Glutathione peroxidase 4 suppresses manganese-dependent oxidative stress to reduce colorectal tumorigenesis.

Glutathione peroxidase 4 suppresses manganese-dependent oxidative stress to reduce 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 Glutathione peroxidase 4 suppresses manganese-dependent oxidative stress to reduce colorectal tumorigenesis. Xiang Xue, Zhaoli Liu, Yanshan Liang, Young-Yon Kwon, Rui Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3837925/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 The role of glutathione peroxidase 4 (GPX4) in ferroptosis and various cancers is well-established; however, its specific contribution to colorectal cancer has been unclear. Surprisingly, in a genetic mouse model of colon tumors, the deletion of GPX4 specifically in colon epithelial cells increased tumor burden but decreased oxidized glutathione. Notably, this specific GPX4 deletion did not enhance susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice with varied iron diets but showed vulnerability in mice with a vitamin E-deficient diet. Additionally, a high manganese diet heightened susceptibility, while a low manganese diet reduced DSS-induced colitis in colon epithelial-specific GPX4-deficient mice. Strikingly, the low manganese diet also significantly reduced colorectal cancer formation in both colon epithelial-specific GPX4-deficient and wildtype mice. Mechanistically, antioxidant proteins, especially manganese-dependent superoxide dismutase (MnSOD or SOD2), correlated with disease severity. Treatment with tempol, a superoxide dismutase mimetic radical scavenger, suppressed GPX4 deficiency-induced colorectal tumors. In conclusion, the study elucidates the critical role of GPX4 in inhibiting colorectal cancer progression by regulating oxidative stress in a manganese-dependent manner. The findings underscore the intricate interactions between GPX4, dietary factors, and their collective influence on colorectal cancer development, providing potential insights for personalized therapeutic strategies. Health sciences/Gastroenterology/Gastrointestinal diseases/Gastrointestinal cancer/Colorectal cancer/Colon cancer Health sciences/Gastroenterology/Gastrointestinal diseases/Gastroenteritis Colorectal cancer Oxidative stress Experimental Colitis Cell Death Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Ferroptosis, a distinctive process among various types of cell death, has garnered significant attention in cancer research. It is a mechanism through which cells, particularly cancer cells, undergo elimination due to the accumulation of excessive iron and lipid peroxides (Stockwell et al. 2017 ). Ferroptosis can be considered an intrinsic defense mechanism against cancer, especially given the heightened vulnerability of many cancer cells to ferroptosis due to their elevated metabolic rate and increased demand for iron and lipids. Consequently, inducing ferroptosis in cancer cells emerges as a promising therapeutic strategy. While ferroptosis holds promise as a cancer treatment strategy, several challenges persist. For example, colorectal cancer, which accumulates substantial iron, has developed mechanisms to resist ferroptosis. Notably, the expression of glutathione peroxidase 4 (GPX4), a key protective protein that reduces lipid peroxides and prevents ferroptosis, is upregulated in colorectal cancer patients undergoing neoadjuvant chemoradiotherapy, correlating with a poor prognosis (Zhang et al. 2022 ). The resistance of colorectal cancer to ferroptosis poses a challenge in cancer treatment. Researchers are actively exploring ways to exploit ferroptosis as a therapeutic approach. Investigations involve the use of small molecules and drugs that either inhibit GPX4 or induce lipid peroxidation to selectively target cancer cells. Additionally, there is ongoing research into combining ferroptosis-inducing agents with traditional cancer therapies, such as chemotherapy or radiation. A comprehensive understanding of the precise mechanisms underlying ferroptosis in cancer cells and the development of effective and safe therapies are ongoing areas of research. For example, a study demonstrated that the inhibition of serine/arginine-rich splicing factor 9 (SRSF9) enhances the sensitivity of colorectal cancer to erastin-induced ferroptosis by reducing GPX4 expression (Wang et al., 2021 ). In a separate study, sodium butyrate was found to promote ferroptosis by inducing lipid reactive oxygen species (ROS) production through the downregulation of GPX4, leading to reduced tumor growth in xenografts and a colitis-associated colorectal tumor model (Wang et al., 2023 ). Furthermore, the GPX4 inhibitor RSL3 was shown to hinder colorectal cancer cell growth by inducing ferroptosis, an effect reversible by overexpressing GPX4 (Sui et al., 2018 ). Moreover, the deficiency of GPX4 in myeloid cells has been demonstrated to actively promote the progression of colorectal cancer (Canli et al., 2017 ). Specifically, this deficiency leads to an augmented production of ROS, instigating genome-wide DNA mutations within intestinal epithelial cells, ultimately fostering tumor invasion. However, no studies have investigated the influence of colon epithelial-specific GPX4 knockout on colorectal cancer progression. Our findings reveal that GPX4 depletion in colorectal epithelial cells unexpectedly enhances colorectal tumorigenesis. Mechanistically, under conditions of high oxidative stress, including vitamin E deficiency, elevated manganese (Mn) exposure, and tumor development, superoxide dismutase (SOD2) is upregulated in the colon tissues of colorectal epithelial GPX4 deficient mice. A low Mn diet and the SOD mimetic radical scavenger Tempol mitigate GPX4 deficiency induced SOD2 expression and colorectal tumor formation. These findings underscore the crucial role of GPX4 in suppressing Mn-dependent oxidative stress to curtail colorectal tumorigenesis. RESULTS Depletion of GPX4 specifically in colon epithelial cells promotes colorectal tumorigenesis. To investigate the role of GPX4 in colorectal tumorigenesis, we generated mice with colon epithelial cell specific Gpx4 depletion. GPX4 deletion was confirmed by genotyping ( Figure S1 A ) and qPCR ( Figure S1 B ). Colorectal cancer was induced as described in the methods, with the treatment scheme illustrated in Figure S1 C . We observed that, compared to Cdx2 CreERT2 Apc F/+ mice, mice with colon epithelial-specific GPX4 depletion ( Cdx2 CreERT2 Apc F/+ Gpx4 F/F ) showed no difference in body weight change compared with GPX4 wildtype mice (Fig. 1 A). However, GPX4 deficient mice exhibited increased colorectal tumor formation under a dissection microscope (Fig. 1 B). The number of total tumors (Fig. 1 C), tumors with a diameter less than 3mm (Fig. 1 D), and tumors with a diameter more than 3mm (Fig. 1 E) were all increased in the GPX4 deficient mice. Additionally, there was an increased tumor burden in the GPX4 deficient mice (Fig. 1 F). H&E staining of colorectal tumors from GPX4 deficient and wildtype mice (Fig. 1 G) indicated a similar disease score in both groups (Fig. 1 H). Ki67 staining (Fig. 1 I), and quantification (Fig. 1 J) suggested increased cell proliferation in the GPX4 deficient mice, while the apoptosis marker CC3 was not changed (Fig. 1 K and 1 L). These results suggest a tumor-inhibiting function of GPX4 in colorectal cancer. Depletion of GPX4 specifically in colon epithelial cells results in reduced oxidized glutathione levels in colorectal tumors. To understand the mechanism behind the enhanced colorectal tumorigenesis in GPX4-deficient mice, we conducted metabolomics analysis on colon tumors from Cdx2 CreERT2 Apc F/+ Gpx4 F/F mice and Cdx2 CreERT2 Apc F/+ mice. Targeted metabolomics revealed two significantly changed metabolites ( Table S2 ): Threonine and Homovanillic acid. In untargeted metabolomics, 41 significantly changed metabolites were identified in the negative mode and 85 in the positive mode ( Table S2 ). Enrichment analysis of these 127 significantly changed metabolites using MetaboAnalyst 6.0 highlighted glutathione metabolism and Vitamin B6 metabolism as the top two enriched metabolite sets (Fig. 2 A). This enrichment is due to decreased levels of oxidized glutathione (Fig. 2 B), glycine (Fig. 2 C), and spermidine (Fig. 2 D) in the colon tumors of Cdx2 CreERT2 Apc F/+ Gpx4 F/F mice compared to those in Cdx2 CreERT2 Apc F/+ mice. These findings suggest a redox imbalance status due to the critical role of GPX4 in glutathione oxidation. GPX4 depletion specifically in colon epithelial cells increases susceptibility to colitis in mice treated with a vitamin E-deficient diet. As inflammation is a high-risk factor for colorectal cancer and intestinal epithelial GPX4 restricts enteritis (Mayr et al, 2020 ), we investigated whether colon epithelial GPX4 deficiency in mice affects their susceptibility to colitis. There was no difference in body weight and colon length between Cdx2 CreERT2 Gpx4 F/F mice and Gpx4 F/F mice after treating mice with tamoxifen and DSS. Despite GPX4 being a well-known ferroptosis inhibitor, no differences were observed in body weight and colon length between Cdx2 CreERT2 Gpx4 F/F mice and Gpx4 F/F mice with different iron concentrations in their diets (3.5ppm, 40ppm, 1000ppm) ( Figure S2 A–S2H ). Similar results were seen after Salmonella treatment ( Figure S2 I and S2J ). A vitamin E supplement diet has been demonstrated to reduce GPX4 deficiency-enhanced ferroptosis in hematopoietic stem and progenitor cells (Hu et al. 2021 ). Interestingly, Cdx2 CreERT2 Gpx4 F/F mice exhibited increased body weight loss (Fig. 3 A) and shorter colon lengths compared to Gpx4 F/F mice (Fig. 3 B) when induced with DSS and treated with a vitamin E-deficient diet. qPCR analysis indicated that the expression level of Gpx4 was significantly decreased in the colon tissues of Cdx2 CreERT2 Gpx4 F/F mice compared to wildtype control ( Figure S3 A ). Interestingly, proinflammatory cytokines including Tnfa (Figure S3 B) , Il6 (Figure S3 C) , Cxcl1 (Figure S3 D) and Ptgs2 (Figure S3 E) , as well as stem cell marker Lgr5 (Figure S3 F) was not changed. However, the anti-inflammatory cytokines including Il10 (Figure S3 G) , Il22 (Figure S3 H) , and transcription factor Foxm1 (Figure S3 I) were significantly decreased in the colon tissues of Cdx2 CreERT2 Gpx4 F/F mice. qPCR analysis revealed a significant decrease in the expression level of Gpx4 in the colon tissues of Cdx2 CreERT2 Gpx4 F/F mice compared to the wildtype control ( Figure S3 A ). Interestingly, proinflammatory cytokines, including Tnfa ( Figure S3 B ), Il6 ( Figure S3 C ), Cxcl1 ( Figure S3 D ), Ptgs2 ( Figure S3 E ), and the stem cell marker Lgr5 ( Figure S3 F ), remained unchanged. However, anti-inflammatory cytokines, including Il10 ( Figure S3 G ), Il22 ( Figure S3 H ), and the transcription factor Foxm1 ( Figure S3 I ), were significantly decreased in the colon tissues of Cdx2 CreERT2 Gpx4 F/F mice. The immunoblot analysis of several major antioxidant proteins, including SOD2, NQO1, and HO-1 (Fig. 3 C), revealed an increase in these proteins in Cdx2 CreERT2 Gpx4 F/F mice, confirming a state of redox imbalance. H&E staining (Fig. 3 D) and pathology scoring (Fig. 3 E) indicated increased inflammation and tissue damage. While the proliferation marker Ki67 remained unchanged (Fig. 3 F and 3 G), an increase in the apoptosis marker CC3 was observed in colon tissues of Cdx2 CreERT2 Gpx4 F/F mice (Fig. 3 H and 3 I). This suggests that GPX4 deficiency contributes to enhanced apoptosis during the colitis process. Together, in the absence of vitamin E, mice deficient in GPX4 exhibit heightened susceptibility to DSS-induced colitis. GPX4 depletion specifically in colon epithelial cells increases intestinal inflammation in mice treated with a high Mn diet. While GPX4 is a selenium-dependent enzyme (Weaver et al, 2022), SOD2 is one of the rare mitochondrial enzymes evolved to use Mn as a cofactor over the more abundant element iron (Naranuntarat et al, 2017). Compared with iron, Mn can catalyze the Fenton reaction more effectively to induce higher ROS production (Cheng et al, 2021 ). We found that when treated with a high Mn diet, Cdx2 CreERT2 Gpx4 F/F mice showed more body weight loss (Fig. 4 A) and shorter colon lengths (Fig. 4 B). Also, there was an increased expression of antioxidant proteins such as SOD2, NQO1, and HO-1 in colon tissues of Cdx2 CreERT2 Gpx4 F/F mice compared to Gpx4 F/F mice (Fig. 4 C). However, H&E staining revealed a similar disease score (Fig. 4 D and 4 E). We found that cell proliferation, as indicated by Ki67 staining, remained unchanged (Fig. 4 F and 4 G). However, there was an observed increase in apoptosis, demonstrated by CC3 staining in the colons of Cdx2 CreERT2 Gpx4 F/F mice (Fig. 4 H and 4 I). These results indicate that in the presence of a high concentration of Mn, GPX4 deficient mice are more susceptible to DSS-induced colitis. GPX4 depletion specifically in colon epithelial cells reduces intestinal inflammation in mice treated with a low Mn diet. To test whether Mn is required for enhanced colonic inflammation due to GPX4 deficiency, we treated mice with a low Mn diet. We observed that Cdx2 CreERT2 Gpx4 F/F mice experienced less body weight loss (Fig. 5 A) and longer colon length (Fig. 5 B) than Gpx4 F/F mice under Mn deficient conditions. Additionally, there was a reduction in the expression of antioxidant proteins such as SOD2, NQO1, and HO-1 in the colon tissue of Cdx2 CreERT2 Gpx4 F/F mice (Fig. 5 C). H&E staining and pathological scoring revealed reduced inflammation in Cdx2 CreERT2 Gpx4 F/F mice when treated with a Mn deficient diet (Fig. 5 D and 5 E). We also noted Ki67 expression remained unchanged (Fig. 5 F and 5 G), while reduced apoptosis through CC3 staining in the colons of Cdx2 CreERT2 Gpx4 F/F mice (Fig. 5 H and 5 I), These results indicate that GPX4 depletion reduces intestinal inflammation in mice treated with a low Mn diet. Mn deficiency reduces colorectal tumor formation in mice. Mn plays a crucial role in intestinal epithelial barrier formation, and a low Mn diet has been linked to an increase in DSS-induced colitis in mice (Choi et al. 2020 ). However, whether Mn influences the progression of colitis-induced colorectal cancer is not clear. To address this question, we induced colorectal tumor formation under three dietary conditions: a control diet, a Mn-deficient diet, and a high Mn diet. Unfortunately, the high Mn diet was associated with reduced mouse survival during DSS treatment ( Figure S4 ). We observed that the Mn-deficient diet did not impact mouse body weight (Fig. 6 A) but significantly reduced colorectal tumor formation (Fig. 6 B). The Mn-deficient diet group exhibited decreased total tumor numbers (Fig. 6 C), smaller tumors (sizes less than 3mm; Fig. 6 D), larger tumors (sizes more than 3 mm; Fig. 6 E), and overall tumor burden (Fig. 6 F). H&E staining revealed lower disease scores in the Mn-deficient diet group (Fig. 6 G and 6 H). Ki67 staining demonstrated reduced tumor cell proliferation (Fig. 6 I and 6 J), while CC3 staining showed no change in apoptosis (Fig. 6 K and 6 L). Western blot analysis indicated reduced levels of the antioxidant protein SOD2 and the Mn efflux protein ZnT10 in the Mn-deficient diet group, while the protein levels of HO-1 and NQO1 remained unchanged (Fig. 6 M). Further analysis of metal levels in colon tumors via laser ablation ICP-MS revealed significantly lower Mn levels, but not other metals, in the Mn-deficient diet group compared to the control diet group ( Figure S5A and S5B ). The Mn-deficient diet fed to Cdx2 CreERT2 Apc F/+ Gpx4 F/F mice showed a reduction in total tumor numbers ( Figure S6A ) and tumor numbers at size 2-3mm ( Figure S6B ) compared to the control diet. However, it led to no significant differences in smaller tumors (sizes less than 1mm; Figure S6C ), tumor numbers at size 1-2mm ( Figure S6D ), and larger tumors (sizes more than 3 mm; Figure S6E ), as well as overall tumor burden ( Figure S6F ). These results suggest that Mn is essential for colorectal tumor formation. The SOD mimetic tempol treatment rescues GPX4-inhibited colorectal tumor formation. To investigate whether GPX4 deficiency-enhanced colorectal cancer depends on oxidative stress, we treated both Cdx2 CreERT2 Apc F/+ and Cdx2 CreERT2 Apc F/+ Gpx4 F/F mice with the general SOD-mimetic agent tempol. We observed no impact of tempol on mouse body weight ( Figure S7A ); however, under the microscope, Cdx2 CreERT2 Apc F/+ Gpx4 F/F mice exhibited increased colorectal tumor formation, a phenomenon that was effectively blocked by tempol treatment (Fig. 7 A). Following tempol treatment, Cdx2 CreERT2 Apc F/+ Gpx4 F/F mice demonstrated a reduction in total tumor number (Fig. 7 B), smaller tumors (sizes less than 3 mm; Fig. 7 C), larger tumors (sizes more than 3 mm; Fig. 7 D), and overall tumor burden (Fig. 7 E). H&E staining further indicated that tempol treatment inhibited the enhanced tumor formation seen in Cdx2 CreERT2 Apc F/+ Gpx4 F/F mice (Fig. 7 F). Furthermore, tempol treatment effectively inhibited the increased cell proliferation observed in Cdx2 CreERT2 Apc F/+ Gpx4 F/F mice (Fig. 7 G and 7 H) and downregulated the expression of antioxidant proteins, including HO-1 and SOD2 (Fig. 7 I). However, CC3 staining revealed no significant change in apoptosis ( Figure S7B and S7C ). In conclusion, our results suggest that GPX4 deficiency may promote colorectal tumor formation by increasing ROS production. Discussion The major findings of our study center around the crucial role of GPX4 in colorectal cancer progression. GPX4, a key player in ferroptosis and cancer, has an unclear role in colorectal cancer. An earlier study reported no significant overall association with colorectal adenoma risk for GPX4 (Peters et al., 2008 ). However, a recent systematic review showed that carriers of the GPX4 (rs173041) T allele were associated with an increased risk of developing colorectal cancer (Barbosa et al., 2022 ). Another most recent study reports that high expression of GPX4, which suppresses ferroptosis, was associated with poorer 5-year overall survival only in KRAS mutant tumors from male colorectal cancer patients (Yan et al., 2023 ). Surprisingly, in our mouse model, GPX4 deletion in colon epithelial cells increased tumor burden. Treatment with tempol suppressed GPX4 deficiency-induced colorectal tumors, highlighting GPX4's crucial role in inhibiting oxidative stress in colorectal cancer progression. Oxidative stress, a common feature in various human diseases, including colorectal cancer, is emerging as a significant contributor to colorectal cancer development. In colitis-associated colorectal cancer mice, oxidative stress is heightened in the colon (Lei et al., 2021 ). GPX4 is the sole enzyme capable of reducing toxic lipid hydroperoxides in biological membranes to the corresponding alcohols using glutathione as the electron donor (Zhang et al., 2021 ). This aligns with our findings that the deletion of GPX4 resulted in decreased oxidized glutathione levels in colorectal tumors. Consistently, transgenic mice overexpressing GPX4 are protected against oxidative stress-induced apoptosis (Ran et al., 2004 ). Interestingly, in our study, apoptosis levels were significantly altered in the acute colitis model but not in the colitis-associated colorectal cancer mouse model, suggesting other compensatory apoptotic factors are involved in colorectal tumors. GPX4 and SOD2 are two of the most important antioxidant defense enzymes that protect cells against oxidative stress (Fan et al., 2011 ). As a selenoprotein, GPX4 has been reported to play a major role in maintaining the oxidative phosphorylation system and protecting mitochondria from oxidative damage in gut epithelial cells (Cole-Ezea et al., 2012 ). One study reported significant two-loci interactions between rs4880 (SOD2) and rs713041 (GPX4), reflecting functional interactions between the gene products (Meplan et al., 2010). Deletion of SOD2 in skeletal muscle leads to no major impairment in whole-body metabolism, which is likely partly explained by a compensatory response that may exist from other redox enzymes, including GPX4 (Zhuang et al., 2021 ). Considering GPX4 deletion didn't worsen DSS-induced colitis with varied iron diets but showed vulnerability with a vitamin E-deficient diet and increased expression of Mn-dependent SOD2, we propose GPX4 and SOD2 coordinately regulate redox homeostasis. ROS levels are higher in the ulcerative colitis region, but ROS scavenging enzyme SOD2 is barely detected in resident macrophages, resulting in distinct ROS vulnerability for inflammatory macrophages and resident macrophages (Du et al., 2023 ). SOD2 upregulation in cancer cells establishes a steady flow of H 2 O 2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting SOD2 expression or inhibiting AMPK suppresses the metabolic switch and dampens the viability of transformed cells, indicating that the SOD2/AMPK axis is critical to support cancer cell bioenergetics (Hart et al., 2015 ). Also, the absence of SOD2 delays p53-induced tumor formation (Case and Domann 2014 ). Moreover, overexpression of miR-212 reduces the levels of SOD2 to block the epithelial mesenchymal transition process during colorectal tumor metastasis (Meng et al., 2013 ). Consistently, we found that antioxidant proteins, especially SOD2, correlated with DSS-induced colitis severity and colorectal tumor numbers in a Mn-dependent manner. The consumption of antioxidant micronutrients, including Mn, does not modulate the effects of smoking on colorectal cancer risk (Hansen et al., 2013 ). However, residing in the proximity of industries releasing Mn increases colorectal cancer risk (García-Pérez et al., 2020 ). In the absence of GPX4, we found a high Mn diet increased susceptibility, while a low Mn diet reduced DSS-induced colitis and significantly decreased colorectal cancer formation. These findings represent conceptual advances in understanding the nuanced impact of GPX4 in colorectal cancer development, shedding light on its role in oxidative stress regulation, particularly in a Mn-dependent manner. The study provides insights into the intricate interplay between GPX4, dietary factors, and their influence on colorectal cancer progression. In line with our results, hepatocyte restricted GPX4 loss does not suppress hepatocellular tumorigenesis (Conche et al., 2023 ). In hepatocellular carcinoma, ferroptosis does not provide a cell-autonomous tumor suppressor function but rather triggers an adaptive immune response, placing ferroptosis upstream of CD8 + T cells. Ferroptosis is a potent anticancer target for the treatment of hepatocellular carcinoma and colorectal cancer liver metastasis in combination with immune checkpoint and myeloid-derived suppressor cell blockade, while primary colorectal cancer is resistant to this combinatorial treatment. Our study found that the low Mn diet significantly curtailed colorectal cancer in both GPX4-deficient and wild-type mice. This provides an alternative strategy to treat colorectal cancer. In conclusion, the study not only unravels the specific mechanisms by which GPX4 influences colorectal cancer but also suggests potential therapeutic strategies. The findings may resonate with researchers and clinicians working in the fields of cancer biology, oxidative stress, and precision medicine. Moreover, the study's consideration of dietary factors adds a practical dimension, addressing potential preventive measures and highlighting the importance of personalized approaches in cancer care. Declarations Conflicts of Interest: The authors declare that there is no conflict of interests. Data Availability Statement: All data are available upon request or through the associated datasets in the manuscript. Author Contributions: Z.L. and X.X. drafted the manuscript and the figures. D.M., R.L., Y. L., Y. K. and S.H. contributed to data acquisition. X.X. designed the topic and edited the final manuscript. Funding: X.X. received partial support from the National Institute of General Medical Sciences (NIGMS, P20 GM130422), Environmental Health and Toxicology Pilot Awards from UNM Center for Native Environmental Health Equity Research (P50 MD015706), and New Mexico Integrative Science Program Incorporating Research in Environmental Sciences (NM-INSPIRES, 1P30ES032755). X.X. also acknowledges support from a Research Program Support Pilot Project Award from UNM comprehensive cancer center (P30CA118100) and the Cardiovascular and Metabolic Disease Research Program Pilot Project Grant from UNMHSC Office of Research Signature Programs. The funders have no role in the design, analysis, and reporting of the study. Acknowledgment: We extend our heartfelt appreciation to GemPharmatech for their generous support in designing the genotyping protocol for our Gpx4 floxed mice. Their collaboration has been instrumental in advancing our research endeavors. References Aghabozorgi AS, Bahreyni A, Soleimani A, et al. Role of adenomatous polyposis coli (APC) gene mutations in the pathogenesis of colorectal cancer; current status and perspectives. Biochimie 2019; 157:64–71. Barbosa P, Abo El-Magd NF, Hesketh J, et al. The Role of rs713041 Glutathione Peroxidase 4 (GPX4) Single Nucleotide Polymorphism on Disease Susceptibility in Humans: A Systematic Review and Meta-Analysis. Int. J. Mol. Sci. 2022;23. Canli Ö, Nicolas AM, Gupta J, et al. Myeloid Cell-Derived Reactive Oxygen Species Induce Epithelial Mutagenesis. Cancer Cell 2017; 32:869-883.e5. Case AJ, Domann FE. Absence of manganese superoxide dismutase delays p53-induced tumor formation. Redox Biol 2014; 2:220–3. Cheng J, Zhu Y, Xing X, Xiao J, Chen H, Zhang H, Wang D, Zhang Y, Zhang G, Wu Z, Liu Y. Manganese-deposited iron oxide promotes tumor-responsive ferroptosis that synergizes the apoptosis of cisplatin. Theranostics 2021; 11(11):5418-5429. Choi E-K, Aring L, Das NK, et al. Impact of dietary manganese on experimental colitis in mice. FASEB J 2020; 34:2929–43. Cole-Ezea P, Swan D, Shanley D, et al. Glutathione peroxidase 4 has a major role in protecting mitochondria from oxidative damage and maintaining oxidative phosphorylation complexes in gut epithelial cells. Free Radic Biol Med 2012; 53:488–97. Conche C, Finkelmeier F, Pešić M, et al. Combining ferroptosis induction with MDSC blockade renders primary tumours and metastases in liver sensitive to immune checkpoint blockade. Gut 2023;72:1774 LP – 1782. Du J, Zhang J, Wang L, et al. Selective oxidative protection leads to tissue topological changes orchestrated by macrophage during ulcerative colitis. Nat Commun 2023;14:3675. El Marjou F, Janssen KP, Chang BHJ, et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis 2004; 39:186–93. Fan Y-Y, Ran Q, Toyokuni S, et al. Dietary Fish Oil Promotes Colonic Apoptosis and Mitochondrial Proton Leak in Oxidatively Stressed Mice. Cancer Prev Res 2011; 4:1267–74. García-Pérez J, Fernández de Larrea-Baz N, Lope V, et al. Residential proximity to industrial pollution sources and colorectal cancer risk: A multicase-control study (MCC-Spain). Environ Int 2020;144:106055. Hare DJ, Kysenius K, Paul B, et al. Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS). JoVE 2017; e55042. Hart PC, Mao M, de Abreu ALP, et al. MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer. Nat Commun 2015; 6:6053. Hansen RD, Albieri V, Tjønneland A, et al. Effects of Smoking and Antioxidant Micronutrients on Risk of Colorectal Cancer. Clin Gastroenterol Hepatol 2013; 11:406-415.e3. Hinoi T, Akyol A, Theisen BK, et al. Mouse Model of Colonic Adenoma-Carcinoma Progression Based on Somatic Apc Inactivation. Cancer Res 2007; 67:9721–30. Hu Q, Zhang Y, Lou H, et al. GPX4 and vitamin E cooperatively protect hematopoietic stem and progenitor cells from lipid peroxidation and ferroptosis. Cell Death Dis 2021; 12:706. Lei L, Yang J, Zhang J, et al. The lipid peroxidation product EKODE exacerbates colonic inflammation and colon tumorigenesis. Redox Biol 2021;42:101880. Liu Z, Villareal L, Goodla L, et al. Iron promotes glycolysis to drive colon tumorigenesis. Biochim Biophys Acta - Mol Basis Dis 2023;166846. Mayr L, Grabherr F, Schwärzler J, et al. Dietary lipids fuel GPX4-restricted enteritis resembling Crohn’s disease. Nat Commun 2020; 11:1775. doi:10.1038/s41467-020-15646-6. Meng X, Wu J, Pan C, et al. Genetic and Epigenetic Down-regulation of MicroRNA-212 Promotes Colorectal Tumor Metastasis via Dysregulation of MnSOD. Gastroenterology 2013; 145:426-436.e6. Méplan C, Hughes DJ, Pardini B, et al. Genetic variants in selenoprotein genes increase risk of colorectal cancer. Carcinogenesis 2010;31:1074–9. Naranuntarat A, Jensen LT, Pazicni S, et al. The Interaction of Mitochondrial Iron with Manganese Superoxide Dismutase *. J Biol Chem 2009; 284:22633–40. Peters U, Chatterjee N, Hayes RB, et al. Variation in the Selenoenzyme Genes and Risk of Advanced Distal Colorectal Adenoma. Cancer Epidemiol Biomarkers Prev 2008; 17:1144–54. Ran Q, Liang H, Gu M, et al. Transgenic Mice Overexpressing Glutathione Peroxidase 4 Are Protected against Oxidative Stress-induced Apoptosis. J Biol Chem 2004; 279:55137–46. Stockwell BR, Friedmann Angeli JP, Bayir H, et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell . 2017;171(2):273-285. Sui X, Zhang R, Liu S, et al. RSL3 Drives Ferroptosis Through GPX4 Inactivation and ROS Production in Colorectal Cancer. Front. Pharmacol. 2018;9. TeSlaa T, Bartman CR, Jankowski CSR, et al. The Source of Glycolytic Intermediates in Mammalian Tissues. Cell Metab 2021; 33:367-378.e5. Wang G, Qin S, Chen L, et al. Butyrate dictates ferroptosis sensitivity through FFAR2-mTOR signaling. Cell Death Dis 2023;14:292. Wang R, Su Q, Yin H, et al. Inhibition of SRSF9 enhances the sensitivity of colorectal cancer to erastin-induced ferroptosis by reducing glutathione peroxidase 4 expression. Int J Biochem Cell Biol 2021; 134:105948. Weaver K, Skouta R. The Selenoprotein Glutathione Peroxidase 4: From Molecular Mechanisms to Novel Therapeutic Opportunities. Biomedicines. 2022;10. doi:10.3390/biomedicines10040891. Yan H, Talty R, Jain A, et al. Discovery of decreased ferroptosis in male colorectal cancer patients with KRAS mutations. Redox Biol 2023;62:102699. Zhang X, Ma Y, Ma J, et al. Glutathione Peroxidase 4 as a Therapeutic Target for Anti-Colorectal Cancer Drug-Tolerant Persister Cells. Front. Oncol. 2022;12. Zhang Y, Swanda R V, Nie L, et al. mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation. Nat Commun 2021; 12:1589. Zhuang A, Yang C, Liu Y, et al. SOD2 in skeletal muscle: New insights from an inducible deletion model. Redox Biol 2021;47:102135. Additional Declarations There is NO Competing Interest. Supplementary Files TableS1Primers.pdf TableS2Metabolitesnamemap.pdf 240102SupplementMaterials.pdf Supple Materials 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. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3837925","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":265793989,"identity":"7b9b4120-342a-46ea-86c5-4fbe0695bccd","order_by":0,"name":"Xiang Xue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYLCCDwwMMgwMCVDeASJ0MM5gYOAhTQszD0laDG7kGD62+WPHY3A8gU26MIdBju9GAn4tkj1njI1z25J5DM48YJOeuY3BWJKQFn72HjPp3AZmHoMbQFt4tzEkbiCkhY2Zx/y3xZ96uJZ6glpAtjAzsB2Ga0kwIOyXY8WSvW3HeSTPPGy25t0mYTjzzAP8WgxuJG/88ONPtRzf8eSDt3m32cjzHSdgCxJgbAASEkQrHwWjYBSMglGABwAAppY+BVv8LisAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4704-1814","institution":"University of New Mexico","correspondingAuthor":true,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Xue","suffix":""},{"id":265793990,"identity":"af6a7f4d-5165-4ee2-9c7a-fe054f01d4f1","order_by":1,"name":"Zhaoli Liu","email":"","orcid":"","institution":"University of New Mexico","correspondingAuthor":false,"prefix":"","firstName":"Zhaoli","middleName":"","lastName":"Liu","suffix":""},{"id":265793991,"identity":"aa7e0a40-4213-4e8f-bdf7-82188e966c34","order_by":2,"name":"Yanshan Liang","email":"","orcid":"","institution":"Harvard T.H. Chan School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Yanshan","middleName":"","lastName":"Liang","suffix":""},{"id":265793992,"identity":"ccbfbd6f-d3df-4331-b7de-9965ce341b15","order_by":3,"name":"Young-Yon Kwon","email":"","orcid":"","institution":"Harvard T.H. Chan School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Young-Yon","middleName":"","lastName":"Kwon","suffix":""},{"id":265793993,"identity":"e949e4c0-5e70-4fdd-bf78-5ff535ad7b3f","order_by":4,"name":"Rui Liu","email":"","orcid":"","institution":"University of New Mexico","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Liu","suffix":""},{"id":265793994,"identity":"22835820-cb2d-45dd-b6ac-b6e3a5886f18","order_by":5,"name":"David Martin","email":"","orcid":"","institution":"UNM","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Martin","suffix":""},{"id":265793995,"identity":"93620619-c794-40ae-8cf4-5b875c8ec0f6","order_by":6,"name":"Sheng Hui","email":"","orcid":"","institution":"Harvard","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Hui","suffix":""}],"badges":[],"createdAt":"2024-01-05 18:06:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3837925/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3837925/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49300197,"identity":"9f8fc71b-5139-4d40-bdb3-0bd7a102f4f9","added_by":"auto","created_at":"2024-01-08 09:17:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2682905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPX4 depletion specifically in colon epithelial cells promotes colorectal tumorigenesis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Body weight curve, (\u003cstrong\u003eB\u003c/strong\u003e) macroscopic colorectal tumor images, (\u003cstrong\u003eC\u003c/strong\u003e) total tumor number, (\u003cstrong\u003eD\u003c/strong\u003e) tumor number for diameter less than 3mm, (\u003cstrong\u003eE\u003c/strong\u003e) tumor number for diameter more than 3mm. (\u003cstrong\u003eF\u003c/strong\u003e) Tumor loading, (\u003cstrong\u003eG\u003c/strong\u003e) H\u0026amp;E staining and (\u003cstrong\u003eH\u003c/strong\u003e) pathological score, (\u003cstrong\u003eI\u003c/strong\u003e) Ki67 staining and (\u003cstrong\u003eJ\u003c/strong\u003e) its quantification. (\u003cstrong\u003eK\u003c/strong\u003e) Cleaved caspase 3 (CC3) staining in colorectal tumor from in \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n=12)\u003cem\u003e \u003c/em\u003eand \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Gpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (n=12)\u003cem\u003e \u003c/em\u003eand (\u003cstrong\u003eL\u003c/strong\u003e) its quantification.\u003cstrong\u003e \u003c/strong\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001, NS not significant. Unpaired Student t test.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/c31a291ba1a79d0168c81e68.jpg"},{"id":49299471,"identity":"f0996012-9a8e-4813-b8a6-649e91c14445","added_by":"auto","created_at":"2024-01-08 09:09:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":824837,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPX4 depletion specifically in colon epithelial cells leads to redox imbalance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Overview of enriched Top 25 metabolites sets in colon tumors from mice with GPX4 depletion specifically in colon epithelial cells. The levels of (\u003cstrong\u003eB\u003c/strong\u003e) reduced oxidized glutathione, (\u003cstrong\u003eC\u003c/strong\u003e) glycine and (\u003cstrong\u003eD\u003c/strong\u003e) spermidine in colon tissues from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n=5) and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Gpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (n=5). *p \u0026lt; 0.05, **p \u0026lt; 0.01, NS not significant. Unpaired Student t test.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/7b00bc928d896ece6a40e4b4.jpg"},{"id":49299475,"identity":"23028068-4260-46ce-b5af-88ac9b2156fa","added_by":"auto","created_at":"2024-01-08 09:09:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2422879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPX4 depletion specifically in colon epithelial cells leads to increased intestinal inflammation when treated with vitamin E deficient diet.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Body weight loss curve, (\u003cstrong\u003eB\u003c/strong\u003e) colon length, (\u003cstrong\u003eC\u003c/strong\u003e) Western blot analysis of antioxidant protein levels of NQO1, HO-1, and SOD2, (\u003cstrong\u003eD\u003c/strong\u003e) H\u0026amp;E staining of colon and (\u003cstrong\u003eE\u003c/strong\u003e) its disease score, (\u003cstrong\u003eF\u003c/strong\u003e) Ki67 staining and (\u003cstrong\u003eG\u003c/strong\u003e) its quantification, (\u003cstrong\u003eH\u003c/strong\u003e) CC3 staining and (\u003cstrong\u003eI\u003c/strong\u003e) its quantification in colon tissues from \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e (Cre-, n=5) and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e (Cre+, n=5) mice. *p \u0026lt; 0.05, **p \u0026lt; 0.01, NS not significant. Unpaired Student t test.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/5a9e6331705da445c821f4e2.jpg"},{"id":49300198,"identity":"355c72f7-19c8-47ee-ae1c-0ae311c7932c","added_by":"auto","created_at":"2024-01-08 09:17:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1996876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPX4 depletion specifically in colon epithelial cells increases intestinal inflammation under a high manganese (Mn) diet treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Body weight loss curve and \u003cstrong\u003e(B) \u003c/strong\u003ecolon length, \u003cstrong\u003e(C)\u003c/strong\u003e Western blot analysis of antioxidant protein levels of NQO1, HO-1 and SOD2, \u003cstrong\u003e(D)\u003c/strong\u003e H\u0026amp;E staining and \u003cstrong\u003e(E)\u003c/strong\u003e pathological score of colon tissues, \u003cstrong\u003e(F)\u003c/strong\u003e Ki67 staining and \u003cstrong\u003e(G)\u003c/strong\u003e its quantification, \u003cstrong\u003e(H)\u003c/strong\u003e CC3 staining and \u003cstrong\u003e(I)\u003c/strong\u003e its quantification in colon tissues from \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e (n=7) and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (n=8). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, NS not significant. Unpaired Student t test.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/d3d3f25ac0f8d4f4a3a7a242.jpg"},{"id":49299478,"identity":"de980869-4c59-439b-82e4-2bafc59a2e69","added_by":"auto","created_at":"2024-01-08 09:09:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2379586,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPX4 depletion specifically in colon epithelial cells reduces intestinal inflammation under a low Mn diet treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Body weight loss curve, \u003cstrong\u003e(B)\u003c/strong\u003e colon length, \u003cstrong\u003e(C)\u003c/strong\u003e Western blot analysis of antioxidant protein levels of NQO1, HO-1 and SOD2,\u003cstrong\u003e (D)\u003c/strong\u003e H\u0026amp;E staining of colon tissues and \u003cstrong\u003e(E)\u003c/strong\u003e pathological score, \u003cstrong\u003e(F)\u003c/strong\u003e Ki67 staining and \u003cstrong\u003e(G)\u003c/strong\u003e its quantification for colon tissues, \u003cstrong\u003e(H)\u003c/strong\u003e CC3 staining and \u003cstrong\u003e(I)\u003c/strong\u003e its quantification in colon tissues from \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e (n=11) and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (n=8). *p \u0026lt; 0.05, **p \u0026lt; 0.01, NS not significant. Unpaired Student t test.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/27d7ede42e350b91ac2b6293.jpg"},{"id":49299477,"identity":"82a1ea7a-a56c-4fcc-9314-753f717893ba","added_by":"auto","created_at":"2024-01-08 09:09:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2387816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMn deficient diet reduces colorectal tumor formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Body weight curve, \u003cstrong\u003e(B)\u003c/strong\u003e macroscopic colorectal tumor images, \u003cstrong\u003e(C) \u003c/strong\u003etotal tumor number, \u003cstrong\u003e(D)\u003c/strong\u003e tumor number for diameter less than 3mm, \u003cstrong\u003e(E) \u003c/strong\u003etumor number for diameter more than 3mm\u003cstrong\u003e,\u003c/strong\u003e and \u003cstrong\u003e(F)\u003c/strong\u003e tumor loading, \u003cstrong\u003e(G)\u003c/strong\u003e H\u0026amp;E staining and \u003cstrong\u003e(H)\u003c/strong\u003e pathological score of colon tumor tissues, \u003cstrong\u003e(I)\u003c/strong\u003e Ki67 staining in colorectal tumor and \u003cstrong\u003e(J) \u003c/strong\u003eits quantification, \u003cstrong\u003e(K)\u003c/strong\u003e CC3 staining in colorectal tumor and \u003cstrong\u003e(L) \u003c/strong\u003eits quantification. \u003cstrong\u003e(M)\u003c/strong\u003e Western blot analysis of antioxidant protein levels of NQO1, HO-1 and SOD2 protein expression in colon tissues from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n=5) and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Gpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e (n=6) mice. *p \u0026lt; 0.05, **p \u0026lt; 0.01, NS not significant. Unpaired Student t test.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/3073238cdd8d11e37008e4fe.jpg"},{"id":49299473,"identity":"e050f98c-cfae-446b-b3ab-1783f4041c74","added_by":"auto","created_at":"2024-01-08 09:09:45","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2727042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOD mimetic tempol treatment reduces colon epithelial cells specific GPX4 deficiency enhanced colorectal tumor formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Macroscopic colorectal tumor images, \u003cstrong\u003e(B) \u003c/strong\u003etotal tumor number, \u003cstrong\u003e(C)\u003c/strong\u003e tumor number for diameter less than 3mm, \u003cstrong\u003e(D) \u003c/strong\u003etumor number for diameter more than 3mm\u003cstrong\u003e,\u003c/strong\u003e and \u003cstrong\u003e(E)\u003c/strong\u003e tumor loading, \u003cstrong\u003e(F)\u003c/strong\u003e H\u0026amp;E staining and \u003cstrong\u003e(G)\u003c/strong\u003e Ki67 staining and \u003cstrong\u003e(H) \u003c/strong\u003eits quantification,\u003cstrong\u003e (I)\u003c/strong\u003e Western blot analysis of antioxidant protein levels of NQO1, HO-1 and SOD2 protein expression of colon tumor tissues from \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n=7) and \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Gpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e (n=7) mice treated with 2% DSS and vehicle control or Tempol. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e mice, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001 vs \u003cem\u003eCdx2\u003c/em\u003e\u003csup\u003e CreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Gpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice, NS not significant. Two-way ANOVA followed by Tukey’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/5faaa5475fd8fff9d664bf4f.jpg"},{"id":52770178,"identity":"eebcbc3b-b0a6-4b30-b023-1444eb4a698a","added_by":"auto","created_at":"2024-03-15 14:29:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1628904,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/edc65375-edb8-4476-bd6e-d7626a3c5b01.pdf"},{"id":49299470,"identity":"30d37c5b-9b8c-473c-9c84-1ccbf0d3a1a1","added_by":"auto","created_at":"2024-01-08 09:09:45","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":34191,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1Primers.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/eb645b8036847866eab5d1bf.pdf"},{"id":49299472,"identity":"ee0e8f4e-645d-4414-b6f4-328fc50fa410","added_by":"auto","created_at":"2024-01-08 09:09:45","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":88194,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2Metabolitesnamemap.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/e4aca869c159bea391e69bac.pdf"},{"id":49299479,"identity":"3735ae2d-8da9-44d8-a209-a021708e6da6","added_by":"auto","created_at":"2024-01-08 09:09:45","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":7069664,"visible":true,"origin":"","legend":"\u003cp\u003eSupple Materials\u003c/p\u003e","description":"","filename":"240102SupplementMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3837925/v1/5d06fe0924da1140cfecfa51.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Glutathione peroxidase 4 suppresses manganese-dependent oxidative stress to reduce colorectal tumorigenesis.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFerroptosis, a distinctive process among various types of cell death, has garnered significant attention in cancer research. It is a mechanism through which cells, particularly cancer cells, undergo elimination due to the accumulation of excessive iron and lipid peroxides (Stockwell et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Ferroptosis can be considered an intrinsic defense mechanism against cancer, especially given the heightened vulnerability of many cancer cells to ferroptosis due to their elevated metabolic rate and increased demand for iron and lipids. Consequently, inducing ferroptosis in cancer cells emerges as a promising therapeutic strategy.\u003c/p\u003e \u003cp\u003eWhile ferroptosis holds promise as a cancer treatment strategy, several challenges persist. For example, colorectal cancer, which accumulates substantial iron, has developed mechanisms to resist ferroptosis. Notably, the expression of glutathione peroxidase 4 (GPX4), a key protective protein that reduces lipid peroxides and prevents ferroptosis, is upregulated in colorectal cancer patients undergoing neoadjuvant chemoradiotherapy, correlating with a poor prognosis (Zhang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe resistance of colorectal cancer to ferroptosis poses a challenge in cancer treatment. Researchers are actively exploring ways to exploit ferroptosis as a therapeutic approach. Investigations involve the use of small molecules and drugs that either inhibit GPX4 or induce lipid peroxidation to selectively target cancer cells. Additionally, there is ongoing research into combining ferroptosis-inducing agents with traditional cancer therapies, such as chemotherapy or radiation.\u003c/p\u003e \u003cp\u003eA comprehensive understanding of the precise mechanisms underlying ferroptosis in cancer cells and the development of effective and safe therapies are ongoing areas of research. For example, a study demonstrated that the inhibition of serine/arginine-rich splicing factor 9 (SRSF9) enhances the sensitivity of colorectal cancer to erastin-induced ferroptosis by reducing GPX4 expression (Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In a separate study, sodium butyrate was found to promote ferroptosis by inducing lipid reactive oxygen species (ROS) production through the downregulation of GPX4, leading to reduced tumor growth in xenografts and a colitis-associated colorectal tumor model (Wang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, the GPX4 inhibitor RSL3 was shown to hinder colorectal cancer cell growth by inducing ferroptosis, an effect reversible by overexpressing GPX4 (Sui et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, the deficiency of GPX4 in myeloid cells has been demonstrated to actively promote the progression of colorectal cancer (Canli et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Specifically, this deficiency leads to an augmented production of ROS, instigating genome-wide DNA mutations within intestinal epithelial cells, ultimately fostering tumor invasion. However, no studies have investigated the influence of colon epithelial-specific GPX4 knockout on colorectal cancer progression.\u003c/p\u003e \u003cp\u003eOur findings reveal that GPX4 depletion in colorectal epithelial cells unexpectedly enhances colorectal tumorigenesis. Mechanistically, under conditions of high oxidative stress, including vitamin E deficiency, elevated manganese (Mn) exposure, and tumor development, superoxide dismutase (SOD2) is upregulated in the colon tissues of colorectal epithelial GPX4 deficient mice. A low Mn diet and the SOD mimetic radical scavenger Tempol mitigate GPX4 deficiency induced SOD2 expression and colorectal tumor formation. These findings underscore the crucial role of GPX4 in suppressing Mn-dependent oxidative stress to curtail colorectal tumorigenesis.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eDepletion of GPX4 specifically in colon epithelial cells promotes colorectal tumorigenesis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the role of GPX4 in colorectal tumorigenesis, we generated mice with colon epithelial cell specific \u003cem\u003eGpx4\u003c/em\u003e depletion. GPX4 deletion was confirmed by genotyping (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e) and qPCR (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). Colorectal cancer was induced as described in the methods, with the treatment scheme illustrated in \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e. We observed that, compared to \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e mice, mice with colon epithelial-specific GPX4 depletion (\u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e) showed no difference in body weight change compared with GPX4 wildtype mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). However, GPX4 deficient mice exhibited increased colorectal tumor formation under a dissection microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The number of total tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), tumors with a diameter less than 3mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), and tumors with a diameter more than 3mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) were all increased in the GPX4 deficient mice. Additionally, there was an increased tumor burden in the GPX4 deficient mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). H\u0026amp;E staining of colorectal tumors from GPX4 deficient and wildtype mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) indicated a similar disease score in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Ki67 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), and quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ) suggested increased cell proliferation in the GPX4 deficient mice, while the apoptosis marker CC3 was not changed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). These results suggest a tumor-inhibiting function of GPX4 in colorectal cancer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDepletion of GPX4 specifically in colon epithelial cells results in reduced oxidized glutathione levels in colorectal tumors.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo understand the mechanism behind the enhanced colorectal tumorigenesis in GPX4-deficient mice, we conducted metabolomics analysis on colon tumors from \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice and \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e mice. Targeted metabolomics revealed two significantly changed metabolites (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e): Threonine and Homovanillic acid. In untargeted metabolomics, 41 significantly changed metabolites were identified in the negative mode and 85 in the positive mode (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Enrichment analysis of these 127 significantly changed metabolites using MetaboAnalyst 6.0 highlighted glutathione metabolism and Vitamin B6 metabolism as the top two enriched metabolite sets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This enrichment is due to decreased levels of oxidized glutathione (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), glycine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and spermidine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) in the colon tumors of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice compared to those in \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e mice. These findings suggest a redox imbalance status due to the critical role of GPX4 in glutathione oxidation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGPX4 depletion specifically in colon epithelial cells increases susceptibility to colitis in mice treated with a vitamin E-deficient diet.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs inflammation is a high-risk factor for colorectal cancer and intestinal epithelial GPX4 restricts enteritis (Mayr et al, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), we investigated whether colon epithelial GPX4 deficiency in mice affects their susceptibility to colitis. There was no difference in body weight and colon length between \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice and \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice after treating mice with tamoxifen and DSS. Despite GPX4 being a well-known ferroptosis inhibitor, no differences were observed in body weight and colon length between \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice and \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice with different iron concentrations in their diets (3.5ppm, 40ppm, 1000ppm) (\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u0026ndash;S2H\u003c/b\u003e). Similar results were seen after \u003cem\u003eSalmonella\u003c/em\u003e treatment (\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eI and S2J\u003c/b\u003e). A vitamin E supplement diet has been demonstrated to reduce GPX4 deficiency-enhanced ferroptosis in hematopoietic stem and progenitor cells (Hu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Interestingly, \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice exhibited increased body weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and shorter colon lengths compared to \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) when induced with DSS and treated with a vitamin E-deficient diet. qPCR analysis indicated that the expression level of \u003cem\u003eGpx4\u003c/em\u003e was significantly decreased in the colon tissues of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice compared to wildtype control (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA\u003c/b\u003e). Interestingly, proinflammatory cytokines including \u003cem\u003eTnfa\u003c/em\u003e \u003cb\u003e(Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB)\u003c/b\u003e, \u003cem\u003eIl6\u003c/em\u003e \u003cb\u003e(Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC)\u003c/b\u003e, \u003cem\u003eCxcl1\u003c/em\u003e \u003cb\u003e(Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD)\u003c/b\u003e and \u003cem\u003ePtgs2\u003c/em\u003e \u003cb\u003e(Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE)\u003c/b\u003e, as well as stem cell marker \u003cem\u003eLgr5\u003c/em\u003e \u003cb\u003e(Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF)\u003c/b\u003e was not changed. However, the anti-inflammatory cytokines including Il10 \u003cb\u003e(Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG)\u003c/b\u003e, \u003cem\u003eIl22\u003c/em\u003e \u003cb\u003e(Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eH)\u003c/b\u003e, and transcription factor \u003cem\u003eFoxm1\u003c/em\u003e \u003cb\u003e(Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eI)\u003c/b\u003e were significantly decreased in the colon tissues of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice. qPCR analysis revealed a significant decrease in the expression level of \u003cem\u003eGpx4\u003c/em\u003e in the colon tissues of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice compared to the wildtype control (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA\u003c/b\u003e). Interestingly, proinflammatory cytokines, including \u003cem\u003eTnfa\u003c/em\u003e (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB\u003c/b\u003e), \u003cem\u003eIl6\u003c/em\u003e (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC\u003c/b\u003e), \u003cem\u003eCxcl1\u003c/em\u003e (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD\u003c/b\u003e), \u003cem\u003ePtgs2\u003c/em\u003e (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE\u003c/b\u003e), and the stem cell marker \u003cem\u003eLgr5\u003c/em\u003e (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF\u003c/b\u003e), remained unchanged. However, anti-inflammatory cytokines, including \u003cem\u003eIl10\u003c/em\u003e (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG\u003c/b\u003e), \u003cem\u003eIl22\u003c/em\u003e (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eH\u003c/b\u003e), and the transcription factor \u003cem\u003eFoxm1\u003c/em\u003e (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eI\u003c/b\u003e), were significantly decreased in the colon tissues of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice. The immunoblot analysis of several major antioxidant proteins, including SOD2, NQO1, and HO-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), revealed an increase in these proteins in \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice, confirming a state of redox imbalance. H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and pathology scoring (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) indicated increased inflammation and tissue damage. While the proliferation marker Ki67 remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), an increase in the apoptosis marker CC3 was observed in colon tissues of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). This suggests that GPX4 deficiency contributes to enhanced apoptosis during the colitis process. Together, in the absence of vitamin E, mice deficient in GPX4 exhibit heightened susceptibility to DSS-induced colitis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGPX4 depletion specifically in colon epithelial cells increases intestinal inflammation in mice treated with a high Mn diet.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWhile GPX4 is a selenium-dependent enzyme (Weaver et al, 2022), SOD2 is one of the rare mitochondrial enzymes evolved to use Mn as a cofactor over the more abundant element iron (Naranuntarat et al, 2017). Compared with iron, Mn can catalyze the Fenton reaction more effectively to induce higher ROS production (Cheng et al, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We found that when treated with a high Mn diet, \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice showed more body weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and shorter colon lengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Also, there was an increased expression of antioxidant proteins such as SOD2, NQO1, and HO-1 in colon tissues of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice compared to \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). However, H\u0026amp;E staining revealed a similar disease score (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). We found that cell proliferation, as indicated by Ki67 staining, remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). However, there was an observed increase in apoptosis, demonstrated by CC3 staining in the colons of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). These results indicate that in the presence of a high concentration of Mn, GPX4 deficient mice are more susceptible to DSS-induced colitis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGPX4 depletion specifically in colon epithelial cells reduces intestinal inflammation in mice treated with a low Mn diet.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo test whether Mn is required for enhanced colonic inflammation due to GPX4 deficiency, we treated mice with a low Mn diet. We observed that \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice experienced less body weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and longer colon length (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) than \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice under Mn deficient conditions. Additionally, there was a reduction in the expression of antioxidant proteins such as SOD2, NQO1, and HO-1 in the colon tissue of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). H\u0026amp;E staining and pathological scoring revealed reduced inflammation in \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice when treated with a Mn deficient diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). We also noted Ki67 expression remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), while reduced apoptosis through CC3 staining in the colons of \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), These results indicate that GPX4 depletion reduces intestinal inflammation in mice treated with a low Mn diet.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMn deficiency reduces colorectal tumor formation in mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMn plays a crucial role in intestinal epithelial barrier formation, and a low Mn diet has been linked to an increase in DSS-induced colitis in mice (Choi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, whether Mn influences the progression of colitis-induced colorectal cancer is not clear. To address this question, we induced colorectal tumor formation under three dietary conditions: a control diet, a Mn-deficient diet, and a high Mn diet. Unfortunately, the high Mn diet was associated with reduced mouse survival during DSS treatment (\u003cb\u003eFigure S4\u003c/b\u003e). We observed that the Mn-deficient diet did not impact mouse body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) but significantly reduced colorectal tumor formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The Mn-deficient diet group exhibited decreased total tumor numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), smaller tumors (sizes less than 3mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), larger tumors (sizes more than 3 mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), and overall tumor burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). H\u0026amp;E staining revealed lower disease scores in the Mn-deficient diet group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Ki67 staining demonstrated reduced tumor cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ), while CC3 staining showed no change in apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Western blot analysis indicated reduced levels of the antioxidant protein SOD2 and the Mn efflux protein ZnT10 in the Mn-deficient diet group, while the protein levels of HO-1 and NQO1 remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). Further analysis of metal levels in colon tumors via laser ablation ICP-MS revealed significantly lower Mn levels, but not other metals, in the Mn-deficient diet group compared to the control diet group (\u003cb\u003eFigure S5A and S5B\u003c/b\u003e). The Mn-deficient diet fed to \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice showed a reduction in total tumor numbers (\u003cb\u003eFigure S6A\u003c/b\u003e) and tumor numbers at size 2-3mm (\u003cb\u003eFigure S6B\u003c/b\u003e) compared to the control diet. However, it led to no significant differences in smaller tumors (sizes less than 1mm; \u003cb\u003eFigure S6C\u003c/b\u003e), tumor numbers at size 1-2mm (\u003cb\u003eFigure S6D\u003c/b\u003e), and larger tumors (sizes more than 3 mm; \u003cb\u003eFigure S6E\u003c/b\u003e), as well as overall tumor burden (\u003cb\u003eFigure S6F\u003c/b\u003e). These results suggest that Mn is essential for colorectal tumor formation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe SOD mimetic tempol treatment rescues GPX4-inhibited colorectal tumor formation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether GPX4 deficiency-enhanced colorectal cancer depends on oxidative stress, we treated both \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice with the general SOD-mimetic agent tempol. We observed no impact of tempol on mouse body weight (\u003cb\u003eFigure S7A\u003c/b\u003e); however, under the microscope, \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice exhibited increased colorectal tumor formation, a phenomenon that was effectively blocked by tempol treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Following tempol treatment, \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice demonstrated a reduction in total tumor number (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), smaller tumors (sizes less than 3 mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), larger tumors (sizes more than 3 mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), and overall tumor burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). H\u0026amp;E staining further indicated that tempol treatment inhibited the enhanced tumor formation seen in \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Furthermore, tempol treatment effectively inhibited the increased cell proliferation observed in \u003cem\u003eCdx2\u003c/em\u003e \u003csup\u003eCreERT2\u003c/sup\u003e \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003eF/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eGpx4\u003c/em\u003e\u003csup\u003eF/F\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH) and downregulated the expression of antioxidant proteins, including HO-1 and SOD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). However, CC3 staining revealed no significant change in apoptosis (\u003cb\u003eFigure S7B and S7C\u003c/b\u003e). In conclusion, our results suggest that GPX4 deficiency may promote colorectal tumor formation by increasing ROS production.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe major findings of our study center around the crucial role of GPX4 in colorectal cancer progression. GPX4, a key player in ferroptosis and cancer, has an unclear role in colorectal cancer. An earlier study reported no significant overall association with colorectal adenoma risk for GPX4 (Peters et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, a recent systematic review showed that carriers of the GPX4 (rs173041) T allele were associated with an increased risk of developing colorectal cancer (Barbosa et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Another most recent study reports that high expression of GPX4, which suppresses ferroptosis, was associated with poorer 5-year overall survival only in KRAS mutant tumors from male colorectal cancer patients (Yan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Surprisingly, in our mouse model, GPX4 deletion in colon epithelial cells increased tumor burden. Treatment with tempol suppressed GPX4 deficiency-induced colorectal tumors, highlighting GPX4's crucial role in inhibiting oxidative stress in colorectal cancer progression.\u003c/p\u003e \u003cp\u003eOxidative stress, a common feature in various human diseases, including colorectal cancer, is emerging as a significant contributor to colorectal cancer development. In colitis-associated colorectal cancer mice, oxidative stress is heightened in the colon (Lei et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). GPX4 is the sole enzyme capable of reducing toxic lipid hydroperoxides in biological membranes to the corresponding alcohols using glutathione as the electron donor (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This aligns with our findings that the deletion of GPX4 resulted in decreased oxidized glutathione levels in colorectal tumors. Consistently, transgenic mice overexpressing GPX4 are protected against oxidative stress-induced apoptosis (Ran et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Interestingly, in our study, apoptosis levels were significantly altered in the acute colitis model but not in the colitis-associated colorectal cancer mouse model, suggesting other compensatory apoptotic factors are involved in colorectal tumors.\u003c/p\u003e \u003cp\u003eGPX4 and SOD2 are two of the most important antioxidant defense enzymes that protect cells against oxidative stress (Fan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). As a selenoprotein, GPX4 has been reported to play a major role in maintaining the oxidative phosphorylation system and protecting mitochondria from oxidative damage in gut epithelial cells (Cole-Ezea et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). One study reported significant two-loci interactions between rs4880 (SOD2) and rs713041 (GPX4), reflecting functional interactions between the gene products (Meplan et al., 2010). Deletion of SOD2 in skeletal muscle leads to no major impairment in whole-body metabolism, which is likely partly explained by a compensatory response that may exist from other redox enzymes, including GPX4 (Zhuang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Considering GPX4 deletion didn't worsen DSS-induced colitis with varied iron diets but showed vulnerability with a vitamin E-deficient diet and increased expression of Mn-dependent SOD2, we propose GPX4 and SOD2 coordinately regulate redox homeostasis.\u003c/p\u003e \u003cp\u003eROS levels are higher in the ulcerative colitis region, but ROS scavenging enzyme SOD2 is barely detected in resident macrophages, resulting in distinct ROS vulnerability for inflammatory macrophages and resident macrophages (Du et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). SOD2 upregulation in cancer cells establishes a steady flow of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting SOD2 expression or inhibiting AMPK suppresses the metabolic switch and dampens the viability of transformed cells, indicating that the SOD2/AMPK axis is critical to support cancer cell bioenergetics (Hart et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Also, the absence of SOD2 delays p53-induced tumor formation (Case and Domann \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Moreover, overexpression of miR-212 reduces the levels of SOD2 to block the epithelial mesenchymal transition process during colorectal tumor metastasis (Meng et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Consistently, we found that antioxidant proteins, especially SOD2, correlated with DSS-induced colitis severity and colorectal tumor numbers in a Mn-dependent manner.\u003c/p\u003e \u003cp\u003eThe consumption of antioxidant micronutrients, including Mn, does not modulate the effects of smoking on colorectal cancer risk (Hansen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, residing in the proximity of industries releasing Mn increases colorectal cancer risk (Garc\u0026iacute;a-P\u0026eacute;rez et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the absence of GPX4, we found a high Mn diet increased susceptibility, while a low Mn diet reduced DSS-induced colitis and significantly decreased colorectal cancer formation. These findings represent conceptual advances in understanding the nuanced impact of GPX4 in colorectal cancer development, shedding light on its role in oxidative stress regulation, particularly in a Mn-dependent manner. The study provides insights into the intricate interplay between GPX4, dietary factors, and their influence on colorectal cancer progression.\u003c/p\u003e \u003cp\u003eIn line with our results, hepatocyte restricted GPX4 loss does not suppress hepatocellular tumorigenesis (Conche et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In hepatocellular carcinoma, ferroptosis does not provide a cell-autonomous tumor suppressor function but rather triggers an adaptive immune response, placing ferroptosis upstream of CD8\u0026thinsp;+\u0026thinsp;T cells. Ferroptosis is a potent anticancer target for the treatment of hepatocellular carcinoma and colorectal cancer liver metastasis in combination with immune checkpoint and myeloid-derived suppressor cell blockade, while primary colorectal cancer is resistant to this combinatorial treatment. Our study found that the low Mn diet significantly curtailed colorectal cancer in both GPX4-deficient and wild-type mice. This provides an alternative strategy to treat colorectal cancer.\u003c/p\u003e \u003cp\u003eIn conclusion, the study not only unravels the specific mechanisms by which GPX4 influences colorectal cancer but also suggests potential therapeutic strategies. The findings may resonate with researchers and clinicians working in the fields of cancer biology, oxidative stress, and precision medicine. Moreover, the study's consideration of dietary factors adds a practical dimension, addressing potential preventive measures and highlighting the importance of personalized approaches in cancer care.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interests.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement:\u003c/h2\u003e\n\u003cp\u003eAll data are available upon request or through the associated datasets in the manuscript.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e\n\u003cp\u003eZ.L. and X.X. drafted the manuscript and the figures. D.M., R.L., Y. L., Y. K. and S.H. contributed to data acquisition. X.X. designed the topic and edited the final manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eX.X. received partial support from the National Institute of General Medical Sciences (NIGMS, P20 GM130422), Environmental Health and Toxicology Pilot Awards from UNM Center for Native Environmental Health Equity Research (P50 MD015706), and New Mexico Integrative Science Program Incorporating Research in Environmental Sciences (NM-INSPIRES, 1P30ES032755). X.X. also acknowledges support from a Research Program Support Pilot Project Award from UNM comprehensive cancer center (P30CA118100) and the Cardiovascular and Metabolic Disease Research Program Pilot Project Grant from UNMHSC Office of Research Signature Programs. The funders have no role in the design, analysis, and reporting of the study.\u003c/p\u003e\n\u003ch2\u003eAcknowledgment:\u003c/h2\u003e\n\u003cp\u003eWe extend our heartfelt appreciation to GemPharmatech for their generous support in designing the genotyping protocol for our \u003cem\u003eGpx4\u003c/em\u003e floxed mice. Their collaboration has been instrumental in advancing our research endeavors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAghabozorgi AS, Bahreyni A, Soleimani A, \u003cem\u003eet al.\u003c/em\u003e Role of adenomatous polyposis coli (APC) gene mutations in the pathogenesis of colorectal cancer; current status and perspectives. \u003cem\u003eBiochimie\u003c/em\u003e 2019; 157:64\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eBarbosa P, Abo El-Magd NF, Hesketh J, \u003cem\u003eet al.\u003c/em\u003e The Role of rs713041 Glutathione Peroxidase 4 (GPX4) Single Nucleotide Polymorphism on Disease Susceptibility in Humans: A Systematic Review and Meta-Analysis. Int. J. Mol. Sci. 2022;23.\u003c/li\u003e\n\u003cli\u003eCanli \u0026Ouml;, Nicolas AM, Gupta J, \u003cem\u003eet al.\u003c/em\u003e Myeloid Cell-Derived Reactive Oxygen Species Induce Epithelial Mutagenesis. \u003cem\u003eCancer Cell\u003c/em\u003e 2017; 32:869-883.e5.\u003c/li\u003e\n\u003cli\u003eCase AJ, Domann FE. Absence of manganese superoxide dismutase delays p53-induced tumor formation. \u003cem\u003eRedox Biol\u003c/em\u003e 2014; 2:220\u0026ndash;3.\u003c/li\u003e\n\u003cli\u003eCheng J, Zhu Y, Xing X, Xiao J, Chen H, Zhang H, Wang D, Zhang Y, Zhang G, Wu Z, Liu Y. Manganese-deposited iron oxide promotes tumor-responsive ferroptosis that synergizes the apoptosis of cisplatin. Theranostics 2021; 11(11):5418-5429.\u003c/li\u003e\n\u003cli\u003eChoi E-K, Aring L, Das NK, \u003cem\u003eet al.\u003c/em\u003e Impact of dietary manganese on experimental colitis in mice. \u003cem\u003eFASEB J\u003c/em\u003e 2020; 34:2929\u0026ndash;43.\u003c/li\u003e\n\u003cli\u003eCole-Ezea P, Swan D, Shanley D, \u003cem\u003eet al.\u003c/em\u003e Glutathione peroxidase 4 has a major role in protecting mitochondria from oxidative damage and maintaining oxidative phosphorylation complexes in gut epithelial cells. \u003cem\u003eFree Radic Biol Med\u003c/em\u003e 2012; 53:488\u0026ndash;97.\u003c/li\u003e\n\u003cli\u003eConche C, Finkelmeier F, Pe\u0026scaron;ić M, \u003cem\u003eet al.\u003c/em\u003e Combining ferroptosis induction with MDSC blockade renders primary tumours and metastases in liver sensitive to immune checkpoint blockade. \u003cem\u003eGut\u003c/em\u003e 2023;72:1774 LP \u0026ndash; 1782.\u003c/li\u003e\n\u003cli\u003eDu J, Zhang J, Wang L, \u003cem\u003eet al.\u003c/em\u003e Selective oxidative protection leads to tissue topological changes orchestrated by macrophage during ulcerative colitis. \u003cem\u003eNat Commun\u003c/em\u003e 2023;14:3675.\u003c/li\u003e\n\u003cli\u003eEl Marjou F, Janssen KP, Chang BHJ, \u003cem\u003eet al.\u003c/em\u003e Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. \u003cem\u003eGenesis\u003c/em\u003e 2004; 39:186\u0026ndash;93.\u003c/li\u003e\n\u003cli\u003eFan Y-Y, Ran Q, Toyokuni S, \u003cem\u003eet al.\u003c/em\u003e Dietary Fish Oil Promotes Colonic Apoptosis and Mitochondrial Proton Leak in Oxidatively Stressed Mice. \u003cem\u003eCancer Prev Res\u003c/em\u003e 2011; 4:1267\u0026ndash;74.\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-P\u0026eacute;rez J, Fern\u0026aacute;ndez de Larrea-Baz N, Lope V, \u003cem\u003eet al.\u003c/em\u003e Residential proximity to industrial pollution sources and colorectal cancer risk: A multicase-control study (MCC-Spain). \u003cem\u003eEnviron Int\u003c/em\u003e 2020;144:106055.\u003c/li\u003e\n\u003cli\u003eHare DJ, Kysenius K, Paul B, \u003cem\u003eet al.\u003c/em\u003e Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS). \u003cem\u003eJoVE\u003c/em\u003e 2017; e55042.\u003c/li\u003e\n\u003cli\u003eHart PC, Mao M, de Abreu ALP, \u003cem\u003eet al.\u003c/em\u003e MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer. \u003cem\u003eNat Commun\u003c/em\u003e 2015; 6:6053.\u003c/li\u003e\n\u003cli\u003eHansen RD, Albieri V, Tj\u0026oslash;nneland A, \u003cem\u003eet al.\u003c/em\u003e Effects of Smoking and Antioxidant Micronutrients on Risk of Colorectal Cancer. \u003cem\u003eClin Gastroenterol Hepatol\u003c/em\u003e 2013; 11:406-415.e3. \u003c/li\u003e\n\u003cli\u003eHinoi T, Akyol A, Theisen BK, \u003cem\u003eet al.\u003c/em\u003e Mouse Model of Colonic Adenoma-Carcinoma Progression Based on Somatic Apc Inactivation. \u003cem\u003eCancer Res\u003c/em\u003e 2007; 67:9721\u0026ndash;30. \u003c/li\u003e\n\u003cli\u003eHu Q, Zhang Y, Lou H, \u003cem\u003eet al.\u003c/em\u003e GPX4 and vitamin E cooperatively protect hematopoietic stem and progenitor cells from lipid peroxidation and ferroptosis. \u003cem\u003eCell Death Dis\u003c/em\u003e 2021; 12:706.\u003c/li\u003e\n\u003cli\u003eLei L, Yang J, Zhang J, \u003cem\u003eet al.\u003c/em\u003e The lipid peroxidation product EKODE exacerbates colonic inflammation and colon tumorigenesis. \u003cem\u003eRedox Biol\u003c/em\u003e 2021;42:101880. \u003c/li\u003e\n\u003cli\u003eLiu Z, Villareal L, Goodla L, \u003cem\u003eet al.\u003c/em\u003e Iron promotes glycolysis to drive colon tumorigenesis. \u003cem\u003eBiochim Biophys Acta - Mol Basis Dis\u003c/em\u003e 2023;166846.\u003c/li\u003e\n\u003cli\u003eMayr L, Grabherr F, Schw\u0026auml;rzler J, \u003cem\u003eet al.\u003c/em\u003e Dietary lipids fuel GPX4-restricted enteritis resembling Crohn\u0026rsquo;s disease. \u003cem\u003eNat Commun\u003c/em\u003e 2020; 11:1775. doi:10.1038/s41467-020-15646-6.\u003c/li\u003e\n\u003cli\u003eMeng X, Wu J, Pan C, \u003cem\u003eet al.\u003c/em\u003e Genetic and Epigenetic Down-regulation of MicroRNA-212 Promotes Colorectal Tumor Metastasis via Dysregulation of MnSOD. \u003cem\u003eGastroenterology\u003c/em\u003e 2013; 145:426-436.e6.\u003c/li\u003e\n\u003cli\u003eM\u0026eacute;plan C, Hughes DJ, Pardini B, \u003cem\u003eet al.\u003c/em\u003e Genetic variants in selenoprotein genes increase risk of colorectal cancer. \u003cem\u003eCarcinogenesis\u003c/em\u003e 2010;31:1074\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eNaranuntarat A, Jensen LT, Pazicni S, \u003cem\u003eet al.\u003c/em\u003e The Interaction of Mitochondrial Iron with Manganese Superoxide Dismutase *. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2009; 284:22633\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003ePeters U, Chatterjee N, Hayes RB, \u003cem\u003eet al.\u003c/em\u003e Variation in the Selenoenzyme Genes and Risk of Advanced Distal Colorectal Adenoma. \u003cem\u003eCancer Epidemiol Biomarkers Prev\u003c/em\u003e 2008; 17:1144\u0026ndash;54. \u003c/li\u003e\n\u003cli\u003eRan Q, Liang H, Gu M, \u003cem\u003eet al.\u003c/em\u003e Transgenic Mice Overexpressing Glutathione Peroxidase 4 Are Protected against Oxidative Stress-induced Apoptosis. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2004; 279:55137\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003eStockwell BR, Friedmann Angeli JP, Bayir H, et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. \u003cem\u003eCell\u003c/em\u003e. 2017;171(2):273-285.\u003c/li\u003e\n\u003cli\u003eSui X, Zhang R, Liu S, \u003cem\u003eet al.\u003c/em\u003e RSL3 Drives Ferroptosis Through GPX4 Inactivation and ROS Production in Colorectal Cancer. Front. Pharmacol. 2018;9. \u003c/li\u003e\n\u003cli\u003eTeSlaa T, Bartman CR, Jankowski CSR, \u003cem\u003eet al.\u003c/em\u003e The Source of Glycolytic Intermediates in Mammalian Tissues. \u003cem\u003eCell Metab\u003c/em\u003e 2021; 33:367-378.e5.\u003c/li\u003e\n\u003cli\u003eWang G, Qin S, Chen L, \u003cem\u003eet al.\u003c/em\u003e Butyrate dictates ferroptosis sensitivity through FFAR2-mTOR signaling. \u003cem\u003eCell Death Dis\u003c/em\u003e 2023;14:292.\u003c/li\u003e\n\u003cli\u003eWang R, Su Q, Yin H, \u003cem\u003eet al.\u003c/em\u003e Inhibition of SRSF9 enhances the sensitivity of colorectal cancer to erastin-induced ferroptosis by reducing glutathione peroxidase 4 expression. \u003cem\u003eInt J Biochem Cell Biol\u003c/em\u003e 2021; 134:105948.\u003c/li\u003e\n\u003cli\u003eWeaver K, Skouta R. The Selenoprotein Glutathione Peroxidase 4: From Molecular Mechanisms to Novel Therapeutic Opportunities. Biomedicines. 2022;10. doi:10.3390/biomedicines10040891.\u003c/li\u003e\n\u003cli\u003eYan H, Talty R, Jain A, \u003cem\u003eet al.\u003c/em\u003e Discovery of decreased ferroptosis in male colorectal cancer patients with KRAS mutations. \u003cem\u003eRedox Biol\u003c/em\u003e 2023;62:102699.\u003c/li\u003e\n\u003cli\u003eZhang X, Ma Y, Ma J, \u003cem\u003eet al.\u003c/em\u003e Glutathione Peroxidase 4 as a Therapeutic Target for Anti-Colorectal Cancer Drug-Tolerant Persister Cells. Front. Oncol. 2022;12.\u003c/li\u003e\n\u003cli\u003eZhang Y, Swanda R V, Nie L, \u003cem\u003eet al.\u003c/em\u003e mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation. \u003cem\u003eNat Commun\u003c/em\u003e 2021; 12:1589.\u003c/li\u003e\n\u003cli\u003eZhuang A, Yang C, Liu Y, \u003cem\u003eet al.\u003c/em\u003e SOD2 in skeletal muscle: New insights from an inducible deletion model. \u003cem\u003eRedox Biol\u003c/em\u003e 2021;47:102135.\u003c/li\u003e\n\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":"Colorectal cancer, Oxidative stress, Experimental Colitis, Cell Death","lastPublishedDoi":"10.21203/rs.3.rs-3837925/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3837925/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe role of glutathione peroxidase 4 (GPX4) in ferroptosis and various cancers is well-established; however, its specific contribution to colorectal cancer has been unclear. Surprisingly, in a genetic mouse model of colon tumors, the deletion of GPX4 specifically in colon epithelial cells increased tumor burden but decreased oxidized glutathione. Notably, this specific GPX4 deletion did not enhance susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice with varied iron diets but showed vulnerability in mice with a vitamin E-deficient diet. Additionally, a high manganese diet heightened susceptibility, while a low manganese diet reduced DSS-induced colitis in colon epithelial-specific GPX4-deficient mice. Strikingly, the low manganese diet also significantly reduced colorectal cancer formation in both colon epithelial-specific GPX4-deficient and wildtype mice. Mechanistically, antioxidant proteins, especially manganese-dependent superoxide dismutase (MnSOD or SOD2), correlated with disease severity. Treatment with tempol, a superoxide dismutase mimetic radical scavenger, suppressed GPX4 deficiency-induced colorectal tumors. In conclusion, the study elucidates the critical role of GPX4 in inhibiting colorectal cancer progression by regulating oxidative stress in a manganese-dependent manner. The findings underscore the intricate interactions between GPX4, dietary factors, and their collective influence on colorectal cancer development, providing potential insights for personalized therapeutic strategies.\u003c/p\u003e","manuscriptTitle":"Glutathione peroxidase 4 suppresses manganese-dependent oxidative stress to reduce colorectal tumorigenesis.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 09:09:40","doi":"10.21203/rs.3.rs-3837925/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":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":27997871,"name":"Health sciences/Gastroenterology/Gastrointestinal diseases/Gastrointestinal cancer/Colorectal cancer/Colon cancer"},{"id":27997872,"name":"Health sciences/Gastroenterology/Gastrointestinal diseases/Gastroenteritis"}],"tags":[],"updatedAt":"2024-03-15T14:21:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-08 09:09:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3837925","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3837925","identity":"rs-3837925","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}