Ferritinophagy Loss Drives Mitochondrial Iron Import and Colorectal Tumorigenesis

Ferritinophagy Loss Drives Mitochondrial Iron Import and Colorectal Tumorigenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ferritinophagy Loss Drives Mitochondrial Iron Import and Colorectal Tumorigenesis Xiang Xue, Hyeoncheol Kim, Luke Villareal, Naiara Santana-Codina, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7483419/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Iron is an essential cofactor for mitochondrial metabolism, yet the regulatory networks linking cellular iron homeostasis to colorectal cancer (CRC) progression remain incompletely understood. Here, we identify nuclear receptor coactivator 4 (NCOA4), a ferritinophagy receptor, as a context-dependent tumor suppressor that coordinates cytosolic and mitochondrial iron handling in CRC. Analysis of human tumors and colon-specific Ncoa4 knockout mice revealed that NCOA4 loss drives tumorigenesis by inducing transferrin receptor–mediated iron uptake and mitochondrial calcium uniporter (MCU)–dependent mitochondrial iron import. This dual iron overload elevates mitochondrial reactive oxygen species, activates STAT3 signaling, and enhances tumor cell proliferation. NCOA4 overexpression reverses these effects, reducing MCU expression and tumor growth. Pharmacological inhibition of MCU, STAT3, or mitochondrial iron transport mitigated tumorigenesis in NCOA4-deficient models. Our findings define an NCOA4–MCU–STAT3 metabolic signaling axis that couples iron metabolism to oncogenic progression and reveal mitochondrial iron handling as a therapeutic vulnerability in CRC. Health sciences/Gastroenterology/Gastrointestinal diseases/Gastrointestinal cancer/Colorectal cancer/Colon cancer Biological sciences/Biochemistry/Metals/Iron Iron Metabolism Colorectal Carcinogenesis Reactive Oxygen Species Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Colorectal cancer (CRC) is a major global health problem and a leading cause of cancer-related deaths in the United States (Rawla et al., 2019 ). By 2040, CRC is projected to have the highest global incidence among all cancers, driven by population aging, growth, and lifestyle-related risk factors (Soerjomataram et al., 2021). Although U.S. incidence declined for decades due to screening and preventive measures, CRC remains the third most common cancer and the third leading cause of cancer death (Siegel et al., 2023). In 2023, an estimated 153,020 new cases and 52,550 deaths were reported, with annual treatment costs expected to reach $ 21 billion by 2030 (Stukalin et al., 2022 ). Despite medical advances, survival for advanced CRC remains poor, underscoring the need for novel therapeutic strategies. Iron, an essential dietary micronutrient, has been linked to CRC risk. Epidemiological studies show that high iron levels increase CRC risk, whereas iron reduction decreases it (Lee et al., 2004 ; Osborne et al., 2010 ; Zacharski et al., 2008 ). Human and mouse colon tumors exhibit elevated iron compared to normal tissue (Pusatcioglu et al., 2014 ; Xue et al., 2016 ), and dietary iron restriction reduces tumor burden in preclinical models (Xue et al., 2012 ), while high iron intake promotes growth (Radulescu et al., 2012 ). However, iron deficiency anemia in many CRC patients limits systemic iron deprivation as a therapy. Mitochondria are central to iron metabolism, producing iron–sulfur clusters and driving ROS generation that supports tumor proliferation (Rouault et al., 2005; Dixon et al., 2014). We previously showed that targeting mitochondrial iron with the FDA-approved chelator deferiprone or reducing ROS with TEMPO suppresses colon tumorigenesis without affecting systemic iron (Xue et al., 2017 ), highlighting mitochondrial iron metabolism as a therapeutic target. Ferritinophagy, mediated by nuclear receptor coactivator 4 (NCOA4), regulates iron release from ferritin for cellular use (Mancias et al., 2014 ). It plays critical roles in erythropoiesis (Santana-Codina et al., 2019 ; Ryu et al., 2017 ) and ferroptosis—an iron-dependent cell death pathway (Dixon et al., 2012 ; Yu et al., 2022 ). Dysregulated ferritinophagy contributes to cancer, including therapy resistance in pancreatic tumors via enhanced mitochondrial iron delivery (Jain et al., 2022). NCOA4’s effects on iron metabolism are tissue-specific, as whole-body knockout reduces systemic iron but increases tissue iron in the liver, spleen, and intestines (Bellelli et al., 2016 ). Liver-specific NCOA4 knockdown increases hepatic iron levels, while overexpression reduces them (Li et al., 2020 ; Li et al., 2022 ). However, its role in colonic iron homeostasis and CRC remains unclear. Here, we show that NCOA4 expression is reduced in human CRC and that colon-specific NCOA4 deletion in mice accelerates tumorigenesis, whereas overexpression suppresses it. NCOA4 deficiency caused cytosolic and mitochondrial iron accumulation, increased ROS, and enhanced TFRC-mediated iron uptake. Proteomics revealed upregulation of the mitochondrial calcium uniporter (MCU), promoting mitochondrial iron loading, ROS production, and activation of oncogenic STAT3 signaling (Xue et al., 2016 ). These findings identify NCOA4 as a key regulator of iron metabolism and CRC progression, with potential as a therapeutic target. Results NCOA4 is decreased in colon tumors and predicts CRC patient survival Bioinformatic analysis revealed that NCOA4 expression is significantly reduced in human colon tumors, with the lowest levels observed in metastatic tissues ( Fig. 1A ). Consistent with these findings, our independent analysis of colorectal cancer (CRC) samples from our institution demonstrated a similar downregulation of NCOA4 mRNA in tumor tissues compared with normal colonic mucosa ( Fig. 1B ). Immunoblotting confirmed reduced NCOA4 protein levels in tumor samples ( Figs. 1C, 1D ), whereas FTH1—a marker of NCOA4 inactivation (Santana-Codina and Mancias, 2018 )—was moderately upregulated in the same samples ( Figs. 1C, 1E ). Reduced NCOA4 mRNA expression has been associated with poorer cancer patient survival (Gu et al., 2022 ). Analysis of a colorectal adenocarcinoma dataset (TCGA, PanCancer Atlas) in cBioPortal (Cerami et al., 2012) revealed that ~ 2.5% of CRC patients (13/526) harbor NCOA4 mutations (missense, truncating, or deep deletions) ( Fig. S1A ). Progression-free survival was significantly lower in patients with NCOA4 mutations compared to those without (p = 0.0102) ( Fig. S1B ). Kaplan–Meier survival curves from the KMPlotter database similarly indicated that low NCOA4 expression is associated with poorer overall survival in CRC patients ( Fig. 1F ), and high FTH1 expression correlated with unfavorable prognosis ( Fig. 1G ). Together, these findings demonstrate that NCOA4 downregulation, accompanied by FTH1 accumulation, is associated with colorectal tumorigenesis and poor clinical outcomes, supporting a critical role for NCOA4 in CRC pathophysiology. Under iron-replete conditions, NCOA4 undergoes proteasomal degradation (Mancias et al., 2015 ). We observed reduced NCOA4 levels in colon tumors from Cdx2 Cre-ERT2 Apc F/+ mice mice (Xue et al., 2016 ) fed a normal chow diet, as well as in normal colon tissues of mice fed a high-iron diet ( Figs. S1C, S1D ). These results suggest that NCOA4 downregulation is conserved between humans and mice, validating mouse models as relevant systems for studying human colon tumorigenesis. Colon-specific NCOA4 deficiency promotes tumor development in mouse models of CRC To investigate the role of NCOA4 in colon tumorigenesis, we generated Cdx2 ERT2-Cre Ncoa4 F/F mice, enabling colon epithelial cell–specific deletion of Ncoa4 . Using a well-established colitis-associated cancer (CAC) model induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) (Thaker et al., 2012 ; Lee et al., 2022 ), we found that both tumor number and burden were significantly increased in Cdx2 ERT2-Cre Ncoa4 F/F mice following tamoxifen (TAM) administration to activate Cre recombinase ( Figs. S2A–S2C ). Histological analysis with hematoxylin and eosin (H&E) staining revealed increased high-grade dysplasia in Ncoa4 -deficient colons compared with controls ( Figs. S2D, S2E ). Immunofluorescence analysis showed increased Ki-67 expression ( Figs. S2D, S2F ), indicating enhanced proliferation, while cleaved caspase-3 (CC3), a marker of apoptosis, showed no significant change ( Figs. S2D, S2G ). To further validate these findings, we generated mice with monoallelic Apc deletion ( Cdx2 ERT2-Cre Ncoa4 F/F Apc F/+ and control Cdx2 ERT2-Cre Ncoa4 +/+ Apc F/+ ). These mice develop colorectal tumors following repeated DSS exposure. After TAM-induced recombination and DSS treatment, tumor number and burden were significantly elevated in Ncoa4 -deficient Apc F/+ mice compared with controls ( Figs. 2A–2C ). H&E staining confirmed more extensive high-grade dysplasia in Cdx2 ERT2-Cre Ncoa4 F/F Apc F/+ mice ( Figs. 2D, 2E ). Immunofluorescence again showed increased Ki-67 and reduced CC3 ( Figs. 2D, 2F, 2G ). Collectively, results from two independent models—AOM/DSS-induced CAC and monoallelic Apc deletion—demonstrate that NCOA4 loss promotes colon tumorigenesis, likely by facilitating tumor initiation and progression through increased epithelial proliferation and dysplastic transformation. Ncoa4 knockout increases intracellular iron, ATP levels, and xenograft tumor growth Given that Ncoa4 deletion enhances colon tumor formation in vivo , we next examined its effects on CRC cell behavior and tumor progression. NCOA4 was stably knocked out in human HCT116 cells, confirmed by qPCR ( Fig. 3A ) and immunoblotting ( Fig. 3B ). Consistent with previous studies (Bellelli et al., 2016 ), NCOA4 knockout resulted in FTH1 upregulation ( Fig. 3B ). Total intracellular iron levels were significantly elevated in knockout cells, as measured by inductively coupled plasma mass spectrometry (ICP-MS; Fig. 3C ). Similarly, Ncoa4 deletion in mouse MC38 CRC cells led to increased FTH1, elevated intracellular iron ( Figs. 3D–3F ), increased ATP levels ( Figs. 3G, 3J ), and enhanced tumor growth in vivo ( Figs. 3H, 3K ), with NCOA4 downregulation confirmed in each case ( Figs. 3I, 3L ). Collectively, these results show that NCOA4 loss causes intracellular iron overload, promoting higher ATP production and tumor growth. NCOA4 depletion increases iron uptake–related but not autophagy-related proteins in colon tumors NCOA4 functions both as a canonical autophagy receptor and a driver of ferritin condensate formation, which are degraded via macroautophagy and endosomal microautophagy (Ohshima et al., 2022 ). Since autophagy supports mitochondrial function by regulating iron metabolism in pancreatic cancer (Mukhopadhyay et al., 2023 ), we examined whether autophagy contributes to enhanced colon tumor growth in the context of Ncoa4 deficiency. Immunoblotting showed that Ncoa4 deletion did not alter key autophagy-related proteins (ATG5, ATG16L, p62, LC3-II/I, p-S6K) in colon tumors ( Fig. S3A ). Furthermore, treatment with the autophagy inhibitor chloroquine failed to suppress increased xenograft tumor growth derived from MC38 sg Ncoa4 cells in C57BL/6 mice ( Fig. S3B ), suggesting that neither canonical autophagy nor ferritinophagy primarily drive tumor growth following Ncoa4 loss. This aligns with previous reports indicating ferritinophagy is not required for colon cancer cell growth (Hasan et al., 2020 ). In contrast, iron regulatory protein 2 (IRP2) and its downstream effector transferrin receptor (TFRC)—key mediators of cellular iron uptake (Kim et al., 2023 ; Zhao et al., 2024 )—were significantly upregulated in Ncoa4 -deficient cells and xenograft tumors ( Figs. 4A, 4B ). Hypoxia-inducible factor 2α (HIF-2α) and its downstream targets, Six-Transmembrane Epithelial Antigen of the Prostate 4 (STEAP4) and divalent metal transporter 1 (DMT1)—which promote a pro-tumorigenic iron-dependent state (Cheng et al., 2013 ; Xue et al., 2016 , 2017 )—were also elevated in Ncoa4 knockout models ( Figs. 4A, 4B ). qPCR analysis confirmed increased Tfrc , Dmt1 , and Steap4 expression in colon tumors from Cdx2 ERT2-Cre Ncoa4 F/F Apc F/+ mice ( Figs. 4C–4E ). Immunoblotting corroborated increased TFRC, HIF-2α, DMT1, and STEAP4 proteins in these tumors ( Fig. 4F ). Since NCOA4 was initially characterized as an androgen receptor (AR) coactivator (Yeh et al., 1996), and AR protein negatively correlates with TFRC levels in human CRC (Firehose Legacy dataset; Fig. S3C ), we hypothesized AR antagonism might influence TFRC expression. However, treatment with the AR antagonist bicalutamide repressed TFRC protein at high doses ( Fig. S3D ), suggesting AR signaling does not significantly mediate Ncoa4 deficiency–induced tumor growth. Together, these data support a model whereby Ncoa4 depletion suppresses ferritinophagy and stabilizes FTH1, causing iron sequestration in ferritin that mimics iron starvation. This triggers IRP2–TFRC and HIF-2α pathways to enhance iron uptake and accumulation in CRC ( Fig. 4G ), promoting tumorigenesis through pro-tumorigenic iron signaling. TFRC depletion abolishes NCOA4 deficiency–enhanced colon tumorigenesis Given the critical role of iron accumulation mediated by the IRP2/TFRC axis in CRC (Lee et al., 2004 ; Shen et al., 2018 ; Yang et al., 2022 ; Kim et al., 2023 ), we tested whether inhibiting iron uptake could mitigate tumorigenesis induced by Ncoa4 deletion. Genetic knockout of Tfrc in Cdx2 ERT2-Cre Ncoa4 F/F Apc F/+ mice abolished tumor formation driven by Ncoa4 loss ( Figs. S4A–S4C ). H&E and immunofluorescence staining showed that increased proliferation (Ki-67) observed in Ncoa4 -deficient tumors was reversed by Tfrc deletion ( Figs. S4D, S4E ). Conversely, apoptosis marker cleaved caspase-3 (CC3) was unchanged in Ncoa4 -deficient tumors but significantly elevated in Ncoa4 / Tfrc double knockout tumors ( Figs. S4D, S4F ). These findings demonstrate that TFRC upregulation is essential for Ncoa4 deletion–induced colorectal tumorigenesis. Ncoa4 knockout robustly increases MCU mRNA and protein levels in mouse colon tissues To explore downstream effects of iron accumulation following Ncoa4 deletion, we performed unbiased quantitative proteomics comparing colon tissues from Cdx2 ERT2-Cre Ncoa4 +/+ and Cdx2 ERT2-Cre Ncoa4 F/F mice ( Table S1 ). Metascape Enriched Ontology Clustering ( Fig. S5A ) and Gene Set Enrichment Analysis ( Fig. 5A ) revealed robust upregulation of mitochondrial proteins, particularly those involved in mitochondrial calcium ion transport. Volcano plot ( Fig. S5B ) and heatmap ( Figs. 5B, 5C ) analyses identified the mitochondrial calcium uniporter (MCU) as one of the most significantly increased proteins. qPCR ( Fig. 5D ) and immunoblot ( Fig. 5E ) analyses confirmed significantly elevated Mcu mRNA and MCU protein levels in colon tissues from Cdx2 ERT2-Cre Ncoa4 F/F mice versus controls. MCU protein was also markedly increased in colon tumors from Cdx2 ERT2-Cre Ncoa4 F/F Apc F/+ mice compared to controls ( Fig. 5F ). These data indicate that Ncoa4 knockout induces MCU upregulation in the colon. AMPK–CREB signaling pathway is required for MCU induction in NCOA4 knockout cells MCU expression is transcriptionally regulated by phosphorylated CREB1 (Takahashi et al., 2019 ; Shanmughapriya et al., 2015 ), which is modulated by phosphorylated AMPK. AMPK activity is influenced by reactive oxygen species (ROS) (Thomson et al., 2008 ; Hwang et al., 2014 ; Agostini et al., 2023 ). Since iron catalyzes ROS production via Fenton chemistry, we assessed intracellular iron and ROS in HCT116 sg NCOA4 cells. Both intracellular iron ( Fig. 6A ) and total ROS ( Fig. 6B ) were elevated. Proteins involved in the p-AMPK/p-CREB1/MCU axis were upregulated in NCOA4 -deleted cells ( Fig. 6C ). Treatment with the AMPK inhibitor Compound C decreased p-AMPK, p-CREB1, and MCU levels in human and mouse Ncoa4 -deficient cells ( Figs. 6D, S4C ). Similarly, the CREB1 inhibitor KG-501 suppressed p-CREB1 and MCU expression ( Figs. 6E, S5D ). MCU regulates mitochondrial calcium uptake and metabolism during CRC progression (Zeng et al., 2018 ; Liu et al., 2020 ) and mediates mitochondrial import of divalent metals including manganese and iron (Wettmarshausen et al., 2018 ; Sripetchwandee et al., 2014 ; Arcos et al., 2025 ). Increased MCU expression in NCOA4 -deficient cells suggests enhanced mitochondrial iron import, elevating mitochondrial ROS (mtROS) (Xue et al., 2017 ). Indeed, both mitochondrial iron and mtROS were elevated in NCOA4 knockout cells but were reduced by the MCU inhibitor minocycline (Schwartz et al., 2013 ; Hu et al., 2024 ; Arcos et al., 2025 ) ( Figs. 6F, 6G ). Treatment with the mitochondrial ROS scavenger mito-tempo decreased mtROS and downregulated p-AMPK, p-CREB1, and MCU protein levels ( Fig. 6H ). These data support a model wherein NCOA4 loss promotes intracellular iron accumulation and ROS production, activating AMPK–CREB1 signaling, which induces MCU expression and further mitochondrial iron and ROS accumulation ( Fig. S5E ). NCOA4 deletion alters oxidative stress response in colon tumors and cells We observed that the master antioxidant regulator nuclear factor erythroid 2-related factor 2 (NRF2) downstream targets—Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1)—were significantly reduced in colon tumors from Ncoa4 knockout mice, while Kelch-like ECH-associated protein 1 (KEAP1) levels remained unchanged ( Fig. S6A ). Similar reductions in HO-1 and NQO1 were seen in MC38 Ncoa4 knockout cells ( Fig. S6B ). Consistently, human NQO1 antioxidant response element (ARE) luciferase reporter activity was decreased in MC38 Ncoa4 knockout cells ( Fig. S6C ). Markers of oxidative stress, including lipid peroxidation marker 4-hydroxynonenal (4-HNE), NQO1, glutathione peroxidase 4 (GPX4), and the cystine/glutamate antiporter xCT were elevated in vitro ( Fig. S6D ). In xenograft tumors, only xCT was reduced significantly, while other oxidative stress markers showed no changes ( Fig. S6E ). This discrepancy may reflect the presence of dietary vitamin E, a lipid peroxidation scavenger abundant in chow diets but absent in culture media. Vitamin E is known to protect against lipid peroxidation and ferroptosis (Hu et al., 2021 ). These findings suggest that increased proliferation in Ncoa4 -deficient cells is balanced by enhanced lipid peroxidation–induced ferroptosis in vitro . In vivo , ferroptosis may be attenuated by antioxidant mechanisms such as dietary vitamin E. Supporting this, feeding mice a vitamin E-deficient diet for one week abolished the tumor-promoting effect of Ncoa4 knockout ( Figs. S6F, S6G ). These data implicate ferroptosis sensitivity in the tumorigenic effects of Ncoa4 deficiency (Anandhan et al., 2023 ). NCOA4 deletion activates STAT3 signaling via MCU-mediated mitochondrial iron accumulation Iron accumulation can activate STAT3 via phosphorylation (Xue et al., 2016 ). Consistent with this, Ncoa4 deletion markedly increased STAT3 activity, as measured by a Sis-Inducible Element (SIE) luciferase assay ( Fig. S7A ). Pharmacological inhibition of MCU with minocycline significantly reduced phosphorylated STAT3 (p-STAT3) expression ( Fig. S7B ) and suppressed enhanced xenograft growth driven by Ncoa4 deficiency ( Figs. S7C, S7D ). Furthermore, treatment with the STAT3 inhibitor NSC78956 or the iron chelator deferiprone (DFP) effectively attenuated tumor growth in Ncoa4 deletion models ( Figs. S7E, S7F ). These results support a model whereby Ncoa4 deletion promotes mitochondrial iron accumulation and oxidative stress via MCU upregulation, activating pro-tumorigenic signaling including STAT3. NCOA4 overexpression inhibits colorectal cancer progression via MCU-dependent mitochondrial iron regulation To further validate NCOA4’s tumor-suppressive role via MCU regulation, we used a mouse model with inducible NCOA4 overexpression ( Cdx2 ERT2-Cre NCOA4 LSL/+ and Cdx2 ERT2-Cre NCOA4 LSL/LSL alleles) combined with Apc F/+ to induce colon tumors via TAM and DSS treatment. Gross colon images showed visibly fewer and smaller tumors in homozygous NCOA4 LSL/LSL mice compared to heterozygous or wild-type controls ( Fig. 7A ). Quantitative tumor counts and average tumor size were significantly reduced in the homozygous group ( Figs. 7B–7D ), with tumor burden also markedly decreased ( Fig. 7E ). qPCR and immunoblot confirmed increased Ncoa4 mRNA and protein expression in Cdx2 ERT2-Cre NCOA4 LSL/+ and Cdx2 ERT2-Cre NCOA4 LSL/LSL mice ( Figs. 7F, 7H ). Notably, only homozygous Cdx2 ERT2-Cre NCOA4 LSL/LSL mice exhibited decreased Mcu mRNA and protein levels ( Figs. 7G, 7H ), indicating a mechanistic link between NCOA4 overexpression and reduced mitochondrial iron uptake. Together, these results support that NCOA4 overexpression inhibits colon tumor development, at least in part, by downregulating MCU and modulating mitochondrial iron metabolism. Discussion Our study uncovers key upstream mechanisms of iron-driven colorectal tumorigenesis and identifies novel therapeutic targets centered on the regulation of ferritinophagy. While previous reports have demonstrated that NCOA4-mediated ferritinophagy can suppress tumor growth in cancers such as pancreatic cancer and acute myeloid leukemia (Santana-Codina et al., 2022 ; Ravichandran et al., 2022 ; Larrue et al., 2024 ), our findings—consistent with a bioinformatic study linking lower NCOA4 levels to poorer prognosis in CRC (Gu et al., 2022 )—reveal a contrasting role for NCOA4 in colorectal cancer. Specifically, we show that loss of NCOA4 increases both cytosolic and mitochondrial iron levels, thereby promoting CRC cell growth and supporting a pro-tumorigenic role for NCOA4 deficiency in the colorectal tumor microenvironment. This work represents one of the first experimental validations of NCOA4 as a context-dependent oncogenic modifier in CRC, challenging the prevailing notion that NCOA4 inhibition is universally tumor-suppressive. Our results underscore the importance of evaluating the role of NCOA4 and ferritinophagy in a cancer-type–specific manner and highlight the therapeutic potential of targeting iron regulatory pathways in colorectal cancer. Mechanistically, loss of NCOA4 impairs ferritinophagy, leading to iron sequestration and a cellular iron starvation response. This response drives the upregulation of iron uptake machinery, including the IRP2–TFRC axis and HIF-2α–DMT1/STEAP4 pathway, which collectively promote intracellular iron accumulation and CRC progression. Our extensive prior work (Xue et al., 2012 , 2016 , 2017 ; Schwartz et al., 2021 ; Kim et al., 2023 ; Villareal et al., 2024 ; Arcos et al., 2025 ) and that of others (Pusatcioglu et al., 2014 ; Radulescu et al., 2012 ) consistently demonstrate that CRC tumors accumulate iron to sustain growth and progression, reinforcing iron metabolism as a promising therapeutic target. Importantly, our data reveal a novel link between NCOA4 deletion and mitochondrial iron overload, resulting in elevated mtROS. Proteomic analyses indicated that canonical mitochondrial iron transporters, mitoferrin-1 (MFRN1) and MFRN2, were not significantly upregulated. Instead, the mitochondrial calcium uniporter (MCU) emerged as one of the most highly expressed proteins associated with mitochondrial iron accumulation. This mitochondrial dysfunction appears to be driven by MCU upregulation, which facilitates not only calcium influx but also mitochondrial iron uptake (Zhang et al., 2019 ; Arcos et al., 2025 ). MCU overexpression and the resulting increase in mtROS serve as second messengers to activate downstream oncogenic pathways—most notably the iron- and ROS-dependent STAT3 signaling cascade (Xue et al., 2016 ; Cao et al., 2020 ). STAT3 activation is well established in cancer and linked to enhanced proliferation, survival, immune evasion, and metastasis (Lee et al., 2019 ; Wang et al., 2022 ; Li et al., 2025 ), further emphasizing the oncogenic potential driven by NCOA4 loss. The reciprocal relationship between NCOA4 and MCU expression is particularly noteworthy. NCOA4 overexpression suppresses CRC tumorigenesis and correlates with reduced MCU levels, suggesting that NCOA4 maintains mitochondrial homeostasis by limiting MCU-mediated iron influx. This balance is critical, as mitochondrial iron and ROS levels are tightly connected to cellular bioenergetics, apoptosis, and redox signaling (Richardson et al., 2010 ; Nakamura and Takada, 2021 ; Zhao et al., 2024 ). Disruption of this equilibrium through NCOA4 deletion leads to metabolic reprogramming conducive to tumor growth (Liu et al., 2022 ). Future studies generating NCOA4 and MCU double knockout mice would be valuable to directly test MCU’s role in mitochondrial iron accumulation in the absence of NCOA4. Our findings underscore the complex interplay between iron metabolism, mitochondrial function, and oncogenic signaling in CRC. They suggest that targeting iron uptake pathways, mitochondrial iron handling, or downstream effectors such as STAT3 could represent viable therapeutic strategies, especially in tumors characterized by NCOA4 loss or low expression. Therapeutic approaches using iron chelators or MCU inhibitors—alone or combined with STAT3 pathway antagonists—warrant further investigation to counteract NCOA4 deficiency–driven tumorigenesis. Our study also indicates that ferritinophagy actively maintains iron homeostasis in CRC cells, with the observed phenotypes likely reflecting long-term consequences of NCOA4 ablation. It would be valuable to assess effects at earlier time points post-NCOA4 deletion—using inducible knockdown systems—to determine whether acute NCOA4 loss produces similar or distinct outcomes on cell growth and iron metabolism. Specifically, it will be important to examine if acute NCOA4 ablation transiently decreases cytosolic iron, triggering compensatory TFRC upregulation and possibly resulting in an overshoot toward iron overload. In summary, our study elucidates a tumor-promoting function of NCOA4 loss in colorectal cancer through dysregulation of iron homeostasis and mitochondrial dynamics. These insights reveal novel molecular targets and pathways for therapeutic development, which may ultimately improve outcomes for patients with NCOA4-deficient colorectal cancers. Materials and Methods Cell Culture Human HCT116 (RRID: CVCL_0291) and mouse MC38 (RRID: CVCL_B288) colorectal cancer cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO₂. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Cat. #11965092, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Cat. #F2442-500ML, Millipore Sigma, Burlington, MA) and 1% penicillin-streptomycin (10,000 U/mL; Cat. #15140122, Thermo Fisher Scientific). To generate stable NCOA4 knockout cell lines (sg NCOA4 ), HCT116 and MC38 cells were transfected with px459-NCOA4 or empty control plasmids using Lipofectamine 2000 (Cat. #11668027, Thermo Fisher Scientific). After transfection, cells were selected with puromycin to establish knockout clones. Animals Animal studies were conducted to address specific research questions, adhering to the Institute of Laboratory Animal Resources guidelines, and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of New Mexico Health Sciences Center (Protocol# 23-201434-HSC), following the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). Mice, encompassing both sexes, were housed in standard cages under a 12-h light–dark cycle, in a temperature-controlled environment, with ad libitum access to a standard chow diet and water unless otherwise specified. Colon-specific NCOA4 knockout and overexpression mouse models were generated by crossing Ncoa4 floxed ( Ncoa4 F/F ) and NCOA4 LSL/LSL mice (Santana-Codina et al., 2022 ) with tamoxifen-inducible Cdx2 ERT2 − Cre mice (Yin et al., 2025 ). These crosses produced Cdx2 ERT2 − Cre Ncoa4 F/F and Cdx2 ERT2 − Cre NCOA4 LSL/LSL mice, allowing selective deletion or overexpression of NCOA4 in the colon epithelium upon tamoxifen treatment. Colorectal cancer (CRC) was modeled using two established approaches. In the colitis-associated cancer (CAC) model, mice received intraperitoneal injections of azoxymethane (AOM) at 1 mg/mL to deliver a total dose of 10 mg/kg over two consecutive days. Beginning the day after AOM injection, tamoxifen was administered at 100 mg/kg for three consecutive days to induce NCOA4 deletion. Following tamoxifen treatment, mice were given 2% dextran sodium sulfate (DSS) in drinking water for seven days to induce colonic inflammation, followed by 14 days of regular water for tissue recovery. This inflammatory and recovery cycle was repeated once. In the second model, CRC was induced by combining monoallelic Apc deletion with DSS treatment. Cdx2 ERT2 − Cre Apc F/+ mice, Cdx2 ERT2 − Cre Ncoa4 F/F Apc F/+ mice and Cdx2 ERT2 − Cre NCOA4 LSL/LSL Apc F/+ mice were generated. After tamoxifen-induced recombination, mice received 2% DSS in drinking water for seven days, followed by 28 days of recovery with regular water. These models allowed assessment of NCOA4’s role in genetically predisposed and inflammation-driven tumorigenesis. For in vivo tumor growth studies, MC38 murine CRC cells with stable NCOA4 knockout were injected subcutaneously into the flanks of syngeneic C57BL/6 mice at 1 × 10⁶ cells per site. Tumors were harvested after two weeks. For pharmacological inhibition studies, treatment began one week after injection once tumors were palpable. Mice were treated with vehicle control, minocycline at 20 mg/kg daily, NSC74859 (a STAT3 inhibitor) at 5 mg/kg intraperitoneally every other day, a Vitamin E-deficient diet (TD.88163, Envigo), a Vitamin E control diet (50 IU, TD.99455, Envigo), or deferiprone (DFP) at 1 mg/mL in drinking water. Treatments lasted for one week. Histology, Immunofluorescence, and DAB-Enhanced Perl’s Iron Staining Formalin-fixed, paraffin-embedded colon tissue sections were processed for hematoxylin and eosin (H&E) staining, immunofluorescence (IF), and iron staining. For H&E staining, sections were deparaffinized and rehydrated through graded ethanol to distilled water. Sections were stained with hematoxylin for two minutes, rinsed in tap water, dipped briefly in bluing solution, and rinsed again for two minutes. Eosin staining was performed for five minutes, followed by dehydration through ethanol series, clearing in xylene, and mounting for histopathological evaluation by a gastrointestinal pathologist (Dr. Martin) in a blind manner. For immunofluorescence, antigen retrieval was performed by incubating sections in 10 mM sodium citrate buffer at sub-boiling temperature for 12 minutes. Slides were cooled to room temperature for two hours and then blocked with 10% normal goat serum in 0.1% Triton X-100 in phosphate-buffered saline (PBS) or Tris-buffered saline (TBS) for one hour. Primary antibodies were diluted in 1% normal goat serum with 0.1% PBST or TBST and incubated overnight at 4°C. After washing, fluorophore-conjugated secondary antibodies diluted in 1% normal goat serum were applied for one hour at room temperature. Slides were washed and mounted using EverBrite™ Mounting Medium (Biotium). Primary antibodies used included cleaved Caspase-3 (CC3, #9664), Ki-67 (#12202), and phospho-STAT3 (p-STAT3, #9145) from Cell Signaling Technology. FerroOrange, Mito-Ferro Green, DCFH-DA, and MitoSOX Staining Cells were seeded at 2 × 10⁵ per well in 24-well plates. After adherence, cells were stained in pre-warmed Hank’s Balanced Salt Solution (HBSS) containing fluorescent probes: 0.5 µM FerroOrange for cytosolic ferrous iron (Dojindo), 1 µM Mito-Ferro Green for mitochondrial iron, 10 µM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA; Cayman Chemical) for total cellular reactive oxygen species (ROS), and 1 µM MitoSOX (Thermo Fisher Scientific) for mitochondrial superoxide. Staining was performed at 37°C for 30 minutes, followed by washing with PBS. Fluorescence images were captured using an Invitrogen™ EVOS™ FL Auto Imaging System. The RFP channel was used for FerroOrange and MitoSOX, while the GFP channel was used for DCFH-DA and Mito-Ferro Green. ImageJ software was used to quantify fluorescence intensity normalized to Hoechst 33342 nuclear staining. Luciferase reporter gene assay To measure luciferase activity, cells were seeded at 5 × 10⁴ per well in 24-well plates and transfected using polyethylenimine (PEI) with either the pGL4.47[luc2P/SIE-RE/Hygro] reporter plasmid containing a STAT3-responsive element alongside a LacZ plasmid for normalization, or the human NQO1-ARE TATA-Inr luciferase reporter plasmid (for MC38 wild-type and sgNcoa4 cells, Yin et al., 2025 ). After 48 hours, cells were lysed in lysis buffer. Luciferase activity was then quantified using a Promega Luciferase Assay Kit (E1500), with 20 µL of supernatant added to a 96-well plate and 100 µL of luciferase reagent per well. Luminescence intensity was measured by a plate reader and normalized to protein concentration. Quantitative Polymerase Chain Reaction (qPCR) Analysis Total RNA was extracted using the IBI Isolate DNA/RNA Reagent Kit following manufacturer’s instructions. For RNA from DSS-treated colon tissues, an additional purification was performed by precipitating RNA with 8 M lithium chloride (LiCl), followed by ethanol washes to improve RNA quality. RNA was resuspended in DEPC-treated water and quantified. Quantitative PCR was conducted using the LightCycler 480 system (Roche Diagnostics) with gene-specific primers listed in Table S2 . Relative gene expression was calculated by the ΔΔCt method using housekeeping genes for normalization. Immunoblotting Analysis Cells and tumor tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer. Lysates were centrifuged, and protein concentration was determined using a BioTek Synergy HTX Multi-Mode Microplate Reader. Equal amounts of protein (10–50 µg) were loaded on SDS-PAGE gels, separated by electrophoresis, and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk and incubated overnight with primary antibodies, followed by incubation with appropriate secondary antibodies for 1.5 hours. The antibodies used are listed in Table S3 . ATP Measurement Cells (5 × 10³) were seeded in white opaque 96-well plates. After 48 hours, an equal volume of CellTiter-Glo® 2D Cell Viability Assay reagent was added. Luminescence was measured after 10 minutes using a SpectraMax M5 Microplate Reader. MTT Assay Cells were seeded at 5 × 10⁴ cells/mL in 24-well plates. After treatments, 125 µL of 5 mg/mL MTT solution was added and incubated at 37°C for 30 minutes. Formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured with a BioTek Synergy HTX Microplate Reader. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Analysis Tissue samples were digested overnight in concentrated nitric acid and diluted with Milli-Q water. Samples were analyzed on an Agilent 7900 ICP-MS instrument at the University of New Mexico Health Sciences Center. Iron content was normalized to tissue weight. Unbiased Quantitative Proteomics Study Ncoa4 F/F and Cdx2 ERT2 − Cre Ncoa4 F/F mice were treated with tamoxifen (100 mg/kg daily for three days) to induce colon-specific Ncoa4 deletion. One week after the final injection, mice were sacrificed, and colonic epithelial cells were isolated by scraping the luminal surface. Proteomic analysis was performed as described previously (Santana-Codina et al., 2022 ). Statistical Analysis Data are presented as mean ± standard deviation. Statistical significance was evaluated using independent or paired t-tests, one-way or two-way ANOVA where appropriate. A p-value of less than 0.05 was considered statistically significant. Declarations Declarations of Interest The authors declare no competing interests. Declaration of Generative AI and AI-Assisted Technologies in the Writing Process During the preparation of this work, the authors used ChatGPT to improve language and readability. Following the use of this tool, the authors carefully reviewed and edited the content and take full responsibility for the final publication. Ethics Statement for Human The study was approved by the University of New Mexico Health Sciences Center Institutional Review Board (protocol #19–131). Human tissue samples were obtained from the University of New Mexico Comprehensive Cancer Center Human Tissue Repository and the Cooperative Human Tissue Network (CHTN). Written informed consent was obtained from all participants, and patient data were anonymized to ensure confidentiality. Ethics Statement for Animal Animal studies were conducted to address specific research questions, adhering to the Institute of Laboratory Animal Resources guidelines, and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of New Mexico Health Sciences Center (Protocol# 23-201434-HSC), following the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). Patient and public involvement Patients and the public were not involved in the design, conduct, reporting, or dissemination plans of this research. Funding X.X. was supported by National Institutes of Health grants R01ES035780, 3P30CA118100-19S3, and P20GM130422; the HSC Pilot Funding Program from the UNM HSC Office of Research; the Dedicated Health Research Funds from the Research Allocation Committee of the UNM School of Medicine; the UNMCCC Research Program Support Pilot Project Award and Off-Setting Cuts Pilot Project (P30CA118100); and the Health Sciences & Main Campus Research Collaboration Seed Grant Award from UNM Rainforest Innovations. This research was also partially supported by the UNM Comprehensive Cancer Center Support Grant NCI P30CA118100 through Graduate Student Support Pilot Grant #1484. L.B.V. received support from The Infectious Disease and Immunity Program at the UNM Health Sciences Center (T32AI007538). C.V.C. was supported by the NIGMS Institutional Research and Academic Career Development Award (K12GM088021) and the UNM METALS Superfund Research Center (P42ES025589). E.R.P. was supported by NIH grants P30CA118100 and P20GM121176. Author Contributions H.K., L.B.V., L.G., C.V.C., and X.X. performed the experiments. E.R.P. provided guidance on androgen receptor studies. N.S. and J.D.M. conducted the unbiased proteomics study. D.R.M. evaluated histological samples. H.K., L.B.V., and X.X. drafted the manuscript and prepared figures. X.X. designed the project and edited the final manuscript. Acknowledgements We thank Dr. Miljan Kuljanin for assistance with mass spectrometry-based proteomics. Data availability statement All data relevant to the study are included in the article or uploaded as online supplemental information. Additional data are available from the corresponding author upon reasonable request. References Agostini F, Bisaglia M, Plotegher N (2023) Linking ROS Levels to Autophagy: The Key Role of AMPK. 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Cancer Res 78:2876–2885 Additional Declarations There is NO Competing Interest. Supplementary Files TableS1XueProteomedataNSC.xlsx Supplemental Table 1 250806SupplementalTable2and3.docx Supplemental Table 2 and 3 250809SupplementaryfiguresV1.docx Supplementary figures Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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12:14:08","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167569,"visible":true,"origin":"","legend":"","description":"","filename":"rs74834192structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/702dfff445be37f550a52f12.xml"},{"id":91986640,"identity":"f5e13f65-cfdf-4f44-ab1e-fe4885dad1ca","added_by":"auto","created_at":"2025-09-23 12:06:08","extension":"html","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":178277,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/5007a5c6d61b2f46a15af8cc.html"},{"id":91986628,"identity":"39e89bc1-9c89-4b25-9f03-86fcf0d34dc1","added_by":"auto","created_at":"2025-09-23 12:06:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":798073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNCOA4 is decreased in colon tumors and predicts CRC patient survival.\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) NCOA4 mRNA expression in human normal (n = 377), tumor (n = 1450), and metastatic (n = 99) colon tissues from the TNMplot database. (\u003cstrong\u003eB\u003c/strong\u003e) qPCR analysis of colorectal tumors (n = 11) and their adjacent normal colon tissues (n = 10). (\u003cstrong\u003eC\u003c/strong\u003e) Immunoblotting analysis and quantification of (\u003cstrong\u003eD\u003c/strong\u003e) NCOA4 and (\u003cstrong\u003eE\u003c/strong\u003e) FTH1 protein levels from colorectal tumors and adjacent normal colon tissues. Kaplan–Meier survival curves of (\u003cstrong\u003eF\u003c/strong\u003e) NCOA4 and (\u003cstrong\u003eG\u003c/strong\u003e) FTH1 gene expression in colorectal tumor patients using data from the KMPlotter database. RFS, relapse-free survival. *p \u0026lt; 0.05, ****p \u0026lt; 0.0001. Unpaired Student’s t-test for B, D, and E.\u003c/p\u003e","description":"","filename":"250809NCOA4figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/d4283b9e109dc479be20b1ab.png"},{"id":91986632,"identity":"9c134132-6d6b-49e3-8b25-ec82978889cc","added_by":"auto","created_at":"2025-09-23 12:06:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2909896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColon-specific NCOA4 knockout increases colon tumorigenesis.\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) Tumor number, (\u003cstrong\u003eB\u003c/strong\u003e) tumor number by size, (\u003cstrong\u003eC\u003c/strong\u003e) tumor burden, (\u003cstrong\u003eD\u003c/strong\u003e) representative histological images, and quantification of (\u003cstrong\u003eE\u003c/strong\u003e) high-grade dysplasia, (\u003cstrong\u003eF\u003c/strong\u003e) Ki-67+ cells, and (\u003cstrong\u003eG\u003c/strong\u003e) cleaved caspase 3 (CC3)+ cells in colon tumors from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e (n = 3-11) and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003emice (n = 3-5). *p \u0026lt; 0.05, **p \u0026lt; 0.01. Unpaired Student’s t-test for A, C, E–G. Two-way ANOVA with Sidak’s multiple comparisons test for B.\u003c/p\u003e","description":"","filename":"250809NCOA4figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/f312c96ae56b82346a6c54f9.png"},{"id":91986630,"identity":"e61beacb-25f5-4a67-b2b9-9e9bf8dfe8c7","added_by":"auto","created_at":"2025-09-23 12:06:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":367280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNCOA4 knockout increases intracellular iron, ATP levels, and xenograft tumor growth in CRC cells.\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) qPCR, (\u003cstrong\u003eB\u003c/strong\u003e) immunoblotting, and (\u003cstrong\u003eC\u003c/strong\u003e) ICP-MS analysis of iron levels in HCT116 cells (n = 3–6) with NCOA4 knockout (sg\u003cem\u003eNCOA4\u003c/em\u003e) or wild-type control (sgEV). (\u003cstrong\u003eD\u003c/strong\u003e) qPCR, (\u003cstrong\u003eE\u003c/strong\u003e) immunoblotting, and (\u003cstrong\u003eF\u003c/strong\u003e) ICP-MS analysis of iron levels in MC38 cells (n = 3–14) with Ncoa4 knockout (sg\u003cem\u003eNcoa4\u003c/em\u003e) or wild-type control (sgEV). (\u003cstrong\u003eG\u003c/strong\u003e) CellTiter-Glo assay for HCT116 sgEV and sg\u003cem\u003eNCOA4\u003c/em\u003e cells. (\u003cstrong\u003eH\u003c/strong\u003e) Tumor weight and (\u003cstrong\u003eI\u003c/strong\u003e) NCOA4 expression in xenograft tumors derived from HCT116 sgNCOA4 (n = 5–8) or sgEV (n = 4–8) cells. (\u003cstrong\u003eJ\u003c/strong\u003e) CellTiter-Glo assay for MC38 sgEV and sg\u003cem\u003eNcoa4\u003c/em\u003e cells. (\u003cstrong\u003eK\u003c/strong\u003e) Tumor weight and (\u003cstrong\u003eL\u003c/strong\u003e) Ncoa4 expression in xenograft tumors derived from MC38 sg\u003cem\u003eNcoa4\u003c/em\u003e (n = 4–5) or sgEV (n = 4–6) cells. Values above blots are mean ± S.D. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. Unpaired Student’s t-test.\u003c/p\u003e","description":"","filename":"250809NCOA4figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/7d1d3254f90063a2755da126.png"},{"id":91986635,"identity":"22922c9f-c6bf-45d0-ac8d-38d51e4619dc","added_by":"auto","created_at":"2025-09-23 12:06:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":812868,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNCOA4 depletion induces iron uptake machinery in CRC.\u003c/strong\u003e\u003cbr\u003e\nImmunoblotting analysis in (\u003cstrong\u003eA\u003c/strong\u003e) HCT116 sgEV and sg\u003cem\u003eNCOA4\u003c/em\u003e cells and (\u003cstrong\u003eB\u003c/strong\u003e) their derived xenografts. qPCR analysis of (\u003cem\u003eC\u003c/em\u003e) Tfrc, (\u003cstrong\u003eD\u003c/strong\u003e) Dmt1, and (\u003cstrong\u003eE\u003c/strong\u003e) Steap4, and (\u003cstrong\u003eF\u003c/strong\u003e) immunoblotting analysis for colon tissues from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003emice (n = 3) and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003emice (n = 3). (\u003cstrong\u003eG\u003c/strong\u003e) Proposed mechanism: NCOA4 depletion represses ferritinophagy and stabilizes FTH1, causing iron sequestration in ferritin and mimicking iron starvation. This induces IRP2-TFRC and HIF-2α-DMT1/STEAP4 signaling, increasing cellular iron uptake and cytosolic free iron. Values above blots are mean ± S.D. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. Unpaired Student’s t-test for A, B, and F. Two-way ANOVA with Sidak’s multiple comparisons test for C–E.\u003c/p\u003e","description":"","filename":"250809NCOA4figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/5a64c094d2c3ae0358be110f.png"},{"id":91986636,"identity":"2138e701-ae57-424f-ad5c-a1e87a4cede9","added_by":"auto","created_at":"2025-09-23 12:06:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":525833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNCOA4 knockout robustly increases mRNA and protein levels of mitochondrial calcium uniporter (MCU) in mouse colon tissues.\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) Gene set enrichment analysis from unbiased quantitative proteomics showing enrichment of mitochondrial calcium ion transport proteins. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap of enriched mitochondrial calcium ion transport proteins. (\u003cstrong\u003eC\u003c/strong\u003e) Quantitative proteomics values for MCU. (\u003cstrong\u003eD\u003c/strong\u003e) qPCR and (\u003cstrong\u003eE\u003c/strong\u003e) immunoblotting analysis of colon tissues from colon epithelial cell-specific NCOA4 knockout mice (\u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e, n = 3–6) and wild-type controls (\u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, n = 3–6). (\u003cstrong\u003eF\u003c/strong\u003e) Immunoblotting analysis of colon tumor tissues from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+ \u003c/sup\u003emice (n = 3) and wild-type controls (n = 3). *p \u0026lt; 0.05, **p \u0026lt; 0.01. Unpaired Student’s t-test.\u003c/p\u003e","description":"","filename":"250809NCOA4figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/a04abd869649647b195b1b15.png"},{"id":91986633,"identity":"9556a213-e357-4b1a-a430-63c99f679dd8","added_by":"auto","created_at":"2025-09-23 12:06:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":617750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMPK-CREB signaling pathway is required for MCU induction in NCOA4 knockout cells.\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) FerroOrange staining quantification, (\u003cstrong\u003eB\u003c/strong\u003e) DCFH-DA staining, and (\u003cstrong\u003eC\u003c/strong\u003e) immunoblotting analysis in human HCT116 sg\u003cem\u003eNCOA4\u003c/em\u003e (n = 3) and sgEV control cells (n = 3). Immunoblotting analysis of HCT116 sg\u003cem\u003eNCOA4\u003c/em\u003eand sgEV cells treated with (\u003cstrong\u003eD\u003c/strong\u003e) AMPK inhibitor compound C or (\u003cstrong\u003eE\u003c/strong\u003e) CREB inhibitor KG-501. (\u003cstrong\u003eF\u003c/strong\u003e) Mitochondrial iron measured by MitoFerroGreen staining and (\u003cstrong\u003eG\u003c/strong\u003e) mitochondrial ROS measured by MitoSOX staining in HCT116 sg\u003cem\u003eNCOA4\u003c/em\u003e and sgEV cells treated with DMSO or MCU inhibitor minocycline. (\u003cstrong\u003eH\u003c/strong\u003e) Immunoblotting analysis of HCT116 sg\u003cem\u003eNCOA4\u003c/em\u003e and sgEV cells treated with or without mitochondrial ROS inhibitor mito-Tempol. Values above blots are mean ± S.D. or means only. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. untreated sgEV; #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001 vs. untreated sgNCOA4. Unpaired Student’s t-test for A–C. Two-way ANOVA with Sidak’s multiple comparisons test for D–H.\u003c/p\u003e","description":"","filename":"250809NCOA4figures6.png","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/5d034f469d8e7440ebd2fb27.png"},{"id":91986638,"identity":"986990ca-6451-419d-b918-cf6096811a92","added_by":"auto","created_at":"2025-09-23 12:06:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1484594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHomozygous overexpression of NCOA4 in mice represses colon tumor growth via MCU suppression.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative gross images; (\u003cstrong\u003eB\u003c/strong\u003e) tumor count; (\u003cstrong\u003eC\u003c/strong\u003e) tumor count based on size; (\u003cstrong\u003eD\u003c/strong\u003e) average tumor size; (\u003cstrong\u003eE\u003c/strong\u003e) tumor burden; qPCR analysis of (\u003cstrong\u003eF\u003c/strong\u003e) \u003cem\u003eNCOA4 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003eG\u003c/strong\u003e)\u003cem\u003e Mcu, \u003c/em\u003e(\u003cstrong\u003eH\u003c/strong\u003e) Immunoblot analysis of colon tumors from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e\u003cem\u003e NCOA4\u003c/em\u003e\u003csup\u003eLSL/+\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e (n = 3-4), \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e\u003cem\u003e NCOA4\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e (n = 3-11), and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e\u003cem\u003e NCOA4\u003c/em\u003e\u003csup\u003eLSL/LSL\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+ \u003c/sup\u003e(n = 3-12). Values above blots are mean ± S.D. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test for B, D, E-H. Two-way ANOVA followed with Sidak’s multiple comparisons test for C.\u003c/p\u003e","description":"","filename":"250809NCOA4figures7.png","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/994a355ca5e8c252bac987b2.png"},{"id":91988499,"identity":"25154695-4721-4387-b1b3-9f558e903dc7","added_by":"auto","created_at":"2025-09-23 12:22:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9558529,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/a460cfa6-dbf6-428d-aa18-1a3a9c072261.pdf"},{"id":91986641,"identity":"fa2874f8-320c-444b-a803-c3a496cb47b3","added_by":"auto","created_at":"2025-09-23 12:06:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3503761,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Table 1\u003c/p\u003e","description":"","filename":"TableS1XueProteomedataNSC.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/f23a0ecb2db7f4fe0bc1f2d3.xlsx"},{"id":91988158,"identity":"d5cb9ad9-5185-44a1-977e-11c02e21a0c2","added_by":"auto","created_at":"2025-09-23 12:14:08","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18697,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Table 2 and 3\u003c/p\u003e","description":"","filename":"250806SupplementalTable2and3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/1ccf0f50efea31f1089ebcb4.docx"},{"id":91986643,"identity":"0709637f-031e-430c-a6b7-ec56e07fae5a","added_by":"auto","created_at":"2025-09-23 12:06:09","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19333704,"visible":true,"origin":"","legend":"Supplementary figures","description":"","filename":"250809SupplementaryfiguresV1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7483419/v1/5237d5f51999adbadb780db0.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Ferritinophagy Loss Drives Mitochondrial Iron Import and Colorectal Tumorigenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is a major global health problem and a leading cause of cancer-related deaths in the United States (Rawla et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). By 2040, CRC is projected to have the highest global incidence among all cancers, driven by population aging, growth, and lifestyle-related risk factors (Soerjomataram et al., 2021). Although U.S. incidence declined for decades due to screening and preventive measures, CRC remains the third most common cancer and the third leading cause of cancer death (Siegel et al., 2023). In 2023, an estimated 153,020 new cases and 52,550 deaths were reported, with annual treatment costs expected to reach \u003cspan\u003e$\u003c/span\u003e21\u0026nbsp;billion by 2030 (Stukalin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite medical advances, survival for advanced CRC remains poor, underscoring the need for novel therapeutic strategies.\u003c/p\u003e\u003cp\u003eIron, an essential dietary micronutrient, has been linked to CRC risk. Epidemiological studies show that high iron levels increase CRC risk, whereas iron reduction decreases it (Lee et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Osborne et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zacharski et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Human and mouse colon tumors exhibit elevated iron compared to normal tissue (Pusatcioglu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xue et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and dietary iron restriction reduces tumor burden in preclinical models (Xue et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), while high iron intake promotes growth (Radulescu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, iron deficiency anemia in many CRC patients limits systemic iron deprivation as a therapy.\u003c/p\u003e\u003cp\u003eMitochondria are central to iron metabolism, producing iron\u0026ndash;sulfur clusters and driving ROS generation that supports tumor proliferation (Rouault et al., 2005; Dixon et al., 2014). We previously showed that targeting mitochondrial iron with the FDA-approved chelator deferiprone or reducing ROS with TEMPO suppresses colon tumorigenesis without affecting systemic iron (Xue et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), highlighting mitochondrial iron metabolism as a therapeutic target.\u003c/p\u003e\u003cp\u003eFerritinophagy, mediated by nuclear receptor coactivator 4 (NCOA4), regulates iron release from ferritin for cellular use (Mancias et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It plays critical roles in erythropoiesis (Santana-Codina et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ryu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and ferroptosis\u0026mdash;an iron-dependent cell death pathway (Dixon et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Dysregulated ferritinophagy contributes to cancer, including therapy resistance in pancreatic tumors via enhanced mitochondrial iron delivery (Jain et al., 2022). NCOA4\u0026rsquo;s effects on iron metabolism are tissue-specific, as whole-body knockout reduces systemic iron but increases tissue iron in the liver, spleen, and intestines (Bellelli et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Liver-specific NCOA4 knockdown increases hepatic iron levels, while overexpression reduces them (Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, its role in colonic iron homeostasis and CRC remains unclear.\u003c/p\u003e\u003cp\u003eHere, we show that NCOA4 expression is reduced in human CRC and that colon-specific NCOA4 deletion in mice accelerates tumorigenesis, whereas overexpression suppresses it. NCOA4 deficiency caused cytosolic and mitochondrial iron accumulation, increased ROS, and enhanced TFRC-mediated iron uptake. Proteomics revealed upregulation of the mitochondrial calcium uniporter (MCU), promoting mitochondrial iron loading, ROS production, and activation of oncogenic STAT3 signaling (Xue et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These findings identify NCOA4 as a key regulator of iron metabolism and CRC progression, with potential as a therapeutic target.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eNCOA4 is decreased in colon tumors and predicts CRC patient survival\u003c/h2\u003e\u003cp\u003eBioinformatic analysis revealed that \u003cem\u003eNCOA4\u003c/em\u003e expression is significantly reduced in human colon tumors, with the lowest levels observed in metastatic tissues (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). Consistent with these findings, our independent analysis of colorectal cancer (CRC) samples from our institution demonstrated a similar downregulation of \u003cem\u003eNCOA4\u003c/em\u003e mRNA in tumor tissues compared with normal colonic mucosa (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e). Immunoblotting confirmed reduced NCOA4 protein levels in tumor samples (\u003cb\u003eFigs.\u0026nbsp;1C, 1D\u003c/b\u003e), whereas FTH1\u0026mdash;a marker of NCOA4 inactivation (Santana-Codina and Mancias, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u0026mdash;was moderately upregulated in the same samples (\u003cb\u003eFigs.\u0026nbsp;1C, 1E\u003c/b\u003e). Reduced \u003cem\u003eNCOA4\u003c/em\u003e mRNA expression has been associated with poorer cancer patient survival (Gu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Analysis of a colorectal adenocarcinoma dataset (TCGA, PanCancer Atlas) in cBioPortal (Cerami et al., 2012) revealed that ~\u0026thinsp;2.5% of CRC patients (13/526) harbor \u003cem\u003eNCOA4\u003c/em\u003e mutations (missense, truncating, or deep deletions) (\u003cb\u003eFig. S1A\u003c/b\u003e). Progression-free survival was significantly lower in patients with \u003cem\u003eNCOA4\u003c/em\u003e mutations compared to those without (p\u0026thinsp;=\u0026thinsp;0.0102) (\u003cb\u003eFig. S1B\u003c/b\u003e). Kaplan\u0026ndash;Meier survival curves from the KMPlotter database similarly indicated that low \u003cem\u003eNCOA4\u003c/em\u003e expression is associated with poorer overall survival in CRC patients (\u003cb\u003eFig.\u0026nbsp;1F\u003c/b\u003e), and high FTH1 expression correlated with unfavorable prognosis (\u003cb\u003eFig.\u0026nbsp;1G\u003c/b\u003e). Together, these findings demonstrate that \u003cem\u003eNCOA4\u003c/em\u003e downregulation, accompanied by FTH1 accumulation, is associated with colorectal tumorigenesis and poor clinical outcomes, supporting a critical role for NCOA4 in CRC pathophysiology.\u003c/p\u003e\u003cp\u003eUnder iron-replete conditions, NCOA4 undergoes proteasomal degradation (Mancias et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We observed reduced NCOA4 levels in colon tumors from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eCre-ERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e mice mice (Xue et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) fed a normal chow diet, as well as in normal colon tissues of mice fed a high-iron diet (\u003cb\u003eFigs. S1C, S1D\u003c/b\u003e). These results suggest that NCOA4 downregulation is conserved between humans and mice, validating mouse models as relevant systems for studying human colon tumorigenesis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eColon-specific NCOA4 deficiency promotes tumor development in mouse models of CRC\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of NCOA4 in colon tumorigenesis, we generated \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice, enabling colon epithelial cell\u0026ndash;specific deletion of \u003cem\u003eNcoa4\u003c/em\u003e. Using a well-established colitis-associated cancer (CAC) model induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) (Thaker et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), we found that both tumor number and burden were significantly increased in \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice following tamoxifen (TAM) administration to activate Cre recombinase (\u003cb\u003eFigs. S2A\u0026ndash;S2C\u003c/b\u003e). Histological analysis with hematoxylin and eosin (H\u0026amp;E) staining revealed increased high-grade dysplasia in \u003cem\u003eNcoa4\u003c/em\u003e-deficient colons compared with controls (\u003cb\u003eFigs. S2D, S2E\u003c/b\u003e). Immunofluorescence analysis showed increased Ki-67 expression (\u003cb\u003eFigs. S2D, S2F\u003c/b\u003e), indicating enhanced proliferation, while cleaved caspase-3 (CC3), a marker of apoptosis, showed no significant change (\u003cb\u003eFigs. S2D, S2G\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo further validate these findings, we generated mice with monoallelic \u003cem\u003eApc\u003c/em\u003e deletion (\u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e and control \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e). These mice develop colorectal tumors following repeated DSS exposure. After TAM-induced recombination and DSS treatment, tumor number and burden were significantly elevated in \u003cem\u003eNcoa4\u003c/em\u003e-deficient \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e mice compared with controls (\u003cb\u003eFigs.\u0026nbsp;2A\u0026ndash;2C\u003c/b\u003e). H\u0026amp;E staining confirmed more extensive high-grade dysplasia in \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e mice (\u003cb\u003eFigs.\u0026nbsp;2D, 2E\u003c/b\u003e). Immunofluorescence again showed increased Ki-67 and reduced CC3 (\u003cb\u003eFigs.\u0026nbsp;2D, 2F, 2G\u003c/b\u003e). Collectively, results from two independent models\u0026mdash;AOM/DSS-induced CAC and monoallelic \u003cem\u003eApc\u003c/em\u003e deletion\u0026mdash;demonstrate that NCOA4 loss promotes colon tumorigenesis, likely by facilitating tumor initiation and progression through increased epithelial proliferation and dysplastic transformation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNcoa4\u003c/b\u003e \u003cb\u003eknockout increases intracellular iron, ATP levels, and xenograft tumor growth\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that \u003cem\u003eNcoa4\u003c/em\u003e deletion enhances colon tumor formation \u003cem\u003ein vivo\u003c/em\u003e, we next examined its effects on CRC cell behavior and tumor progression. \u003cem\u003eNCOA4\u003c/em\u003e was stably knocked out in human HCT116 cells, confirmed by qPCR (\u003cb\u003eFig.\u0026nbsp;3A\u003c/b\u003e) and immunoblotting (\u003cb\u003eFig.\u0026nbsp;3B\u003c/b\u003e). Consistent with previous studies (Bellelli et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), NCOA4 knockout resulted in FTH1 upregulation (\u003cb\u003eFig.\u0026nbsp;3B\u003c/b\u003e). Total intracellular iron levels were significantly elevated in knockout cells, as measured by inductively coupled plasma mass spectrometry (ICP-MS; \u003cb\u003eFig.\u0026nbsp;3C\u003c/b\u003e). Similarly, \u003cem\u003eNcoa4\u003c/em\u003e deletion in mouse MC38 CRC cells led to increased FTH1, elevated intracellular iron (\u003cb\u003eFigs.\u0026nbsp;3D\u0026ndash;3F\u003c/b\u003e), increased ATP levels (\u003cb\u003eFigs.\u0026nbsp;3G, 3J\u003c/b\u003e), and enhanced tumor growth \u003cem\u003ein vivo\u003c/em\u003e (\u003cb\u003eFigs.\u0026nbsp;3H, 3K\u003c/b\u003e), with NCOA4 downregulation confirmed in each case (\u003cb\u003eFigs.\u0026nbsp;3I, 3L\u003c/b\u003e). Collectively, these results show that NCOA4 loss causes intracellular iron overload, promoting higher ATP production and tumor growth.\u003c/p\u003e\n\u003ch3\u003eNCOA4 depletion increases iron uptake–related but not autophagy-related proteins in colon tumors\u003c/h3\u003e\n\u003cp\u003eNCOA4 functions both as a canonical autophagy receptor and a driver of ferritin condensate formation, which are degraded via macroautophagy and endosomal microautophagy (Ohshima et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Since autophagy supports mitochondrial function by regulating iron metabolism in pancreatic cancer (Mukhopadhyay et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we examined whether autophagy contributes to enhanced colon tumor growth in the context of \u003cem\u003eNcoa4\u003c/em\u003e deficiency.\u003c/p\u003e\u003cp\u003eImmunoblotting showed that \u003cem\u003eNcoa4\u003c/em\u003e deletion did not alter key autophagy-related proteins (ATG5, ATG16L, p62, LC3-II/I, p-S6K) in colon tumors (\u003cb\u003eFig. S3A\u003c/b\u003e). Furthermore, treatment with the autophagy inhibitor chloroquine failed to suppress increased xenograft tumor growth derived from MC38 sg\u003cem\u003eNcoa4\u003c/em\u003e cells in C57BL/6 mice (\u003cb\u003eFig. S3B\u003c/b\u003e), suggesting that neither canonical autophagy nor ferritinophagy primarily drive tumor growth following \u003cem\u003eNcoa4\u003c/em\u003e loss. This aligns with previous reports indicating ferritinophagy is not required for colon cancer cell growth (Hasan et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, iron regulatory protein 2 (IRP2) and its downstream effector transferrin receptor (TFRC)\u0026mdash;key mediators of cellular iron uptake (Kim et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u0026mdash;were significantly upregulated in \u003cem\u003eNcoa4\u003c/em\u003e-deficient cells and xenograft tumors (\u003cb\u003eFigs.\u0026nbsp;4A, 4B\u003c/b\u003e). Hypoxia-inducible factor 2α (HIF-2α) and its downstream targets, Six-Transmembrane Epithelial Antigen of the Prostate 4 (STEAP4) and divalent metal transporter 1 (DMT1)\u0026mdash;which promote a pro-tumorigenic iron-dependent state (Cheng et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Xue et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u0026mdash;were also elevated in \u003cem\u003eNcoa4\u003c/em\u003e knockout models (\u003cb\u003eFigs.\u0026nbsp;4A, 4B\u003c/b\u003e). qPCR analysis confirmed increased \u003cem\u003eTfrc\u003c/em\u003e, \u003cem\u003eDmt1\u003c/em\u003e, and \u003cem\u003eSteap4\u003c/em\u003e expression in colon tumors from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e mice (\u003cb\u003eFigs.\u0026nbsp;4C\u0026ndash;4E\u003c/b\u003e). Immunoblotting corroborated increased TFRC, HIF-2α, DMT1, and STEAP4 proteins in these tumors (\u003cb\u003eFig.\u0026nbsp;4F\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eSince NCOA4 was initially characterized as an androgen receptor (AR) coactivator (Yeh et al., 1996), and AR protein negatively correlates with TFRC levels in human CRC (Firehose Legacy dataset; \u003cb\u003eFig. S3C\u003c/b\u003e), we hypothesized AR antagonism might influence TFRC expression. However, treatment with the AR antagonist bicalutamide repressed TFRC protein at high doses (\u003cb\u003eFig. S3D\u003c/b\u003e), suggesting AR signaling does not significantly mediate \u003cem\u003eNcoa4\u003c/em\u003e deficiency\u0026ndash;induced tumor growth.\u003c/p\u003e\u003cp\u003eTogether, these data support a model whereby \u003cem\u003eNcoa4\u003c/em\u003e depletion suppresses ferritinophagy and stabilizes FTH1, causing iron sequestration in ferritin that mimics iron starvation. This triggers IRP2\u0026ndash;TFRC and HIF-2α pathways to enhance iron uptake and accumulation in CRC (\u003cb\u003eFig.\u0026nbsp;4G\u003c/b\u003e), promoting tumorigenesis through pro-tumorigenic iron signaling.\u003c/p\u003e\n\u003ch3\u003eTFRC depletion abolishes NCOA4 deficiency–enhanced colon tumorigenesis\u003c/h3\u003e\n\u003cp\u003eGiven the critical role of iron accumulation mediated by the IRP2/TFRC axis in CRC (Lee et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we tested whether inhibiting iron uptake could mitigate tumorigenesis induced by \u003cem\u003eNcoa4\u003c/em\u003e deletion. Genetic knockout of \u003cem\u003eTfrc\u003c/em\u003e in \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e mice abolished tumor formation driven by \u003cem\u003eNcoa4\u003c/em\u003e loss (\u003cb\u003eFigs. S4A\u0026ndash;S4C\u003c/b\u003e). H\u0026amp;E and immunofluorescence staining showed that increased proliferation (Ki-67) observed in \u003cem\u003eNcoa4\u003c/em\u003e-deficient tumors was reversed by \u003cem\u003eTfrc\u003c/em\u003e deletion (\u003cb\u003eFigs. S4D, S4E\u003c/b\u003e). Conversely, apoptosis marker cleaved caspase-3 (CC3) was unchanged in \u003cem\u003eNcoa4\u003c/em\u003e-deficient tumors but significantly elevated in \u003cem\u003eNcoa4\u003c/em\u003e/\u003cem\u003eTfrc\u003c/em\u003e double knockout tumors (\u003cb\u003eFigs. S4D, S4F\u003c/b\u003e). These findings demonstrate that TFRC upregulation is essential for \u003cem\u003eNcoa4\u003c/em\u003e deletion\u0026ndash;induced colorectal tumorigenesis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNcoa4\u003c/b\u003e \u003cb\u003eknockout robustly increases MCU mRNA and protein levels in mouse colon tissues\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore downstream effects of iron accumulation following \u003cem\u003eNcoa4\u003c/em\u003e deletion, we performed unbiased quantitative proteomics comparing colon tissues from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (\u003cb\u003eTable S1\u003c/b\u003e). Metascape Enriched Ontology Clustering (\u003cb\u003eFig. S5A\u003c/b\u003e) and Gene Set Enrichment Analysis (\u003cb\u003eFig.\u0026nbsp;5A\u003c/b\u003e) revealed robust upregulation of mitochondrial proteins, particularly those involved in mitochondrial calcium ion transport. Volcano plot (\u003cb\u003eFig. S5B\u003c/b\u003e) and heatmap (\u003cb\u003eFigs.\u0026nbsp;5B, 5C\u003c/b\u003e) analyses identified the mitochondrial calcium uniporter (MCU) as one of the most significantly increased proteins. qPCR (\u003cb\u003eFig.\u0026nbsp;5D\u003c/b\u003e) and immunoblot (\u003cb\u003eFig.\u0026nbsp;5E\u003c/b\u003e) analyses confirmed significantly elevated \u003cem\u003eMcu\u003c/em\u003e mRNA and MCU protein levels in colon tissues from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice versus controls. MCU protein was also markedly increased in colon tumors from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e mice compared to controls (\u003cb\u003eFig.\u0026nbsp;5F\u003c/b\u003e). These data indicate that \u003cem\u003eNcoa4\u003c/em\u003e knockout induces MCU upregulation in the colon.\u003c/p\u003e\n\u003ch3\u003eAMPK–CREB signaling pathway is required for MCU induction in NCOA4 knockout cells\u003c/h3\u003e\n\u003cp\u003eMCU expression is transcriptionally regulated by phosphorylated CREB1 (Takahashi et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shanmughapriya et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which is modulated by phosphorylated AMPK. AMPK activity is influenced by reactive oxygen species (ROS) (Thomson et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hwang et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Agostini et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Since iron catalyzes ROS production via Fenton chemistry, we assessed intracellular iron and ROS in HCT116 sg\u003cem\u003eNCOA4\u003c/em\u003e cells. Both intracellular iron (\u003cb\u003eFig.\u0026nbsp;6A\u003c/b\u003e) and total ROS (\u003cb\u003eFig.\u0026nbsp;6B\u003c/b\u003e) were elevated. Proteins involved in the p-AMPK/p-CREB1/MCU axis were upregulated in \u003cem\u003eNCOA4\u003c/em\u003e-deleted cells (\u003cb\u003eFig.\u0026nbsp;6C\u003c/b\u003e). Treatment with the AMPK inhibitor Compound C decreased p-AMPK, p-CREB1, and MCU levels in human and mouse \u003cem\u003eNcoa4\u003c/em\u003e-deficient cells (\u003cb\u003eFigs.\u0026nbsp;6D, S4C\u003c/b\u003e). Similarly, the CREB1 inhibitor KG-501 suppressed p-CREB1 and MCU expression (\u003cb\u003eFigs.\u0026nbsp;6E, S5D\u003c/b\u003e). MCU regulates mitochondrial calcium uptake and metabolism during CRC progression (Zeng et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and mediates mitochondrial import of divalent metals including manganese and iron (Wettmarshausen et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sripetchwandee et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Arcos et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Increased MCU expression in \u003cem\u003eNCOA4\u003c/em\u003e-deficient cells suggests enhanced mitochondrial iron import, elevating mitochondrial ROS (mtROS) (Xue et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Indeed, both mitochondrial iron and mtROS were elevated in \u003cem\u003eNCOA4\u003c/em\u003e knockout cells but were reduced by the MCU inhibitor minocycline (Schwartz et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Arcos et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) (\u003cb\u003eFigs.\u0026nbsp;6F, 6G\u003c/b\u003e). Treatment with the mitochondrial ROS scavenger mito-tempo decreased mtROS and downregulated p-AMPK, p-CREB1, and MCU protein levels (\u003cb\u003eFig.\u0026nbsp;6H\u003c/b\u003e). These data support a model wherein \u003cem\u003eNCOA4\u003c/em\u003e loss promotes intracellular iron accumulation and ROS production, activating AMPK\u0026ndash;CREB1 signaling, which induces MCU expression and further mitochondrial iron and ROS accumulation (\u003cb\u003eFig. S5E\u003c/b\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eNCOA4 deletion alters oxidative stress response in colon tumors and cells\u003c/h2\u003e\u003cp\u003eWe observed that the master antioxidant regulator nuclear factor erythroid 2-related factor 2 (NRF2) downstream targets\u0026mdash;Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1)\u0026mdash;were significantly reduced in colon tumors from \u003cem\u003eNcoa4\u003c/em\u003e knockout mice, while Kelch-like ECH-associated protein 1 (KEAP1) levels remained unchanged (\u003cb\u003eFig. S6A\u003c/b\u003e). Similar reductions in HO-1 and NQO1 were seen in MC38 \u003cem\u003eNcoa4\u003c/em\u003e knockout cells (\u003cb\u003eFig. S6B\u003c/b\u003e). Consistently, human NQO1 antioxidant response element (ARE) luciferase reporter activity was decreased in MC38 \u003cem\u003eNcoa4\u003c/em\u003e knockout cells (\u003cb\u003eFig. S6C\u003c/b\u003e). Markers of oxidative stress, including lipid peroxidation marker 4-hydroxynonenal (4-HNE), NQO1, glutathione peroxidase 4 (GPX4), and the cystine/glutamate antiporter xCT were elevated \u003cem\u003ein vitro\u003c/em\u003e (\u003cb\u003eFig. S6D\u003c/b\u003e). In xenograft tumors, only xCT was reduced significantly, while other oxidative stress markers showed no changes (\u003cb\u003eFig. S6E\u003c/b\u003e). This discrepancy may reflect the presence of dietary vitamin E, a lipid peroxidation scavenger abundant in chow diets but absent in culture media. Vitamin E is known to protect against lipid peroxidation and ferroptosis (Hu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These findings suggest that increased proliferation in \u003cem\u003eNcoa4\u003c/em\u003e-deficient cells is balanced by enhanced lipid peroxidation\u0026ndash;induced ferroptosis \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, ferroptosis may be attenuated by antioxidant mechanisms such as dietary vitamin E. Supporting this, feeding mice a vitamin E-deficient diet for one week abolished the tumor-promoting effect of \u003cem\u003eNcoa4\u003c/em\u003e knockout (\u003cb\u003eFigs. S6F, S6G\u003c/b\u003e). These data implicate ferroptosis sensitivity in the tumorigenic effects of \u003cem\u003eNcoa4\u003c/em\u003e deficiency (Anandhan et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eNCOA4 deletion activates STAT3 signaling via MCU-mediated mitochondrial iron accumulation\u003c/h3\u003e\n\u003cp\u003eIron accumulation can activate STAT3 via phosphorylation (Xue et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Consistent with this, \u003cem\u003eNcoa4\u003c/em\u003e deletion markedly increased STAT3 activity, as measured by a Sis-Inducible Element (SIE) luciferase assay (\u003cb\u003eFig. S7A\u003c/b\u003e). Pharmacological inhibition of MCU with minocycline significantly reduced phosphorylated STAT3 (p-STAT3) expression (\u003cb\u003eFig. S7B\u003c/b\u003e) and suppressed enhanced xenograft growth driven by \u003cem\u003eNcoa4\u003c/em\u003e deficiency (\u003cb\u003eFigs. S7C, S7D\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, treatment with the STAT3 inhibitor NSC78956 or the iron chelator deferiprone (DFP) effectively attenuated tumor growth in \u003cem\u003eNcoa4\u003c/em\u003e deletion models (\u003cb\u003eFigs. S7E, S7F\u003c/b\u003e). These results support a model whereby \u003cem\u003eNcoa4\u003c/em\u003e deletion promotes mitochondrial iron accumulation and oxidative stress via MCU upregulation, activating pro-tumorigenic signaling including STAT3.\u003c/p\u003e\n\u003ch3\u003eNCOA4 overexpression inhibits colorectal cancer progression via MCU-dependent mitochondrial iron regulation\u003c/h3\u003e\n\u003cp\u003eTo further validate NCOA4\u0026rsquo;s tumor-suppressive role via MCU regulation, we used a mouse model with inducible NCOA4 overexpression (\u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNCOA4\u003c/em\u003e\u003csup\u003eLSL/+\u003c/sup\u003e and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNCOA4\u003c/em\u003e\u003csup\u003eLSL/LSL\u003c/sup\u003e alleles) combined with \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e to induce colon tumors via TAM and DSS treatment. Gross colon images showed visibly fewer and smaller tumors in homozygous \u003cem\u003eNCOA4\u003c/em\u003e\u003csup\u003eLSL/LSL\u003c/sup\u003e mice compared to heterozygous or wild-type controls (\u003cb\u003eFig.\u0026nbsp;7A\u003c/b\u003e). Quantitative tumor counts and average tumor size were significantly reduced in the homozygous group (\u003cb\u003eFigs.\u0026nbsp;7B\u0026ndash;7D\u003c/b\u003e), with tumor burden also markedly decreased (\u003cb\u003eFig.\u0026nbsp;7E\u003c/b\u003e). qPCR and immunoblot confirmed increased \u003cem\u003eNcoa4\u003c/em\u003e mRNA and protein expression in \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNCOA4\u003c/em\u003e\u003csup\u003eLSL/+\u003c/sup\u003e and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNCOA4\u003c/em\u003e\u003csup\u003eLSL/LSL\u003c/sup\u003e mice (\u003cb\u003eFigs.\u0026nbsp;7F, 7H\u003c/b\u003e). Notably, only homozygous \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2-Cre\u003c/sup\u003e \u003cem\u003eNCOA4\u003c/em\u003e\u003csup\u003eLSL/LSL\u003c/sup\u003e mice exhibited decreased \u003cem\u003eMcu\u003c/em\u003e mRNA and protein levels (\u003cb\u003eFigs.\u0026nbsp;7G, 7H\u003c/b\u003e), indicating a mechanistic link between NCOA4 overexpression and reduced mitochondrial iron uptake. Together, these results support that NCOA4 overexpression inhibits colon tumor development, at least in part, by downregulating MCU and modulating mitochondrial iron metabolism.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study uncovers key upstream mechanisms of iron-driven colorectal tumorigenesis and identifies novel therapeutic targets centered on the regulation of ferritinophagy. While previous reports have demonstrated that NCOA4-mediated ferritinophagy can suppress tumor growth in cancers such as pancreatic cancer and acute myeloid leukemia (Santana-Codina et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ravichandran et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Larrue et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), our findings\u0026mdash;consistent with a bioinformatic study linking lower NCOA4 levels to poorer prognosis in CRC (Gu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u0026mdash;reveal a contrasting role for NCOA4 in colorectal cancer. Specifically, we show that loss of NCOA4 increases both cytosolic and mitochondrial iron levels, thereby promoting CRC cell growth and supporting a pro-tumorigenic role for NCOA4 deficiency in the colorectal tumor microenvironment.\u003c/p\u003e\u003cp\u003eThis work represents one of the first experimental validations of NCOA4 as a context-dependent oncogenic modifier in CRC, challenging the prevailing notion that NCOA4 inhibition is universally tumor-suppressive. Our results underscore the importance of evaluating the role of NCOA4 and ferritinophagy in a cancer-type\u0026ndash;specific manner and highlight the therapeutic potential of targeting iron regulatory pathways in colorectal cancer.\u003c/p\u003e\u003cp\u003eMechanistically, loss of NCOA4 impairs ferritinophagy, leading to iron sequestration and a cellular iron starvation response. This response drives the upregulation of iron uptake machinery, including the IRP2\u0026ndash;TFRC axis and HIF-2α\u0026ndash;DMT1/STEAP4 pathway, which collectively promote intracellular iron accumulation and CRC progression. Our extensive prior work (Xue et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Schwartz et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Villareal et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Arcos et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and that of others (Pusatcioglu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Radulescu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) consistently demonstrate that CRC tumors accumulate iron to sustain growth and progression, reinforcing iron metabolism as a promising therapeutic target.\u003c/p\u003e\u003cp\u003eImportantly, our data reveal a novel link between NCOA4 deletion and mitochondrial iron overload, resulting in elevated mtROS. Proteomic analyses indicated that canonical mitochondrial iron transporters, mitoferrin-1 (MFRN1) and MFRN2, were not significantly upregulated. Instead, the mitochondrial calcium uniporter (MCU) emerged as one of the most highly expressed proteins associated with mitochondrial iron accumulation. This mitochondrial dysfunction appears to be driven by MCU upregulation, which facilitates not only calcium influx but also mitochondrial iron uptake (Zhang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Arcos et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). MCU overexpression and the resulting increase in mtROS serve as second messengers to activate downstream oncogenic pathways\u0026mdash;most notably the iron- and ROS-dependent STAT3 signaling cascade (Xue et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Cao et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). STAT3 activation is well established in cancer and linked to enhanced proliferation, survival, immune evasion, and metastasis (Lee et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), further emphasizing the oncogenic potential driven by NCOA4 loss.\u003c/p\u003e\u003cp\u003eThe reciprocal relationship between NCOA4 and MCU expression is particularly noteworthy. NCOA4 overexpression suppresses CRC tumorigenesis and correlates with reduced MCU levels, suggesting that NCOA4 maintains mitochondrial homeostasis by limiting MCU-mediated iron influx. This balance is critical, as mitochondrial iron and ROS levels are tightly connected to cellular bioenergetics, apoptosis, and redox signaling (Richardson et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Nakamura and Takada, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Disruption of this equilibrium through NCOA4 deletion leads to metabolic reprogramming conducive to tumor growth (Liu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Future studies generating NCOA4 and MCU double knockout mice would be valuable to directly test MCU\u0026rsquo;s role in mitochondrial iron accumulation in the absence of NCOA4.\u003c/p\u003e\u003cp\u003eOur findings underscore the complex interplay between iron metabolism, mitochondrial function, and oncogenic signaling in CRC. They suggest that targeting iron uptake pathways, mitochondrial iron handling, or downstream effectors such as STAT3 could represent viable therapeutic strategies, especially in tumors characterized by NCOA4 loss or low expression. Therapeutic approaches using iron chelators or MCU inhibitors\u0026mdash;alone or combined with STAT3 pathway antagonists\u0026mdash;warrant further investigation to counteract NCOA4 deficiency\u0026ndash;driven tumorigenesis.\u003c/p\u003e\u003cp\u003eOur study also indicates that ferritinophagy actively maintains iron homeostasis in CRC cells, with the observed phenotypes likely reflecting long-term consequences of NCOA4 ablation. It would be valuable to assess effects at earlier time points post-NCOA4 deletion\u0026mdash;using inducible knockdown systems\u0026mdash;to determine whether acute NCOA4 loss produces similar or distinct outcomes on cell growth and iron metabolism. Specifically, it will be important to examine if acute NCOA4 ablation transiently decreases cytosolic iron, triggering compensatory TFRC upregulation and possibly resulting in an overshoot toward iron overload.\u003c/p\u003e\u003cp\u003eIn summary, our study elucidates a tumor-promoting function of NCOA4 loss in colorectal cancer through dysregulation of iron homeostasis and mitochondrial dynamics. These insights reveal novel molecular targets and pathways for therapeutic development, which may ultimately improve outcomes for patients with NCOA4-deficient colorectal cancers.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture\u003c/h2\u003e\u003cp\u003eHuman HCT116 (RRID: CVCL_0291) and mouse MC38 (RRID: CVCL_B288) colorectal cancer cell lines were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. Cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Cat. #11965092, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Cat. #F2442-500ML, Millipore Sigma, Burlington, MA) and 1% penicillin-streptomycin (10,000 U/mL; Cat. #15140122, Thermo Fisher Scientific). To generate stable NCOA4 knockout cell lines (sg\u003cem\u003eNCOA4\u003c/em\u003e), HCT116 and MC38 cells were transfected with px459-NCOA4 or empty control plasmids using Lipofectamine 2000 (Cat. #11668027, Thermo Fisher Scientific). After transfection, cells were selected with puromycin to establish knockout clones.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e Animal studies were conducted to address specific research questions, adhering to the Institute of Laboratory Animal Resources guidelines, and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of New Mexico Health Sciences Center (Protocol# 23-201434-HSC), following the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). Mice, encompassing both sexes, were housed in standard cages under a 12-h light\u0026ndash;dark cycle, in a temperature-controlled environment, with ad libitum access to a standard chow diet and water unless otherwise specified.\u003c/p\u003e\u003cp\u003eColon-specific NCOA4 knockout and overexpression mouse models were generated by crossing \u003cem\u003eNcoa4\u003c/em\u003e floxed (\u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e) and \u003cem\u003eNCOA4\u003c/em\u003e\u003csup\u003eLSL/LSL\u003c/sup\u003e mice (Santana-Codina et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) with tamoxifen-inducible \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e mice (Yin et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These crosses produced \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e \u003cem\u003eNCOA4\u003c/em\u003e\u003csup\u003eLSL/LSL\u003c/sup\u003e mice, allowing selective deletion or overexpression of NCOA4 in the colon epithelium upon tamoxifen treatment.\u003c/p\u003e\u003cp\u003eColorectal cancer (CRC) was modeled using two established approaches. In the colitis-associated cancer (CAC) model, mice received intraperitoneal injections of azoxymethane (AOM) at 1 mg/mL to deliver a total dose of 10 mg/kg over two consecutive days. Beginning the day after AOM injection, tamoxifen was administered at 100 mg/kg for three consecutive days to induce NCOA4 deletion. Following tamoxifen treatment, mice were given 2% dextran sodium sulfate (DSS) in drinking water for seven days to induce colonic inflammation, followed by 14 days of regular water for tissue recovery. This inflammatory and recovery cycle was repeated once. In the second model, CRC was induced by combining monoallelic \u003cem\u003eApc\u003c/em\u003e deletion with DSS treatment. \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e mice, \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e mice and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e \u003cem\u003eNCOA4\u003c/em\u003e\u003csup\u003eLSL/LSL\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003eF/+\u003c/sup\u003e mice were generated. After tamoxifen-induced recombination, mice received 2% DSS in drinking water for seven days, followed by 28 days of recovery with regular water. These models allowed assessment of NCOA4\u0026rsquo;s role in genetically predisposed and inflammation-driven tumorigenesis.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e tumor growth studies, MC38 murine CRC cells with stable NCOA4 knockout were injected subcutaneously into the flanks of syngeneic C57BL/6 mice at 1 \u0026times; 10⁶ cells per site. Tumors were harvested after two weeks. For pharmacological inhibition studies, treatment began one week after injection once tumors were palpable. Mice were treated with vehicle control, minocycline at 20 mg/kg daily, NSC74859 (a STAT3 inhibitor) at 5 mg/kg intraperitoneally every other day, a Vitamin E-deficient diet (TD.88163, Envigo), a Vitamin E control diet (50 IU, TD.99455, Envigo), or deferiprone (DFP) at 1 mg/mL in drinking water. Treatments lasted for one week.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eHistology, Immunofluorescence, and DAB-Enhanced Perl\u0026rsquo;s Iron Staining\u003c/h2\u003e\u003cp\u003eFormalin-fixed, paraffin-embedded colon tissue sections were processed for hematoxylin and eosin (H\u0026amp;E) staining, immunofluorescence (IF), and iron staining. For H\u0026amp;E staining, sections were deparaffinized and rehydrated through graded ethanol to distilled water. Sections were stained with hematoxylin for two minutes, rinsed in tap water, dipped briefly in bluing solution, and rinsed again for two minutes. Eosin staining was performed for five minutes, followed by dehydration through ethanol series, clearing in xylene, and mounting for histopathological evaluation by a gastrointestinal pathologist (Dr. Martin) in a blind manner.\u003c/p\u003e\u003cp\u003eFor immunofluorescence, antigen retrieval was performed by incubating sections in 10 mM sodium citrate buffer at sub-boiling temperature for 12 minutes. Slides were cooled to room temperature for two hours and then blocked with 10% normal goat serum in 0.1% Triton X-100 in phosphate-buffered saline (PBS) or Tris-buffered saline (TBS) for one hour. Primary antibodies were diluted in 1% normal goat serum with 0.1% PBST or TBST and incubated overnight at 4\u0026deg;C. After washing, fluorophore-conjugated secondary antibodies diluted in 1% normal goat serum were applied for one hour at room temperature. Slides were washed and mounted using EverBrite\u0026trade; Mounting Medium (Biotium). Primary antibodies used included cleaved Caspase-3 (CC3, #9664), Ki-67 (#12202), and phospho-STAT3 (p-STAT3, #9145) from Cell Signaling Technology.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eFerroOrange, Mito-Ferro Green, DCFH-DA, and MitoSOX Staining\u003c/h2\u003e\u003cp\u003eCells were seeded at 2 \u0026times; 10⁵ per well in 24-well plates. After adherence, cells were stained in pre-warmed Hank\u0026rsquo;s Balanced Salt Solution (HBSS) containing fluorescent probes: 0.5 \u0026micro;M FerroOrange for cytosolic ferrous iron (Dojindo), 1 \u0026micro;M Mito-Ferro Green for mitochondrial iron, 10 \u0026micro;M 2\u0026rsquo;,7\u0026rsquo;-dichlorodihydrofluorescein diacetate (DCFH-DA; Cayman Chemical) for total cellular reactive oxygen species (ROS), and 1 \u0026micro;M MitoSOX (Thermo Fisher Scientific) for mitochondrial superoxide. Staining was performed at 37\u0026deg;C for 30 minutes, followed by washing with PBS. Fluorescence images were captured using an Invitrogen\u0026trade; EVOS\u0026trade; FL Auto Imaging System. The RFP channel was used for FerroOrange and MitoSOX, while the GFP channel was used for DCFH-DA and Mito-Ferro Green. ImageJ software was used to quantify fluorescence intensity normalized to Hoechst 33342 nuclear staining.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLuciferase reporter gene assay\u003c/h2\u003e\u003cp\u003eTo measure luciferase activity, cells were seeded at 5 \u0026times; 10⁴ per well in 24-well plates and transfected using polyethylenimine (PEI) with either the pGL4.47[luc2P/SIE-RE/Hygro] reporter plasmid containing a STAT3-responsive element alongside a LacZ plasmid for normalization, or the human NQO1-ARE TATA-Inr luciferase reporter plasmid (for MC38 wild-type and sgNcoa4 cells, Yin et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). After 48 hours, cells were lysed in lysis buffer. Luciferase activity was then quantified using a Promega Luciferase Assay Kit (E1500), with 20 \u0026micro;L of supernatant added to a 96-well plate and 100 \u0026micro;L of luciferase reagent per well. Luminescence intensity was measured by a plate reader and normalized to protein concentration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative Polymerase Chain Reaction (qPCR) Analysis\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using the IBI Isolate DNA/RNA Reagent Kit following manufacturer\u0026rsquo;s instructions. For RNA from DSS-treated colon tissues, an additional purification was performed by precipitating RNA with 8 M lithium chloride (LiCl), followed by ethanol washes to improve RNA quality. RNA was resuspended in DEPC-treated water and quantified. Quantitative PCR was conducted using the LightCycler 480 system (Roche Diagnostics) with gene-specific primers listed in \u003cb\u003eTable S2\u003c/b\u003e. Relative gene expression was calculated by the ΔΔCt method using housekeeping genes for normalization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eImmunoblotting Analysis\u003c/h2\u003e\u003cp\u003eCells and tumor tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer. Lysates were centrifuged, and protein concentration was determined using a BioTek Synergy HTX Multi-Mode Microplate Reader. Equal amounts of protein (10\u0026ndash;50 \u0026micro;g) were loaded on SDS-PAGE gels, separated by electrophoresis, and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk and incubated overnight with primary antibodies, followed by incubation with appropriate secondary antibodies for 1.5 hours. The antibodies used are listed in \u003cb\u003eTable S3\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eATP Measurement\u003c/h2\u003e\u003cp\u003eCells (5 \u0026times; 10\u0026sup3;) were seeded in white opaque 96-well plates. After 48 hours, an equal volume of CellTiter-Glo\u0026reg; 2D Cell Viability Assay reagent was added. Luminescence was measured after 10 minutes using a SpectraMax M5 Microplate Reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eMTT Assay\u003c/h2\u003e\u003cp\u003eCells were seeded at 5 \u0026times; 10⁴ cells/mL in 24-well plates. After treatments, 125 \u0026micro;L of 5 mg/mL MTT solution was added and incubated at 37\u0026deg;C for 30 minutes. Formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured with a BioTek Synergy HTX Microplate Reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eInductively Coupled Plasma Mass Spectrometry (ICP-MS) Analysis\u003c/h2\u003e\u003cp\u003eTissue samples were digested overnight in concentrated nitric acid and diluted with Milli-Q water. Samples were analyzed on an Agilent 7900 ICP-MS instrument at the University of New Mexico Health Sciences Center. Iron content was normalized to tissue weight.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eUnbiased Quantitative Proteomics Study\u003c/h2\u003e\u003cp\u003e\u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003eERT2\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e \u003cem\u003eNcoa4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice were treated with tamoxifen (100 mg/kg daily for three days) to induce colon-specific Ncoa4 deletion. One week after the final injection, mice were sacrificed, and colonic epithelial cells were isolated by scraping the luminal surface. Proteomic analysis was performed as described previously (Santana-Codina et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical significance was evaluated using independent or paired t-tests, one-way or two-way ANOVA where appropriate. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n\u003ch2\u003eDeclarations of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n\u003ch2\u003eDeclaration of Generative AI and AI-Assisted Technologies in the Writing Process\u003c/h2\u003e\n\u003cp\u003eDuring the preparation of this work, the authors used ChatGPT to improve language and readability. Following the use of this tool, the authors carefully reviewed and edited the content and take full responsibility for the final publication.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n\u003ch2\u003eEthics Statement for Human\u003c/h2\u003e\n\u003cp\u003eThe study was approved by the University of New Mexico Health Sciences Center Institutional Review Board (protocol #19\u0026ndash;131). Human tissue samples were obtained from the University of New Mexico Comprehensive Cancer Center Human Tissue Repository and the Cooperative Human Tissue Network (CHTN). Written informed consent was obtained from all participants, and patient data were anonymized to ensure confidentiality.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n\u003ch2\u003eEthics Statement for Animal\u003c/h2\u003e\n\u003cp\u003eAnimal studies were conducted to address specific research questions, adhering to the Institute of Laboratory Animal Resources guidelines, and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of New Mexico Health Sciences Center (Protocol# 23-201434-HSC), following the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n\u003ch2\u003ePatient and public involvement\u003c/h2\u003e\n\u003cp\u003ePatients and the public were not involved in the design, conduct, reporting, or dissemination plans of this research.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eX.X. was supported by National Institutes of Health grants R01ES035780, 3P30CA118100-19S3, and P20GM130422; the HSC Pilot Funding Program from the UNM HSC Office of Research; the Dedicated Health Research Funds from the Research Allocation Committee of the UNM School of Medicine; the UNMCCC Research Program Support Pilot Project Award and Off-Setting Cuts Pilot Project (P30CA118100); and the Health Sciences \u0026amp; Main Campus Research Collaboration Seed Grant Award from UNM Rainforest Innovations. This research was also partially supported by the UNM Comprehensive Cancer Center Support Grant NCI P30CA118100 through Graduate Student Support Pilot Grant #1484. L.B.V. received support from The Infectious Disease and Immunity Program at the UNM Health Sciences Center (T32AI007538). C.V.C. was supported by the NIGMS Institutional Research and Academic Career Development Award (K12GM088021) and the UNM METALS Superfund Research Center (P42ES025589). E.R.P. was supported by NIH grants P30CA118100 and P20GM121176.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eH.K., L.B.V., L.G., C.V.C., and X.X. performed the experiments. E.R.P. provided guidance on androgen receptor studies. N.S. and J.D.M. conducted the unbiased proteomics study. D.R.M. evaluated histological samples. H.K., L.B.V., and X.X. drafted the manuscript and prepared figures. X.X. designed the project and edited the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank Dr. Miljan Kuljanin for assistance with mass spectrometry-based proteomics.\u003c/p\u003e\n\u003ch3\u003eData availability statement\u003c/h3\u003e\n\u003cp\u003eAll data relevant to the study are included in the article or uploaded as online supplemental information. Additional data are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgostini F, Bisaglia M, Plotegher N (2023) Linking ROS Levels to Autophagy: The Key Role of AMPK. 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Cancer Res 78:2876\u0026ndash;2885\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Iron Metabolism, Colorectal Carcinogenesis, Reactive Oxygen Species","lastPublishedDoi":"10.21203/rs.3.rs-7483419/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7483419/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIron is an essential cofactor for mitochondrial metabolism, yet the regulatory networks linking cellular iron homeostasis to colorectal cancer (CRC) progression remain incompletely understood. Here, we identify nuclear receptor coactivator 4 (NCOA4), a ferritinophagy receptor, as a context-dependent tumor suppressor that coordinates cytosolic and mitochondrial iron handling in CRC. Analysis of human tumors and colon-specific \u003cem\u003eNcoa4\u003c/em\u003e knockout mice revealed that NCOA4 loss drives tumorigenesis by inducing transferrin receptor\u0026ndash;mediated iron uptake and mitochondrial calcium uniporter (MCU)\u0026ndash;dependent mitochondrial iron import. This dual iron overload elevates mitochondrial reactive oxygen species, activates STAT3 signaling, and enhances tumor cell proliferation. NCOA4 overexpression reverses these effects, reducing MCU expression and tumor growth. Pharmacological inhibition of MCU, STAT3, or mitochondrial iron transport mitigated tumorigenesis in NCOA4-deficient models. Our findings define an NCOA4\u0026ndash;MCU\u0026ndash;STAT3 metabolic signaling axis that couples iron metabolism to oncogenic progression and reveal mitochondrial iron handling as a therapeutic vulnerability in CRC.\u003c/p\u003e","manuscriptTitle":"Ferritinophagy Loss Drives Mitochondrial Iron Import and Colorectal Tumorigenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 12:06:03","doi":"10.21203/rs.3.rs-7483419/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"439990d7-94a8-43b0-8976-5f4650be63a6","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54256952,"name":"Health sciences/Gastroenterology/Gastrointestinal diseases/Gastrointestinal cancer/Colorectal cancer/Colon cancer"},{"id":54256953,"name":"Biological sciences/Biochemistry/Metals/Iron"}],"tags":[],"updatedAt":"2025-09-23T12:06:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 12:06:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7483419","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7483419","identity":"rs-7483419","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}