acids Gut 68 322 334

    Article  CAS  Google Scholar 

  6. K Hoffmeyer A Raggioli S Rudloff R Anton A Hierholzer I Valle Del K Hein R Vogt R Kemler 2012 Wnt/beta-catenin signaling regulates telomerase in stem cells and cancer cells Science 336 1549 1554

    Article  CAS  Google Scholar 

  7. S Rebouissou A Franconi J Calderaro E Letouze S Imbeaud C Pilati JC Nault G Couchy A Laurent C Balabaud 2016 Genotype-phenotype correlation of CTNNB1 mutations reveals different ss-catenin activity associated with liver tumor progression Hepatology 64 2047 2061

    Article  CAS  Google Scholar 

  8. J Li X Ma D Chakravarti S Shalapour RA DePinho 2021 Genetic and biological hallmarks of colorectal cancer Genes Dev 35 787 820

    Article  CAS  Google Scholar 

  9. P Morice A Leary C Creutzberg N Abu-Rustum E Darai 2016 Endometrial cancer Lancet 387 1094 1108

    Article  Google Scholar 

  10. A Hayashi J Hong CA Iacobuzio-Donahue 2021 The pancreatic cancer genome revisited Nat Rev Gastroenterol Hepatol 18 469 481

    Article  Google Scholar 

  11. PJ Morin AB Sparks V Korinek N Barker H Clevers B Vogelstein KW Kinzler 1997 Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC Science 275 1787 1790

    Article  CAS  Google Scholar 

  12. W Liu X Dong M Mai RS Seelan K Taniguchi KK Krishnadath KC Halling JM Cunningham LA Boardman C Qian 2000 Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating beta-catenin/TCF signalling Nat Genet 26 146 147

    Article  CAS  Google Scholar 

  13. S Satoh Y Daigo Y Furukawa T Kato N Miwa T Nishiwaki T Kawasoe H Ishiguro M Fujita T Tokino 2000 AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1 Nat Genet 24 245 250

    Article  CAS  Google Scholar 

  14. E Cerami J Gao U Dogrusoz BE Gross SO Sumer BA Aksoy A Jacobsen CJ Byrne ML Heuer E Larsson 2012 The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data Cancer Discov 2 401 404

    Article  Google Scholar 

  15. J Gao BA Aksoy U Dogrusoz G Dresdner B Gross SO Sumer Y Sun A Jacobsen R Sinha E Larsson 2013 Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal Sci Signal 6 pl1

    Article  Google Scholar 

  16. H Liao X Li L Zhao Y Wang X Wang Y Wu X Zhou W Fu L Liu HG Hu YG Chen 2020 A PROTAC peptide induces durable beta-catenin degradation and suppresses Wnt-dependent intestinal cancer Cell Discov 6 35

    Article  CAS  Google Scholar 

  17. R Mathur L Sehgal FK Braun Z Berkova J Romaguerra M Wang MA Rodriguez L Fayad SS Neelapu F Samaniego 2015 Targeting Wnt pathway in mantle cell lymphoma-initiating cells J Hematol Oncol 8 63

    Article  Google Scholar 

  18. B Bilir O Kucuk CS Moreno 2013 Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells J Transl Med 11 280

    Article  Google Scholar 

  19. S Dandekar E Romanos-Sirakis F Pais T Bhatla C Jones W Bourgeois SP Hunger EA Raetz ML Hermiston R Dasgupta 2014 Wnt inhibition leads to improved chemosensitivity in paediatric acute lymphoblastic leukaemia Br J Haematol 167 87 99

    Article  CAS  Google Scholar 

  20. L Ji W Qian L Gui Z Ji P Yin GN Lin Y Wang B Ma WQ Gao 2020 Blockade of beta-catenin-induced ccl28 suppresses gastric cancer progression via inhibition of treg cell infiltration Cancer Res 80 2004 2016

    Article  CAS  Google Scholar 

  21. Y Zhang X Wang 2020 Targeting the Wnt/beta-catenin signaling pathway in cancer J Hematol Oncol 13 165

    Article  Google Scholar 

  22. M Kahn 2014 Can we safely target the WNT pathway? Nat Rev Drug Discov 13 513 532

    Article  CAS  Google Scholar 

  23. F Liu X Gai Y Wu B Zhang X Wu R Cheng B Tang K Shang N Zhao W Deng 2022 Oncogenic β-catenin stimulation of AKT2-CAD-mediated pyrimidine synthesis is targetable vulnerability in liver cancer Proc Natl Acad Sci U S A 119 e2202157119

    Article  CAS  Google Scholar 

  24. S Pushpakom F Iorio PA Eyers KJ Escott S Hopper A Wells A Doig T Guilliams J Latimer C McNamee 2019 Drug repurposing: progress, challenges and recommendations Nat Rev Drug Discov 18 41 58

    Article  CAS  Google Scholar 

  25. Z Tang B Kang C Li T Chen Z Zhang 2019 GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis Nucleic Acids Res 47 W556 W560

    Article  CAS  Google Scholar 

  26. DS Chandrashekar SK Karthikeyan PK Korla H Patel AR Shovon M Athar GJ Netto ZS Qin S Kumar U Manne 2022 UALCAN: an update to the integrated cancer data analysis platform Neoplasia 25 18 27

    Article  CAS  Google Scholar 

  27. JA Castro-Mondragon R Riudavets-Puig I Rauluseviciute RB Lemma L Turchi R Blanc-Mathieu J Lucas P Boddie A Khan N Manosalva Perez 2022 JASPAR 2022: the 9th release of the open-access database of transcription factor binding profiles Nucleic Acids Res 50 D165 D173

    Article  CAS  Google Scholar 

  28. S Entezari SM Haghi N Norouzkhani B Sahebnazar F Vosoughian D Akbarzadeh M Islampanah N Naghsh M Abbasalizadeh N Deravi 2022 Iron chelators in treatment of iron overload J Toxicol 2022 4911205

    Article  Google Scholar 

  29. SV Torti FM Torti 2013 Iron and cancer: more ore to be mined Nat Rev Cancer 13 342 355

    Article  CAS  Google Scholar 

  30. B Hassannia P Vandenabeele T Vanden Berghe 2019 Targeting ferroptosis to iron out cancer Cancer Cell 35 830 849

    Article  CAS  Google Scholar 

  31. Y Wang L Zheng W Shang Z Yang T Li F Liu W Shao L Lv L Chai L Qu 2022 Wnt/beta-catenin signaling confers ferroptosis resistance by targeting GPX4 in gastric cancer Cell Death Differ https://doi.org/10.1038/s41418-022-01008-w

    Article  Google Scholar 

  32. P Hatzis LG Flier van der MA Driel van V Guryev F Nielsen S Denissov IJ Nijman J Koster EE Santo W Welboren 2008 Genome-wide pattern of TCF7L2/TCF4 chromatin occupancy in colorectal cancer cells Mol Cell Biol 28 2732 2744

    Article  CAS  Google Scholar 

  33. AK Mayank V Pandey AA Vashisht WD Barshop S Rayatpisheh T Sharma T Haque DN Powers JA Wohlschlegel 2019 An oxygen-dependent interaction between FBXL5 and the CIA-targeting complex regulates iron homeostasis Mol Cell 75 382–393 e385

    Google Scholar 

  34. T Moroishi M Nishiyama Y Takeda K Iwai KI Nakayama 2011 The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo Cell Metab 14 339 351

    Article  CAS  Google Scholar 

  35. AD Read RE Bentley SL Archer KJ Dunham-Snary 2021 Mitochondrial iron-sulfur clusters: structure, function, and an emerging role in vascular biology Redox Biol 47 102164

    Article  CAS  Google Scholar 

  36. US Food and Drug Administration. https://www.fda.gov/media/155049/download.

  37. KJ Weigel SG Lynch SM LeVine 2014 Iron chelation and multiple sclerosis ASN Neuro 6 e00136

    Google Scholar 

  38. K Bajbouj J Shafarin M Hamad 2018 High-dose deferoxamine treatment disrupts intracellular iron homeostasis, reduces growth, and induces apoptosis in metastatic and nonmetastatic breast cancer cell lines Technol Cancer Res Treat 17 1533033818764470

    Article  CAS  Google Scholar 

  39. Y Kuang W Guo J Ling D Xu Y Liao H Zhao X Du H Wang M Xu H Song 2019 Iron-dependent CDK1 activity promotes lung carcinogenesis via activation of the GP130/STAT3 signaling pathway Cell Death Dis 10 297

    Article  Google Scholar 

  40. Z Estrov A Tawa XH Wang ID Dube H Sulh A Cohen EW Gelfand MH Freedman 1987 In vitro and in vivo effects of deferoxamine in neonatal acute leukemia Blood 69 757 761

    Article  CAS  Google Scholar 

  41. T Yamasaki S Terai I Sakaida 2011 Deferoxamine for advanced hepatocellular carcinoma N Engl J Med 365 576 578

    Article  CAS  Google Scholar 

  42. A Mandala A Armstrong B Girresch J Zhu A Chilakala S Chavalmane K Chaudhary P Biswas J Ogilvie JP Gnana-Prakasam 2020 Fenofibrate prevents iron induced activation of canonical Wnt/beta-catenin and oxidative stress signaling in the retina NPJ Aging Mech Dis 6 12

    Article  CAS  Google Scholar 

  43. S Radulescu MJ Brookes P Salgueiro RA Ridgway E McGhee K Anderson SJ Ford DH Stones TH Iqbal C Tselepis OJ Sansom 2016 Luminal Iron levels govern intestinal tumorigenesis after apc loss in vivo Cell Rep 17 2805 2807

    Article  CAS  Google Scholar 

  44. GS Coombs AA Schmitt CA Canning A Alok IC Low N Banerjee S Kaur V Utomo CM Jones S Pervaiz 2012 Modulation of Wnt/beta-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer Oncogene 31 213 225

    Article  CAS  Google Scholar 

  45. S Song T Christova S Perusini S Alizadeh RY Bao BW Miller R Hurren Y Jitkova M Gronda M Isaac 2011 Wnt inhibitor screen reveals iron dependence of beta-catenin signaling in cancers Cancer Res 71 7628 7639

    Article  CAS  Google Scholar 

  46. NM Bastide F Chenni M Audebert RL Santarelli S Tache N Naud M Baradat I Jouanin R Surya DA Hobbs 2015 A central role for heme iron in colon carcinogenesis associated with red meat intake Cancer Res 75 870 879

    Article  CAS  Google Scholar 

  47. C Chapoutot M Esslimani Z Joomaye J Ramos P Perney C Laurent P Fabbro-Peray D Larrey J Domergue F Blanc 2000 Liver iron excess in patients with hepatocellular carcinoma developed on viral C cirrhosis Gut 46 711 714

    Article  CAS  Google Scholar 

  48. C Niederau R Fischer A Purschel W Stremmel D Haussinger G Strohmeyer 1996 Long-term survival in patients with hereditary hemochromatosis Gastroenterology 110 1107 1119

    Article  CAS  Google Scholar 

  49. Y Muto T Moroishi K Ichihara M Nishiyama H Shimizu H Eguchi K Moriya K Koike K Mimori M Mori 2019 Disruption of FBXL5-mediated cellular iron homeostasis promotes liver carcinogenesis J Exp Med 216 950 965

    Article  CAS  Google Scholar 

  50. AJ Schwartz JW Goyert S Solanki SA Kerk B Chen C Castillo PP Hsu BT Do R Singhal MK Dame 2021 Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells Nat Metab 3 969 982

    Article  CAS  Google Scholar 

  51. Z Gu H Wang J Xia Y Yang Z Jin H Xu J Shi I Domenico De G Tricot F Zhan 2015 Decreased ferroportin promotes myeloma cell growth and osteoclast differentiation Cancer Res 75 2211 2221

    Article  CAS  Google Scholar 

  52. X Xue SK Ramakrishnan K Weisz D Triner L Xie D Attili A Pant B Gyorffy M Zhan C Carter-Su 2016 Iron Uptake via DMT1 integrates cell cycle with JAK-STAT3 signaling to promote colorectal tumorigenesis Cell Metab 24 447 461

    Article  CAS  Google Scholar 

  53. J Kato K Miyanishi M Kobune T Nakamura K Takada R Takimoto Y Kawano S Takahashi M Takahashi Y Sato 2007 Long-term phlebotomy with low-iron diet therapy lowers risk of development of hepatocellular carcinoma from chronic hepatitis C J Gastroenterol 42 830 836

    Article  CAS  Google Scholar 

  54. M Arredondo MT Nunez 2005 Iron and copper metabolism Mol Aspects Med 26 313 327

    Article  CAS  Google Scholar 

  55. S Dong S Liang Z Cheng X Zhang L Luo L Li W Zhang S Li Q Xu M Zhong 2022 ROS/PI3K/Akt and Wnt/beta-catenin signalings activate HIF-1alpha-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer J Exp Clin Cancer Res 41 15

    Article  CAS  Google Scholar 

  56. A Kaidi AC Williams C Paraskeva 2007 Interaction between beta-catenin and HIF-1 promotes cellular adaptation to hypoxia Nat Cell Biol 9 210 217

    Article  CAS  Google Scholar 

  57. L Tacchini L Bianchi A Bernelli-Zazzera G Cairo 1999 Transferrin receptor induction by hypoxia. HIF-1-mediated transcriptional activation and cell-specific post-transcriptional regulation J Biol Chem 274 24142 24146

    Article  CAS  Google Scholar 

  58. CN Lok P Ponka 1999 Identification of a hypoxia response element in the transferrin receptor gene J Biol Chem 274 24147 24152

    Article  CAS  Google Scholar 

  59. H Ding CZ Yan H Shi YS Zhao SY Chang P Yu WS Wu CY Zhao YZ Chang XL Duan 2011 Hepcidin is involved in iron regulation in the ischemic brain PLoS ONE 6 e25324

    Article  CAS  Google Scholar 

  60. RR Fagundes AR Bourgonje S Hu R Barbieri BH Jansen N Sinnema T Blokzijl CT Taylor RK Weersma KN Faber G Dijkstra 2022 HIF1alpha-dependent induction of TFRC by a combination of intestinal inflammation and systemic iron deficiency in inflammatory bowel disease Front Physiol 13 889091

    Article  Google Scholar 

  61. M Fiorillo F Toth M Brindisi F Sotgia MP Lisanti 2020 Deferiprone (DFP) targets cancer stem cell (CSC) propagation by inhibiting mitochondrial metabolism and inducing ROS production Cells 9 6 1526

    Article  Google Scholar 

  62. JH Choi JS Kim YW Won J Uhm BB Park YY Lee 2016 The potential of deferasirox as a novel therapeutic modality in gastric cancer World J Surg Oncol 14 77

    Article  Google Scholar 

  63. LM Bystrom ML Guzman S Rivella 2014 Iron and reactive oxygen species: friends or foes of cancer cells? Antioxid Redox Signal 20 1917 1924

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge support from Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College.

Funding

This work was supported by Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (CIFMS) (2021-1-I2M-018), Haihe Laboratory of Cell Ecosystem Innovation Fund (HH22KYZX0012), National Natural Science Foundation of China (81730078, 81872287).

Author information

Author notes

  1. Yuting Wu and Shuhui Yang have equally contributed to this work

Authors and Affiliations

  1. State Key Laboratory of Medical Molecular Biology, Haihe Laboratory of Cell Ecosystem, Department of Physiology, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, 5 Dong Dan San Tiao, Beijing, China

    Yuting Wu, Shuhui Yang, Luyang Han, Kezhuo Shang, Xiaochen Gai, Weiwei Deng, Fangming Liu & Hongbing Zhang

  2. Department of Physiology, School of Life Science, China Medical University, Shenyang, China

    Baohui Zhang

Authors
  1. Yuting Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuhui Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Luyang Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kezhuo Shang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Baohui Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiaochen Gai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Weiwei Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fangming Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hongbing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YW and SY designed and performed most experiments, interpreted, analyzed the data, and wrote the manuscript. LH performed xenograft tumor experiments. BZ performed drug screening and cell line establishment. KS performed flow cytometry experiment. XG and WD assisted the study. FL and HZ supervised this study and revised the article. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Fangming Liu or Hongbing Zhang.

Ethics declarations

Ethical approval and consent to participate

Our study was approved by the Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College (ethical code: ACUC-A02-2022–057). Experimental procedures were compliant with the regulation of Beijing Administration Office of Laboratory Animal.

Consent for publication

Consent to publish has been obtained from all authors.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

Figure S1. Iron addition has no impact on cell proliferation of WT and β-cateninΔ(ex3)/+ MEFs. Figure S2. β-catenin-activated MEFs are resistant to ferroptosis induction. Figure S3. β-catenin is positively correlated with IRP2 and TfR1. Table S1. Mutations of cell lines.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wu, Y., Yang, S., Han, L. et al. β-catenin-IRP2-primed iron availability to mitochondrial metabolism is druggable for active β-catenin-mediated cancer. J Transl Med 21, 50 (2023). https://doi.org/10.1186/s12967-023-03914-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12967-023-03914-0

Keywords

Journal of Translational Medicine

ISSN: 1479-5876

Contact us