CAFs-derived exosomes inhibits ferroptosis via GALNT14-mediated O-GalNAcylation of SLC7A11 in colorectal cencer

CAFs-derived exosomes inhibits ferroptosis via GALNT14-mediated O-GalNAcylation of SLC7A11 in colorectal cencer | 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 CAFs-derived exosomes inhibits ferroptosis via GALNT14-mediated O-GalNAcylation of SLC7A11 in colorectal cencer huayang pan, Jialu Guan, Lingfeng Guo, Shuo Ning, Wenxi Zhao, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8266112/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract In the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs), the dominant stromal component, actively shape cancer progression through exosomal communication. Here, we identify hsa_circ_0003892 ( circLDLR ), a CAF-derived circRNA, as a key factor linked to poor prognosis in colorectal cancer (CRC). During CAF–CRC interaction, circLDLR is packaged into exosomes and transferred to tumor cells, enhancing proliferation and metastasis largely by reducing ferroptosis susceptibility. Mechanistic exploration revealed that circLDLR stabilizes Polypeptide N-Acetylgalactosaminyltransferase 14 (GALNT14) by shielding it from ZNRF2-mediated ubiquitin degradation, leading to elevated GALNT14 protein levels. Elevated GALNT14 promotes O-GalNAcylation of Solute Carrier Family 7 Member 11 (SLC7A11) at Ser26, facilitating its membrane localization, thereby suppressing ferroptosis in CRC cells. Moreover, EIF4A3, an RNA-binding protein (RBP), contributes to circLDLR biogenesis within CAFs. Taken together, our study reveals that CAFs-derived circLDLR can confer ferroptosis resistance and boost the progression of CRC, which are mainly dependent on increasing the stability of GALNT14 and enhancing O-GalNAcylation-mediated membrane localization of SLC7A11, thus, disrupting circLDLR transfer between CAFs and CRC cells may offer a promising approach for CRC therapy. Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer Biological sciences/Cancer/Cancer microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION CRC remains one of the most prevalent and lethal malignancies worldwide, with its progression and metastasis being major contributors to patient mortality 1 . Components of the tumor microenvironment (TME) interact with malignant cells, contributing to the hallmark of cancer 2 . CAFs are among the most abundant and versatile components within the TME. They are histologically prominent and biologically important in CRC initiation, progression, and metastasis 3, 4 . Growing evidence have demonstrated that CAFs can promote cancer cells proliferation, invasion, and resistance to therapy via secreting exosomes that facilitate intercellular communication within the TME 5 . Exosomes are critical cellular communicators. Accumulating evidences have reported that exosomes secreted by stromal cells play a pivotal role in cancer progression by encapsulating a variety of proteins, lipid, mRNAs, microRNAs, lncRNAs, circRNA and transferring to cancer cells 6, 7 . Among the molecules carried by exosomes, circRNAs have attracted significant attention due to their characteristics. Beyond the role as miRNA sponges, circRNAs have been shown to regulate protein stability, modulate signaling pathways, and influence cellular processes such as proliferation, migration, and apoptosis 8, 9 . However, the mechanism that how circRNAs contribute to CRC progression, particularly through interactions with the TME, warrants further investigation. Ferroptosis is an iron-dependent programmed death pathways, its molecular properties distinguish it from other types of programmed cell death 10 . Dysregulation of ferroptosis has been implicated in the progression of various diseases, including cancer 11 . Glycosylation represents a major form of protein post-translational modification (PTM), in which polysaccharides are transferred to specific amino acid residues in proteins via glycosyltransferases. Glycosylation plays crucial roles in protein stabilization. O-glycosylation is polysaccharides bonded to the hydroxyl group of the oxygen atom of serine or threonine residues. O-GalNAcylation, also known as O-glycosylation mucins, is initiated by polypeptide N-acetylgalactosaminyl-transferase. This complex mechanism involves more than 20 different peptide GalNAc transferase enzymes. O-GalNAcylation is expressed across multiple tumor types and plays critical roles in tumor metastasis, immune evasion and metabolic reprogramming. In this study, we identified circLDLR as a significantly upregulated circRNA driving the progression of CRC. We further demonstrated that circLDLR is highly expressed in CAFs, where it promotes CRC progression through exosomes-packaged circLDLR is internalized by CRC cells. Mechanistically, circLDLR stabilizes GALNT14, a key regulator of protein O-GalNAcylation, by inhibiting its ubiquitin-mediated degradation, thereby enhancing the membrane localization of SLC7A11 and suppressing ferroptosis in CRC cells. RESULTS The expression and characteristics of circLDLR To identify abnormally expressed circRNAs in CRC, two independent CRC datasets (GSE126094, GSE147597) from the Gene Expression Omnibus (GEO) database were selected and analyzed. 7 differentially expressed circRNAs upregulated in CRC were found (Fig. 1A, Fig. S1 ). Subsequently, thoese circRNAs were chosen for further detection. Using qRT-PCR analysis of 43 pairs of CRC samples, it was validated that hsa_circ_0003892 exhibited the most significant differential expression (Fig. 1B). To further analyze the relationship between circLDLR expression and clinicopathologic features in CRC patients, we examined 43 CRC samples and found that high circLDLR expression was positively correlated with tumor size, lymph node metastasis, and stage (Fig. S2 A, Table S1 ). According to circBase, circLDLR is located on human chromosome 19 and is generated from the back-splicing of 13–16 exons of the LDLR gene. The back-splice junction site was confirmed by Sanger sequencing in CRC cells (Fig. 1C). Then, we designed divergent primers and convergent primers, which were used to amplify in gDNA and cDNA, respectively. As shown in Fig. 1D, agarose gel electrophoresis revealed that circLDLR could only be amplified from cDNA using the divergent primers. Additionally, due to the closed loop structure of circLDLR , its expression level was not affected by RNase R treatment, whereas the expression of linear LDLR significantly decreased (Fig. 1E). To better understand the source of circLDLR , we performed FISH analysis on CRC tissues and adjacent normal tissues, finding that circLDLR was predominantly localized in the cytoplasm of CRC stromal cells, in sharp contrast to the epithelium (Fig. 1F). CircLDLR FISH and Vimentin immunofluorescence co-localization experiments conclusively demonstrated that circLDLR is predominantly localized within CAFs of tumor tissues (Fig. 1G). CAFs and NFs were isolated from CRC tissues and adjacent normal tissues, and adherent in culture with aspindle-shaped morphology (Fig. 1H). Herein, Western blot was performed to further confirm the purity and phenotype of NFs and CAFs. The results showed that in CAFs, the expression levels of FAP and α-SMA were notably higher than in NFs, while no differential expression of Vimentin (Fig. 1I, K). Compared with other cells, FISH revealed that circLDLR had higher expression level in CAFs (Fig. 1J). Next, CAFs and NFs were isolated from 43 pairs of CRC patients, and circLDLR were detected by qRT-PCR. It was found that the expression level of circLDLR in CAFs was significantly increased compared to NFs (Fig. S2 B). Therefore, elevated circLDLR in CAFs may be significantly related to the occurrence and development of CRC. The aberrant expression of circLDLR in CAFs exacerbates CRC progression The level of circLDLR in primary fibroblasts and CRC cells lines were detected by qRT-PCR and showed that the level was highest in CAFs. Among the CRC cell lines, HCT116 exhibited relatively high expression, while RKO show relatively low expression (Fig. S3 A). To further identify the function of CAFs in CRC, CRC cells were co-cultured with CAFs and NFs. It was found that CAFs dramatically promoted the proliferation, migration and invasion abilities of CRC cells. Furthermore, compared with the NFs group, CAFs co-culture notably potentiated the viability of CRC cells (Fig. S3 B-D). To further verify whether the oncogenic effect of CAFs was mainly relied on circLDLR . Firstly, the circLDLR in CAFs was deleted or overexpressed, and then transfected CAFs were co-cultured with CRC cells. As expected, overexpression of circLDLR in CAFs notably facilitated the growth of CRC cells, while circLDLR deletion generated the opposite effect (Fig. S3 E-G). Next, qRT-PCR was utilized to determine circLDLR in CAFs and co-cultured CRC cells. As shown in Fig. S3 H, I, silencing circLDLR in CAFs notably reduced the expression of circLDLR in CAFs and co-cultured CRC cells, whereas ectopic expression of circLDLR in CAFs exhibited contrary effects. Taken together, we hypothesized that circLDLR may shuttle from CAFs to CRC cells, thereby aggravated the malignant behaviors of CRC cells. Exosomal circLDLR shuttles from CAFs to CRC cells Given that recent evidence have reported CAFs could exacerbate cancer progression 12 , exosomes may play an important role in crosstalk between CAFs and cancer cells 13 , which inspired us to suppose whether exosomes are essential in circLDLR transfer. Exosomes in CAFs/NFs conditioned medium were purified by ultracentrifugation then confirmed by transmission electron microscopy and nanoparticle tracking analysis (NTA) (Fig. 2A). The exosomes were observed to be cup-shaped structure with diameters of 100 nm. Further, exosome markers CD81, CD63, TSG101 and Calnexin were detected by Western blotting analysis (Fig. 2B). CAFs derived exosomes were labeled with PKH67. Subsequently, PKH67-labeled exosomes were internalized by co-cultured CRC cells. (Fig. 2C). Further, CRC cells were co-cultured with CAFs transfected with cy3-tagged circLDLR . As shown in Fig. 2D, E, the fluorescently labeled circLDLR was notably increased in CRC cells, but the elevation could be abrogated by GW4869 (an inhibitor of exosomes secretion). The level of circLDLR in GW4869-treated CAFs remained stable (Fig. 2F). Additionally, in order to better characterize the biological function of circLDLR in CRC cells, circLDLR of CAFs was overexpressed or deleted, then CAFs derived exosomes were purified and incubated with CRC cells, as we expected, in ablation/overexpression group, the promoting effect on the progression of CRC cells correspondingly attenuated or potentiated (Fig. 2G, H, Fig. S4 A, B), consistent with the results of CAFs co-cultured CRC cells. Further, the results of qRT-PCR revealed that overexpression of circLDLR increased levels of circLDLR in CAF exosomes and cancer cells, whereas deletion of circLDLR exerted the opposite effect (Fig. 2I, J). Taken together, these results demonstrates that CAFs-derived exosomal circLDLR can be transferred to CRC cells and facilitate progression of CRC cells. CAFs-derived circLDLR facilitates the progression of CRC cells by suppressing ferroptosis To better characterize of CAFs-derived circLDLR in CRC, circLDLR were deleted in CAFs, and then exosomes enriched from CAFs were co-cultured with CRC cells. Subsequently, those cells were treated with various programmed cell death inhibitors (including ferroptosis, necroptosis, apoptosis and autophagy) to explore the possible cell death pattern that modulated by circLDLR . Intriguingly, Lip-1 and Fer-1, two inhibitors of ferroptosis, reversed the increase death of CRC cells when added CAFs (sh- circLDLR )-derived exosomes, but not Nec-1(necroptosis inhibitor), Z-VAD(apoptosis inhibitor) and CQ (autophagy inhibitor) (Fig. 3A). These results indicated that circLDLR of CAFs-derived exosomes might be involved in the progresssion of ferroptosis in CRC cells. Furthermore, CRC cells were treated with erastin and detected the proliferation ability, the result showed that CAFs (sh-NC)-derived exosomes and Lip-1 significantly decreased erastin-induced cell death, but not CAFs (sh- circLDLR )-derived exosomes (Fig. 3B). Additionally, we checked the concentration of the relative intracellular lipid peroxidation, mitochondrial superoxide (MitoSOX) and Mitochondrial Membrane Potential (MMP), CAFs (sh-NC)-derived exosomes and Lip-1 increased those levels of HCT116 and RKO cells (Fig. 3C-E, Fig. S5 A-C). As we all known, the intracellular Fe 2 + and Iron are a pool of redox-active iron and essentional for triggering oxidative damage. As shown in Fig. 3F, G and Fig. S5 D, E, Fe 2+ and Iron levels were higher in erastin-treated cells than control, whereas CAFs (sh-NC)-derived exosomes and Lip-1 could reverse these alterations. Transmission electron microscope (TEM) analysis further verified that CAFs (sh-NC)-derived exosomes particularly restored typical morphological features of ferroptosis, including shrunken mitochondria with elevated membrane density (Fig. 3H, Fig. S5 F). Invasion, migration and colony formation assays also validated the effect of CAFs (sh-NC)-derived exosomes (Fig. 3I, J Fig. S5 G, H). Taken together, we conclude that the circLDLR from CAFs-derived exosomes promote CRC cells growth via conferring ferroptosis resistance. CircLDLR stabilizes GALNT14 via protecting it against ZNRF2-mediated ubiquitin/proteasome-mediated degradation To dissect how circLDLR regulates cell ferroptosis, we first determined the subcellular localization of circLDLR by nuclear and cytoplasmic fractionation as well as FISH examination. The results indicated that circLDLR predominantly localized in the cytoplasm of CRC cells (Fig. 4A). The results of RIP assays showed that the circular RNA ciRS-7, function as competitive endogenous RNA (ceRNA), was significantly enriched by AGO2, but not circLDLR (Fig. 4B). Then, we conducted circRNA pull-down and MS assays to examine potential proteins bind with circLDLR (Fig. 4C, Table S2 ). Among those proteins, GALNT14 aroused our interest, and the binding between circLDLR and GALNT14 was further confirmed by RIP assays (Fig. 4D). Then, we performed IHC staining to reveal that GALNT14 presented the higher level in CRC tissue and predominantly localized in the cytoplasm (Fig. 4E). In addition, we performed RNA FISH-immunofluorescence analysis and found circLDLR co-localized with GALNT14 in the cytoplasm (Fig. 4F). When circLDLR was deleted/overexpressed in CRC cells, as expected, the expression of circLDLR was positiveiy correlated with GALNT14 protein level but not mRNA level (Fig. 4G). To further address the molecular basis underlying the interaction between circLDLR and GALNT14, we designed four mutants based on the structural composition of circLDLR (formed by backsplicing of four exons). Mutation of the circLDLR 296-466nt significantly decreased GALNT14 enrichment by circLDLR (Fig. 4H), indicating that the 296-466nt sequences is essential for the circLDLR -GALNT14 interaction. Next, we investigated the mechanism of circLDLR regulating GALNT14 level. Firstiy, CRC cells were treated with CHX to block protein synthesis for indicated times. The result of western blot revealed that circLDLR could stabilize GALNT14. (Fig. 4I). Next, we found that the effect of circLDLR depletion on GALNT14 protein levels was not affected by the lysosome inhibitor CQ but could be abolished by proteasome inhibitors MG-132 (Fig. 4J). Additionally, overexpression of wild-type circLDLR decreased the level of Ubiquitination on GALNT14, whereas overexpression of the mutant circLDLR had no effect (Fig. 4K). Thus, these findings suggest that circLDLR plays a central role in GALNT14 protein degradation through the ubiquitination/proteasome pathway. K48-, K29-and K63-linked polyubiquitin chains are three main types of polyubiquitin linkage. We constructed vectors carrying K48-only, K29-only and K63-only respectively and bearing HA tags. Ubiquitination assays suggested that K48-linked polyubiquitin of GALNT14 was reduced by circLDLR overexpression, while other polyubiquitin linkage had no evidently alteration (Fig. 4L). In sum, these findings validate that circLDLR promotes GALNT14 expression via protecting it from degradation of the K48-linked ubiquitin-proteasome pathway. Considering the E3 ubiquitin ligase is essential for the ubiquitination reaction, UbiBrowser 2.0 was applied to determine the potential E3 ligases might be involved in GALNT14 degradation (Fig. S6 A). Meanwhile, STARBASE database showed that ZNRF2 have a lower level in cancer than in normal tissue (Fig. S6 B). Thus, we supposed that ZNRF2 is the E3 ubiquitin ligase for GALNT14. To confirm whether ZNRF2 participates in regulating the ubiquitination effect of GALNT14, CO-IP assays were conducted, and the reciprocal interaction between ZNRF2 and GALNT14 was confrmed (Fig. 4M). The results of western blot showed that ZNRF2 overexpression significantly reduced the level of GALNT14, and could be rescued by MG-132 (Fig. 4N). CRC cells were treated with CHX for indicated times, and western blot analysis was implemented. The result showed that ZNRF2 overexpression also shortened the half-life of GALNT14 protein (Fig. 4O). In addition, we transfected K63-only, K48-only, K63R, K48R mutant ubiquitin and overexpressed ZNRF2, and found that ZNRF2 only dictated the K48-linked polyubiquitin of GALNT14 (Fig. 4P). Therefore, these results suggest that ZNRF2 is the E3 ubiquitin ligase which binds with GALNT14, and degrades them through the K48-linked polyubiquitin of GALNT14. CircLDLR confers ferroptosis resistance by upregulating GALNT14 According to the results in Fig. 4G and 4P, circLDLR and ZNRF2 both affect the ubiquitination degradation of GALNT14 through K48-linked ubiquitination. So, to further verity whether the effect of ZNRF2 on GALNT14 protein could be influenced by circLDLR , we conducted CO-IP and PLA assays. As we expected, the interaction between ZNRF2 and GALNT14 was disrupted by circLDLR overexpression (Fig. 5A, B). CircLDLR ablation could enhance the K48-linked ubiquitination of GALNT14 protein, ZNRF2 silencing could rescue the degradation GALNT14 protein caused by circLDLR ablation through the K48-linked ubiquitin–proteasome pathway (Fig. 5C). To further explore whether circLDLR influenced cancer cells ferroptosis through GALNT14, CRC cells were treated with erastin. The obtained data showed that migration, invasion and colony abilities were accelerated by circLDLR , while this influence was abolished by GALNT14 deletion (Fig. S7 A-D). In parallel, several main characteristics of ferroptosis (Lipid peroxidation level, relative levels of MMP and MitoSOX, relative Fe 2+ and Iron levels) were changed by circLDLR overexpression, and the inhibitory effect of circLDLR on ferroptosis could be reversed by GALNT14 detection (Fig. 5D-M). Above all, these evidences concomitantly indicate that circLDLR markedly dampens the sensitivity of CRC cells to ferroptosis, which is mainly dependent on GALNT14. GALNT14 impairs ferroptosis via O-GalNAcylation of SLC7A11 and its membrane localization To explore the molecular mechanism underlying the inhibition of ferroptosis in CRC cells by GALNT14. Firstly, we overexpressed/deleted GALNT14 in CRC cells, the level of O-GalNAcylation increased/decreased respectively (Fig. S8A, B). Then, GALNT14 overexpression stable transfection CRC cell lines were established,and then co-cultured with ferroptosis inducers of various pathways (including System Xc−, GCL, GPX4, and FSP1). GALNT14 overexpression was found to rescue the ferroptosis only by inducer targeting System Xc−, but not by inducers targeting GCL, GPX4, and FSP1 (Fig. 6A). As we all known, the uptake of cystine and the synthesis of GSH play important roles in the process of ferroptosis 14 – 16 . The two critical functions of System Xc − were diminished significantly by GALNT14 ablation. When cells were supplemented with the downstream products of System Xc−, Acetylcysteine (NAC) and GSH, ferroptosis induced by GALNT14 ablation was markedly alleviated in CRC cells (Fig. 6B, C). SLC7A11, as the most important functional subunit of System Xc−, proper membrane localization is essential for the functionality of SLC7A11. Previous studies have found that O-GlcNAcylation of SLC7A11 can alter its activity, but the specific mechanism remains unclear. As a member of polypeptide N-acetyl- α-galactosaminyltransferases family, GALNT14 mediated O-GalNAcylation. These findings inspired us to doubt whether SLC7A11 can be regulated by GALNT14.Western blot assay and immunofluorescence assay showed that GALNT14 did not affect the total level of SLC7A11 protein, but altered the membrane localization of SLC7A11 (Fig. 6D, E). CO-IP assays and PLA were conducted in CRC cell and 293T cell, the results confirmed that SLC7A11 could interacte with GALNT14 (Fig. 6F, G). Meanwhile, GALNT14 overexpression/deletion could significantly increase/decrease the O-GalNAcylation level of SLC7A11 (Fig. 6H). These results consistently confirmed the regulatory role of GALNT14 in the membrane localization of SLC7A11. Further, the mucin-type O-glycosylation sites of SLC7A11 were predicted using NetOGlyc-4.0. The database showed that S8, T9, S26 and T218 might be the O-GalNAcylation sites of SLC7A11, meanwhile, N314, predicted using NetNGlyc-1.0, might be the N-glycosylation site (Fig. 6I, Table S3 ). Then, S8R, T9R, S26R, T218R mutation vectors were constructed respectively and generating HA-tags, N314R mutation vectors serve as conrol. CO-IP and VVL lectin blot experiments verified that only S26R reduced the interaction of SLC7A11and GALNT14 (Fig. 6J). Then, a serious of assays with these mutant O-GalNAcylation sites of SLC7A11 in GALNT14-OE and SLC7A11-KO stably transfected cells were performed. As we expected, the proliferation, migration and invasion experiments showed that only S26 mutant site could not reverse the promoting effect of ferroptosis initiated by SLC7A11-KO (Fig. S9A, B). CRC cells transfected with S26R also could not rescue the O-GalNAcylation level of SLC7A11 and the level of GSH and Cystine uptake which reduced by SLC7A11-KD (Fig. 6K-M). Western blot and immunofluorescence assays showed that S26 was the key site on the membrane localization of SLC7A11 (Fig. 6N, O). Collectively, the results illustrate that GALNT14 induces O-GalNAcylation of SLC7A11 at the S26 site to promote its membrane localization, and further confers ferroptosis resistance in CRC cells. Biogenesis of circLDLR is Facilitated by EIF4A3 Prior research had demonstrated that the biogenesis of exon-derived circRNAs can be regulated by RBPs through their interactions with flanking intron sequences of circRNAs 17 . EIF4A3, QKI and FUS had been shown to facilitate circRNA biogenesis 18 – 20 . To investigate its potential role in circLDLR formation, we employed CircInteractome to predict RBP-binding sites within the flanking regions of circLDLR . We found 7 putative binding sites for EIF4A3 in the downstream of the circLDLR pre-mRNA (Fig. S10A). Due to sequence overlaps, these sites were named as a-c , and the sequence on circLDLR named as d (Fig. S10B). This binding specificity was further validated by RNA pull-down assays (Fig. S10C). Notably, EIF4A3 overexpression significantly upregulated circLDLR expression in CAFs, while its deletion reduced circLDLR levels (Fig. S10D). To further explore the expression of EIF4A3 in CRC patients, an IHC assay was conducted CRC tissues and adjacent normal tissues, the result revealed that the expression of EIF4A3 was higher in CRC tissues than in adjacent normal tissues (Fig. S10E) and that the expression of EIF4A3 and circLDLR was positively correlated in CRC patients (Fig. S10F).Collectively, these results establish that EIF4A3 as a positive regulator of circLDLR biogenesis through direct binding to its flanking intronic regions. CircLDLR in CAFs promotes cancer growth and metastasis in vivo To further investigate the impact of CAF-exosomes circLDLR on cancer progression in vivo , two human CRC cell lines were employed to establish tumor model. In subcutaneous xenograft model, circLDLR stably overexpressed and deleted CAFs were constructed. Then, CRC cells were co-injected with or without CAFs into the nude mice subcutaneously. As shown in Fig. 7A, B, co-transplanted with CAFs accelerated subcutaneous tumor growth. This pro-tumorigenic effect was enhanced when co-transplanted with CAFs OE- circLDLR , whereas co-transplanted with CAFs KD- circLDLR displayed the opposite effect. We further investigated the effect of circLDLR on CRC metastasis in vivo . CAFs OE- circLDLR further accelerated the liver metastasis of CRC cells in vivo , as indicated by more tumor nodules, and more serious colorectal tissue damage (Fig. 7C). However, CAFs KD- circLDLR reversed the lung metastasis of CRC cells in vivo (Fig. 7D). In addition, we performed IHC and HE assays. IHC staining showed the result that CAFs sh-circ/CAFs OE-circ decreased/increased the expression of Ki67 and GALNT14 in subcutaneous tumors, whereas the 4HNE exhibited the opposite level (Fig. 7E-G). Moreover, HE staining confirmed the morphology of tumor tissues in lung and liver metastasis groups (Fig. 7H). Collectively, these results revealed that circLDLR is essential for CAFs to potentiate the growth and metastasis of CRC in vivo . DISCUSSION As key cellular components of TME, CAFs play a pivotal role in the TME. Accumulating studies have revealed that CAFs can regulate tumor growth and metastasis through the secretion of non-coding RNAs 21 , 22 . Ferroptosis, as an iron-dependent form of regulated cell death, plays a critical role in colorectal cancer progression. Emerging evidence suggests that CAFs are involved in tumor progression, but the precise mechanisms in regulating the interplay between CAFs and ferroptosis remain elusive. In this study, we demonstrate that EIF4A3-facilitated circLDLR is predominantly shuttled from CAFs to CRCcells via exosomes, subsequently, circLDLR protects against ferroptosis by impairing SLC7A11 membrane localization in CRC cells, thereby facilitating tumor progression and metastasis. With the advancs of research, their functional roles of circRNA in cellular processes have been increasingly elucidated, particularly their critical function of tumor progression and oncogenesis 8 , 9 , 23 . A substantial body of research has established that CAFs-derived exosomes influence the progression of various cancers 6 , 13 , 24 . Notably, exosome-packaged circRNAs contribute significantly to these regulatory effects 7 , 25 – 27 . Beyond functioning as ceRNAs, increasing lines of evidence have demonstrated that circular RNAs can directly bind to proteins and regulate the expression of their target proteins 28 – 31 . Here, GALNT14 was identified as a promising target of circLDLR . We further demonstrated that circLDLR binds GALNT14 through its specific functional domain, thereby disrupting the interaction between GALNT14 and the E3 ubiquitin ligase ZNRF2, decelerating ZNRF2-mediated K48-linked polyubiquitination and degradation, ultimately leading to elevated GALNT14 expression. Numerous evidences highlight RBPs as critical regulators of circRNA biogenesis. As a key RBP, Eukaryotic Translation Initiation Factor 4A3 (EIF4A3) has been shown to modulate multiple circRNAs 19 , 32 , 33 . In this study, bioinformatic predictions identified circLDLR as a potential EIF4A3 target. In CAFs, we validated EIF4A3 positive regulation of circLDLR . However, whether EIF4A3 directly modulates circLDLR back-splicing remains to be further elucidated. O-linked glycosylation is one common type of Glycosylation 34 . Aberrant glycosylation endows tumor cells with resistance to cell death. Whereas most studies have focused on the role of O-GlcNAcylation in tumors, the function of O-GalNAcylation (mucin-type O-glycosylation) in tumor progression remains largely ignored. Although the two modifications belong to O-linked glycosylation, certain functional differences exist between the two modifications, both modifications target serine or threonine residues. GALNT14 is a pivotal O-glycosyltransferase implicated in tumor progression through its regulation of protein O-glycosylation in various cancers 35 , 36 . In HCC, GALNT14-mediated O-GalNAcylation at serine-161 of PHB2 activates the IGF1R cascade, thereby enhancing cell proliferation, migration, and therapy resistance 37 . Similarly, in LUAD, GALNT14-driven O-GalNAcylation suppresses endogenous ROS production to promote tumor aggressiveness 38 . Previous studies have confirmed that ferroptosis, play critical roles in the initiation and progression of colorectal cancer 39 – 41 . In this study, we identified SLC7A11 as a novel O-GalNAcylated substrate of GALNT14. While prior research primarily focused on expression levels of SLC7A11 (unlike FSP1 studies that emphasized N-myristoylation-dependent membrane localization for ferroptosis regulation 42 ), emerging evidence now highlights the critical role of SLC7A11 membrane trafficking in ferroptosis regulation 43 , 44 . Recent report indicate that O-GlcNAcylation sustains SLC7A11 activity to suppress ferroptosis 45 , even if the mechanistic basis remains unclear. Intriguingly, our work reveals that GALNT14 mediates O-GalNAcylation at S26 of SLC7A11, promoting its membrane localization and functional activation without altering total SLC7A11 abundance—thereby inhibiting ferroptosis and driving CRC progression. This discovery diverges from the canonical paradigm of glycosylation-mediated protein stabilization in ferroptosis regulation, instead establishing a novel mechanism where O-GalNAc modification orchestrates SLC7A11 spatial redistribution to modulate ferroptosis susceptibility. Our finding expands the understanding of O-GalNAcylation in ferroptosis regulation and provide new directions for targeting SLC7A11 membrane dynamics in cancer therapy. In conclusion, our study reveals a novel CAF-to-CRC signaling axis: EIF4A3 upregulates circLDLR in CAFs, where circLDLR is packaged into exosomes and transferred to CRC cells. Within CRC cells, circLDLR binds and stabilizes GALNT14 by protecting against ZNRF2-mediated ubiquitination and degradation. Then, the accumulated GALNT14 catalyzes O-GalNAcylation at S26 site of SLC7A11, promoting its membrane localization to inhibit ferroptosis—ultimately driving CRC growth and metastasis. These findings highlight the importance of in tumor-stromal interactions and provide a rational basis for developing targeted therapies to disrupt the CAFs-CRC cells crosstalk. METHODS Cell culture Human HCT116, HT29, RKO, LOVO, SW620, SW480 and HEK 293T cells were purchased from Procell (Wuhan,China). Human NCM460 cell was purchased from Fuheng Biology (Shanghai, China). Human HCT116 and HT29 cell lines were cultured in McCoy’s 5A Complete medium (Procell, #PM150710B). Human RKO cell line was cultured in MEM medium (Gibco,#11095080). Human LOVO cell line were cultured in Ham’s F-12K complete medium (Procell, #CM-0144). SW620, SW480, HEK 293T and NCM460 cells were cultured in DMEM medium (Gibco,#11965092). MEM and DMEM medium plus 10% fetal bovine serum (FBS) (TIANHANG, #13011 − 8611) and 1% penicillin/streptomycin (SEVEN BIOTECH, #SC118-01). Cell lines were incubated at 37°C with 5% CO 2 . Mycoplasma contamination was confirmed to be negative in cells. After thawing the frozen stocks, cells were generally passaged fewer than 10 times. Human CRC tissue specimens Primary CRC specimens and paired non-cancerous tissues were obtained from the First Affiliated Hospital of Harbin Medical University. The characteristics of the patients are listed in Table S1 . The study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University, and written informed consent was provided by the patients. Isolation and culture of patient-derived NFs and CAFs CAFs were isolated from CRC tissues of patients, and NFs were separated from the paired adjacent normal area. Briefly, the fresh paired tissues were washed three times with PBS containing 100 U/ ml penicillin and 100 µg/ml streptomycin. Then, the tissues were chopped into small pieces and digested in collagenase II (Gibco, #17101015) at 37℃. All cells were collected and resuspended in DMEM/F12 medium (Gibco, #11320033) containing 15% FBS. The suspensions were fostered in DMEM/F12 medium supplemented with 15% FBS for about 48 hours. After stable adherence of the cells, DMEM/F12 medium was used to wash out the excess tissues and dead cells, then DMEM/F12 containing 15% FBS was added for subsequent cell culture. Cell morphology was measured by microscopical measurement, and fibroblast identity was verified by detection of specific markers (α-SMA, FAP, and vimentin). All primary fibroblasts used in this study were less than three passages. Co-culture assay For co-culture experiments, CAFs or NFs were cultured with CRC cells using 0.4µm Transwell inserts (Corning, USA). Specifically, 6×10⁴ CRC cells were seeded into the lower chamber, while 2×10⁴ CAFs/NFs were plated in the upper chamber. After 48 hours of co-culture, CRC cells from the lower chamber were collected for following analyses. Isolation and identification of exosomes CAFs or NFs were cultured in serum-free DMEM/F12 medium for 48 hours to generate conditioned medium (CM). The CM was sequentially centrifuged at 4°C: first at 2,000 × g for 30 min (to remove dead cells), then at 10,000 × g for 30 min (to eliminate cell debris and large vesicles), and finally at 120,000 × g for 70 min (to pellet exosomes). The resulting pellet was washed with 1× PBS to reduce protein contamination, and filtered with 0.22-µm filters. Purified vesicles were characterized by Nanoparticle tracking analysis (NTA) using ZetaView PMX 110 ((Particle Metrix, Meerbusch, Germany) for size distribution,Transmission electron microscopy (FEI Tecnai G2 Spirit, Thermo Scientific, USA) for morphological validation, and Western blotting of exosomal markers. Exosomes labeling and tracing Isolated exosomes were fluorescently labeled with PKH67 (MCE, #257277-27-3), then added to the culture supernatant. After 24 hours of co-incubation with CRC cells, the nuclei were stained with DAPI (Beyotime, #P0131), and exosome uptake was evaluated using confocal microscopy (Zeiss, Germany). Fluorescence in situ hybridization (FISH) Cellular localization of circLDLR was determined using FISH probe and kits (RiboBio, Guangzhou, China). Briefly, fixed and permeabilized cells were incubated with prehybridization buffer for 30 min at 37°C. Cy3-labeled circLDLR probe was mixed with pre-warmed hybridization buffer and incubated with cells overnight at 37°C in the dark. Post-hybridization washes were performed with wash buffer and PBS. Nuclei were stained with DAPI, and images were analyzed using confocal microscopy. Proximity ligation assay (PLA) The DuoLink® In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma-Aldrich) was used to detect interacting proteins. Briefly, CRC cells were fixed with 4% paraformaldehyde. The cells were washed with PBS and permeabilized using 0.1% Triton X-100 in PBS for 20 min. After blocking, the cells were incubated with primary antibodies at 4°C overnight. The subsequent step was incubating the pre-diluted anti-rabbit plus and anti-mouse minus probes at 37°C for 1h. Then, cells were incubated with 1× ligase for 30 min and 1× polymerase for 100 min at 37°C. Finally, coverslips were mounted on the slide with Duolink® In situ Mounting Medium with DAPI. Western blot assay Proteins extracted from cells or exosomes were separated by 10% SDS-PAGE and transferred onto 0.22µm PVDF membranes. Membranes were blocked with 5% fat-free milk for 1 hour at room temperature, followed by incubation with primary antibodies at 4°C overnight. After three washes with TBST, membranes were probed with species-matched secondary antibodies for 1 hour at room temperature. Following three additional TBST washes to remove nonspecific binding, target proteins were visualized using an Odyssey infrared imaging system (LI-COR Biosciences, USA). For lectin blot analysis, membranes were incubated with 20 µg/mL Vicia Villosa Lectin (VVL; Vector Labs, #B-1235-2) or Peanut Agglutinin (PNA; Vector Labs, #B-1075-5) in PBS for 30 min at room temperature. After wash with TPBS (PBS containing 0.05% Tween-20), membranes were incubated with ABC-HRP kit for 30 min at room temperature. Following additional TPBS washes, target glycoproteins were detected using enhanced chemiluminescence (ECL). The primary and secondary antibodies are listed in Table S4 . Immunoprecipitation (IP) and co-immunoprecipitation (CO-IP) Total cellular proteins were extracted using Cell Lysis Buffer for Western and IP (Beyotime, #P0013). Firstly, 500 µL of antibody working solution or normal IgG working solution was added and incubated with A/G magnetic beads (MCE, #HY-K0202) for 1 hour at room temperature on a rotary mixer. Subsequently, 2 mg of total protein was incubated overnight at 4°C with protein A/G magnetic beads or protein-agarose bound VVL (Vectorlabs, #AL-1233). The magnetic beads or agarose beads were then washed three times with TBST, and the bound proteins were eluted using loading buffer for Western blot analysis. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA from tissues and cells was isolated using TRIzol reagent (Invitrogen), while exosomal RNA was isolated with the miRNeasy Mini Kit (QIAGEN, #217004). Their complementary DNA (cDNA) synthesis was performed using the PrimeScript RT reagent kit (TaKaRa, Shiga, Japan). qPCR was carried out with FastStart Universal SYBR Green Master Mix (ROX) (Roche, Switzerland) and primers (sequences in Table S5 ), using GAPDH as the endogenous control. Immunohistochemistry (IHC) and hematoxylin–eosin (H&E) staining Tissue samples were fixed in 4% paraformaldehyde (PFA), embedded, sectioned, and subsequently stained with various antibodies, including: Ki67(proteintech, #27309), GALNT14 (proteintech, #16939), 4HNE (Abmart, #PC6313) for IHC. For metastatic lesion analysis, liver and lung tissues were similarly fixed, embedded, sectioned, and stained with H&E. All images were captured using an Olympus microscope and processed using NDP.view2 imaging software. CCK-8 assay Cell viability was measured using the CCK-8 assay. Briefly, cells were seeded in 96-well plates at a density of 3×10³ cells per well. At indicated time points, 10 µL of CCK-8 solution (SEVEN BIOTECH, #SC119) was added to each well, followed by incubation at 37°C with 5% CO₂ for 4 hours. Absorbance at 450 nm (OD₄₅₀) was measured using a microplate reader. Cell viability was assessed daily through OD₄₅₀ measurements. Colony formation assay CRC cells were seeded at a density of 1500 cells/well into six-well plates for 2 weeks. Thereafter, the colonies were stained with 0.1% crystal violet and counted. Migration and invasion assays Falcon Transwell inserts (8-µm pore, Corning, USA) were used for migration assays. CRC cells were serum-starved for 24 hours, trypsinized, and resuspended in serum-free medium. Cells (4×10 4 in 200 µL serum-free medium) were seeded into the upper chamber of Transwell inserts. The lower chamber contained 700 µL medium with 10% FBS as chemoattractant. After 24 hours, non-migrated cells on the upper membrane surface were removed with cotton swabs. Migrated cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified. For the invasion assay, cells were inserted into the upper chamber of the Matrigel invasion chamber and subsequently treated as described for the transwell migration assay. Lipid Peroxidation measurement Lipid Peroxidation levels were assessed using the Lipid Peroxidation Assay Kit with BODIPY 581/591 C11 (Beyotime, #S0043S). Approximately 2 × 105 transfected cells/well were seeded in twelve-well plates overnight. Thereafter, cells were treated with 10µM erastin (MCE, #HY-15763) and 1µM liproxstatin-1 (Lip-1) (MCE, #HY-12726)for 48 h.BODIPY 581/591 C11 was added to cells in the dark for 20 min at 37°C, followed by washing with PBS. Thereafter, fluorescence imaging was performed using a ZEISS confocal microscope. Iron assay To quantify the intracellular levels of total iron and ferrous iron, an iron assay kit (APPLYGEN, #E1042) and a Ferrous Ion Assay Kit (Beyotime, #S1070S) were used according to the manufacturer's instructions. Mitochondrial membrane potential (MMP) measurement The MMP was measured using theJC-1 Mitochondrial Membrane Potential Assay Kit (MCE, #HY-K0601), according to the manufacturer’s instructions. Mitochondrial superoxide measurement Mitochondrial superoxide levels were determined using MitoSOX Red mitochondrial superoxide indicator for live-cell imaging (MCE, #HY-D1055), according to the manufacturer’s instructions. RNA pull-down assay Biotinylated full-length RNA probes and fragmented circLDLR probes were synthesized by GenePharma Co., Ltd (Shanghai, China). Streptavidin Magnetic Beads (MCE, #HY-K0208) were pre-cleared by incubating with transfected cell lysates for 1 hour at 4°C to reduce non-specific binding. Biotinylated probes were then incubated with the beads for 60 minutes at room temperature to form probe-bead complexes. These complexes were subsequently incubated with transfected cell lysates overnight at 4°C. After magnetic separation, the beads were washed five times with buffer. RNA-protein complexes were eluted and analyzed by Western blotting. The probes used in RNA pull-down are provided in Table S5 . RNA immunoprecipitation (RIP) RIP experiments were performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Co-precipitated RNA was detected by qRT-PCR. The primers used in RIP are provided in Table S5 . Mice xenograft model All experimental procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital of Harbin Medical University. Female 4–6 weeks old BALB/c nude mice were purchased from Huachuang Sino Pharma Tech Co., Ltd (Jiangsu, China) and maintained under SPF conditions in a controlled environment of 12 h of light and darkness, 50–70% humidity, 23 ± 2°C, and animals had free access to food and water. Mice were randomly grouped (n = 6 per group) after 1 week of acclimation, and the luciferase-labeled lentivirus was transfected into CAFs and selected with puromycin (2µg/ml) for 10 days. CRC (3 × 10 6 ) cells either alone or mixed with CAFs (Vector/sh- circLDLR /OE- circLDLR ) at the ratio of 1:1 in 200 µl PBS were subcutaneously injected into nude mices. Tumors were measured every 5 d for 20 days, volume (mm 3 ) = length × width 2 × 0.52. The mice were sacrificed after 20 days and the tumors were excised and weighed. For the lung metastasis models, Luciferase-labeled 1 × 10 6 HCT116 either alone or mixed with CAFs (Vector/sh- circLDLR ) at the ratio of 1:1 in 100 µl PBS were injected into the tail veins of nude mice (n = 6/group). The mice were euthanized 6 weeks later, and the lungs were surgically dissected for further investigation. To establish a liver metastasis models, Luciferase-labeled 2 × 10 6 RKO either alone or mixed with CAFs (Vector/OE- circLDLR ) at the ratio of 1:1 in 200 µl PBS were injected into the spleens of nude mice. Briefly, nude mice (n = 6/group) were anesthetized, and a lateral incision was made to expose the spleen. Mixed cells were resuspended in 200 µL PBS and injected into the spleen using a microsyringe. The wounds were closed with 4 − 0 sutures. The mice were euthanized, and liver samples were excised for further analysis after 3 weeks. Before the lung and liver metastasis model mice were euthanized, the IVIS Lumina imaging station (Caliper Life Sciences, Hopkinton, MA, USA) was used for bioluminescence imaging after intraperitoneal injection of 150 µl D-luciferin (15mg/ml) (Beyotime, #ST196). Lastly, the primary tumors of subcutaneous xenograft models, lungs and livers of metastasis models were obtained and fixed with 4% formalin. The level of ki67, GALNT14 and 4HNE were detected by IHC. The metastatic area in the lungs and livers were carefully examined by H&E staining. Statistical analysis All statistical analyses were performed using GraphPad Prism 8.0 (San Diego, CA, USA). Bioinformatics analysis was performed with R (version 4.2.1) ( http://www.R-project.org/ ). The number of biological replicates for each experiment was indicated in the corresponding figure legend. All values represented the mean ± standard deviation (S.D) and were derived from a minimum of three independent biological replicates. P values are calculated by unpaired two-sided t -test. In figures, statistical comparisons between the control group and the experimental group are denoted as follows: ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Declarations Data availability The data that support the findings of this study are available from the corresponding author on request. COMPETING INTERESTS The authors declare that they have no competing interests. ETHICS APPROVAL AND CONSENT TO PARTICIPATE This study was approved by the institutional ethics review board of the First Affiliated Hospital of Harbin Medicine University (no. IRB-AF/SC-04/02.2) and by the Animal Ethics Committee of the First Affiliated Hospital of Harbin Medicine University (no. 2023089). Participants gave informed consent to participate in the study before taking part. AUTHOR CONTRIBUTIONS H.Y.P supervised the project. H.Y.P and J.L.G conceived and designed the study. J.L.G, L.F.G and S.N performed experiments. J.L.G and W.X.Z contributed to data analysis and generated figures. H.Y.P and J.L.G wrote the manuscript. All authors reviewed and approved the manuscript for submission. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 82303270), the Excellent Young Medical Talents Training Fund of the First Affiliated Hospital of Harbin Medical University (Grant No. 2021Y06), Heilongjiang Postdoctoral Scientific Research Developmental Fund (Grant No. LBH-Q18089), the University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (Grant No. UNPYSCT-2018070), Chen Xiao-Ping Foundation For The Development Of Science And Technology Of Hubei Province (Grant No. CXPJJH121001-2021029). The Open Fund of Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Harbin, China (Grant No. GPKF202507) References Dekker, E, Tanis, PJ, Vleugels, J, Kasi, PM, Wallace, MB. Colorectal cancer. Lancet 2019;394:1467–1480. 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Supplementary Files TableS1.pdf Table S1 TableS2.pdf Table S2 TableS3.pdf Table S3 TableS4.pdf Table S4 TableS5.pdf Table S5 originalwesternblots.pdf original western blots Supplementalfigure.pdf Supplemental figure Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 04 Feb, 2026 Review # 2 received at journal 02 Feb, 2026 Review # 1 received at journal 28 Jan, 2026 Reviewer # 2 agreed at journal 20 Jan, 2026 Reviewer # 1 agreed at journal 12 Jan, 2026 Reviewers invited by journal 07 Dec, 2025 Submission checks completed at journal 03 Dec, 2025 Editor assigned by journal 03 Dec, 2025 First submitted to journal 03 Dec, 2025 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. 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figure","description":"","filename":"Supplementalfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8266112/v1/76875ec7216dcf4ebbc53991.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"CAFs-derived exosomes inhibits ferroptosis via GALNT14-mediated O-GalNAcylation of SLC7A11 in colorectal cencer","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCRC remains one of the most prevalent and lethal malignancies worldwide, with its progression and metastasis being major contributors to patient mortality\u003csup\u003e1\u003c/sup\u003e. Components of the tumor microenvironment (TME) interact with malignant cells, contributing to the hallmark of cancer\u003csup\u003e2\u003c/sup\u003e.\u0026nbsp;CAFs are among the most abundant and versatile components within the TME.\u0026nbsp;They are histologically prominent and biologically important in CRC initiation, progression, and metastasis\u003csup\u003e3, 4\u003c/sup\u003e. Growing evidence have demonstrated that CAFs can promote cancer cells proliferation, invasion, and resistance to therapy via secreting exosomes that facilitate intercellular communication within the TME\u003csup\u003e5\u003c/sup\u003e. Exosomes are critical cellular communicators. Accumulating evidences have reported that exosomes secreted by stromal cells play a pivotal role in cancer progression by encapsulating a variety of proteins, lipid, mRNAs, microRNAs, lncRNAs, circRNA and transferring to cancer cells\u003csup\u003e6, 7\u003c/sup\u003e. Among the molecules carried by exosomes, circRNAs have attracted significant attention due to their characteristics.\u003c/p\u003e\n\u003cp\u003eBeyond the role as miRNA sponges, circRNAs have been shown to regulate protein stability, modulate signaling pathways, and influence cellular processes such as proliferation, migration, and apoptosis\u003csup\u003e8, 9\u003c/sup\u003e. However, the mechanism that how circRNAs contribute to CRC progression, particularly through interactions with the TME, warrants further investigation.\u003c/p\u003e\n\u003cp\u003eFerroptosis is an iron-dependent programmed death pathways, its molecular properties distinguish it from other types of programmed cell death\u003csup\u003e10\u003c/sup\u003e. Dysregulation of ferroptosis has been implicated in the progression of various diseases, including cancer\u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eGlycosylation represents a major form of protein post-translational modification (PTM), in which polysaccharides are transferred to specific amino acid residues in proteins via glycosyltransferases. Glycosylation plays crucial roles in protein stabilization. O-glycosylation is polysaccharides bonded to the hydroxyl group of the oxygen atom of serine or threonine residues. O-GalNAcylation, also known as\u0026nbsp;O-glycosylation mucins, is initiated by polypeptide N-acetylgalactosaminyl-transferase. This complex mechanism involves more than 20 different peptide GalNAc transferase enzymes. O-GalNAcylation is expressed across multiple tumor types and plays critical roles in tumor metastasis, immune evasion and metabolic reprogramming.\u003c/p\u003e\n\u003cp\u003eIn this study, we identified\u003cem\u003e\u0026nbsp;circLDLR\u003c/em\u003e as a significantly upregulated circRNA driving the progression of CRC. We further demonstrated that \u003cem\u003ecircLDLR\u0026nbsp;\u003c/em\u003eis highly expressed in CAFs, where it promotes CRC progression through exosomes-packaged \u003cem\u003ecircLDLR\u0026nbsp;\u003c/em\u003eis internalized by CRC cells. Mechanistically, \u003cem\u003ecircLDLR\u0026nbsp;\u003c/em\u003estabilizes GALNT14, a key regulator of protein O-GalNAcylation, by inhibiting its ubiquitin-mediated degradation, thereby enhancing the membrane localization of SLC7A11 and suppressing ferroptosis in CRC cells.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eThe expression and characteristics of\u003c/b\u003e \u003cb\u003ecircLDLR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify abnormally expressed circRNAs in CRC, two independent CRC datasets (GSE126094, GSE147597) from the Gene Expression Omnibus (GEO) database were selected and analyzed. 7 differentially expressed circRNAs upregulated in CRC were found (Fig.\u0026nbsp;1A, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Subsequently, thoese circRNAs were chosen for further detection. Using qRT-PCR analysis of 43 pairs of CRC samples, it was validated that \u003cem\u003ehsa_circ_0003892\u003c/em\u003e exhibited the most significant differential expression (Fig.\u0026nbsp;1B). To further analyze the relationship between \u003cem\u003ecircLDLR\u003c/em\u003e expression and clinicopathologic features in CRC patients, we examined 43 CRC samples and found that high \u003cem\u003ecircLDLR\u003c/em\u003e expression was positively correlated with tumor size, lymph node metastasis, and stage (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). According to circBase, \u003cem\u003ecircLDLR\u003c/em\u003e is located on human chromosome 19 and is generated from the back-splicing of 13\u0026ndash;16 exons of the LDLR gene. The back-splice junction site was confirmed by Sanger sequencing in CRC cells (Fig.\u0026nbsp;1C). Then, we designed divergent primers and convergent primers, which were used to amplify in gDNA and cDNA, respectively. As shown in Fig.\u0026nbsp;1D, agarose gel electrophoresis revealed that \u003cem\u003ecircLDLR\u003c/em\u003e could only be amplified from cDNA using the divergent primers. Additionally, due to the closed loop structure of \u003cem\u003ecircLDLR\u003c/em\u003e, its expression level was not affected by RNase R treatment, whereas the expression of linear LDLR significantly decreased (Fig.\u0026nbsp;1E).\u003c/p\u003e\u003cp\u003eTo better understand the source of \u003cem\u003ecircLDLR\u003c/em\u003e, we performed FISH analysis on CRC tissues and adjacent normal tissues, finding that \u003cem\u003ecircLDLR\u003c/em\u003e was predominantly localized in the cytoplasm of CRC stromal cells, in sharp contrast to the epithelium (Fig.\u0026nbsp;1F). \u003cem\u003eCircLDLR\u003c/em\u003e FISH and Vimentin immunofluorescence co-localization experiments conclusively demonstrated that \u003cem\u003ecircLDLR\u003c/em\u003e is predominantly localized within CAFs of tumor tissues (Fig.\u0026nbsp;1G). CAFs and NFs were isolated from CRC tissues and adjacent normal tissues, and adherent in culture with aspindle-shaped morphology (Fig.\u0026nbsp;1H). Herein, Western blot was performed to further confirm the purity and phenotype of NFs and CAFs. The results showed that in CAFs, the expression levels of FAP and α-SMA were notably higher than in NFs, while no differential expression of Vimentin (Fig.\u0026nbsp;1I, K). Compared with other cells, FISH revealed that \u003cem\u003ecircLDLR\u003c/em\u003e had higher expression level in CAFs (Fig.\u0026nbsp;1J). Next, CAFs and NFs were isolated from 43 pairs of CRC patients, and \u003cem\u003ecircLDLR\u003c/em\u003e were detected by qRT-PCR. It was found that the expression level of \u003cem\u003ecircLDLR\u003c/em\u003e in CAFs was significantly increased compared to NFs (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Therefore, elevated \u003cem\u003ecircLDLR\u003c/em\u003e in CAFs may be significantly related to the occurrence and development of CRC.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe aberrant expression of\u003c/b\u003e \u003cb\u003ecircLDLR\u003c/b\u003e \u003cb\u003ein CAFs exacerbates CRC progression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe level of \u003cem\u003ecircLDLR\u003c/em\u003e in primary fibroblasts and CRC cells lines were detected by qRT-PCR and showed that the level was highest in CAFs. Among the CRC cell lines, HCT116 exhibited relatively high expression, while RKO show relatively low expression (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). To further identify the function of CAFs in CRC, CRC cells were co-cultured with CAFs and NFs. It was found that CAFs dramatically promoted the proliferation, migration and invasion abilities of CRC cells. Furthermore, compared with the NFs group, CAFs co-culture notably potentiated the viability of CRC cells (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB-D).\u003c/p\u003e\u003cp\u003eTo further verify whether the oncogenic effect of CAFs was mainly relied on \u003cem\u003ecircLDLR\u003c/em\u003e. Firstly, the \u003cem\u003ecircLDLR\u003c/em\u003e in CAFs was deleted or overexpressed, and then transfected CAFs were co-cultured with CRC cells. As expected, overexpression of \u003cem\u003ecircLDLR\u003c/em\u003e in CAFs notably facilitated the growth of CRC cells, while \u003cem\u003ecircLDLR\u003c/em\u003e deletion generated the opposite effect (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE-G). Next, qRT-PCR was utilized to determine \u003cem\u003ecircLDLR\u003c/em\u003e in CAFs and co-cultured CRC cells. As shown in Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eH, I, silencing \u003cem\u003ecircLDLR\u003c/em\u003e in CAFs notably reduced the expression of \u003cem\u003ecircLDLR\u003c/em\u003e in CAFs and co-cultured CRC cells, whereas ectopic expression of \u003cem\u003ecircLDLR\u003c/em\u003e in CAFs exhibited contrary effects. Taken together, we hypothesized that \u003cem\u003ecircLDLR\u003c/em\u003e may shuttle from CAFs to CRC cells, thereby aggravated the malignant behaviors of CRC cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExosomal\u003c/b\u003e \u003cb\u003ecircLDLR\u003c/b\u003e \u003cb\u003eshuttles from CAFs to CRC cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that recent evidence have reported CAFs could exacerbate cancer progression\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, exosomes may play an important role in crosstalk between CAFs and cancer cells\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, which inspired us to suppose whether exosomes are essential in \u003cem\u003ecircLDLR\u003c/em\u003e transfer. Exosomes in CAFs/NFs conditioned medium were purified by ultracentrifugation then confirmed by transmission electron microscopy and nanoparticle tracking analysis (NTA) (Fig.\u0026nbsp;2A). The exosomes were observed to be cup-shaped structure with diameters of 100 nm. Further, exosome markers CD81, CD63, TSG101 and Calnexin were detected by Western blotting analysis (Fig.\u0026nbsp;2B). CAFs derived exosomes were labeled with PKH67. Subsequently, PKH67-labeled exosomes were internalized by co-cultured CRC cells. (Fig.\u0026nbsp;2C). Further, CRC cells were co-cultured with CAFs transfected with cy3-tagged \u003cem\u003ecircLDLR\u003c/em\u003e. As shown in Fig.\u0026nbsp;2D, E, the fluorescently labeled \u003cem\u003ecircLDLR\u003c/em\u003e was notably increased in CRC cells, but the elevation could be abrogated by GW4869 (an inhibitor of exosomes secretion). The level of \u003cem\u003ecircLDLR\u003c/em\u003e in GW4869-treated CAFs remained stable (Fig.\u0026nbsp;2F). Additionally, in order to better characterize the biological function of \u003cem\u003ecircLDLR\u003c/em\u003e in CRC cells, \u003cem\u003ecircLDLR\u003c/em\u003e of CAFs was overexpressed or deleted, then CAFs derived exosomes were purified and incubated with CRC cells, as we expected, in ablation/overexpression group, the promoting effect on the progression of CRC cells correspondingly attenuated or potentiated (Fig.\u0026nbsp;2G, H, Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA, B), consistent with the results of CAFs co-cultured CRC cells. Further, the results of qRT-PCR revealed that overexpression of \u003cem\u003ecircLDLR\u003c/em\u003e increased levels of \u003cem\u003ecircLDLR\u003c/em\u003e in CAF exosomes and cancer cells, whereas deletion of \u003cem\u003ecircLDLR\u003c/em\u003e exerted the opposite effect (Fig.\u0026nbsp;2I, J). Taken together, these results demonstrates that CAFs-derived exosomal \u003cem\u003ecircLDLR\u003c/em\u003e can be transferred to CRC cells and facilitate progression of CRC cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCAFs-derived\u003c/b\u003e \u003cb\u003ecircLDLR\u003c/b\u003e \u003cb\u003efacilitates the progression of CRC cells by suppressing ferroptosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo better characterize of CAFs-derived \u003cem\u003ecircLDLR\u003c/em\u003e in CRC, \u003cem\u003ecircLDLR\u003c/em\u003e were deleted in CAFs, and then exosomes enriched from CAFs were co-cultured with CRC cells. Subsequently, those cells were treated with various programmed cell death inhibitors (including ferroptosis, necroptosis, apoptosis and autophagy) to explore the possible cell death pattern that modulated by \u003cem\u003ecircLDLR\u003c/em\u003e. Intriguingly, Lip-1 and Fer-1, two inhibitors of ferroptosis, reversed the increase death of CRC cells when added CAFs (sh-\u003cem\u003ecircLDLR\u003c/em\u003e)-derived exosomes, but not Nec-1(necroptosis inhibitor), Z-VAD(apoptosis inhibitor) and CQ (autophagy inhibitor) (Fig.\u0026nbsp;3A). These results\u003c/p\u003e\u003cp\u003eindicated that \u003cem\u003ecircLDLR\u003c/em\u003e of CAFs-derived exosomes might be involved in the progresssion of ferroptosis in CRC cells. Furthermore, CRC cells were treated with erastin and detected the proliferation ability, the result showed that CAFs (sh-NC)-derived exosomes and Lip-1 significantly decreased erastin-induced cell death, but not CAFs (sh-\u003cem\u003ecircLDLR\u003c/em\u003e)-derived exosomes (Fig.\u0026nbsp;3B). Additionally, we checked the concentration of the relative intracellular lipid peroxidation, mitochondrial superoxide (MitoSOX) and Mitochondrial Membrane Potential (MMP), CAFs (sh-NC)-derived exosomes and Lip-1 increased those levels of HCT116 and RKO cells (Fig.\u0026nbsp;3C-E, Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA-C). As we all known, the intracellular Fe\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;and Iron are a pool of redox-active iron and essentional for triggering oxidative damage. As shown in Fig.\u0026nbsp;3F, G and Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eD, E, Fe\u003csup\u003e2+\u003c/sup\u003e and Iron levels were higher in erastin-treated cells than control, whereas CAFs (sh-NC)-derived exosomes and Lip-1 could reverse these alterations. Transmission electron microscope (TEM) analysis further verified that CAFs (sh-NC)-derived exosomes particularly restored typical morphological features of ferroptosis, including shrunken mitochondria with elevated membrane density (Fig.\u0026nbsp;3H, Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eF). Invasion, migration and colony formation assays also validated the effect of CAFs (sh-NC)-derived exosomes (Fig.\u0026nbsp;3I, J Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eG, H). Taken together, we conclude that the \u003cem\u003ecircLDLR\u003c/em\u003e from CAFs-derived exosomes promote CRC cells growth via conferring ferroptosis resistance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircLDLR\u003c/b\u003e \u003cb\u003estabilizes GALNT14 via protecting it against ZNRF2-mediated ubiquitin/proteasome-mediated degradation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo dissect how \u003cem\u003ecircLDLR\u003c/em\u003e regulates cell ferroptosis, we first determined the subcellular localization of \u003cem\u003ecircLDLR\u003c/em\u003e by nuclear and cytoplasmic fractionation as well as FISH examination. The results indicated that \u003cem\u003ecircLDLR\u003c/em\u003e predominantly localized in the cytoplasm of CRC cells (Fig.\u0026nbsp;4A). The results of RIP assays showed that the circular RNA ciRS-7, function as competitive endogenous RNA (ceRNA), was significantly enriched by AGO2, but not \u003cem\u003ecircLDLR\u003c/em\u003e (Fig.\u0026nbsp;4B). Then, we conducted circRNA pull-down and MS assays to examine potential proteins bind with \u003cem\u003ecircLDLR\u003c/em\u003e (Fig.\u0026nbsp;4C, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Among those proteins, GALNT14 aroused our interest, and the binding between \u003cem\u003ecircLDLR\u003c/em\u003e and GALNT14 was further confirmed by RIP assays (Fig.\u0026nbsp;4D). Then, we performed IHC staining to reveal that GALNT14 presented the higher level in CRC tissue and predominantly localized in the cytoplasm (Fig.\u0026nbsp;4E). In addition, we performed RNA FISH-immunofluorescence analysis and found \u003cem\u003ecircLDLR\u003c/em\u003e co-localized with GALNT14 in the cytoplasm (Fig.\u0026nbsp;4F). When \u003cem\u003ecircLDLR\u003c/em\u003e was deleted/overexpressed in CRC cells, as expected, the expression of \u003cem\u003ecircLDLR\u003c/em\u003e was positiveiy correlated with GALNT14 protein level but not mRNA level (Fig.\u0026nbsp;4G). To further address the molecular basis underlying the interaction between \u003cem\u003ecircLDLR\u003c/em\u003e and GALNT14, we designed four mutants based on the structural composition of \u003cem\u003ecircLDLR\u003c/em\u003e (formed by backsplicing of four exons). Mutation of the \u003cem\u003ecircLDLR\u003c/em\u003e 296-466nt significantly decreased GALNT14 enrichment by \u003cem\u003ecircLDLR\u003c/em\u003e (Fig.\u0026nbsp;4H), indicating that the 296-466nt sequences is essential for the \u003cem\u003ecircLDLR\u003c/em\u003e-GALNT14 interaction. Next, we investigated the mechanism of \u003cem\u003ecircLDLR\u003c/em\u003e regulating GALNT14 level. Firstiy, CRC cells were treated with CHX to block protein synthesis for indicated times. The result of western blot revealed that \u003cem\u003ecircLDLR\u003c/em\u003e could stabilize GALNT14. (Fig.\u0026nbsp;4I). Next, we found that the effect of circLDLR depletion on GALNT14 protein levels was not affected by the lysosome inhibitor CQ but could be abolished by proteasome inhibitors MG-132 (Fig.\u0026nbsp;4J). Additionally, overexpression of wild-type circLDLR decreased the level of Ubiquitination on GALNT14, whereas overexpression of the mutant circLDLR had no effect (Fig.\u0026nbsp;4K). Thus, these findings suggest that \u003cem\u003ecircLDLR\u003c/em\u003e plays a central role in GALNT14 protein degradation through the ubiquitination/proteasome pathway. K48-, K29-and K63-linked polyubiquitin chains are three main types of polyubiquitin linkage. We constructed vectors carrying K48-only, K29-only and K63-only respectively and bearing HA tags. Ubiquitination assays suggested that K48-linked polyubiquitin of GALNT14 was reduced by \u003cem\u003ecircLDLR\u003c/em\u003e overexpression, while other polyubiquitin linkage had no evidently alteration (Fig.\u0026nbsp;4L). In sum, these findings validate that \u003cem\u003ecircLDLR\u003c/em\u003e promotes GALNT14 expression via protecting it from degradation of the K48-linked ubiquitin-proteasome pathway.\u003c/p\u003e\u003cp\u003eConsidering the E3 ubiquitin ligase is essential for the ubiquitination reaction, UbiBrowser 2.0 was applied to determine the potential E3 ligases might be involved in GALNT14 degradation (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA). Meanwhile, STARBASE database showed that ZNRF2 have a lower level in cancer than in normal tissue (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eB). Thus, we supposed that ZNRF2 is the E3 ubiquitin ligase for GALNT14. To confirm whether ZNRF2 participates in regulating the ubiquitination effect of GALNT14, CO-IP assays were conducted, and the reciprocal interaction between ZNRF2 and GALNT14 was confrmed (Fig.\u0026nbsp;4M). The results of western blot showed that ZNRF2 overexpression significantly reduced the level of GALNT14, and could be rescued by MG-132 (Fig.\u0026nbsp;4N). CRC cells were treated with CHX for indicated times, and western blot analysis was implemented. The result showed that ZNRF2 overexpression also shortened the half-life of GALNT14 protein (Fig.\u0026nbsp;4O). In addition, we transfected K63-only, K48-only, K63R, K48R mutant ubiquitin and overexpressed ZNRF2, and found that ZNRF2 only dictated the K48-linked polyubiquitin of GALNT14 (Fig.\u0026nbsp;4P). Therefore, these results suggest that ZNRF2 is the E3 ubiquitin ligase which binds with GALNT14, and degrades them through the K48-linked polyubiquitin of GALNT14.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircLDLR\u003c/b\u003e \u003cb\u003econfers ferroptosis resistance by upregulating GALNT14\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAccording to the results in Fig.\u0026nbsp;4G and 4P, \u003cem\u003ecircLDLR\u003c/em\u003e and ZNRF2 both affect the ubiquitination degradation of GALNT14 through K48-linked ubiquitination. So, to further verity whether the effect of ZNRF2 on GALNT14 protein could be influenced by \u003cem\u003ecircLDLR\u003c/em\u003e, we conducted CO-IP and PLA assays. As we expected, the interaction between ZNRF2 and GALNT14 was disrupted by \u003cem\u003ecircLDLR\u003c/em\u003e overexpression (Fig.\u0026nbsp;5A, B). \u003cem\u003eCircLDLR\u003c/em\u003e ablation could enhance the K48-linked ubiquitination of GALNT14 protein, ZNRF2 silencing could rescue the degradation GALNT14 protein caused by \u003cem\u003ecircLDLR\u003c/em\u003e ablation through the K48-linked ubiquitin\u0026ndash;proteasome pathway (Fig.\u0026nbsp;5C). To further explore whether \u003cem\u003ecircLDLR\u003c/em\u003e influenced cancer cells ferroptosis through GALNT14, CRC cells were treated with erastin. The obtained data showed that migration, invasion and colony abilities were accelerated by \u003cem\u003ecircLDLR\u003c/em\u003e, while this influence was abolished by GALNT14 deletion (Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eA-D). In parallel, several main characteristics of ferroptosis (Lipid peroxidation level, relative levels of MMP and MitoSOX, relative Fe\u003csup\u003e2+\u003c/sup\u003e and Iron levels) were changed by circLDLR overexpression, and the inhibitory effect of \u003cem\u003ecircLDLR\u003c/em\u003e on ferroptosis could be reversed by GALNT14 detection (Fig.\u0026nbsp;5D-M). Above all, these evidences concomitantly indicate that \u003cem\u003ecircLDLR\u003c/em\u003e markedly dampens the sensitivity of CRC cells to ferroptosis, which is mainly dependent on GALNT14.\u003c/p\u003e\n\u003ch3\u003eGALNT14 impairs ferroptosis via O-GalNAcylation of SLC7A11 and its membrane localization\u003c/h3\u003e\n\u003cp\u003eTo explore the molecular mechanism underlying the inhibition of ferroptosis in CRC cells by GALNT14. Firstly, we overexpressed/deleted GALNT14 in CRC cells, the level of O-GalNAcylation increased/decreased respectively (Fig. S8A, B). Then, GALNT14 overexpression stable transfection CRC cell lines were established,and then co-cultured with ferroptosis inducers of various pathways (including System Xc\u0026minus;, GCL, GPX4, and FSP1). GALNT14 overexpression was found to rescue the ferroptosis only by inducer targeting System Xc\u0026minus;, but not by inducers targeting GCL, GPX4, and FSP1 (Fig.\u0026nbsp;6A). As we all known, the uptake of cystine and the synthesis of GSH play important roles in the process of ferroptosis\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The two critical functions of System Xc\u0026thinsp;\u0026minus;\u0026thinsp;were diminished significantly by GALNT14 ablation. When cells were supplemented with the downstream products of System Xc\u0026minus;, Acetylcysteine (NAC) and GSH, ferroptosis induced by GALNT14 ablation was markedly alleviated in CRC cells (Fig.\u0026nbsp;6B, C). SLC7A11, as the most important functional subunit of System Xc\u0026minus;, proper membrane localization is essential for the functionality of SLC7A11. Previous studies have found that O-GlcNAcylation of SLC7A11 can alter its activity, but the specific mechanism remains unclear. As a member of polypeptide N-acetyl- α-galactosaminyltransferases family, GALNT14 mediated O-GalNAcylation. These findings inspired us to doubt whether SLC7A11 can be regulated by GALNT14.Western blot assay and immunofluorescence assay showed that GALNT14 did not affect the total level of SLC7A11 protein, but altered the membrane localization of SLC7A11 (Fig.\u0026nbsp;6D, E). CO-IP assays and PLA were conducted in CRC cell and 293T cell, the results confirmed that SLC7A11 could interacte with GALNT14 (Fig.\u0026nbsp;6F, G). Meanwhile, GALNT14 overexpression/deletion could significantly increase/decrease the O-GalNAcylation level of SLC7A11 (Fig.\u0026nbsp;6H). These results consistently confirmed the regulatory role of GALNT14 in the membrane localization of SLC7A11. Further, the mucin-type O-glycosylation sites of SLC7A11 were predicted using NetOGlyc-4.0. The database showed that S8, T9, S26 and T218 might be the O-GalNAcylation sites of SLC7A11, meanwhile, N314, predicted using NetNGlyc-1.0, might be the N-glycosylation site (Fig.\u0026nbsp;6I, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Then, S8R, T9R, S26R, T218R mutation vectors were constructed respectively and generating HA-tags, N314R mutation vectors serve as conrol. CO-IP and VVL lectin blot experiments verified that only S26R reduced the interaction of SLC7A11and GALNT14 (Fig.\u0026nbsp;6J). Then, a serious of assays with these mutant O-GalNAcylation sites of SLC7A11 in GALNT14-OE and SLC7A11-KO stably transfected cells were performed. As we expected, the proliferation, migration and invasion experiments showed that only S26 mutant site could not reverse the promoting effect of ferroptosis initiated by SLC7A11-KO (Fig. S9A, B). CRC cells transfected with S26R also could not rescue the O-GalNAcylation level of SLC7A11 and the level of GSH and Cystine uptake which reduced by SLC7A11-KD (Fig.\u0026nbsp;6K-M). Western blot and immunofluorescence assays showed that S26 was the key site on the membrane localization of SLC7A11 (Fig.\u0026nbsp;6N, O). Collectively, the results illustrate that GALNT14 induces O-GalNAcylation of SLC7A11 at the S26 site to promote its membrane localization, and further confers ferroptosis resistance in CRC cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBiogenesis of\u003c/b\u003e \u003cb\u003ecircLDLR\u003c/b\u003e \u003cb\u003eis Facilitated by EIF4A3\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrior research had demonstrated that the biogenesis of exon-derived circRNAs can be regulated by RBPs through their interactions with flanking intron sequences of circRNAs\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. EIF4A3, QKI and FUS had been shown to facilitate circRNA biogenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To investigate its potential role in \u003cem\u003ecircLDLR\u003c/em\u003e formation, we employed CircInteractome to predict RBP-binding sites within the flanking regions of \u003cem\u003ecircLDLR\u003c/em\u003e. We found 7 putative binding sites for EIF4A3 in the downstream of the \u003cem\u003ecircLDLR\u003c/em\u003e pre-mRNA (Fig. S10A). Due to sequence overlaps, these sites were named as \u003cem\u003ea-c\u003c/em\u003e, and the sequence on \u003cem\u003ecircLDLR\u003c/em\u003e named as \u003cem\u003ed\u003c/em\u003e (Fig. S10B). This binding specificity was further validated by RNA pull-down assays (Fig. S10C). Notably, EIF4A3 overexpression significantly upregulated \u003cem\u003ecircLDLR\u003c/em\u003e expression in CAFs, while its deletion reduced \u003cem\u003ecircLDLR\u003c/em\u003e levels (Fig. S10D). To further explore the expression of EIF4A3 in CRC patients, an IHC assay was conducted CRC tissues and adjacent normal tissues, the result revealed that the expression of EIF4A3 was higher in CRC tissues than in adjacent normal tissues (Fig. S10E) and that the expression of EIF4A3 and \u003cem\u003ecircLDLR\u003c/em\u003e was positively correlated in CRC patients (Fig. S10F).Collectively, these results establish that EIF4A3 as a positive regulator of \u003cem\u003ecircLDLR\u003c/em\u003e biogenesis through direct binding to its flanking intronic regions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircLDLR\u003c/b\u003e \u003cb\u003ein CAFs promotes cancer growth and metastasis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the impact of CAF-exosomes \u003cem\u003ecircLDLR\u003c/em\u003e on cancer progression \u003cem\u003ein vivo\u003c/em\u003e, two human CRC cell lines were employed to establish tumor model. In subcutaneous xenograft model, \u003cem\u003ecircLDLR\u003c/em\u003e stably overexpressed and deleted CAFs were constructed. Then, CRC cells were co-injected with or without CAFs into the nude mice subcutaneously. As shown in Fig.\u0026nbsp;7A, B, co-transplanted with CAFs accelerated subcutaneous tumor growth. This pro-tumorigenic effect was enhanced when co-transplanted with CAFs OE-\u003cem\u003ecircLDLR\u003c/em\u003e, whereas co-transplanted with CAFs KD-\u003cem\u003ecircLDLR\u003c/em\u003e displayed the opposite effect. We further investigated the effect of \u003cem\u003ecircLDLR\u003c/em\u003e on CRC metastasis \u003cem\u003ein vivo\u003c/em\u003e. CAFs OE-\u003cem\u003ecircLDLR\u003c/em\u003e further accelerated the liver metastasis of CRC cells \u003cem\u003ein vivo\u003c/em\u003e, as indicated by more tumor nodules, and more serious colorectal tissue damage (Fig.\u0026nbsp;7C). However, CAFs KD-\u003cem\u003ecircLDLR\u003c/em\u003e reversed the lung metastasis of CRC cells \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;7D). In addition, we performed IHC and HE assays. IHC staining showed the result that CAFs sh-circ/CAFs OE-circ decreased/increased the expression of Ki67 and GALNT14 in subcutaneous tumors, whereas the 4HNE exhibited the opposite level (Fig.\u0026nbsp;7E-G). Moreover, HE staining confirmed the morphology of tumor tissues in lung and liver metastasis groups (Fig.\u0026nbsp;7H). Collectively, these results revealed that \u003cem\u003ecircLDLR\u003c/em\u003e is essential for CAFs to potentiate the growth and metastasis of CRC \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAs key cellular components of TME, CAFs play a pivotal role in the TME. Accumulating studies have revealed that CAFs can regulate tumor growth and metastasis through the secretion of non-coding RNAs\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Ferroptosis, as an iron-dependent form of regulated cell death, plays a critical role in colorectal cancer progression. Emerging evidence suggests that CAFs are involved in tumor progression, but the precise mechanisms in regulating the interplay between CAFs and ferroptosis remain elusive. In this study, we demonstrate that EIF4A3-facilitated \u003cem\u003ecircLDLR\u003c/em\u003e is predominantly shuttled from CAFs to CRCcells via exosomes, subsequently, \u003cem\u003ecircLDLR\u003c/em\u003e protects against ferroptosis by impairing SLC7A11 membrane localization in CRC cells, thereby facilitating tumor progression and metastasis.\u003c/p\u003e\u003cp\u003eWith the advancs of research, their functional roles of circRNA in cellular processes have been increasingly elucidated, particularly their critical function of tumor progression and oncogenesis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. A substantial body of research has established that CAFs-derived exosomes influence the progression of various cancers\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Notably, exosome-packaged circRNAs contribute significantly to these regulatory effects\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Beyond functioning as ceRNAs, increasing lines of evidence have demonstrated that circular RNAs can directly bind to proteins and regulate the expression of their target proteins\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Here, GALNT14 was identified as a promising target of \u003cem\u003ecircLDLR\u003c/em\u003e. We further demonstrated that \u003cem\u003ecircLDLR\u003c/em\u003e binds GALNT14 through its specific functional domain, thereby disrupting the interaction between GALNT14 and the E3 ubiquitin ligase ZNRF2, decelerating ZNRF2-mediated K48-linked polyubiquitination and degradation, ultimately leading to elevated GALNT14 expression. Numerous evidences highlight RBPs as critical regulators of circRNA biogenesis. As a key RBP, Eukaryotic Translation Initiation Factor 4A3 (EIF4A3) has been shown to modulate multiple circRNAs\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In this study, bioinformatic predictions identified \u003cem\u003ecircLDLR\u003c/em\u003e as a potential EIF4A3 target. In CAFs, we validated EIF4A3 positive regulation of \u003cem\u003ecircLDLR\u003c/em\u003e. However, whether EIF4A3 directly modulates \u003cem\u003ecircLDLR\u003c/em\u003e back-splicing remains to be further elucidated.\u003c/p\u003e\u003cp\u003eO-linked glycosylation is one common type of Glycosylation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Aberrant glycosylation endows tumor cells with resistance to cell death. Whereas most studies have focused on the role of O-GlcNAcylation in tumors, the function of O-GalNAcylation (mucin-type O-glycosylation) in tumor progression remains largely ignored. Although the two modifications belong to O-linked glycosylation, certain functional differences exist between the two modifications, both modifications target serine or threonine residues. GALNT14 is a pivotal O-glycosyltransferase implicated in tumor progression through its regulation of protein O-glycosylation in various cancers\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In HCC, GALNT14-mediated O-GalNAcylation at serine-161 of PHB2 activates the IGF1R cascade, thereby enhancing cell proliferation, migration, and therapy resistance\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Similarly, in LUAD, GALNT14-driven O-GalNAcylation suppresses endogenous ROS production to promote tumor aggressiveness\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Previous studies have confirmed that ferroptosis, play critical roles in the initiation and progression of colorectal cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In this study, we identified SLC7A11 as a novel O-GalNAcylated substrate of GALNT14. While prior research primarily focused on expression levels of SLC7A11 (unlike FSP1 studies that emphasized N-myristoylation-dependent membrane localization for ferroptosis regulation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e), emerging evidence now highlights the critical role of SLC7A11 membrane trafficking in ferroptosis regulation\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Recent report indicate that O-GlcNAcylation sustains SLC7A11 activity to suppress ferroptosis\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, even if the mechanistic basis remains unclear. Intriguingly, our work reveals that GALNT14 mediates O-GalNAcylation at S26 of SLC7A11, promoting its membrane localization and functional activation without altering total SLC7A11 abundance\u0026mdash;thereby inhibiting ferroptosis and driving CRC progression. This discovery diverges from the canonical paradigm of glycosylation-mediated protein stabilization in ferroptosis regulation, instead establishing a novel mechanism where O-GalNAc modification orchestrates SLC7A11 spatial redistribution to modulate ferroptosis susceptibility. Our finding expands the understanding of O-GalNAcylation in ferroptosis regulation and provide new directions for targeting SLC7A11 membrane dynamics in cancer therapy.\u003c/p\u003e\u003cp\u003eIn conclusion, our study reveals a novel CAF-to-CRC signaling axis: EIF4A3 upregulates \u003cem\u003ecircLDLR\u003c/em\u003e in CAFs, where \u003cem\u003ecircLDLR\u003c/em\u003e is packaged into exosomes and transferred to CRC cells. Within CRC cells, \u003cem\u003ecircLDLR\u003c/em\u003e binds and stabilizes GALNT14 by protecting against ZNRF2-mediated ubiquitination and degradation. Then, the accumulated GALNT14 catalyzes O-GalNAcylation at S26 site of SLC7A11, promoting its membrane localization to inhibit ferroptosis\u0026mdash;ultimately driving CRC growth and metastasis. These findings highlight the importance of in tumor-stromal interactions and provide a rational basis for developing targeted therapies to disrupt the CAFs-CRC cells crosstalk.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eHuman HCT116, HT29, RKO, LOVO, SW620, SW480 and HEK 293T cells were purchased from Procell (Wuhan,China). Human NCM460 cell was purchased from Fuheng Biology (Shanghai, China). Human HCT116 and HT29 cell lines were cultured in McCoy\u0026rsquo;s 5A Complete medium (Procell, #PM150710B). Human RKO cell line was cultured in MEM medium (Gibco,#11095080). Human LOVO cell line were cultured in Ham\u0026rsquo;s F-12K complete medium (Procell, #CM-0144). SW620, SW480, HEK 293T and NCM460 cells were cultured in DMEM medium (Gibco,#11965092). MEM and DMEM medium plus 10% fetal bovine serum (FBS) (TIANHANG, #13011\u0026thinsp;\u0026minus;\u0026thinsp;8611) and 1% penicillin/streptomycin (SEVEN BIOTECH, #SC118-01). Cell lines were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Mycoplasma contamination was confirmed to be negative in cells. After thawing the frozen stocks, cells were generally passaged fewer than 10 times.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHuman CRC tissue specimens\u003c/h3\u003e\n\u003cp\u003ePrimary CRC specimens and paired non-cancerous tissues were obtained from the First Affiliated Hospital of Harbin Medical University. The characteristics of the patients are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University, and written informed consent was provided by the patients.\u003c/p\u003e\n\u003ch3\u003eIsolation and culture of patient-derived NFs and CAFs\u003c/h3\u003e\n\u003cp\u003eCAFs were isolated from CRC tissues of patients, and NFs were separated from the paired adjacent normal area. Briefly, the fresh paired tissues were washed three times with PBS containing 100 U/ ml penicillin and 100 \u0026micro;g/ml streptomycin. Then, the tissues were chopped into small pieces and digested in collagenase II (Gibco, #17101015) at 37℃. All cells were collected and resuspended in DMEM/F12 medium (Gibco, #11320033) containing 15% FBS. The suspensions were fostered in DMEM/F12 medium supplemented with 15% FBS for about 48 hours. After stable adherence of the cells, DMEM/F12 medium was used to wash out the excess tissues and dead cells, then DMEM/F12 containing 15% FBS was added for subsequent cell culture. Cell morphology was measured by microscopical measurement, and fibroblast identity was verified by detection of specific markers (α-SMA, FAP, and vimentin). All primary fibroblasts used in this study were less than three passages.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCo-culture assay\u003c/h2\u003e\u003cp\u003eFor co-culture experiments, CAFs or NFs were cultured with CRC cells using 0.4\u0026micro;m Transwell inserts (Corning, USA). Specifically, 6\u0026times;10⁴ CRC cells were seeded into the lower chamber, while 2\u0026times;10⁴ CAFs/NFs were plated in the upper chamber. After 48 hours of co-culture, CRC cells from the lower chamber were collected for following analyses.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIsolation and identification of exosomes\u003c/h3\u003e\n\u003cp\u003eCAFs or NFs were cultured in serum-free DMEM/F12 medium for 48 hours to generate conditioned medium (CM). The CM was sequentially centrifuged at 4\u0026deg;C: first at 2,000 \u0026times; g for 30 min (to remove dead cells), then at 10,000 \u0026times; g for 30 min (to eliminate cell debris and large vesicles), and finally at 120,000 \u0026times; g for 70 min (to pellet exosomes). The resulting pellet was washed with 1\u0026times; PBS to reduce protein contamination, and filtered with 0.22-\u0026micro;m filters. Purified vesicles were characterized by Nanoparticle tracking analysis (NTA) using ZetaView PMX 110 ((Particle Metrix, Meerbusch, Germany) for size distribution,Transmission electron microscopy (FEI Tecnai G2 Spirit, Thermo Scientific, USA) for morphological validation, and Western blotting of exosomal markers.\u003c/p\u003e\n\u003ch3\u003eExosomes labeling and tracing\u003c/h3\u003e\n\u003cp\u003eIsolated exosomes were fluorescently labeled with PKH67 (MCE, #257277-27-3), then added to the culture supernatant. After 24 hours of co-incubation with CRC cells, the nuclei were stained\u003c/p\u003e\u003cp\u003ewith DAPI (Beyotime, #P0131), and exosome uptake was evaluated using confocal microscopy (Zeiss, Germany).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eFluorescence in situ hybridization (FISH)\u003c/h2\u003e\u003cp\u003eCellular localization of \u003cem\u003ecircLDLR\u003c/em\u003e was determined using FISH probe and kits (RiboBio, Guangzhou, China). Briefly, fixed and permeabilized cells were incubated with prehybridization buffer for 30 min at 37\u0026deg;C. Cy3-labeled \u003cem\u003ecircLDLR\u003c/em\u003e probe was mixed with pre-warmed hybridization buffer and incubated with cells overnight at 37\u0026deg;C in the dark. Post-hybridization washes were performed with wash buffer and PBS. Nuclei were stained with DAPI, and images were analyzed using confocal microscopy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eProximity ligation assay (PLA)\u003c/h2\u003e\u003cp\u003eThe DuoLink\u0026reg; In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma-Aldrich) was used to detect interacting proteins. Briefly, CRC cells were fixed with 4% paraformaldehyde. The cells were washed with PBS and permeabilized using 0.1% Triton X-100 in PBS for 20 min. After blocking, the cells were incubated with primary antibodies at 4\u0026deg;C overnight. The subsequent step was incubating the pre-diluted anti-rabbit plus and anti-mouse minus probes at 37\u0026deg;C for 1h. Then, cells were incubated with 1\u0026times; ligase for 30 min and 1\u0026times; polymerase for 100 min at 37\u0026deg;C. Finally, coverslips were mounted on the slide with Duolink\u0026reg; In situ Mounting Medium with DAPI.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot assay\u003c/h2\u003e\u003cp\u003eProteins extracted from cells or exosomes were separated by 10% SDS-PAGE and transferred onto 0.22\u0026micro;m PVDF membranes. Membranes were blocked with 5% fat-free milk for 1 hour at room temperature, followed by incubation with primary antibodies at 4\u0026deg;C overnight. After three washes with TBST, membranes were probed with species-matched secondary antibodies for 1 hour at room temperature. Following three additional TBST washes to remove nonspecific binding, target proteins were visualized using an Odyssey infrared imaging system (LI-COR Biosciences, USA). For lectin blot analysis, membranes were incubated with 20 \u0026micro;g/mL Vicia Villosa Lectin (VVL; Vector Labs, #B-1235-2) or Peanut Agglutinin (PNA; Vector Labs, #B-1075-5) in PBS for 30 min at room temperature. After wash with TPBS (PBS containing 0.05% Tween-20), membranes were incubated with ABC-HRP kit for 30 min at room temperature. Following additional TPBS washes, target glycoproteins were detected using enhanced chemiluminescence (ECL). The primary and secondary antibodies are listed in Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eImmunoprecipitation (IP) and co-immunoprecipitation (CO-IP)\u003c/h2\u003e\u003cp\u003eTotal cellular proteins were extracted using Cell Lysis Buffer for Western and IP (Beyotime, #P0013). Firstly, 500 \u0026micro;L of antibody working solution or normal IgG working solution was added and incubated with A/G magnetic beads (MCE, #HY-K0202) for 1 hour at room temperature on a rotary mixer. Subsequently, 2 mg of total protein was incubated overnight at 4\u0026deg;C with protein A/G magnetic beads or protein-agarose bound VVL (Vectorlabs, #AL-1233). The magnetic beads or agarose beads were then washed three times with TBST, and the bound proteins were eluted using loading buffer for Western blot analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eRNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA from tissues and cells was isolated using TRIzol reagent (Invitrogen), while exosomal RNA was isolated with the miRNeasy Mini Kit (QIAGEN, #217004). Their complementary DNA\u003c/p\u003e\u003cp\u003e(cDNA) synthesis was performed using the PrimeScript RT reagent kit (TaKaRa, Shiga, Japan). qPCR was carried out with FastStart Universal SYBR Green Master Mix (ROX) (Roche, Switzerland) and primers (sequences in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), using GAPDH as the endogenous control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry (IHC) and hematoxylin\u0026ndash;eosin (H\u0026amp;E) staining\u003c/h2\u003e\u003cp\u003eTissue samples were fixed in 4% paraformaldehyde (PFA), embedded, sectioned, and subsequently stained with various antibodies, including: Ki67(proteintech, #27309), GALNT14 (proteintech, #16939), 4HNE (Abmart, #PC6313) for IHC. For metastatic lesion analysis, liver and lung tissues were similarly fixed, embedded, sectioned, and stained with H\u0026amp;E. All images were captured using an Olympus microscope and processed using NDP.view2 imaging software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCCK-8 assay\u003c/h2\u003e\u003cp\u003eCell viability was measured using the CCK-8 assay. Briefly, cells were seeded in 96-well plates at a density of 3\u0026times;10\u0026sup3; cells per well. At indicated time points, 10 \u0026micro;L of CCK-8 solution (SEVEN BIOTECH, #SC119) was added to each well, followed by incubation at 37\u0026deg;C with 5% CO₂ for 4 hours. Absorbance at 450 nm (OD₄₅₀) was measured using a microplate reader. Cell viability was assessed daily through OD₄₅₀ measurements.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eColony formation assay\u003c/h2\u003e\u003cp\u003eCRC cells were seeded at a density of 1500 cells/well into six-well plates for 2 weeks. Thereafter, the colonies were stained with 0.1% crystal violet and counted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMigration and invasion assays\u003c/h2\u003e\u003cp\u003eFalcon Transwell inserts (8-\u0026micro;m pore, Corning, USA) were used for migration assays. CRC cells were serum-starved for 24 hours, trypsinized, and resuspended in serum-free medium. Cells (4\u0026times;10\u003csup\u003e4\u003c/sup\u003e in 200 \u0026micro;L serum-free medium) were seeded into the upper chamber of Transwell inserts. The lower chamber contained 700 \u0026micro;L medium with 10% FBS as chemoattractant. After 24 hours, non-migrated cells on the upper membrane surface were removed with cotton swabs. Migrated cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified. For the invasion assay, cells were inserted into the upper chamber of the Matrigel invasion chamber and subsequently treated as described for the transwell migration assay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eLipid Peroxidation measurement\u003c/h2\u003e\u003cp\u003eLipid Peroxidation levels were assessed using the Lipid Peroxidation Assay Kit with BODIPY 581/591 C11 (Beyotime, #S0043S). Approximately 2 \u0026times; 105 transfected cells/well were seeded in twelve-well plates overnight. Thereafter, cells were treated with 10\u0026micro;M erastin (MCE, #HY-15763) and 1\u0026micro;M liproxstatin-1 (Lip-1) (MCE, #HY-12726)for 48 h.BODIPY 581/591 C11 was added to cells in the dark for 20 min at 37\u0026deg;C, followed by washing with PBS. Thereafter, fluorescence imaging was performed using a ZEISS confocal microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eIron assay\u003c/h2\u003e\u003cp\u003eTo quantify the intracellular levels of total iron and ferrous iron, an iron assay kit (APPLYGEN, #E1042) and a Ferrous Ion Assay Kit (Beyotime, #S1070S) were used according to the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eMitochondrial membrane potential (MMP) measurement\u003c/h2\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eThe MMP was measured using theJC-1 Mitochondrial Membrane Potential Assay Kit\u003c/h2\u003e\u003cp\u003e(MCE, #HY-K0601), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eMitochondrial superoxide measurement\u003c/h2\u003e\u003cp\u003eMitochondrial superoxide levels were determined using MitoSOX Red mitochondrial superoxide indicator for live-cell imaging (MCE, #HY-D1055), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eRNA pull-down assay\u003c/h2\u003e\u003cp\u003eBiotinylated full-length RNA probes and fragmented \u003cem\u003ecircLDLR\u003c/em\u003e probes were synthesized by GenePharma Co., Ltd (Shanghai, China). Streptavidin Magnetic Beads (MCE, #HY-K0208) were pre-cleared by incubating with transfected cell lysates for 1 hour at 4\u0026deg;C to reduce non-specific binding. Biotinylated probes were then incubated with the beads for 60 minutes at room temperature to form probe-bead complexes. These complexes were subsequently incubated with transfected cell lysates overnight at 4\u0026deg;C. After magnetic separation, the beads were washed five times with buffer. RNA-protein complexes were eluted and analyzed by Western blotting. The probes used in RNA pull-down are provided in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eRNA immunoprecipitation (RIP)\u003c/h2\u003e\u003cp\u003e RIP experiments were performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer\u0026rsquo;s instructions. Co-precipitated RNA was detected by qRT-PCR. The primers used in RIP are provided in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eMice xenograft model\u003c/h2\u003e\u003cp\u003e All experimental procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital of Harbin Medical University. Female 4\u0026ndash;6 weeks old BALB/c nude mice were purchased from Huachuang Sino Pharma Tech Co., Ltd (Jiangsu, China) and maintained under SPF conditions in a controlled environment of 12 h of light and darkness, 50\u0026ndash;70% humidity, 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and animals had free access to food and water. Mice were randomly grouped (n\u0026thinsp;=\u0026thinsp;6 per group) after 1 week of acclimation, and the luciferase-labeled lentivirus was transfected into CAFs and selected with puromycin (2\u0026micro;g/ml) for 10 days. CRC (3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) cells either alone or mixed with CAFs (Vector/sh-\u003cem\u003ecircLDLR\u003c/em\u003e/OE-\u003cem\u003ecircLDLR\u003c/em\u003e) at the ratio of 1:1 in 200 \u0026micro;l PBS were subcutaneously injected into nude mices. Tumors were measured every 5 d for 20 days, volume (mm\u003csup\u003e3\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;length \u0026times; width\u003csup\u003e2\u003c/sup\u003e \u0026times; 0.52. The mice were sacrificed after 20 days and the tumors were excised and weighed.\u003c/p\u003e\u003cp\u003eFor the lung metastasis models, Luciferase-labeled 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e HCT116 either alone or mixed with CAFs (Vector/sh-\u003cem\u003ecircLDLR\u003c/em\u003e) at the ratio of 1:1 in 100 \u0026micro;l PBS were injected into the tail veins of nude mice (n\u0026thinsp;=\u0026thinsp;6/group). The mice were euthanized 6 weeks later, and the lungs were surgically dissected for further investigation.\u003c/p\u003e\u003cp\u003eTo establish a liver metastasis models, Luciferase-labeled 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e RKO either alone or mixed with CAFs (Vector/OE-\u003cem\u003ecircLDLR\u003c/em\u003e) at the ratio of 1:1 in 200 \u0026micro;l PBS were injected into the spleens of nude mice. Briefly, nude mice (n\u0026thinsp;=\u0026thinsp;6/group) were anesthetized, and a lateral incision was made to expose the spleen. Mixed cells were resuspended in 200 \u0026micro;L PBS and injected into the spleen using a microsyringe. The wounds were closed with 4\u0026thinsp;\u0026minus;\u0026thinsp;0 sutures. The mice were euthanized, and liver samples were excised for further analysis after 3 weeks.\u003c/p\u003e\u003cp\u003eBefore the lung and liver metastasis model mice were euthanized, the IVIS Lumina imaging station (Caliper Life Sciences, Hopkinton, MA, USA) was used for bioluminescence imaging after intraperitoneal injection of 150 \u0026micro;l D-luciferin (15mg/ml) (Beyotime, #ST196). Lastly, the primary tumors of subcutaneous xenograft models, lungs and livers of metastasis models were obtained and fixed with 4% formalin. The level of ki67, GALNT14 and 4HNE were detected by IHC. The metastatic area in the lungs and livers were carefully examined by H\u0026amp;E staining.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using GraphPad Prism 8.0 (San Diego, CA, USA). Bioinformatics analysis was performed with R (version 4.2.1) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.R-project.org/\u003c/span\u003e\u003cspan address=\"http://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The number of biological replicates for each experiment was indicated in the corresponding figure legend. All values represented the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (S.D) and were derived from a minimum of three independent biological replicates. \u003cem\u003eP\u003c/em\u003e values are calculated by unpaired two-sided \u003cem\u003et\u003c/em\u003e-test. In figures, statistical comparisons between the control group and the experimental group are denoted as follows: ns no significance, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author on request.\u003c/p\u003e\u003c/div\u003e\u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003cp\u003e This study was approved by the institutional ethics review board of the First Affiliated Hospital of Harbin Medicine University (no. IRB-AF/SC-04/02.2) and by the Animal Ethics Committee of the First Affiliated Hospital of Harbin Medicine University (no. 2023089). Participants gave informed consent to participate in the study before taking part.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e\u003cp\u003eH.Y.P supervised the project. H.Y.P and J.L.G conceived and designed the study. J.L.G, L.F.G and S.N performed experiments. J.L.G and W.X.Z contributed to data analysis and generated figures. H.Y.P and J.L.G wrote the manuscript. All authors reviewed and approved the manuscript for submission.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 82303270), the Excellent Young Medical Talents Training Fund of the First Affiliated Hospital of Harbin Medical University (Grant No. 2021Y06), Heilongjiang Postdoctoral Scientific Research Developmental Fund (Grant No. LBH-Q18089), the University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (Grant No. UNPYSCT-2018070), Chen Xiao-Ping Foundation For The Development Of Science And Technology Of Hubei Province (Grant No. CXPJJH121001-2021029). The Open Fund of Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Harbin, China (Grant No. GPKF202507)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDekker, E, Tanis, PJ, Vleugels, J, Kasi, PM, Wallace, MB. Colorectal cancer. 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Targeting USP8 Inhibits O-GlcNAcylation of SLC7A11 to Promote Ferroptosis of Hepatocellular Carcinoma via Stabilization of OGT. Adv Sci (Weinh) 2023;10:e2302953.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8266112/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8266112/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs), the dominant stromal component, actively shape cancer progression through exosomal communication. Here, we identify \u003cem\u003ehsa_circ_0003892\u003c/em\u003e (\u003cem\u003ecircLDLR\u003c/em\u003e), a CAF-derived circRNA, as a key factor linked to poor prognosis in colorectal cancer (CRC). During CAF–CRC interaction, \u003cem\u003ecircLDLR \u003c/em\u003eis packaged into exosomes and transferred to tumor cells, enhancing proliferation and metastasis largely by reducing ferroptosis susceptibility. Mechanistic exploration revealed that \u003cem\u003ecircLDLR \u003c/em\u003estabilizes Polypeptide N-Acetylgalactosaminyltransferase 14 (GALNT14) by shielding it from ZNRF2-mediated ubiquitin degradation, leading to elevated GALNT14 protein levels. Elevated GALNT14 promotes O-GalNAcylation of Solute Carrier Family 7 Member 11 (SLC7A11) at Ser26, facilitating its membrane localization, thereby suppressing ferroptosis in CRC cells. Moreover, EIF4A3, an RNA-binding protein (RBP), contributes to \u003cem\u003ecircLDLR \u003c/em\u003ebiogenesis within CAFs. Taken together, our study reveals that CAFs-derived \u003cem\u003ecircLDLR \u003c/em\u003ecan confer ferroptosis resistance and boost the progression of CRC, which are mainly dependent on increasing the stability of GALNT14 and enhancing O-GalNAcylation-mediated membrane localization of SLC7A11, thus, disrupting circLDLR transfer between CAFs and CRC cells may offer a promising approach for CRC therapy.\u003c/p\u003e","manuscriptTitle":"CAFs-derived exosomes inhibits ferroptosis via GALNT14-mediated O-GalNAcylation of SLC7A11 in colorectal cencer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 18:50:09","doi":"10.21203/rs.3.rs-8266112/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-02-04T14:24:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-02T20:09:01+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-29T00:36:31+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-20T16:10:33+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-12T16:17:50+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-12-08T04:35:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-03T14:23:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-03T05:23:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2025-12-03T05:23:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"120df093-c3bb-4ec8-87c4-bced505415a8","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":59244428,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer"},{"id":59244429,"name":"Biological sciences/Cancer/Cancer microenvironment"}],"tags":[],"updatedAt":"2026-04-18T06:20:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-10 18:50:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8266112","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8266112","identity":"rs-8266112","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}